WO2017066845A1 - Organisms with modified growth and performance characteristics and methods of making them - Google Patents

Abstract

Disclosed are methods and constructs for improving plant growth characteristics by inhibiting or abrogating expression in a plant of a plant defense-related gene. Also disclosed are plants having modulated expression of a plant defense-related gene, which plants have an improved growth characteristic relative to a corresponding control plant.

Description

TITLE OF THE INVENTION

"ORGANISMS WITH MODIFIED GROWTH AND PERFORMANCE CHARACTERISTICS AND METHODS OF MAKING THEM"

FIELD OF THE INVENTION

[0001] This application claims priority to Australian Provisional Application No.

2015904348 entitled "Organisms with modified growth characteristics and methods of making them" filed 23 October 2015, the contents of which are incorporated herein by reference in their entirety.

[0002] This invention relates generally to methods and constructs for improving plant growth characteristics by inhibiting orabrogating expression in a plant of a plant defense-related gene. The present invention also concerns plants having modulated expression of a plant defense- related gene, which plants have an improved growth characteristic relative to a corresponding control plant.

[0003] Bibliographicdetails of various references referred to by number herein are listed at the end of the specification.

BACKGROUND OF THE INVENTION

[0004] In view of the rapidly growing world population, a majorgoal and challenge of agricultural research is to increase the efficiency of agriculture. Classical genetics and cultivation methods have played a significant role in developing new plant varieties with improved economic, agronomic and horticultural traits and these methods have been supplemented in recent times by biotechnological techniques.

[0005] A trait of particular economic interest is increased yield. Yield is normally defined as the measurable produce of economic value from a crop. Individual plant parts directly contribute to yield based on their number, size and/or weight and quality. A range of factors may influence yield, examples of which include the number and size of the organs, vegetative bio mass (root and/orshoot biomass), reproductive organs, and/orto propagules (such as seeds) of a plant, plant architecture (for example, the number of branches), seed production and leaf senescence yield. Root development, nutrient uptake, pathogen resistance, stress tolerance and early vigor may also be important factors in determining yield. Optimizing the abovementioned factors may therefore contribute to increasing crop yield.

[0006] Seed yield, whether assessed by seed size, seed mass orseed number, is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. They are also important for industrial processesthat rely sugars, oils and many metabolites derived from seeds.

[0007] Early vigor is another important trait. Plants having early vigor show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (with the crop growing in uniform manner, i.e., with the majority of plants reaching the various stages of development at substantially the same time), and often betterand higher yield.

Therefore, early vigor may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.

[0008] Increased biomass is also an economically important trait. Plant biomass is yield for forage crops like alfalfa, silage corn and hay. Many proxies foryield have been used in grain crops. Chief amongst theseare estimates of plantsize. Plantsizecan be measured in many ways depending on speciesand developmental stage, but include total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, flower size, fruit size, rosette diameter, leaf length, root length, root mass, tiller number and leaf number. Plant size at an early developmental stage will typically correlate with plant size later in development. A larger plant with a greater leaf area can typically absorb more light and carbon dioxide than a smaller plantand therefore will likely gain a greater weight during thesame period. There is a strong genetic component to plant size and growth rate, and thus for a range of diverse genotypes plant size underone environmental condition is likely to correlate with size underanother. In this way a standard environment is used as a proxy forthe diverse and dynamic environments encountered at different locations and times by crops in the field.

[0009] Crop yield may therefore be increased by optimizing one or more of the above- mentioned factors.

SUMMARY OF THE INVENTION

[0010] The present invention arises in part from the determination that laboratory

(LAB) strains of Nicotiana benthamiana, which are susceptible to infection by a vast range of viruses, have significantly increased early vigor and increased seed size, as compared to N.

benthamiana strains that are resistant to those viruses. In particular, the present inventors have found that this increased susceptibility to viral infection and increased early vigor in the LAB strain results from a loss of function mutation of an endogenous plant defense-related gene for RNA- dependent RNA polymerase (RDR), which is not shared by the viral resistant strains. Surprisingly, the present inventors have also found that silencing a functional allele of an RDR gene in a wild strain of N. benthamiana not only renders the plant susceptible to viral infection but also significantly results in increased early vigor, increased yield, increased seed size, increased leaf size, increased flower size, increased trichomes, increased plant bio mass, and increased growth rate of the plant, relative to no n -genetically modified control plants of the wild strain. Based on these findings, the present inventors propose that production of genetically modified plants with loss or inhibition of expression of a plant defense-related gene such as a RDR gene, will result in a marked enhancement of at least one growth characteristic of the plant, as described hereafter.

[0011] Accordingly, in one aspect, the present invention provides methodsfor improving a growth characteristic of a plant, as compared to a control plant. These methods generally comprise, consist orconsist essentially of inhibiting expression in the plant of at least one plant defense-related (PDR) gene and/or inhibiting activity of an expression product (e.g., RNA or polypeptide) of the at least one PDR gene. Representative PDR genes include genes involved in wounding and programmed cell death, pathogenesis resistance (PR), RNA interference, salicylic- acid -media ted defenses, jas mo nic-acid-dependent defenses or ethylene-dependent responses, genes linked and/or regulated byabscisicacid (ABA), flavonoid biosynthesis, auxin, cytokinin, brassinosteroids and/or gibberellins as well as disease resistance (R) genes. In specific embodiments, the PDR gene is an RNA-dependent RNA polymerase (RDR) gene, non-limiting examples of which include RDR1, RDR2, RDR3, RDR5 and RDR6, preferably RDR 1. Suitably, the methods comprise introducing a genetic modification in the genome of the plant, which results in partial orcomplete loss of function of the PDR gene. The genetic modification may be achieved using any suitable technique, non-limiting examples of which include site-directed mutagenesis, random chemical mutagenesis, transposon mutagenesis, T-DNA insertion, homologous recombination, targeted induced local lesions in genomes (TILLING) and genome editing. In specific embodiments, the genetic modification comprises introducing into the genome of the plant a modulator nucleic acid sequence encoding an expression product that inhibits expression of the PDR gene, orthat inhibits activity of an expression product of the PDR gene. In illustrative examples of this type, the expression product is a functional nucleicacid (e.g., siRNA, shRNA, miRNA, nucleic acid aptamers, ribozymes, riboswitches, Ul adaptors, molecular beacons, transcriptional factor-binding regions, etc.) that inhibits expression of the PDR gene. In other illustrative examples, the expression product is a PDR-inhibiting protein (e.g., an antibodyor antibody fragment that binds to and inhibits the activity of the PDR). The improved growth characteristic is suitablyselectedfromanyone ormore of increased vigor, increased yield, increased seed size, increased leaf size, increased flowersize, increased trichomes, increased plant bio mass, and increased growth rate relative to the control plant. Suitably, the increased vigor includes increased early vigor. The increased seed size may include an increased surface area, volume, or weight of seed. Suitably, the increased plant bio mass includes any one or more of increased plant size, increased stalk size, increased fruit size, increased root size and increased leaf size. The increased growth rate suitably includes increased growth rate of a plant at one ormore stages of plant development, including embryonic stage, seedling stage, vegetative stage, juvenile stage, reproductive stage, and ripening stage or plant development. In some embodiments, the methods further comprise selecting a plant having an improved growth characteristic relative to the control plant.

[0012] Another aspect of the present invention provides constructs for improving a growth characteristic of a plant. These constructs generally comprise, consist or consist essentially of a control sequence that is operable in a plant cell and that is operably connected to a modulator nucleic acid sequence encoding an expression product that inhibits expression of the PDR gene, or that inhibits activity of an expression product of the PDR gene. The control sequence is suitably capable of facilitating constitutive expression of the modulator nucleicacid sequence. Alternatively, the control sequence may be capable of facilitating organ-specific or tissue-specific expression of the modulator nucleicacid sequence. In otherembodiments, the control sequence is capable of facilitating development-specific expression of the modulator nucleicacid sequence.

[0013] In yet anotheraspect, plant cells are provided thatcomprise a genetic modification, which results in partial orcomplete loss of function of a PDR gene. In some embodiments, the plant cells further comprise a nucleicacid construct comprising a control sequence that is operable in the plant cells and that is operably connected to a nucleotide sequence of interest.

[0014] Still anotheraspect of the present invention provides plants having an improved growth characteristic. These plants generally comprise, consist or consist essentially of a genetic modification, which results in partial orcomplete loss of function of a PDR gene. Suitably, the plant is selected from an embryo, a seed, a seedling, a juvenile plant or a mature plant. In some embodiments, the plants include cells that comprise a nucleicacid construct comprising a control sequence that is operable in the plant cells and that is operably connected to a nucleotide sequence of interest.

[0015] In a further aspect, the present invention provides harvestable parts or progeny of the plants as broadly described above and elsewhere herein, wherein the harvestable part or the progeny comprises the genetic modification. In specific embodiments, the harvestable part is selected from a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, spore, cutting, nut, or root.

[0016] Another aspect ofthe present invention provides a seed comprising a genetic modification, which results in partial orcomplete loss of function of a PDR gene. In some embodiments, the seed further comprises a nucleicacid construct comprising a control sequence that is operable in a plant cell and that is opera bly connected to a nucleotide sequence of interest.

[0017] A further aspect ofthe present invention provides methods of producing a plant with an improved growth characteristic. These methods comprise, consist or consist essentially of introducing a genetic modification in a plant cell, which results in partial orcomplete loss of function of a PDR gene, and regenerating a plant with a partial or complete loss of function ofthe endogenous PDR gene fromthe plant cell.

[0018] In still another aspect, expression systems are provided for expressing a target nucleic acid sequence in a plant cell. These systems generally comprise, consist or consist essentially of a first expression system component (e.g., comprising one or more expression cassettesor constructs) and a second expression system component (e.g., comprising one or more expression cassettes or constructs), wherein the target nucleic acid sequence is expressible from the first expression system component, and wherein a modulator nucleic acid sequence is expressible fromthe second expression system component, wherein the modulator nucleic acid sequence encodes an expression product that inhibits expression of a PDR gene, or that inhibits activity of an expression product ofthe PDR gene. In related aspects, the present invention also provides plant cells and plants that comprise an expression system as broadly described above and elsewhere herein, as well as harvestable parts or progeny of such plants, wherein the harvestable partorthe progeny comprises an expression system as broadly described aboveand elsewhere herein.

[0019] Another aspect ofthe present invention provides methods for expressing a nucleic acid sequence of interest in a plant. These methods generally comprise, consist or consist essentially of co-expressing a nucleic acid sequence of interest and a modulator nucleicacid sequence in cells ofthe plant, wherein expression ofthe modulator nucleicacid sequence produces an expression product that inhibits expression of a PDR gene or that inhibits activity of an expression product of a PDR gene. In some embodiments, the methods further comprise exposing the plant to one ormore stimuli that stimulate orenhance expression ofthe nucleicacid sequence of interest, the modulator nucleicacid sequence or both the nucleicacid sequence of interest and the modulator nucleicacid sequence. Suitably, the nucleicacid sequence of interest encodes a polypeptide of interest and in illustrative examples of this type, the methods further comprise harvesting, isolating or purifying the polypeptide of interest fromthe plant or plant part.

[0020] Since the plants ofthe present invention comprise a genetic modification that results in a partial orcomplete loss of function of a PDR gene, they may be more susceptible than plants that do not have the genetic modification to bioticorabioticstress. Such plants can be used as diagnostic or'sentinel" plants to provide early warning that nearby plants are being stressed so that appropriate actions can be taken. As such, the plants ofthe present invention alsofind utility as'sentinels'forenvironmental monitoring including providing warning ofthe presence of bioticor abiotic stressors, and can allow for appropriate protective measures and/orto prevent exposure to a dangerous condition. Accordingly, anotheraspect ofthe present invention provides methodsfor monitoring a population of plants for exposure to a stress condition (e.g., biotic or abiotic stress condition) orcombination of stress conditions. Such methods can be performed, forexample, by introducing into the population of plants a sentinel plant that comprises, consists or consists essentially of a genetic modification, which results in partial or complete loss of function of a PDR gene, and examining the sentinel plant for susceptibility to the stress condition or combination of stress conditions, which is indicative of exposure of the population of plants to the stress condition or combination of stress conditions. In some embodiments, the sentinel plant is located at or near the edge of the plant population. In other embodiments, the sentinel plant is located at or near the centerof the plant population.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a graphical, photographicand schematic representation showing characterization of N. Benthamiana isolates, (a) Geographical map of N. Benthamiana distribution showing collection sites and habitat zones. Red pins: NT, Northern Territory; NWA, north Western Australia; QLD, Western Queensland; WA, Western Australia and SA, Southern Australia. Green pin: Granites site - collection site of LAB isolate. Desertzone (Australian Bureau of Meteorology: www. a ustralia .gov. a u) is indicated in beige (b) N. benthamiana var. LAB, 16C, SA, NT, QLD, NWA and WA, 6 weeks old plantletsand cauline mature leaves and representative flower symmetry and corolla shape, (c) Identification of RDRl insertion by PCR analyze on N. benthamiana isolates. Total DNA was extracted from each isolate and PCR was carried out using RDRl specific primers as described in materials and methods section, (d) Sequence alignment of the RDRl gene for N. benthamiana var. SA, LAB, NT, NWA, QLD and WA, (restricted to sequence showing the site of the insertion), generated using Geneious.The insertion in N. benthamiana var. SA, 16C and LAB represents the 72 bp loss-of-function mutation. SNPs variation that modify the amino acid sequence are indicated by colored bar.

[0022] Figure 2 is a graphical, photographicand schematic representation showing N. benthamiana isolates response to virus infection correlated to theirgenotype variations forthe RDRl gene. (a)/V. benthamiana var. LAB x QLD hybrids 1) homozygous forthe dysfunctional RDRl, 2) heterozygous, and 3) homozygousforthe functional RDRl) 40 days after inoculation with TMV-U1. (b) Symptoms severity triggered by viruses inN. benthamiana isolates. Severity: x: occasional mild curling of leaves, infrequent chlorotic lesions; xx: moderate leaf curling, frequent chlorotic lesions, mild impaired growth; xxx/xxxx: severe leaf curling, extensive chlorosis lesions, highly impaired growth; xxxxx: death of the plants; (c) Identification of RDRl insertion by PCR analyze on N. benthamiana var. LAB x QLD hybrids, (d) N. benthamiana WA transformed with an artificial microRNA targeting theRDRl gene 11 days after inoculation withTMV-Ul and (e) corresponding relative RDRl RNA transcript level quantification, (f) Plantlets of different isolates 20 DPI with TMV-GFP and leaves 11 DPI with PVX-GFP and (g) Graphic representation of PVX-GFP spread from 0 DPI to 21 DPI, showing that RDRl restricts the rate of virus spread.

[0023] Figure 3 is a graphical and schematic representation showing RDRl insertion in the benthamiana species from the genus Nicotiana dated at 800,000 years ago. (a) Nicotiana species polar tree constructed from concatenated Gsll, MatK and AdhC sequences. Nicotiana section suaveolentes in blue, Nicotiana benthamiana species in red (b) N. benthamiana isolates phylogeny subjected to molecular clock analyses tree. The tree was calibrated with a constant substitution rate of 4.23 E-9 with an sd of 1.8 E-9 substitutions/site/year. Estimated date of divergence at each node is presented in years ago (c) N. benthamiana isolate divergence RAxML tree, (branch labels are bootstrap support values based on 1000 replicates). Both trees (b) and (c) were constructed from 21 concatenated sequences (listed in Table 2). All of the nodes in all of the trees have strong support (PP » 1). (d) Overlay of phylogeneticdistances of isolates (as in c) over geographicdistances (Map of Northern Australia) between isolates.

[0024] Figure 4 is a graphical, photographicand schematic representation showing flower structures, seeds germination and early vigor, (a) Representation of the 3 types of herkogamy found among the N. benthamiana isolates: approach (left), neutral/homostyly (center) and reverse (right) herkogamy; (b) N. benthamiana flowering timelines. Time from seed germination to first flower development (blue); first flowerto maximal flowering (red) and maximal flowering to first seed setting (mature capsule) for N. benthamiana LAB, SA, WA and QLD lines (c) seed and shoot variation of N. benthamiana LAB, WA and #5WA lines (d) seed germination of N. benthamiana LAB, WA, QLD, #5WAand #11WA lines 4 days after sowing.

[0025] Figure 5 is a photographic representation showing N. benthamiana isolates response to virus infection. N. benthamiana isolates 20 days after inoculation withTYLCV (top) and TMV-U1 (bottom).

[0026] Figure 6 is a graphical and schematic representation showing RNAi genes remain intact and conserved among N. benthamiana isolates with the exception of RDRl . (a) RNAi genes copy number amongst the isolates, (b) Scatter plots showing all pair-wise comparisons ofTPM values of RNAi transcripts (top) and Spearman's correlation values and associated p-values (highlighted in grey) calculated from log TPM values of RNAi transcripts identified (bottom) from each isolate. TPM values were generated fromthe RSEM software. Correlations were calculated using the cor() and cor.test() functions in the R statistical package, (c) Alignment of RDRl LAB and Wild isolates from our Nicotiana benthamiana Atlas tool website (www. benthHome.com).

[0027] Figure 7 is a schematic representation showing sequences alignments of RDRl for 51 plant species including N. benthamiana wilds and LAB like isolates (restricted to sequence showing thesite ofthe insertion) generated using Geneious.

[0028] Figure 8 is a schematic representation showing location of the potential original 72 bp sequence insertion of RDRl . (a) RDRl insertion sequence like position in the N. benthamiana LAB genome assembly, (b) Representation ofthe sequences retrieved forthe 72bp insertion from the scaffold Nbv0.5scaffold3298 {RDRl gene), Nbv0.5scaffold954and wild N. benthamiana RDR sequence, (c) Protein translation ofthe sequences shown in B.

[0029] Figure 9 is a graphical representation showing variation of Herkogamy displayed among N. benthamiana isolates. Distance separating the four upperanthers fromthe sigma.

Reverse Herkogamy: presentation ofthe anthers above the stigma, Approach Herkogamy:

presentation ofthe anthers below the stigma.

[0030] Figure 10 is a graphical representation showing controlled deterioration treatment on N. benthamiana isolates. 3 replicates of 50 seeds per isolates were equilibrated at 23% moisture content and subsequently incubated in hermetically sealed foil bags at 45° C. The seeds were removed after24, 72 and 120h and tested fortheirgermination.

[0031] Figure 11 is a photographic representation demonstrating that silencing of RDRl in N. tabacum plants results in substantially larger seed pods, relative to the seed pods of control plants.

[0032] Figure 12 is a graphical representation showing that silencing of RDRl in N. tabacum plants results in significantly higher average seed weights per pods, as compared to the average seeds weights of control plants. DETAILED DESCRIPTION OF THE INVENTION

1 Definitions

[0033] Unless defined otherwise, a II technica I and scientific terms used herein havethe same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materialssimilarorequivalentto those described herein can be used in the practice ortesting of the present invention, preferred methods and materials are described. Forthe purposes of the present invention, the following terms are defined below.

[0034] The articles "a" and "an" are used herein to referto one orto more than one {i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Thus, for example, the term" RDR gene" also includes a plurality of RDR genes.

[0035] Further, the term"about", as used herein when referring to a measurable value such as an amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length and the like, is meant to encompass variationsof ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount, dose, time, temperature, activity, level, number, frequency, percentage, dimension, size, amount, weight, position, length andthe like.

[0036] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the u per and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0037] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0038] As used herein, the term "antibody" includes within its scope polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2 and Fv), including the products of a Fab or other immunoglobulin expression library. With respect to antibodies, the term, "immunologically specific" or "specific" refers to antibodies that bind to one ormore epitopes ofa protein of interest, but which do not substantially recognizeand bind other molecules in a sample containing a mixed population of antigenic biological molecules. Screening assays to determine binding specificity of an antibodyare well known and routinely practiced in the art. Fora comprehensive discussion ofsuch assays, see Harlow etal. (Eds.), ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter6.

[0039] The term "antisense" refers to a nucleotide sequence whose sequence of nucleotide residues is in reverse 5'to 3'orientation in relation to the sequence of deoxynucleotide residues in a sense strand ofa nucleicacid (e.g., DNA or RNA) duplex. A "sense strand" ofa DNA duplex refers to a strand in a DNA duplex which is transcribed by a cell in its natural state into a "sense mRNA."Thus an "antisense' sequence is a sequence having the same sequence as the non- coding strand in a DNA duplex. The term"antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, in other words, at the 5' no n -coding sequence, 3' no n -coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. "Ribozyme" refers to a catalytic RNA and includes sequence-specificendoribonucleases. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein.

[0040] "Autonomous" or"c/s" replication refers to replication of a replicon that contains all cis- and trans-acting sequences (such as the replication gene (rep)) required for replication.

[0041] "Cells","hostcells","transformed hostcells","regenerable host cells" and the like are terms that not only refer to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications mayoccurin succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scopeof the termas used herein.

[0042] The terms "c/s-acting element", "c/s-acting sequence" or"c/s-regulatory region" are used interchangeably herein to mean any sequence of nucleotides, which modulates transcriptional activity of an operably linked promoter and/or expression of an operably linked nucleotide sequence. Those skilled in the art will be aware that a cis -sequence may be ca able of activating, silencing, enhancing, repressing orotherwisealtering the level of expression and/or cell-type-specificity and/or developmental specificity of any nucleotide sequence, including coding and non-coding sequences.

[0043] "Chromosomally-integrated", as used herein, refers to the integration of a heterologous nucleic acid sequence, typically in the form of a construct, into a host DNA by covalent bonds.

[0044] By "coding sequence" is meantany nucleicacid sequence that contributes to the code forthe polypeptide product of a gene orforthe final mRNA product of a gene (e.g. the mRNA product of a gene following splicing). By contrast, the term"non-coding sequence" refers to any nucleic acid sequence thatdoes not contribute to the code forthe polypeptide product of a gene or for the final mRNA product of a gene.

[0045] As used herein the term "co-expression", "co-expressing" and the like meanthat nucleotide sequences coding for two or more nucleicacid sequences are expressed in the same host cell, suitably concurrently {i.e., the expression of a nucleotide sequence and that of another overlap with each other) orsequentially within a short enough period of time that the effective result is equivalent to that obtained when all nucleotide sequences are expressed concurrently.

[0046] As used herein, "complementary" polynucleotides a re those that are capable of hybridizing via base pairing according to the standard Watson -Crick complementarity rules.

Specifically, purines will base pair with pyrimidines to forma combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, oradenine paired with uracil (A:U) in the case of RNA. For example, the sequence "A-G-T" binds to the

complementary sequence "T-C-A." It is understood that two polynucleotides may hybridize to each othereven if they are not completely orfully complementary to each other, provided that each has at leastone regionthat is substantially complementary to the other. The terms "complementary" or "complementarity", as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single stranded molecules (also referred to herein as "nucleobase polymers") may be "partial", in which only some ofthe nucleobases base pair, or it may be "complete" when total complementarity exists between the singlestranded molecules eitheralong the full length ofthe molecules oralong a portion or region ofthe single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. The term "complementary" includes within its scope nucleic acid sequences that are "fully complementary", "substantially complementary" or"partially complementary". As used herein, the ternrTfully complementary" indicates that 100% ofthe nucleobases in a particular nucleobase polymer a re able to engage in base -pairing with another nucleobase polymer. The term "substantially complementary", as used herein, indicates that at least at about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% ofthe nucleobases in a particular nucleobase polymerare able to engage in base-pairing with another nucleobase polymer. As used herein, the term "partially complementary" indicates that at least at about 50%, 55% or 60% ofthe nucleobases in a particular nucleobase polymerare able to engage in base-pairing with another nucleobase polymer. The terms "substantially complementary" and "partially complementary" can also mean that two nucleic acid sequences can hybridize under high stringency or medium stringency conditions and such conditions are well known in the art.

[0047] Throughout this specification, unless the context requires otherwise, the words

"comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps orelements. Thus, use ofthe term"comprising" and the like indicates thatthe listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whateverfollows the phrase

"consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed afterthe phrase, and limited to otherelements that do not interfere with orcontribute to the activity or action specified in the disclosure forthe listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that otherelements are optional and mayor may not be present depending upon whether or not they affect the activityor action ofthe listed elements.

[0048] The terms "conditional expression", "conditionally expressed" "conditionally expressing" and the like refer to the ability to activate or suppress expression of a gene of interest by the presence orabsence of a stimulus or other signal (e.g., chemical, light, hormone, stress, or a pathogen). In specific embodiments, conditional expression of a nucleic acid sequence of interest is dependent on the presence of an inducerorthe absenceof an inhibitor.

[0049] As used herein, the term "concurrent stimulation", "concurrently stimulated" and the like means that the stimulation of a regulated promoter and that of another promoter overlap with each other.

[0050] "Constitutive expression", as used herein, refers to expression using a constitutive or regulated promoter. "Conditional" and "regulated expression" referto expression controlled by a regulated promoter. [0051] "Constitutive promoter" refers to an unregulated promoterthat directs expression of an operably linked transcribable sequence in many or all tissues of a plant regardless of the surrounding environment and suitably at all times.

[0052] The term "construct" refers to a recombinant genetic molecule including one or more isolated nudeicacid sequences f ro m d iffe re nt sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nudeicacid molecule and include any construct that contains (1) nudeicacid sequences, including regulatory and coding sequences that are not found together in nature {i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cos mid, virus, autonomously re licating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nudeicacid molecule where one ormore nudeicacid molecules have been operably linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nudeicacid sequence of interest that is also contained in the construct, such as, for example, a target nudeicacid sequence ora modulator nudeicacid sequence. Such elements may include control elements such as a promoterthat is operably linked to (so as to direct transcription of) the nudeicacid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryoticand eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nudeicacid molecules, such as two or more separate vectors. An "expression construct" generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, forexample, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. Forthe practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see forexample, MolecularCloning: A Laboratory Manual, 3^rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0053] As used herein, the term"contiguous" in the context of a nudeicacid sequence means thatthe sequence is a single sequence, uninterrupted by any intervening sequence or sequences.

[0054] As used herein, the term"control plant" refers to a plant cell, an explant, seed, plant component, plant tissue, plant organ, or whole plant used to compare against transgenicor genetically modified plant forthe purpose of identifying an enhanced phenotype ora desirable trait in the transgenicor genetically modified plant. A "control plant" may in some cases be a transgenic plant line that comprises an empty vectoror markergene, but does not contain the recombinant polynucleotide of interest that is present in the transgenicor genetically modified plant being evaluated. A control plant may be a plant of the same line or variety as the transgenicor genetically modified plant being tested, or it may be another line or variety, such as a plant known to have a specific phenotype, characteristic, or known genotype. A suitable control plant would include a genetically unaltered or non-transgenic plant of the parental line used to generate a transgenic plant herein.

[0055] By "control sequence", "control elemenf'and the like is meant nucleic acid sequences (e.g., DNA) necessary for expression of an opera bly linked coding and/or non-coding sequences in a particular host cell. Control sequences include nucleotide sequences located upstream, within, ordownstreamof a nucleic acid sequence of interest (which maycomprise coding and/or non-coding sequences), and which influence the transcription, RNA processing or stability, or translation of the associated nudeicacid sequence of interest, eitherdirectly or ind irectly. The control seq uences that a re suitable for proka ryotic cells for exa mple, include a promoter, and optionallya cs-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for euka ryotic cells include transcriptional control sequencessuch as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers, introns, Rep recognition elements, intergenic regions, polyadenylation signal sequences, internal ribosome binding sites (IRES), nucleic acid sequencesthat modulate mRNA stability, as well as targeting sequences thattarget a product encoded by a transcribed polynucleotide to an intra cellular compartment within a cell or to the extracellular environment. Control sequences include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.

[0056] By "corresponds to"or"corresponding to" is meant a nudeicacid sequence that displays substantial sequence identity to a reference nudeicacid sequence (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to all or a portion of the reference nudeicacid sequence) or an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence (e.g., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99% oreven upto 100% sequence similarity or identity to all ora portion of the reference amino acid sequence).

[0057] The terms "decrease," "reduce", "inhibit" and their grammatical equivalents are used interchangeably herein to referto "reduction or substantial elimination" of endogenous gene expression and/or polypeptide levels and/or polypeptide activity, relative to a control plant. The reduction or substantial elimination is in increasing order of preference at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reduced, as compared to that of a control plant.

[0058] "Dominant negative" refers to a gene product that adversely affects, blocks or abrogates the function of a normal, wild-type gene product when co -expressed with the wild type gene product within the same cell even whenthe cell is heterozygous (wild -type and dominant negative). Expression of the dominant negative mutant generally results in a decrease in normal function of the wild-type gene product.

[0059] The term "double stranded RNA" or"dsRNA", as used herein, refers to a ribonucleic acid containing at least a region of nucleotides that are in a double stranded conformation. The double stranded RNA may be a single nucleotide polymer with one or more region(s) of self-complementarity such that nucleotides in one segment of the polymer base pair with nucleotides in anothersegment of the polymer. Alternatively, the double stranded RNA may include two nucleotide polymers that have one or more region(s) of complementarity to each other. The double stranded RNA will typically comprise a duplex region comprising two a nti -parallel nucleic acid strands that a re partially, substantially or fully complementary, as defined herein. As used herein, a "strand" refers to a contiguous sequence of nucleotides and reference herein to "two strands" includes the strands being, or each forming a part of, separate nucleotide polymers or molecules, orthe strands being covalently interconnected, e.g., by a linker, to form but one nucleotide polymer or molecule. At least one strand can include a region which is sufficiently complementary to a target sequence. Such strand is termed the "a ntisense strand". A second strand comprised in the double stranded RNA, which comprises a region complementary to the a ntisense strand, is termed the "sense strand". However, a double stranded RNA can also be formed from a single RNA molecule which is at least partly self-complementary, forming a duplex region, e.g., a hairpin or panhandle. In such case, the term "strand" refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule. The term "a ntisense strand" refers to the strand of a double stranded RNA which includes a region that is complementary (typically substantially or fully complementary) to a sequence of nucleotides ("target sequence") located within the RNA transcript of target gene. This strand is also known as a "guide" sequence, and is used in a functioning RISC complex to guide the complex to the correct RNA (e.g., mRNA) for cleavage. As used herein, the term"region of complementarity" refers to the region on the a ntisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully

complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3' terminus. The term "sense strand", as used herein, refers to the strand of a double stranded RNA that includes a region that is substantially complementary to a region of the antisense strand. This strand is also known as an "anti-guide"sequence because it contains the same sequence of nucleotides as the target sequence and therefore binds specifically to the guide sequence.

[0060] As used herein, the terms "encode", "encoding" and the like referto the capacity of a nucleic acid to provide foranother nucleicacid ora polypeptide. Forexample, a nucleicacid sequence is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequenceand a non-coding sequence. Thus, the terms "encode", "encoding"and the like include a RNA product resulting fromtranscription of a DNA molecule, a protein resulting from translation of a RNA molecule, a protein resulting fromtranscription of a DNA molecule to form a RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide a RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0061] The term "endogenous" refers to any polynucleotide or polypeptide which is present and/or naturally expressed within a host organism or cell thereof. Forexample, an "endogenous" nucleic acid refers to a nucleic acid molecule or nucleotide sequence that is naturally found in the cell into which an expression system component of the invention is introduced.

[0062] As used herein, the term "episome" or "replicon" refers to a DNA or RNA virus or a vectorthat undergoes episomal replication in host cells {e.g., plant cells). It contains c/s-acting viral sequences, such as the Rep recognition element (also commonly referred to as a "replication origin"), necessary for replication. It mayor may not contain trans -acting sequences necessary for replication, such as the viral replication genes (for example, the AC1 and AL1 genes in ACMVand TGMV Geminiviruses, respectively). It may or may notcontain a nucleicacid sequence of interest for expression in the host cell.

[0063] "Episomal replication"and "replicon replication" a re used interchangeably herein to refer to replication of replicons, suitably DNA or RNA viruses or virus-derived replicons, that are not stably introduced in a host (e.g., chromosomally-integrated). Episomal replication generally requires the presence of viral replication protein(s) essential for replication, is independentof chromosomal replication, and results in the production of multiple copies of virus or replicons per host genome copy.

[0064] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression may also refer to the production of protein. Thus, as will be clearfrom the context, expression of a coding sequence results fromtranscription and translation of the coding sequence. Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence.

[0065] As used herein, the term "express ion cassette" refers to a polynucleotide sequence capable of effecting expression of a gene of interest (e.g., a target nucleicacid sequence, a modulator nucleicacid sequence etc.) in a host cell. Expression cassettes include at least one control sequence (e.g., a promoter,enhancer,transcriptionterminatorand the like) opera bly linked with the gene of interest, which can be in the form of a contiguous or non -contiguous nucleic acid entity as defined herein. "Overexpression" refers to the level of expression in transgenic organisms that exceeds levels of expression in normal or untransformed organisms. The expression cassette may be naturally present in a host cell or may be part of a construct.

[0066] The term "expression system" refers to any nucleic acid based approach or system for expressing one or more nucleic acids of interest. Where expression of two or more nucleic acid sequences of interest is desired, the expression system will generally comprise a component ("expression system components") for expression of each nucleicacid sequence of interest. Such components may comprise one or more expression cassettes for expressing an individual nucleic acid sequence of interest. Where more than one expression cassette is used to express a nucleicacid sequence of interest, the expression cassettes may be on the same construct or vectororon different constructs orvectors. The expression cassettes may be endogenous or heterologous with respect to the host cell in which they reside or a re proposed to reside, provided that at least one them (e.g., used to expressthe modulator nucleicacid sequence) of the expression system is heterologous with respect to the host cell. In specific embodiments, at least one component of the expression system is in the form of a binary expression system. As used herein, the term "binary expression system" describes an expression system component comprised of two constructs, at least one of which is chromosomally integrated. In specific embodiments, the binary expression system component is a binary viral expression system component comprising a first construct and a second construct in which the first construct comprises an inactive replicon or a proreplicon from which a nucleic acid sequence of interest is expressible in a host cell and the second construct comprises a regulated promoter opera bly-linked to a transactivating gene. The inactive replicon or proreplicon and a chimeric transactivating gene, functioning together, will effect re licon replication and expression of the nucleic acid sequence of interest in a host cell (e.g., a plant cell) in a regulated manner. Both constructs may be stably introduced into the host cell (e.g., chromosomally-integrated) and may be inherited independently. Stimulating the regulated promoter driving the transactivating gene releases the replicon fromthe chromosome and its subsequent episomal replication. The release can be physical excision of the replicon fromthe chromosome involving site-specific recombination, a replicative release from a master chromosomal copy of a proreplicon in the presence of the replication protein, ortranscriptional release froma masterchromosomal copy ofan amplicon.

[0067] As used herein, the terms "fragment" or"portion" when used in reference to a nucleic acid molecule or nucleotide sequence will be understood to mean a nucleicacid molecule or nucleotide sequence of reduced length relative to a reference nucleicacid molecule or nucleotide sequence and comprising, consisting essentially of and/orconsisting of a nucleotide sequence of contiguous nucleotides identical or homologous (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% identical) to the reference nucleicacid ornucleotide sequence. Such a nucleicacid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.

[0068] The term "functional nucleic acid" as used herein refers to a nucleicacid having specific biological functions in vivo or in cells, such as enzymatic functions, catalytic functions, or biologically inhibiting orenhancing functions (e.g., inhibition orenhancement of transcription or translation). Specific examples include siRNA, shRNA, miRNA (including pri-miRNA and pre- miRNA), nucleicacid aptamers (including RNAaptamers and DNA aptamers), ribozymes (including deoxyribozymes), riboswitches, Ul adaptors, molecular beacons, and transcriptional factor-binding regions.

[0069] As used herein, the term"gene" refers to a nucleicacid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA, and the like. Genes mayor may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements including promoters, enhancers, termination sequences and 5' and 3' untranslated regions). A gene may be "isolated" by which is meant a nucleic acid molecule that is su stantially or essentially free fro m components normally found in association with the nucleic acid molecule in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleicacid molecule. Reference to a "gene" also includes within its scope reference to genes having a contiguous sequence, thus defining contiguous nucleic acid entities, as defined herein, or a non-contiguous sequence thus defining a non-contiguous nucleic acid entity as defined herein. In certain embodiments, the term"gene" includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5'and 3' non- coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control sequences such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control sequences. The gene sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extra chromosomal maintenance orfor introduction into a host.

[0070] "Genome" as used herein includes the nuclear and/or plastid genome, and therefore includes introduction of the nucleic acid into, for example, the chloroplast genome.

[0071] The terms "growing" or"regeneration" as used herein mean growing a whole, differentiated plant from a plant cell, a group of plant cells, a plant part (including seeds), ora plant piece (e.g., from a protoplast, callus, or tissue part).

[0072] As used herein, the term"harvesting"and grammatical variations thereof means and includes an act of removing useful plant parts from plants, including crop plants.

[0073] The term "heterologous" as used herein with reference to nucleic acids refers to a nucleic acid molecule or nucleotide sequence that either originates from another species or is from the same species or organism but is modified from either its original form or the form primarily expressed in the cell. Thus, a nucleotide sequence derived from an organism or species different from that of the cell into which the nucleotide sequence is introduced, is heterologous with respect to that cell and the cell's descendants. Such nucleotide sequences are also referred to herein as "foreign" nucleotide sequences. In addition, a heterologous nucleotide sequence includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g. present in a different copy number, and/or under the control of different regulatory sequences than that found in the native state of the nucleic acid molecule. The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleicacid may be recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleicacid encoding a peptide sequence from another source. Similarly, a "heterologous" protein indicates that the protein comprises two or more subsequences that are notfound in the same relationship to each other in nature {e.g., a fusion protein).

[0074] The term "homolog" refers to any gene that is related to a reference gene by descent from a common ancestral DNA sequence. The term"ortholog" refers to homo logs in different species that evolved fro ma common ancestral gene by speciation. Typically, orthologs retain the same orsimilar function despite differences in their primary structure (mutations). The term "para log" refers to ho mo logs in the same species that evolved by genetic duplication of a common ancestral gene. In many cases, paralogs exhibit related (but not always identical functions). As used herein, the term homolog encompasses both orthologs and paralogs. To the extent that a particular species has evolved multiple related genes from an ancestral DNA sequence shared with another species, the termortholog can encompass the term para log. The terms "homolog", "ortholog" and "paralog" also include within their scope expression products [e.g., NA or protein) of the "homolog", "ortholog"or"paralog"genes.

[0075] The term "host" refers to any organism, or cell thereof, whether eukaryotic or prokaryotic into which a construct of the invention can be introduced, particularly, hosts in which RNA silencing occurs. In particularembodiments,the term"host" refers to eukaryotes, including unicellulareukaryotes such asyeast and fungi as well as multicellular eukaryotes such as: plants, illustrative examples of which include angiosperms, gymnosperms, monocotyledons (monocots) and dicotyledons (dicots), and animals non-limiting examples of which include invertebrate animals {e.g., insects, cnidarians, echinoderms, nematodes, etc.); eukaryotic parasites (e.g., malarial parasites, such as Plasmodium falciparum, helminths, etc.); vertebrate animals (e.g., fish, amphibian, reptile, bird, mammal); and mammals (e.g., rodents, primates such as humansand non-human primates). Thus, the term "host cell" suitably encompasses cells of s uch eukaryotes as well as cell lines derived fromsuch eukaryotes.

[0076] The terms "increase", "improve" and their grammatical equivalents are used interchangeably herein and encompass at least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15%or20%, more preferably 25%, 30%, 35% or40% more yield and/orgrowth in comparison to a control plant.

[0077] The term "increased yield" as defined herein is taken to mean an increase in any one or more of the following, each relative to control plants: (i) increased bio mass (weight) of one or more parts ofa plant, particularly a boveg round (harvestable) parts, increased root biomassor increased bio mass of any other harvestable part; (ii) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (iii) increased numberofflowers per plant; (iv) increased number of (filled) seeds; (v) increased seed filling rate (which is expressed asthe ratio between the number of filled seeds divided by the total number of seeds); (vi) increased seed size, which may also influence the composition of seeds; (vii) increased seed volume, which mayalso influence the composition of seeds (including oil, protein and carbohydrate total content and composition); (viii) increased individual seed area; (ix) increased individual seed length and/orseed perimeter; (x) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, overthe total biomass; and (xi) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/orseed weight. An increasedTKW mayalso result from an increase in embryo size and/or endosperm size. An increase in yield may also result in modified architecture, or may occur as a result of modified architecture.

[0078] "Inducible promoter", as used herein, refers to those regulated promoters that can be turned on in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, ora pathogen.

[0079] "Introducing" in the context ofa host cell including an animal cell, animal part, and/or animal organ, plant cell, plant part and/or plant organ means contacting a nucleic acid molecule with the animal cell, animal part, and/oranimal organ, orwith the plant, plant part, and/or plant cell in such a manner that the nucleic acid molecule gains access to the interior of the animal cell, animal part, and/oranimal organ, or the plant cell, plant part and/or plant organ. Where more than one nucleicacid molecule is to be introduced these nucleicacid molecules can be assembled as part ofa single polynucleotide or nucleicacid construct, oras separate

polynucleotide or nucleicacid constructs, and can be located on the same or different nucleicacid constructs. Accordingly, these polynucleotides can be introduced into host cells in a single transformation event, in separate transformation events, or, e.g., as part ofa breeding protocol. Thus, the term "transformation" as used herein refers to the introduction ofa heterologous nucleic acid into a cell. Transformation ofa cell may be stable ortransient. "Transient transformation" in the context ofa polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome or heritable extrachromosomal element of the cell. By "stably introducing" or"stably introduced" in the context ofa polynucleotide introduced into a cell, it is intended that the introduced polynucleotide is stably incorporated or integrated into the genome or stable extra -chromosomal element of the cell, and thus the cell is stably transformed with the polynucleotide. "Stable transformation" or "stably transformed" as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleicacid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. Stable transformation as used herein can also referto a nucleicacid molecule that is maintained extrachromosomally, for example, as a minichromosome.

[0080] The term "intron" refers to a nucleotide sequence within or adjacent to a coding sequence that is removed by RNA splicing, and necessarily contains sequences required for splicing, such as a 3' splice site and a 5' splice site. Reference to introns includes reference to intact introns and split introns, such as an intron split into two regions: a 3' region comprising a 3' splice site, and a 5' region comprising a 5' splice site.

[0081] As used herein, the ternrTloss of function" refers to less or no function of a gene product/protein compared to the wild type. Loss of function of a PDR includes within its scope a PDR gene that has less or no biological function compared to the wild -type PDR, or a PDR polypeptide that has lessor no biological function or activity compared to the wild -type PDR polypeptide. Loss of function can be caused by transcriptional, post-transcription, orpost translational mechanisms or by binding of an antagonist or inhibitor molecule to an expression product {e.g., RNA or polypeptide) of a RDR gene. Loss of function may also be caused by loss of function mutation resulting froma point mutation {e.g., a substitution, a missense mutation, ora nonsense mutation), an insertion, and/ora deletion in a RDR polypeptide or a nucleicacid sequence encoding a RDR polypeptide.

[0082] The term "meganuclease" generally refers to a naturally-occurring homing endonucleasethat binds double-stranded DNAat a recognition sequencethat is greaterthan 12 base pairs and encompasses the corresponding intron insertion site. Naturally-occurring meganucleases can be monomeric {e.g., I-Scel) ordimeric (e.g., I-Crel). The term meganuclease, as used herein, can be used to referto monomeric meganucleases, dimeric meganucleases, orto the monomers which associate to forma dimeric meganuclease.

[0083] The term "microRNA" or"miRNA" refers to small, noncoding RNA molecules that have been found in a diverse array of eukaryotes, including plants. miRNA precursors share a characteristic secondary structure, forming short 'hairpin' RNAs. The term "miRNA" includes processed sequences aswellas corresponding long primary transcripts (pri-miRNAs) and processed precursors (pre-miRNAs). Geneticand biochemical studies have indicated that miRNAs are processed to their mature forms by Dicer, a RNAse III family nuclease, and function through RNA- mediated interference (RNAi) and related pathways to regulate the expression of target genes (Hannon (2002) Nature 418, 244-251; Pasquinelli, era/. (2002) Annu. Rev. Cell. Dev. Biol. 18, 495-513). miRNAs may be configured to permit experimental manipulation of gene expression in cells as synthetic silencing triggers 'short hairpin RNAs' (shRNAs) (Paddison era/. (2002) Cancer Cell 2, 17-23). Silencing by shRNAs involves the RNAi machinery and correlates with the production ofsmall interfering RNAs (siRNAs), which are a signature of RNAi.

[0084] The term "non-coding" refers to sequences of nucleicacid molecules that do not encode part or all of an expressed protein. No n -coding sequences include but are not limited to introns, enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions. Thus, the term "5'-non-coding region" shall be taken in its broadest context to include all nucleotide sequences which are derived from the upstream region of a gene. Such regions may include an intron, e.g., an intron. As used herein, the term "3' no n -coding region" refers to nucleic acid sequences located downstream of a coding sequence and include polyadenylation recognition sequences (normally limited to eukaryotes) and other sequences encoding regulatory elements capable of affecting mRNA processing or gene expression. The polyadenylation signal (normally limited to eukaryotes) is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0085] As used herein, the term "nucleic acid sequence" or"nucleotide sequence" refers to a hetero polymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded ordouble stranded. The terms "nucleotide sequence" "nudeicacid", "nudeicacid molecule", "oligonucleotide" a nd "polynucleotide" a re also used interchangeably herein to referto a heteropolymer of nucleotides, and include RNAor DNA that is linearor branched, single or double stranded, ora hybrid thereof. The term also encompasses RNA/DNA hybrids.. Nucleic acid sequences provided herein are presented herein in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR 1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.

[0086] The term"operablyconnected"or"operably linked"as used herein refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. Forexample,a control sequence (e.g., a promoter) "operably linked" to a nucleotide sequence of interest (e.g., a coding and/or non-coding sequence) refers to positioning and/or orientation of the control sequence relative to the nucleotide sequence of interest to permit expression of that sequence under conditions compatible with the control sequence. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct its expression. Thus, for example, intervening non -coding sequences (e.g., untranslated, yet transcribed, sequences) can be present between a promoter and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. Likewise, "operably connecting" a c/s-acting sequence to a promoter encompasses positioning and/or orientation of the c/s-acting sequence relative to the promoterso that the c/s-acting sequence regulates (e.g., inhibits, abrogates, stimulates or enhances) promoter activity. Alternatively, "operably connecting" non-contiguous nudeicacid sequences of a non- contiguous nudeicacid entity encompasses rearrangement (e.g., positioning and/or orientation) of the non-contiguous nudeicacid sequences relative to each otherso that (1) the reassembled nucleic acid sequencesformthe sequenceof a contiguous nudeicacid entity (e.g., a contiguous target or modulator nudeicacid entity)and optionally (2) if the non-contiguous nudeicacid sequenceseach comprise a coding sequence, each coding sequence is Ίη -frame' with another to produce a complete open reading frame corresponding the coding sequence of the contiguous nucleic acid entity.

[0087] As used herein, "organ-specific" or "tissue -specific" control sequence is one that is capable of preferentially controlling or facilitating expression of an o erably linked coding and/or non-coding sequences in certain organs ortissues, such as the leaves, roots, seed tissue etc. For example, a "root-specific control sequence" such as a "root -specific promoter" is a promoter that is transcriptionally active predominantly in plant roots, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts. Control sequences able to control or facilitate expression in certain cells only are referred to herein as "cell- specific".

[0088] As used herein, "planf'and "differentiated plant" refer to a whole plant or plant part containing differentiated plant cell types, tissues and/or organ systems. Plantlets and seeds are also included within the meaning of the foregoing terms. Plants included in the invention are any plants amenable to transformation techniques, including angios perms, gymnosperms, monocotyledons (monocots) and dicotyledons (dicots). Non-limiting examples of monocot plantsof the present invention include sugarcane, corn, ba rley, rye, oats, wheat, rice, flax, millet, sorghum, grasses, banana, onion, asparagus, lily, coconut, and the like.

[0089] As used herein, "plant cell" refers to a structural and physiological unit of the plant, which comprises a cell wall and also may referto a protoplast, gamete-producing cell, orcell which regenerates into whole plants. A plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue ora plantorgan.

[0090] The term "plant defense-related gene"or"PDR" as used herein refers to genes that combat or defend against an abiotic or biotic stress. PDRs may be expressed constitutively or stimulated or upregulated in responsean abioticor bioticstress. The term"abioticstress"as used herein refers to a non-living stress that typically affects plant health and includes, without limitation, lack of oxygen, ultraviolet radiation, heat, cold, drought, flood, nitrogen, high wind, salinityand osmotic stress. By contrast, the ternrTbiotic stress"as used herein refers to a stress that occurs as a result of damage done to plants by a living organism, e.g., plant pathogens such as but not limited to insects, nematodes, bacteria, fungi, oomycetes, protozoa, viruses, viroids, or any combination thereof, as well as herbivory by pests.

[0091] These whose expression is stimulated or up-regulated in response to living (biotic) agents, including pathogens such as viruses, fungi and bacteria, or by environmental (abiotic) factors such as nutrient deficiency, drought, lack of oxygen, excessive temperature, or pollution.

[0092] As used herein, the term "plant part" includes but is not limited to embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant cells including plant cells that are intact in plants and/orparts of plants, plant protoplasts, plant tissues, plant cell tissue cultures, plant ca Mi, plant clumps, and the like.

[0093] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant.

[0094] "Polypeptide", "peptide", "protein" and "proteinaceous molecule" are used interchangeably herein to referto molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogueof a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes within its scope two or more complementing or interactive polypeptides comprising different parts or portions (e.g., polypeptide domains, polypeptide chains etc.) of a polypeptide of the present invention, wherein the individual complementing polypeptides together reconstitute the activity of the different parts or portions to forma functional polypeptide.

[0095] The term "promoter" refers to a nucleotide sequence, usually upstream (5') to a transcribable sequence, which controls the expression of the transcribable sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoterthat is a short nucleicacid sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which control elements [e.g., c/^'s-acting elements) are added forcontrol of expression. "Pro moter"also refers to a nucleotide sequence that includes a minimal promoter plus control elements (e.g., as-acting elements) that are capable of controlling the expression of a coding sequence orfunctional RNA.

This type of promotersequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a nucleic acid sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal orflipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific nucleic acid-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, oreven be comprised of synthetic nucleic acid segments. A promoter may also contain nucleicacid sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological ordevelopmental conditions. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters." In the presence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A"minimal or core promoter" thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.

[0096] "Promoter activity" refers to the ability ofa promoterto drive expression of a nucleic acid sequence operably linked to the promoter. Promoter activity of a sequence can be assessed by operably linking the sequence to a reporter gene, and determining expression of the reporter.

[0097] The term "regulated promoter" refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and include tissue- specific, developmentally-specificand inducible promoters. Such promoters, therefore, facilitate conditional expression of a nucleicacid sequence of interest. The term"regulated promoter" includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. Different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various types useful in host cells are constantly being discovered. Since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleicacid fragments of different lengths may have identical promoter activity. Illustrative regulated promoters include but are not limited to safener-inducible promoters, promoters derived from the tetracycline-inducible system, promoters derived from salicylate- inducible systems, promoters derived fromalcohol-inducible systems, promoters derived from glucocorticoid-inducible systems, promoters derived from pathogen-inducible systems, promoters derived from carbohydrate inducible systems, promoters derived from hormone inducible systems, promoters derived from antibiotic inducible systems, promoters derived from metal inducible systems, promoters derived from heat shock inducible systems, and promoters derived from ecdysome-inducible systems.

[0098] As used herein, the terms "RNA interfere nee" and "RNAi" referto sequence- specific, post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) in animals and plants, initiated by double stranded RNAthat is homologous in sequence to the silenced gene. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs) triggered by dsRNAfragnnents cleaved from longerdsRNA which direct the degradative mechanismto other RNA sequences having closely homologous sequences. As practiced as a technology, RNAi can be initiated by human intervention to reduce or even silence the expression of target genes using either exogenously synthesized dsRNA or dsRNA transcribed in the cell (e.g., synthesized as a sequence that forms a short hairpin structure). The terms "RNA interference"and "RNAi" are used interchangeably herein to referto "RNA silencing" (also referred to herein as " RNA-mediated gene silencing") as the result of RNAi is the inhibition or "silencing" at the RNA level of the expression of a corresponding gene or nucleic acid sequence of interest. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0099] As used herein, the terms "small interfering RNA" and "short interfering RNA" ("siRNA") referto a short RNA molecule, generally a double stranded RNA molecule about 10-50 nucleotides in length (the term"nucleotides" including nucleotide analogs), preferably between about 15-25 nucleotides in length. In most cases, the siRNA is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Such siRNA can have overhanging ends (e.g., 3'-overhangs of 1, 2, or 3 nucleotides (or nucleotide analogs). Such siRNA can mediate RNA interference.

[0100] As used in connection withthe present invention, the term"shRNA", and in some embodiments the terms "double stranded RNA molecule", dsRNA and the like, referto a RNA molecule having a stem-loop structure. The stem-loop structure includes two mutually complementary sequences, where the respective orientations and the degree of complementarity allow base pairing between the two sequences. The mutually complementary sequences are linked by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.

[0101] The term "sequence identity" as used herein refers to the extent that sequences are identicalon a nucleotide-by-nucleotide basis oran amino acid-by-amino acid basis overa window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences overthe window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue

(e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the numberof matched positions by the total numberof positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Forthe purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Useful methodsfor determining sequence identityare also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo et al. (Applied Math 48:1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md.20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol.215:403-410 (1990)); version 2.0 orhigherof BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and for polynucleotide sequence BLASTN can be used to determine sequence identity.

[0102] Terms used to describe sequence relationships between two or more polynucleotides include "reference sequence", "comparison window", "sequence identity", and "percentage of sequence identity". A "reference sequence" is at least 12 but frequently at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length. Becausetwo polynucleotides may each comprise (1) a sequence {i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides overa "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 contiguous positions, orat least about 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000 or 3000 contiguous positions in which a sequence is compared to a reference sequence of the same numberof contiguous positions afterthe two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of less than about 20%, 15%, 10% or 5% as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul etal., 1997, Nuc. Acids Res.25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel etal., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0103] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of a RNA molecule formed during the transcription of a RNA from a gene or nucleic acid sequence of interest, including RNA (e.g., mRNA) that is a product of RNA processing of a primary transcription product.

[0104] "Tissue-specific promoter", as used herein, refers to regulated promoters that are not expressed in all cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo orcotyledon), orspecific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds orfruit, in fully differentiated leaf, or at the onset of senescence. [0105] The terms "trans-acting sequence" and "frans-acting element" refer to DNAor RNA sequences, whose function does not require them to be on the same molecule. A non-limiting example of a trans -acting sequence is a rep gene (AC1 or AL1 in ACMV orTGMV Geminiviruses, respectively), which can function in replication without being on the replicon.

[0106] As used herein, the term "trans-activation" refers to switching on of gene expression or replicon replication by the expression of another (regulatory) gene in trans.

[0107] The term "transformation" means alteration ofthe genotypeof a host bythe introduction of a heterologous nucleic acid, such as the first and/or second constructs ofthe invention.

[0108] As used herein, the terms "transformed" and"transgenic" referto any organism including an animal, animal part, plant, plant cell, callus, plant tissue, or plant part that contains a II or part of at least one construct ofthe invention. In some embodiments, all or part of at least one construct of the invention is stably introduced into a chromosome orstable extra-chromosomal element, so that it is passed on to successive generations.

[0109] The term "transgene" as used herein, refers to any nucleotide sequence used in the transformation of a plant, animal, or otherorganism. Thus, a transgene can be a coding sequence, a non-coding sequence, a cDNA, a gene orfragment or portion thereof, a genomic sequence, a regulatory element and the like. A "transgenic" organism, such as a transgenic plant, transgenic microorganism, or transgenic animal, is an organism into which a transgene has been delivered or introduced and the transgene can be expressed in the transgenic organism to produce a product, the presence of which can impart an effect and/ora phenotype in the organism.

[0110] As used herein, the term "transient expression" refers to expression in cells in which a transgene is introduced into a host cell, but notselected forits stable maintenance. Non- limiting methods of introducing the transgene include viral infection, agrobacterium-mediated transformation, electropo ration, and biolistic bombardment

[0111] As used herein, the term"5' untranslated region" or"5' UTR" refers to a sequence located upstream (I.e., 5') of a coding region. Typically, a 5' UTR is located downstream (i.e., 3') to a promoter region and 5'of a coding region downstream of the promoter region. Thus, such a sequence, while transcribed, is upstreamof the translation initiation codon and therefore is generally not translated into a portion ofthe polypeptide product.

[0112] The term "3' untranslated region" or"3' UTR" refers to a nucleotide sequence downstream (i.e., 3') of a coding sequence. It generally extends fromthe first nucleotide after the stop codon of a coding sequence to just before the poly(A) tail ofthe corresponding transcribed mRNA. The 3' UTR may contain sequences that regulate translation efficiency, mRNA stability, mRNA targeting and/or polyadenylation.

[0113] The term "upregulated", as used herein, should be interpreted in the most general sense possible. For example, a gene may be "upregulated" if it is expressed at a level significantly and detectably higher (i.e., for example, 1.5-10 fold) than the natural or baseline expression rate.

[0114] By "vector" is meant a nucleicacid molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector typically contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome ofthe defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosoma I entity, the replication of which is independent of chromosomal replication, e.g., a linearorclosed circular plasmid, an

extra chromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vectorsystem may comprise a single vectoror plasmid, two or more vectors or plasmids, which together contain the total DNAto be introduced into the genome of the host cell, or a trans poson. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection markersuch as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[0115] The terms "wild -type", "natural", "native" and the like with respect to an organism, polypeptide, or nucleic acid sequence, that the organism polypeptide, or nucleic acid sequence is naturally occurring oravailable in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.

[0116] The term "yield" in general means a measurable produce of economic value, necessarily relatedto a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, whereas the actual yield is the yield peracre for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted acres. Increased seed yield may manifest itself as one or more of the following: a) an increase in seed bio mass (total seed weight) which may be on an individual seed basis and/or per plant and/or per square meter; b) increased number of flowers per plant; c) increased number of (filled) seeds; d) increased seed filling rate (which is expressed as the ratio between the number of filled seeds divided by the total number of seeds); e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the total biomass; and f) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/orseed weight, and mayalso result froman increase in embryo and/orendospermsize. An increase in seed yield mayalso be manifested as an increase in seed size and/orseed volume. Furthermore, an increase in seed yield mayalso manifest itself as an increase in seed area and/orseed length and/orseed width and/orseed perimeter. Increased yield may also result in modified architecture, or may occur because of modified architecture.

[0117] As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated in the absence of any underscoring or italicizing. For example, "RDR" shall mean a RDR gene, whereas "RDR" shall indicate the protein product of the "RDR" gene.

[0118] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.

2 Methods for enhancing yield-related traits in plants through loss of function of a PDR gene

[0119] The present invention is predicated in part on the determination that plant defense-related systems foradvancing survival of a plant can markedly undermine the growth or bio mass that is potentially achievable by the plant. Based on this determination, the present inventors considerthat one or more growth characteristics of a plant can be significantly improved by decreasing the level or activity of a PDR polypeptide or by inhibiting expression of an endogenous PDR gene of the plant, as compared to control plants. In particular, plants with a loss of function of a PDR gene exhibit improvement in at least one growth characteristic (during at least part of their life cycle), relative to the growth characteristic of corresponding wild type plants at a corresponding stage in their life cycle. The improved growth characteristic may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. A plant having an improved growth characteristic may exhibit early vigor, yield, seed size, leaf size, flower size, trichome number and plant bio mass and may even exhibit early flowering. The improvement in growth characteristic may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. An improved growth characteristic during the early stages in the life cycle of a plant may reflect enhanced vigor. The improvement in growth characteristic may include increased growth rate of the plant. Forexample, an increased growth rate may alterthe harvestcycle of a plant allowing plants to be sown laterand/or harvested soonerthan would otherwise be possible. If the growth rate is sufficiently increased, it may allow forthe sowing of further seeds ofthe same plantspecies (forexample sowing and harvesting of rice plants followed by sowing and harvesting offurther rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow forthe sowing of further seeds of different plants species (forexample the sowing and harvesting of rice plants followed by, forexample, the sowing and optional harvesting of soy bean, potato orany other suitable plant). Harvesting additional times from the same rootstock in the case of some plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual bio mass production per unit area (due to an increase in the numberof times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild -type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined byderiving various parameters fromgrowth curves plotting growth experiments, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.

2.1 PDR genes

[0120] Any endogenous gene involved in plant defense systems is contemplated for targeted loss offunction to improve at least one growth characteristic of a plant of interest. Such PDR genes can be selected forexample from genes involved in wounding and programmed cell death, pathogenesis resistance (PR), RNA interference, salicylic-acid-mediated defenses, jasmonic- acid-dependent defenses or ethylene-dependent responses, genes linked and/or regulated by abscisicacid (ABA), flavonoid biosynthesis, auxin, cytokinin, brassinosteroids and/or gibberellins as well as disease resistance (R) genes. Representative examples of PDR genes are listed in TABLE 5. Numerous plant PDR nucleic acid and encoded amino acid sequences that can be used as a basis for targeting are disclosed in the art for a plethora of plant species, non-limiting examples of which are listed in the National Center for Biotechnology Information (NCBI) databases GenBankand GenPept as well as genome specific data bases. Non-limiting examples of genomic information for PDR genes listed in TABLE 5 is set out in TABLE 6. The present invention also contemplates homologs, orthologs and paralogs of thesePDR genes. Exemplary PDR nucleotide sequences and encoded PDR amino acid sequences are described in TABLES 7 and 8, respectively, and set forth in SEQ ID NO: 1-784. In specific embodiments, the PDR gene targeted for loss of function is a RDR gene illustrative examplesof which include RDR1, RDR2, RDR3, RDR5 and RDR6. Representative nucleotide sequences for targeted loss of function include, but are not restricted to, the nucleotide sequences setforth in Accession numbers: Gb AY574374.1/ Nbv5.1tr623087 {RDR1 ) ; Gb

AY722009 Nbv5.1tr6212914 (RDR2); GbAt2gl9910 (RDR3); Nbv5.1tr6236934 (RDR5); and GbAY722008.1/ Nbv5.1tr6205507 / Nbv5.16391026 (RDR6). In a preferred embodiment, the RDR gene fortargeted loss of function is RDR1.

2.2 Methods of producing loss of function of a PDR gene

[0121] Decreasing the level or activity of a PDR polypeptide or inhibiting expression of an endogenous PDR gene may be accomplished using any suitable technique but will typically be achieved through introduction of a genetic modification in the genome of the plant, which results in partial or complete loss of function of the PDR gene. The genetic modification may be achieved using any appropriate method and numerous such methods are known to the skilled practitioner, non-limiting examples of which include site-directed mutagenesis, random chemical mutagenesis, transposon mutagenesis, T-DNA insertion, homologous recombination, targeted induced local lesions in genomes (TILLING) and genome editing.

2.2.1 Chemical mutagenesis

[0122] Methods for random mutation of genes are well known in the art, see, e.g., U.S. Pat. No.5,830,696. Forexample, mutagenscan be usedto randomly mutate a RDR gene.

Mutagens include, e.g., ultraviolet light orgamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, aloneor in combination, to induce DNA breaks amenable to repair by recombination. Other chemical mutagens include, forexample, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other mutagens are analoguesof nucleotide precursors, e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, oracridine. These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.

2.2.2 Homologous Recombination

[0123] Alternatively, at least one genomic copy of a PDR gene may be modified in the genome of the plant by homologous recombination as further illustrated in Paszkowski etal. (1998, EMBO Journal 7:4021-26). This technique usesthe property of homologous sequences to recognize each other and to exchange nucleotide sequences between each by a process known in the art as homologous recombination. Homologous recombination can occur between the chromosomal copy of a nucleotide sequence in a cell and an incoming copy of the nucleotide sequence introduced in the cell by transformation. Specific modifications are thus accurately introduced in the

chromosomal copy of the PDR gene. In some embodiments, a control sequence of the PDR gene is modified. The existing control sequence may be modified [e.g., mutation ordeletion) to thereby reduce expression of the PD? gene, thus inhibiting orabolishing its expression. In other embodiments, a PDR nucleotide sequence is modified by deletion of a part of the nucleotide sequence orthe entire nucleotide sequence, or by mutation. Expression of a mutated PDR polypeptide in a plant cell is also contemplated in the present invention. Refinements of this technique to disruptendogenous plant genes have been described (Kempin etal., 1997. Nature 389:802-803 and Miao and Lam, 1995. Plant J., 7:359-365). In other embodiments, a mutation in the chromosomal copy of a nucleotide sequence is introduced by transforming a cell with a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. An additional feature of the oligonucleotide is for example the presence of 2'-0-methylation at the RNA residues. The RNA DNA sequence is designed to align with the sequence of a chromosomal copy of a PDR nucleotide sequence and to contain the desired nucleotide change. For example, this technique is further illustrated in U.S. Pat. No.5,501,967 and Zhu etal. (1999, Proc. Natl. Acad. Sci. USA 96: 8768-8773).

2.2.3 Dominant-Negative Mutants

[0124] Alternatively, the activity of a PDR polypeptide may be changed. This is achieved by expression of dominant negative mutants of PDR proteins in transgenic plants, leading to the loss of activity of the endogenous PDR protein.

2.2.4 Antibodies

[0125] In other embodiments, an antibody that is specific foran endogenous PDR protein is produced in a plant cell by introduction of a construct from which the antibody is expressible. Exemplary antibodies for use in the practice of the present invention include monoclonal antibodies, Fv, Fab, Fab' and F(ab')2 immunoglobulin fragments, as well as synthetic antibodies such as but not limited to single domain antibodies (DABs), synthetic stabilized Fv fragments, e.g., single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodiesand multivalent antibodies such as dia bodies and multi-scFv or engineered human equivalents. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are a Iso well known in the art. In illustrative examples, antibodies can be made by conventional immunization (e.g., polyclonal sera and hybridomas) with isolated, purified or recombinant peptides or proteins corresponding to at least a portion of an endogenous PDR polypeptide, oras recombinant fragments corresponding to at least a portion of an PDR endogenous polypeptide, usually expressed in Escherichia coil, after selection from phage display or ribosome display libraries {e.g., available fromCambridge Antibody Technology, Biolnvent,

Affitech and Biosite). Knowledge of the antigen-binding regions [e.g., complementarity-determining regions) of such antibodies can be used to prepare synthetic antibodies as described for example above.

2.2.5 Aptamers

[0126] In other embodiments, the activity of a PDR polypeptide is inhibited by expressing in transgenic plants nucleicacid ligands, so-called aptamers, which specifically bind to the protein. Aptamers are preferentially obtained by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. In the SELEX method, a candidate mixture of single stranded nucleic acids having regionsof randomized sequence is contacted with the protein and those nucleic acids having an increased affinity to the target are partitioned from the remainder of the candidate mixture. The partitioned nucleicacids are amplified to yield a ligand enriched mixture. After several iterations a nucleicacid with optimal affinity to the polypeptide is obtained and is used for expression in transgenic plants. This method is further illustrated in U.S. Pat. No.

5,270,163.

2.2.6 Insertion Mutagenesis

[0127] In some embodiments, insertion mutagenesis is used to inhibit expression of a PDR gene. Insertion mutagenesis facilitates direct reverse genetic screens by providing a physical link to a gene of interest. In plants both T-DNA and transposon insertion mutagens have been utilized as insertion mutagens (Winkler etal., 1998. Methods Mol. Biol. 82:129-136, Martienssen, 1998. Proc. Natl. Acad. Sci. USA 95:2021-2026). T-DNA insertions within any given gene can be detected by polymerase chain reaction (PC ) methods utilizing one gene s ecific primer and one T- DNA specific prime r (Winkle ret a/., 1998. Plant Physiol.3:743-750, and Krysan etal., 1999. Plant Cell 11 :2283-2290). Specific PCR product is formed only when a T-DNA element has inserted either within or close to the gene of interest. Due to the exponential nature of PCR amplification, it is possible to screen many thousands of independently transformed Arabidopsis mutants by sample pooling (Krysan etal., 1999, supra). Once a T-DNA pool is identified with an insertion in the gene of interest, the process of isolating a single plant with that insertion requires de-convolution of the pool architecture.

2.2.7 Genome Editing and Induced Mutagenesis

[0128] In general, methods to modify or a Iter the host endogenous genomic DNA are available. This includes altering the host native DNAsequenceora pre-existing transgenic sequence including regulatory elements, coding and non-coding sequences. These methodsare also useful in targeting nucleicacids to pre -engineered target recognition sequences in the genome. As an example, the genetically modified cell or plant described herein, is generated using "custom" meganucleases produced to modify plant genomes (see, e.g., WO 2009/114321; Gao et al., 2010. Plant Journal 1 : 176-187). Another site -directed engineering is through the use of zinc fingerdomain recognition coupled with the restriction properties of restriction enzyme (see, e.g., Urnoveta/., 2010. Nat Rev Genet. ll(9):636-46; Shukla etal., 2009. Nature 459(7245):437-41). Alternatively, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease system may be used for genome editing to produce a loss of function in a PDR gene. CRISPR/Cas is a recently engineered nuclease system based on a bacterial systemthat can be used forgenome engineering. It is based on part of the adaptive immune response of many bacteria and Archea . When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNAs (crRNA) by the 'immune' response. This crRNA then associates, through a region of partial complementarity, with another type of RNA called tra crRNA to guide the Cas9 nuclease to a region homologous to the crRNA in the target DNA called a "protospacer". Cas9 cleaves the DNAto generate blunt ends at the DSB at sites specified by a

20-nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both the crRNA and the tra crRNA for site specific DNA recognition and cleavage. This system has now been engineered such that the crRNA and tra crRNA can be combined into one molecule (the "single guide RNA"), and the crRNA equivalent portion of the single guide RNA can be engineered to guide the Cas9 nuclease to target any desired sequence (see Jinek etal. (2012) Science 337, p.816- 821, Jinek etal. (2013), eLife 2:e00471,and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system can be engineered to create a double -stranded break (DSB) at a desired target PDR gene in a plant genome, and repair of the DSB can be influenced by the use of repair inhibitors to cause an increase in error prone repair.

2.2.8 Zinc Finger-Mediated Genome Editing

[0129] As an example, a plant or plant cell with a loss of function of a PDR gene may be generated using a zincfinger nuclease-mediated genome editing process. The process for editing a chromosomal sequence includes forexample: (a) introducing into a cell at least one nucleic acid encoding a zincfinger nuclease that recognizes a target sequence in the chromosomal sequence and is able to cleave a site in the chromosomal sequence, and, optionally, (i) at least one donor polynucleotidethat includes a sequence for integration flanked by an upstream sequence and a downstreamsequencethatexhibitsubstantial sequence identity with eithersideof the cleavage site, or (ii) at least one exchange polynucleotide comprising a sequence that is substantially identical to a portion of the chromosomal sequence at the cleavage site and which further comprises at least one nucleotide change; and (b) culturing the cell to allow expression of the zincfinger nuclease such that the zincfinger nuclease introduces a double -stranded break into the chromosomal sequence, and wherein the double -stranded break is repaired by (i) a nonhomologous end-joining repair process such that an inactivating mutation is introduced into the chromosomal sequence, or (ii) a homo logy-directed repair process such that the sequence in the donor polynucleotide is integrated into the chromosomal sequence orthe sequence in the exchange polynucleotide is exchanged with the portion of the chromosomal sequence.

[0130] A zincfinger nuclease includes a DNA binding domain (i.e., zincfinger) and a cleavage domain (i.e., nuclease). The nucleicacid encoding a zincfinger nuclease may include DNA or NA. Zinc finger binding domains may be engineered to recognize and bind to any nucleicacid sequence of choice. See, forexample, Beerli ei al., 2002. Nat. Biotechnol. 20:135-141; Pabo etal., 2011. Ann. Rev. Biochem.70:313-340; Choo etal., 2000. Curr. Opin. Struct. Biol. 10:411-416; Doyoneta/., 2008. Nat. Biotechnol.26:702-708; Santiago etal., 2008. Proc. Natl. Acad. Sci. USA 105:5809-5814; Urnovet al., 2010. Nat Rev Genet. ll(9):636-46; and Shukla etal., 2009. Nature 459 (7245):437-41. An engineered zincfinger binding domain may have a novel binding specificity compared to a naturally-occurring zincfinger protein. As an example, the algorithm of described in U.S. Pat. No.6,453,242 may be used to design a zincfinger binding domain to target a preselected sequence. Nondegenerate recognition code tables may also be used to design a zinc finger binding domain to target a specific sequence (Sera etal., 2002. Biochemistry 41 :7074- 7081). Tools for identifying potential target sites in DNA sequences and designing zincfinger binding domains may be used (Mandell etal., 2006. Nuc. Acid Res.34:W516-W523; Sander etal., 2007. Nuc. Acid Res.35:W599-W605).

[0131] An exemplary zincfinger DNA binding domain recognizes and bindsa sequence having at leastabout 80% sequence identity with the desired target sequence. In other embodiments, the sequence identity may be about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0132] A zincfinger nuclease also includes a cleavage domain. The cleavage domain portion of the zincfinger nucleases may be obtained from any endo nuclease or exo nuclease. Non- limiting examples of endo nucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2010-2011 Catalog, New England Biolabs, Beverly, Mass.; and Belfort etal., 1997. Nuc. Adds Res.25:3379- 3388. Additional enzymes that cleave DNA are known [e.g., 51 Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). One or more of these enzymes (or functional fragments thereof) may be used as a source of cleavage domains.

2.2.9 Meganudease-Based Genome Editing

[0133] Anotherexample for genetically modifying a plant or plant cell to have a loss of function of a PDR gene is by using "custom" meganucleases produced to modify plant genomes (see e.g., WO 2009/114321; Gao eta!., 2010. Plant Journal 1:176-187. Naturally-occurring meganucleases, for example, from the LAGLIDADG family, have been used to promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells and mice. Engineered meganucleases such as, for example, LIG-34 meganucleases, which recognize and cut a 22 bp DNA sequence found in the genome of Zea mays (maize) are known (see e.g., US 20110113509).

2.2.10 TAL Endonucleases (TALEN)

[0134] TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats. A transcription activator-like (TAL) effector-DNA modifying enzymes (TALE or TALEN) are also used to engineer genetic changes. See e.g., US20110145940, Boch etal., 2009. Science 326(5959): 1509-12. Fusions of TAL effectors to the Fokl nuclease provideTALENs that bind and cleave DNA at specific locations. Target specificity is determined by developing customized amino acid repeats in the TAL effectors.

2.2.11 TILLING

[0135] The effects of the invention may also be reproduced using the technique of

TILLING. "TILLING" is an abbreviation of "Targeted Induced Local Lesions In Genomes", which is a mutagenesis technology useful to generate and/or identify nucleic acids encoding proteins with modified expression and/or activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoterforexample). In specific embodiments, these mutant variants exhibit lower PDR activity than that exhibited by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp.16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner J and CasparT (1998) In J Ma rtinez-Za ater, J Salinas, eds, Methods on Molecular Biology, Vol.82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected asan extra peak in the chroma to gram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods forTILLING are well known in the art (McCallum et al., 2000. Nat Biotechnol 18: 455-457; reviewed by Stemple, 2004. Nat RevGenet.5(2): 145-50).

2.2.12 Functional RNA-mediated Gene Silencing

[0136] In specific embodiments, functional RNA-mediated gene silencing methods are used forreducing orsubstantiallyeliminating orabolishing expression a PDR gene in a plant. Suitably, in these embodiments, a sufficient length of substantially contiguous nucleotides of a PDR nucleic acid sequence is required. In order to perform gene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer nucleotides, a Iternatively this may be as much as the entire PDR gene (including the 5' and/or 3' UTR, either in part or in whole). The stretch of substantially contiguous nucleotides maybe derived fromthe nucleicacid encoding the protein of interest (target gene), orfrom any nucleic acid capable of encoding an ortholog, paralog or homolog ofthe PDR protein of interest. Suitably, the stretch of substantially contiguous nucleotides is capable offorming hydrogen bondswith the target gene (eithersenseorantisensestrand), more preferably, the stretch of substantially contiguous nucleotides has, in increasing orderof preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene (either sense orantisense strand). A nucleic acid sequence encoding a (functional) polypeptide is not a requirement for the various methods discussed herein for the reduction or substantial elimination of expression of an endogenous gene.

[0137] This reduction orsubstantial elimination of expression may be achieved using routine tools and techniques. A preferred method forthe reduction orsubstantial elimination of endogenousRDR gene expression is by introducing and expressing in a plant a construct into which the nucleicacid (in this case a stretch of substantially contiguous nucleotides derived from a RDR gene of interest, orfrom any nucleic acid capable of encoding an ortholog, paralog or homolog of anyone ofthe RDR protein of interest) is cloned as an inverted repeat (in part or completely), separated by a spacer (non-coding DNA).

[0138] In such a preferred method, expression of an endogenous PDR gene is reduced or substantially eliminated through RNA-mediated silencing using an inverted repeat of a nucleic acid or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the PDR gene of interest, orfrom any nucleic acid ca able of encoding an ortholog, paralog or homolog ofthe PDR protein of interest), preferably capable offorming a hairpin structure. The inverted repeat is cloned in a suitable nucleicacid construct comprising control sequences. A non- coding DNA nucleic acid sequence (a spacer, forexample a matrix attachment region fragment (MAR), an intron,a polylinker, etc.) is located between the two inverted nucleicacids forming the inverted repeat. After transcription ofthe inverted repeat, a chimeric RNA with a self- complementary structure is formed (partial orcomplete). This double -stranded RNA structure is referred to as the hairpin RNA (hpRNA). The hpRNA is processed by the plant into siRNAs that are incorporated into an RNA-induced silencing complex (RISC). The RISC further cleaves the mRNA transcripts, thereby substantially reducing the numberof mRNA transcripts to be translated into polypeptides. For further genera I details see forexample, Grierson etal. (1998) WO 98/53083; Waterhouse etal. (1999) WO 99/53050).

[0139] Performance ofthe methodsofthe invention does not relyon introducing and expressing in a planta construct into which the nucleicacid is cloned as an inverted repeat, but any one or more of several well-known'gene silencing" methods may be used to achieve the same effects such as spraying dsRNA (w/wo nanoparticles).

[0140] One such method forthe reduction of endogenous gene expression is RNA- mediated silencing of gene expression (downregulation). Silencing in this case is triggered in a plant by a double stranded RNA sequence (dsRNA) that is substantially similar to the target endogenous gene. This dsRNA is further processed by the plant into about 20 to about 26 nucleotides called short interfering RNAs (siRNAs). The siRNAs are incorporated into an RNA- induced silencing complex (RISC) that cleaves the mRNA transcript ofthe endogenous target gene, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. In specific embodiments, the double stranded RNAsequence correspondsto a PDR targetgeneof interest.

[0141] Another example of a RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from a PDR gene of interest, orfrom any nucleic acid capable of encoding an ortholog, paralog or homo log of a PDR protein of interest) in a sense orientation into a plant. "Sense orientation" refers to a DNA sequencethat is homologousto an mRNA transcript thereof.

Introduced into a plant would therefore be at least one copy of the nucleicacid sequence. The additional nucleicacid sequence will reduce expression ofthe endogenous PDRgene, giving rise to a phenomenon known as co -suppression. The reduction of gene expression will be more pronounced if several additional copies of a nucleicacid sequence are introduced into the plant, as there is a positive correlation between high transcript levels and the triggering of co -suppression.

[0142] Another example of a RNA silencing method involves the useof antisense nucleic acid sequences. An "antisense" nucleicacid sequence comprisesa nucleotide sequencethat is complementary to a "sense" nucleicacid sequence encoding a protein, i.e., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA transcript sequence, suitably a PDR mRNA transcript sequence. The antisense nucleicacid sequence is preferably complementary to the endogenous geneto be silenced. The complementarity may be located in the coding region and/or in the non-coding region of a PDR gene.

[0143] Antisense nucleicacid sequences can be designed according to the rules of Watson and Crick base pairing. The antisense nucleicacid sequence may be complementary to the entire PDR nucleic acid sequence (in this case a stretch of substantially contiguous nucleotides derived from a PDR gene of interest, orfrom any nucleic acid capable of encoding an ortholog, paralog or homo log of a PDR protein of interest), but may also be an oligonucleotide that is antisense to only a part ofthe nucleic acid sequence (including the mRNA 5' and 3' UTR). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a PDR polypeptide. The length of a suitable antisense oligonucleotide sequence is known in the art and may start fro ma bout 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less.

[0144] The antisense nucleicacid sequence is suitably produced using a suitable expression construct into which a PDR nucleic acid sequence has been subcloned in an antisense orientation {i.e., RNA transcribed from the inserted nucleicacid will be of an antisense orientation to a target nucleicacid of interest). In specific embodiments, production of antisense nucleicacid sequences in plants occurs by means of a stably integrated nucleicacid construct comprising a promoter, an opera bly linked antisense oligonucleotide, and a terminator.

[0145] The nucleic acid molecules used for silencing in the methods ofthe invention (whether introduced into a plant or generated in situ) hybridize with or bind to mRNA transcripts and/or genomic DNA encoding a polypeptide to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/ortranslation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case ofan antisense nucleicacid sequence which binds to DNA duplexes, through specific interactions in the major groove ofthe double helix. Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleicacid sequences can be modified to target selected cells and then administered systemically. Forexample, for systemic administration, antisense nucleic acid sequences can be modified such that they specifically bind to receptors orantigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid sequences can also be delivered to cells using the constructs described herein.

[0146] According to a further aspect, the antisense nucleicacid sequence is an a- a no me ric nucleic acid sequence. An a-anomeric nucleicacid sequence forms specific double - stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier etai, 1987. NuclAc Res 15: 6625-6641). The antisense nucleic acid sequence may also comprise a 2'-0-methylribonucleotide (Inoue etal., 1987. NuclAc Res 15, 6131-6148) ora chimeric RNA-DNA analogue (Inoue era/., 1987. FEBS Lett.215,327-330).

[0147] The reduction or substantial elimination of expression of an endogenous PDR gene may also be performed using ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that a re capableof cleaving a single -stranded nucleicacid sequence, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, 1988. Nature334, 585-591) can be used to cata lytica My cleave mRNA transcripts encoding a polypeptide, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. A ribozyme having s ecificity for a RDR nucleic acid sequence can be designed (see for example: Cech era/. U.S. Pat. No.4,987,071; andCechera/. U.S. Pat. No.5,116,742). Alternatively, mRNA transcripts corresponding to a RDR nucleic acid sequence can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel and Szostak, 1993. Science 261, 1411-1418). The use of ribozymes forgene silencing in plants is known in the art (e.g., Atkins er a/., 1994. WO 94/00012; Lenneera/., 1995. WO 95/03404; Lutziger era/., 2000. WO 00/00619; Prinsen era/., 1997. WO 97/13865 and Scott era/., 1997. WO 97/38116).

[0148] A further approach to gene silencing is by targeting nucleicacid sequences complementary to the regulatory region of the gene (e.g., the pro mote rand/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See Helene, C, Anticancer Drug Res.6, 569-84, 1991; Helene era/., Ann. N.Y. Acad.Sci. 660, 27-361992; and Maher, L. J. Bioassays 14, 807-15, 1992.

[0149] Artificial and/or natural microRNAs (miRNAs) may be used to knockout gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long. They function primarily to regulate gene expression and/or mRNA translation. Most plant microRNAs (miRNAs) have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein. MiRNAs serve as the specificity components of RISC, since they base -pair to target nucleic acids, mostly mR As, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes. [0150] Artificial microRNAs (amiRNAs), which are typically 21 nucleotides in length, can be genetically engineered specifically to negatively regulategene expressionof single ormultiple genes of interest. Determinants of plant micro RNA target selection are well known in the art. Empirical parameters fortarget recognition have been defined and can be used to aid in the design of specific amiRNAs, (Schwa eta/., Dev. Cell 8, 517-527, 2005). Convenient tools fordesign and generation of amiRNAs and their precursors are also available to the public (Schwab era/., Plant Cell 18, 1121-1133,2006).

[0151] Other functional RNA molecules that are useful for mediating loss of function of a PDR gene in plant cells include molecular beacons, riboswitches and Ul adaptors. Molecular beacons are hairpin-shaped single-stranded nucleicacids having a stem structure and a loop structure, aswellasa fluorophore and quencher. Molecular beacons have utility as probes for confirming the existence of a sequence complementary to a loop moiety. The fluorophore of the molecular beacon is generally quenched because of the short distance between thefluorophore and quencher. If a loop moiety contains a complementary sequence, however, the loop moiety hybridizes to the complementary sequence. This opens the hairpin structure, the fluorophore is separated fromthe quencher, and fluorescence is thus detected. At least one nucleotide (DNA and/or RNA) or a nucleic acid analogue may be present at one or both ends of a molecular beacon in the base sequence of a single -stranded nucleic acid fragment. The number of nucleotides or the like at one or both ends of the molecular beacon is not particularly limited. If such end is to be ligated to the aforementioned hairpin-shaped DNA, such number is preferably between 1 and 20. In accordance with the present invention, the target molecule of the molecular beacon is suitably a PDR mRNA.

[0152] A riboswitch is a cs-acting element existing in a non-translational region at the 5' end of mRNA orthe like, and it functions as a metabolite-sensitive gene switch. The riboswitch directly binds to a low-molecular-weight organic compound orthe like to alterthe mRNA conformation and regulates the gene expression. At least one nucleotide (DNA and/or RNA) or a nucleic acid analogue may be present at one or both ends of the riboswitch in the base sequence of a single-stranded nucleic acid fragment. The number of nucleotides orthe like at one or both ends of the riboswitch is not particularly limited. If such end is to be ligated to the aforementioned hairpin-shaped DNA, such number is preferably between 1 and 20. The target molecule of the riboswitch is not particularly limited.

[0153] A Ul adaptor is a bifunctional single-stranded nucleic acid consisting of about 25 bases, and it comprises a 5'-target domain complementary to the 3'-end exon in the mRNA precursor of the targetgeneand a 3'-Ul domain having a sequence complementary to the 5' region of Ul snRNA (Goraczniak R. er a/., 2009, Nat. Biotechnol., 27: 257-263). Upon introduction of the Ul adaptor into an organism, Ul snRNP containing Ul snRNA binds to a region in the vicinity of a poly A signal of the mRNA precursorof the target gene, and polyadenylation of such mRNA is specifically inhibited. As a result, the mRNA precursorof the target gene (e.g., a RDR gene) is unstabilized and then degraded in the nucleus. Thus, gene silencing takes place. At least one nucleotide (DNA and/or RNA) or a nucleic acid analogue may be present at one or both ends of the Ul adaptor in the base sequence of a single -stranded nucleic acid fragment. The number of nucleotides or the like at one or both ends of the Ul adaptor is not particularly limited. If such an end is to be ligated to the aforementioned hairpin-shaped DNA, such number is preferably between 1 and 20. [0154] For optimal performance, the gene silencing techniques used for reducing expression in a plant of an endogenous PDR gene requires the use of nucleic acid sequences from monocotyledonous plants for transformation of monocotyledonous plants, and from dicotyledonous plants fortransformation of dicotyledonous plants. Preferably, a nucleicacid sequence fromany given plant species is introduced into that same species. For example, a nucleicacid sequence from rice is transformed into a rice plant. However, it is not an absolute requirement that the nucleic acid sequence to be introduced originates from the same plant species as the plant in which it will be introduced. It is sufficient that there is substantial homology between the endogenous target gene and the nucleicacid to be introduced.

[0155] Described above are examples of various methods for reducing or substantially eliminating expression in a plant of an endogenous PDR gene. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve reduction of expression of an endogenous PDR gene in a whole plant or in parts thereof through the use of an appropriate promoter, for example.

3 Constructs for expression of a nucleic acid sequence of interest in a plant with a loss of function of a PDR gene

[0156] The present invention is also directed to expression of a nucleicacid sequence of interest in a plant with a loss of function of a PDR gene. It is envisaged that the improved growth characteristic imparted to the plantthrough a reductionorsubstantiallyelimination of expression of a PDR gene will serve to increase yield of an expression product ofthe nucleic acid sequence of interest. In some embodimentsthe expression ofthe nucleicacid sequence of interest and inhibition of expression ofthe PDR gene is facilitated using a expression systemthat comprises at leasttwo expression system components, in which a first expression system component expresses the nucleicacid sequence of interestand a second expression system component expresses a modulator nucleic acid sequence that inhibits expression ofthe PDR gene orthat inhibits activity of an expression product {e.g., polypeptide product) ofthe PDR gene.

[0157] The modulator nucleicacid sequence is typically opera bly connected in an expression cassette to at least one control sequence or regulatory element, including

transcriptional regulatory elements such as promoters. The choice of promoter will vary depending on the temporal and spatial requirements forexpression ofthe modulator nucleicacid sequence, and also depending on the host cell in which this sequence is desired to be expressed. In some cases, expression in multiple tissues is desirable. While in others, tissue -specific expression is desirable. The promoter may be constitutive or inducible, as discussed for example below.

Expression ofthe modulator nucleicacid sequence can also be controlled at the level of replication.

[0158] In some embodiments, the modulator nucleicacid sequence is in the form of a contiguous nucleicacid entity that encodes an intact or uninterrupted functional NA molecule. Alternatively, the modulator nucleicacid sequence may be in the form of a non-contiguous nucleic acid entity or split gene which comprises a plurality of spaced nucleicacid subsequences, each encoding different portions ofthe functional RNA molecule, wherein the spaced nucleic acid subsequences are capableof rearranging (e.g., by replication or recombination) to forma contiguous nucleic acid entity that encodes an intact functional RNA molecule. 3.1 Nucleic acid sequences of interest

[0159] Nucleic acid sequences of interest will generally be genes of interest that are reflective of the commercial markets and interests of those involved in the development of transgenic hosts and host cells and are generally dependent on the use or uses to which they are put. In specific embodiments, a gene of interest suitably provides a beneficial agronomic trait to plants (e.g., crop plants), illustrative examples of which include nucleicacid sequences that modulate herbicide resistance (U.S. Pat. No.5,633,435; U.S. Pat. No.5,463,175), increased yield (U.S. Pat. No.5,716,837), insect control (U.S. Pat. No.6,063,597; U.S. Pat. No.6,063,756; U.S. Pat. No.6,093,695; U.S. Pat. No.5,942,664; U.S. Pat. No.6,110,464), fungal disease resistance (U.S. Pat. No.5,516,671; U.S. Pat. No.5,773,696; U.S. Pat. No.6,121,436; and U.S. Pat. No. 6,316,407, and U.S. Pat. No.6,506,962), virus resistance (U.S. Pat. No.5,304,730 and U.S. Pat. No.6,013,864), nematode resistance (U.S. Pat. No.6,228,992), bacterial disease resistance (U.S. Pat. No.5,516,671), starch production (U.S. Pat. No.5,750,876 and U.S. Pat. No.6,476,295), modified oils production (U.S. Pat. No.6,444,876), high oil production (U.S. Pat. No.5,608,149 and U.S. Pat. No.6,476,295), modified fatty acid content (U.S. Pat. No.6,537,750), high protein production (U.S. Pat. No.6,380,466), fruit ripening (U.S. Pat. No.5,512,466), enhanced animal and human nutrition (U.S. Pat. No.5,985,605 and U.S. Pat. No.6,171,640), biopolymers (U.S. Pat. No.5,958,745 and US Patent Publication No. US20030028917), environmental stress resistance (U.S. Pat. No.6,072,103), pharmaceutical peptides (U.S. Pat. No.6,080,560), improved processing traits (U.S. Pat. No.6,476,295), improved digestibility (U.S. Pat. No.6,531,648) low raffinose (U.S. Pat. No.6,166,292), industrial enzyme production (U.S. Pat. No.5,543,576), improved flavor (U.S. Pat. No.6,011,199), nitrogen fixation (U.S. Pat. No.5,229,114), hybrid seed production (U.S. Pat. No.5,689,041), and biofuel production (U.S. Pat. No.5,998,700). The patent documents disclosing these nucleic acid sequences are hereby incorporated by reference herein in their entirety.

[0160] The gene of interest mayencodea markerthat when expressed imparts a distinct phenotype to the plant host expressing the markerand thus allows such transformed plant host to be distinguished fromthose that do not have the marker. Such a nucleotide sequence may encode eithera selectable orscreenable marker, dependingon whetherthe marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, orthe like), oron whetherthe marker is simply a trait that one can identify through observation ortesting, such as by screening (e.g., the R-locus trait, color, fluorescence, etc.).

[0161] The present invention also contemplates genes of interest for expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein. Two non- limiting examples are antisense RNA and RNA with ribozyme activity. Both may serve possible functions in reducing or eliminating expression of native orintroduced plantgenes. Forexample, genes may be constructed or isolated, which when transcribed, produce antisense RNAthat is complementary to all or part(s) of a targeted messenger RNA(s). The antisense RNA reduces production of the polypeptide product of the messenger RNA. The polypeptide product may be any protein encoded by the plant genome. The aforementioned genes will be referred to as antisense genes. An antisense gene may thus be introduced into a plant by transformation methods to produce a novel transgenic plant with reduced expression of a selected protein of interest. For example, the protein may be an enzyme that catalyzes a reaction in the plant. Reduction of the enzyme activity may reduce or eliminate products of the reaction which include any enzymatically synthesized compound in the plant such as fatty acids, amino acids, carbohydrates, nucleicacids and the like. Alternatively, the protein may be a storage protein, such as a zein, ora structural protein, the decreased expression of which may lead to changes in seed amino acid composition or plant morphological changes respectively. The possibilities cited above are provided only by way of example and do not represent the full range of applications. Alternatively, genes may also be constructed or isolated, which when transcribed produce RNA enzymes, or ribozymes, which can act as endoribo nucleases and catalyze the cleavage of RNA molecules with selected sequences. The cleavage of selected messenger RNAs can result in the reduced production of their encoded polypeptide products.

[0162] Other genes of interest include genes that code for therapeutic proteins such as but not limited to cytokines and receptors (such as interleukins 1-36 and interferons, as well as their receptors), growth factorsand receptors (such as such as epidermal growth factor (EGF), acid fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor AA, AB, and BB(PDGF AA, AB and BB), insulin-like growth factor (IGF), transforming growth factor (TGF) and their receptors, human serum albumin, a -fetoprotein, antibodies (such as full length immunoglobulins comprising two light and two heavy chains, Fabs, scFvs (single chain variable fragment), camelid-type antibodies, antibody fragments, antibody fragment-fusions, antibody-receptor fusions, etc.), chemokines, hematopoietic growth factors (such as GM-CSF, G- CSF, ere), coagulation factors, complement factors, steroid hormones and their receptors (such as glucocorticoid hormones, mineralo cortical hormones, sexual steroid hormones, etc. and their receptors), matrix proteins (such as fibronectin, collagen, vitronectin, etc.), other bioactive peptides (such as adrenocorticotropic hormone and fragments, angiotensin and related peptides, atrial natriuretic peptides, bradykinin and related peptides, chemo tactic peptides, dynorphin and related peptides, endorphins and β-lipotropin fragments, enkephalin and related peptides, enzyme inhibitors, gastrointestinal peptides, growth hormone releasing peptides, luteinizing hormone releasing hormone and related peptides, melanocyte stimulating hormone and related peptides, neurotensin and related peptides, opioid peptides, oxytocin, vasopressin, vasotocin and related peptides, parathyroid hormone and fragments, protein kinase related peptides (including PKC), somatostatin and related pe tides, substance Pand related peptides, toxins, conditional toxins, antigens, tumor suppressor proteins, membrane proteins, vasoactive proteins and peptides, and anti-viral proteins. Alternatively, the genes of interest may encode industrial enzymes, representative examples of which include lipases, proteases, cellulases, pectinases, amylases, esterases, oxidoreductases, transferases, lactases, isome rases, and invertases.

3.2 Control sequences

[0163] The nucleic acid sequence of interest and/or modulator nucleic acid sequence of the invention ("effector nucleic acid sequences") are operably connected to at least one control sequence or regulatory element including a promoter for driving their expression. Useful promoters include those that are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, tissue-specific, and spatio-temporally regulated. Where expression in specific tissues or organs is desired, tissue-specific promoters may be used. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory elements of choice. Where continuous expression is desired throughout the cells of a host, constitutive promotersare utilized. Additional regulatorysequences upstream and/or downstream from the core promoter sequence may be included in expression cassettes to bring about varying levels of expression of effector nucleic acid sequences in a transgenic host.

3.2.1 Promoters

[0164] The choice of the promoter will vary upon the host in which the expression system of the invention is introduced and it shall be understood that the present invention contemplates any promoter that is operable in a chosen host. In specific embodiments, the hosts are selected from plants, animals and yeast.

[0165] Promoters contemplated by the present invention may be native to a host plant or may be derived from an alternative source, where the promoter is functional in the host plant. Numerous promoters that are active in plant cells have been described in the literature. The choice of plant promoter will generally vary depending on the temporal and spatial requirements for expression, and also depending on the target plant species. In some cases, expression in multiple tissues is desirable. While in others, tissue-specific, e.g., leaf-specific, expression is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters areselected forexpression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the effector nucleic acid sequences in the desired host cell.

[0166] These promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmental^ regulated, spatially-regulated, chemically regulated, stress- responsive, tissue-specific, viral and synthetic promoters. Promoter sequences a re known to be strong or weak. A strong promoter provides for a high level of gene expression, whereas a weak promoter providesfora very low level of gene expression. An inducible promoter is a promoter that provides forthe turning on and off of gene expression in response to an exogenous ly added agent, orto an environmental or developmental stimulus.

[0167] Within a plant promoter region there are several domains that are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the "core promoter region" containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and representsa transcription initiation sequence that defines the transcription start point forthe structural gene.

[0168] The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore, the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences upstreamof the core constitute the remainderof the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemicals, hormones).

[0169] A range of naturally-occurring promoters is known to be operable in plants and have been used to drive the expression of heterologous (both foreign and endogenous) genes in plants: forexample,the constitutive 35S cauliflower mosaic virus (CaMV) promoter, the ripening- enhanced tomato polygalacturonase promoter (Bird ef a/., 1988), the E8 promoter (Diekman & Fischer, 1988) and the fruit specific 2A1 promoter (Pear era/., 1989)and many others, e.g., U2 and U5 snRNA promoters from maize, the promoter from alcohol dehydrogenase, the Z4 promoter from a gene encoding the Z422 kD zein protein, the Z10 promoterfrom a gene encoding a 10 kD zein protein, a Z27 promoterfrom a gene encoding a 27 kD zein protein, the A20 promoterfrom the gene encoding a 19 kD-zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene and the actin promoterfrom rice, e.g., the actin 2 promoter (WO 00/70067); seed specific promoters, such as the phaseolin promoterfrom beans, may also be used. The nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the nucleicacid sequence orencoded polypeptide to be synthesized only when the crop plantsare treated with the inducing chemicals. Chemical induction of gene expression is detailed in EP 0332104 (to Ciba -Geigy) and U.S. Pat. No.5,614,395. A preferred promoter for chemical induction is the tobacco PR-la promoter.

[0170] Examples of some constitutive promoters which have been described include the rice actin 1 (Wang etal., 1992; U.S. Pat. No.5,641,876), CaMV35S (Odell etal., 1985), CaMV 19S (Lawton era/., 1987), nos, Adh, sucrose synthase; and the ubiquitin promoters.

[0171] Examples of tissue specific promoters which have been described includethe lectin (Vodkin, 1983; Lindstrom era/., 1990) corn alcohol dehydrogenase 1 (Vogel era/., 1989;

Dennis etal., 1984), corn light harvesting complex (Simpson, 1986; Bansal etal., 1992), corn heat shock protein (Odell era/., 1985), pea small su bun it RuBP carboxylase (Poulsen era/., 1986), Ti plasmid mannopine synthase (Langridge etal., 1989), Ti plasmid nopaline synthase (Lang ridge er a/., 1989), petunia chalcone isomerase (vanTunen era/., 1988), bean glycine rich protein 1 (Keller era/., 1989), truncated CaMV 35S (Odell er al., 1985), potato patatin (Wenzler etal., 1989), root cell (Yamamoto etal., 1990), maize zein (Reina er al., 1990; Kriz era/., 1987; Wandelt era/., 1989; Langridge era/., 1983; Reina era/., 1990), globulin-1 (Belanger era/., 1991), . alpha. - tubulin, cab (Sullivan er al., 1989), PEPCase (Hudspeth &Grula, 1989), R gene complex-associated promoters (Chandlerera/., 1989), histone, and chalcone synthase promoters (Franken era/., 1991). Tissue specificenhancersare described in Fromm er al. (1989).

[0172] Inducible promoters that have been described include the ABA- and turgor- inducible promoters, the promoterof the auxin-binding protein gene (Schwob etal., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston era/., 1988), the MPI proteinase inhibitor promoter (Cordero era/., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohlerera/., 1995; Quigley er al., 1989; Martinez era/., 1989).

[0173] Several othertissue-specific regulated genes and/or promoters have been reported in plants. These include genes encoding the seed storage proteins (such as napin, cruciferin, β-conglycinin, and phaseolin) zein oroil body proteins (such as oleosin), orgenes involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase. And fatty acid desaturases (fad 2-1)), and othergenes expressed during embryo development (such as Bce4, see, for example, EP 255378 and Kridl er al., 1991). Particularly useful for seed -specific expression is the pea vicilin promoter (Czako era/., 1992). (See also U.S. Pat. No.5,625,136, herein incorporated by reference). Other useful promoters for expression in mature leaves a re those that a re switched on at the onsetof senescence, such as the SAG promoterfrom Arabidopsis (Gan era/., 1995).

[0174] A class of fruit-specific promoters expressed at or during antithesis through fruit development, at least until the beginning of ripening, is discussed in U.S. Pat. No.4,943,674. cDNA clones that are preferentially expressed in cotton fiber have been isolated (John etal., 1992). cDNA clones from tomato displaying differential expression during fruit development have been isolated and characterized (Ma nsson etal., 1985, Slater ei a/., 1985). The promoterfor polygalacturonase gene is active in fruit ripening. The polygalacturonase gene is described in U.S. Pat. No.4,535,060, U.S. Pat. No.4,769,061, U.S. Pat. No.4,801,590, and U.S. Pat. No.

5,107,065, which disclosuresare incorporated herein by reference.

[0175] Other examples of tissue-specific promoters include those that direct expression in leaf cells following damageto the leaf (for example, from chewing insects), in tubers (for example, patatin gene promoter), and in fiber cells (an example of a develop mentally-regulated fibercell protein is E6 (John et al., 1992). The E6 gene is mostactive in fiber, although low levels of transcripts are found in leaf, ovule and flower.

[0176] Examples of other plant promoters reported in the literature include the chloroplast glutamine synthetase GS2 promoterfrom pea, the chloroplast fructose-1,6- biphosphatase (FBPase) promoterfrom wheat, the nuclear photosynthetic ST-LS1 promoterfrom potato, the serine/threonine kinase (PAL) promoterandthe glucoamylase (CHS) promoterfrom Arabidopsis thaliana. Also reported to be active in photosynthetically active tissues are the ribulose-l,5-bisphosphate carboxylase (RbcS) promoterfrom eastern larch (Larix laricina), the promoterforthe cab gene, cab6, from pine, the promoter for the Cab-1 genefrom wheat, the promoterforthe CAB-1 gene fromspinach, the promoterforthe cablRgenefrom rice, the pyruvate, orthophosphate dikinase (PPDK) promoterfrom corn, the promoterforthe tobacco Lhcbl*2 gene, the Arabidopsis thaliana SUC2 sucrose-H+ symporter and the promoterforthe thylakoid membrane proteins fromspinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS).

Other promoters forthe chlorophyll a/b-binding proteins may also be utilised in the invention, such as the promoters forthe LhcB gene and PsbPgene from white mustard.

[0177] The tissue-specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using the diphtheria toxin sequence. One ca n also achieve tissue-specific expression with "leaky" expression by a combination of different tissue- specific promoters (Beals etal., 1997). Othertissue-specificpromoterscan be isolated byone skilled in the art (see U.S. Pat. No.5,589,379). Several inducible promoters ("gene switches") have been reported. Manyare described in the review byGatz (1996) and Gatz (1997). These include tetracycline re ressor system, Lac repressor system, copper-inducible systems, salicylate- inducible systems (such as the PRla system), glucocorticoid (Aoyama et al., 1997) and ecdysome- inducible systems. Also included are the benzene sulfonamide (U.S. Pat. No.5,364,780) and alcohol (WO 97/06269 and WO 97/06268) inducible systems and glutathione S-transferase promoters. Otherstudies have focused on genes inducibly regulated in response to environmental stress or stimuli such as increased salinity. Drought, pathogen and wounding. (Graham etal., 1985; Graham ei al., 1985, Smith etal., 1986). Accumulation of metallocarboxypeptidase-inhibitor protein has been reported in leaves of wounded potato plants (Graham etal., 1981). Other plant genes have been reported to be induced methyl jasmonate, elicitors, heat-shock, anaerobic stress, or herbicide safeners.

[0178] In some embodiments, the promoter is selected from a gamma zein promoter, an oleosin olel6 promoter, a globulinl promoter, an actin I promoter, an actin cl promoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5 promoter, a globulinl promoter, a b- 32, ADPG-pyrophosphorylase promoter, an Ltpl promoter, an Ltp2 promoter, an oleosin olel7 promoter, an oleosin olel8 promoter, an actin 2 promoter, a pollen-specific protein promoter, a pollen -specific pectate lyase promoter, an anther-specific protein promoter, an anther-specific gene RTS2 promoter, a pollen-specific gene promoter, a tapeturn-specificgene promoter, tapetum- specificgene RAB24 promoter, a anthranilate synthase alpha subunit promoter, an alpha zein promoter, an anthranilate synthase beta subunit promoter, a dihydrodipicolinate synthase promoter, a Thil promoter, an alcohol dehydrogenase promoter, a cab binding protein promoter, an H3C4 promoter, a RUBISCO SS starch branching enzyme promoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, a regulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, a cellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteine hydrolase promoter, a superoxide dismutase promoter, a C-kinase receptor promoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRINA promoter, a glucose-6 phosphate isomerase promoter, a pyrophosphate-fructose 6-phosphatelphosphotransferase promoter, an ubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDa photosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDa vacuolar ATPase subunit promoter, a metallothionein-like protein promoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA- and ripening- inducible-like protein promoter, a phenylalanine ammonia lyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteine hydrolase promoter, an a -tubulin promoter, a cab promoter, a PEPCase promoter, an Rgene promoter, a lectin promoter, a light harvesting complex promoter, a heat shock protein promoter, a chalcone synthase promoter, a zein promoter, a globulin-1 promoter, an ABA promoter, an auxin-binding protein promoter, a UDP glucose flavonoid glycosyl-transferase gene promoter, an INTI promoter, an actin promoter, an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35S promoter, a CaMV 19S promoter, a histone promoter, a turgor-inducible promoter, a pea small subunit RuBP carboxylase promoter, a Ti plasmid mannopine synthase promoter, Ti plasmid nopaline synthase promoter, a petunia chalcone isomerase promoter, a bean glycine rich protein I promoter, a CaMV35S transcript promoter, a potato patatin promoter, or a S-E9 small subunit RuBP carboxylase promoter.

[0179] In some embodiments, the promoter is an alcohol dehydrogenase promoter

(e.g., derived from Aspergillus nidulans such as AlcAP).

3.2.2 Other Regulatory Elements

[0180] In addition to promoters, a variety of 5' and 3'transcriptional regulatory sequences is also available for use in expressing an effector nucleic acid sequence of the invention. 3.2.3 Transcription terminators

[0181] The effector nucleic acid sequences of the present invention will typically be opera bly linked to a 3' non-translated sequence that functions in cells to terminate transcription and/orto cause addition of a polyadenylated nucleotide sequence to the 3'end ofthe RNA sequence transcribed from the relevant effector nucleic acid sequences. Thus, a 3' non -translated sequence refers to that portion of a gene comprising a nucleic acid segment that contains a transcriptional termination signal and/or a polyadenylation signal and any other regulatory signals {e.g., translational termination signals) capable of effecting mRINA processing or gene expression. The polyadenylation signal is characterised by modulating the addition of polyadenylicacid tracts to the 3' end ofthe mRNA precursor. Polyadenylation signals are commonly recognised bythe presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon. The 3' non-translated regulatory sequence desirably includes from a bout 50 to 1,000 nucleotide base pairs and contains transcriptional and translational termination sequences. [0182] Exemplary 3' non-translated sequences that a re operable in plants include the CaMV35S terminator, the tml terminator, the nopaline synthase terminator, the pea rbcS E9 terminator, the terminatorforthe T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or to ma to, although other 3' elements known to those of skill in the art can also be employed. Alternatively, one also could use a gamma coixin, oleosin 3 orotherterminatorfromthe genus Co/^'x. Exemplary 3' elements include those from the nopaline synthase gene of Agrobacterium tumefaciens (Bevan et al., 1983), the terminatorforthe T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or to ma to. Non- limiting examples of transcription terminators useful in animal cells (e.g., mammalian cells), include those derived from viruses including SV40, as described in Sambrook etal., supra, as well as growth hormone transcriptional terminators (see, e.g., U.S. Pat. No.5,122,458), and the like. Suitable transcriptional terminators for use in yeast include, but are not limited to FBAt, GPDt, GPMt, ERGlOt, GALlt, CYC1, and ADH1 transcription terminators.

3.2.4 Untranslated leader sequences

[0183] As the nucleicacid sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can influence gene expression, one may also wish to employ a particular leader sequence. Suitable leader sequences are contemplated to include those which comprise sequences predicted to direct optimum expression of the attached gene, i.e., to include a suitable consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure. Sequences that a re derived from genes that are highly expressed in plants will be most preferred.

3.2.5 Introns

[0184] Additional sequencesthat have been found to enhance gene expression in transgenic plants include intron sequences (e.g., fromAdhl, bronzel, actinl, actin 2 (WO 00/760067), orthe sucrose synthase intron) and viral leadersequences (e.g., from TMV, MCMV and AMV). For example, a number of non -translated leader sequences derived from viruses are known to enhance expression. Specifically, leader sequences fromTobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g., Gallie et al., 1987; Skuzeski et al., 1990). Other leaders known in the art include but are not limited to: Picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5 noncoding region) (Elroy-Stein etal., 1989); Potyvirus leaders, for example, TEV leader (Tobacco Etch Virus); MDMV leader(Maize Dwarf Mosaic Virus); Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak etal., 1991); Untranslated leaderfromthe coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling et al., 1987; Tobacco mosaic virus leader (TMV), (Gallie et al., 1989; and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel etal., 1991. See also, Della-Cioppa etal., 1987.

[0185] Introns for use in the present invention contain the required 3' and 5' splice sites to facilitate splicing at the intron/exon junction and subsequent removal of the intron sequence during transcription. Intronsthat are recognized and spliced by plant cellular machinery are well known in the art and any such intron of functional fragment can be used in the methods and transgenic plants of the present invention. Exemplary introns for use in the present methods include those from plants, such as the intron from potato light-inducible tissue specific ST-LS1 gene, as well as synthetic plant introns (see e.g. Goodall etal., (1990) Plant Mol Biol.14(5):727- 33). In the constructs of the present invention that comprise a split genes and 3' and 5' regions of an intron, the 3' and 5' regions ofan intron can be 3'and 5' regions of a single intron orcan be a 3' region of one intron and a 5' region of another intron, providing the 3'and 5' regions contain the necessary splice sitesforsplicing. Regulatory elements such as Adh intron 1 (Callis etal., 1987), sucrose synthase intron (Vasil etal., 1989) orTMV omega element (Gallie, et al., 1989), may further be included where desired.

3.2.6 Enhancers

[0186] Examples of enhancers include elements from the CaMV35S promoter, octopine synthase genes (Ellis etal., 1987), the rice actin I gene, the maize alcohol dehydrogenase gene (Callis et al., 1987), the maize shrunken I gene (Vasil etal., 1989), TMV Omega element (Gallie et al., 1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma ef al., 1988).

[0187] Enhancers that may be used for enhancing expression in animal hosts [e.g., mammalian hosts) include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.

3.2.7 Site-specific recombinase activation

[0188] Regulated expression ofan effector nucleicacid sequence of the present invention can also be regulated by other genetic strategies including recombinase-mediated gene activation in which a blocking nucleic acid sequence comprising transcription termination sequence bound by site-specific sequences ("recombinase recognition sites") is placed between a promoter and the effector nucleicacid sequence, thereby blocking the expression of the effector nucleicacid sequence fromthe promoter. The blocking nucleic acid sequence can be removed by expression of a coding sequence fora site-specific recombinase that mediates excisionof the blocking sequence, thereby resulting in the expression of the effector nucleicacid sequence. In this case, the recombinase gene, the effector nucleicacid sequence, or both can be underthe control of tissue- specific, developmental-specific or inducible promoters. Illustrative recombinases, which are site- specific, include Cre, modified Cre, Dre, Hp, FLP-wild type (wt), FLP-L, FLPe, Flpo or phiC31. Non- limiting examples of recombinase recognition sites include loxP, FRT, raxand attP/B.

Recombination may be effected by any art-known method, e.g., the method of Doetschman etal. (1987, Nature 330:576-578); the method of Thomas etal. (1986, Cell 44:419-428); the Cre-loxP recombination system (Sternberg and Hamilton, 1981, J. Mol. Biol. 150:467-486; Lakso etal., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236); the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman etal., 1991, Science 251:1351-1355; Lyznik etal., 1996, Nucleic Acids Res. 24(19):3784-3789); the Cre-loxP-tetracycline control switch (Gossenand Bujard, 1992, Proc. Natl. Acad. Sci. USA 89:5547-51); and ligand-regulated recombinase system (Kellendonk er al., 1999, J. Mol. Biol. 285:175-82). Desirably, the recombinase is highly active, e.g., the Cre-loxP orthe FLPe system, and has enhanced thermostability (Rodrguez etal., 2000, Nature Genetics 25:139-40).

[0189] In specific embodiments, site-specific recombination is used for reconstituting a functional rep gene in an ancillary construct that comprises the repgene in a non-contiguous form. Reconstitutionof the rep gene leads to production of a Rep protein in trans for the replication ofan associated proreplicon. 3.2.8 tRNA suppressor genes

[0190] An alternate genetic strategy is the use of tRNA suppressor gene. Forexample, the regulated expression of a tRNA suppressor gene can conditionally control expressionof an effector nucleic acid sequence containing an appropriate termination codon as described by Ulmaso et al. 1997. Again, either the tRNA suppressor gene, the effector nucleic acid sequence, or both can be under the control of tissue-specific, developmental-specific or inducible promoters.

3.2.9 Site-specific replicase activation

[0191] In specific embodiments, expression of an effector nucleic acid sequence of the invention is regulated using replicase-mediated gene activation. In these embodiments, the effector nucleic acid sequence, which may be in the form of a contiguous nucleic acid entity or a non-contiguous nucleic acid entity, is expressed using a binary expression system that comprises a proreplicon and a regulated transactivating replication gene (rep). The proreplicon generally comprises c/s-acting sequences (e.g., viral sequences) flanking the effector nucleic acid sequence, which are required for replication, but is incapableof episomal replication in cells (e.g., plant cells) because it lacks a functional rep gene(s) essential for replication. Under appropriate stimulus, the transactivating rep gene expresses the replication protein (Rep) (e.g., viral Rep) missing in the proreplicon and allows the release of a replicon fromthe proreplicon and its episomal replication in a cell autonomous manner. Typically the replication elements are derived from viruses, as described forexample below. Non-liming examples of such binary expression systems are described by Dale etal. (U.S. Pat. No.7,863,430), Dugdale etal. (2013), Yadav (U.S. Pat. No.

6,077,992) and Yadav eta/. (U.S. Pat. No.6,632980 and U.S. Pat. Appl. Pub. No.2004/0092017), each of which is incorporated by reference herein in their entirety.

[0192] Thus, replicon replication can be targeted to specific cells by controlling the expression of replication protein(s) to those cells. The proreplicon embodiments of the present invention are particularly advantageousforexpressing effector nucleic acid sequences in plant hosts. Plants are generally sensitive to cellular toxicity and/or the detrimental effect of viral replication and/or replication protein(s) in earlystages of plant growth and differentiation that involve cell division and differentiation. Thus, controlling the expression of the replication protein and replicon replication entirely or largely to non-dividing, terminally-differentiated cells will reduce the detrimental effect of replicon replication on plant growth and development. Examples of such terminally-differentiated cells include, but are not limited to, the storage cells of seed and root tissues and mature leaf cells. Furthermore, the proreplicon when introduced into a plant host serves as a master copyfor replicons not only in different generations but also in the same generation if cell divisions occurafterthe onset of episomal replication. This strategy will also solve the problem of episomal instability through cell divisions, since episomes are unstable in the absenceof selection. Furthermore, replicon replication is expected to achieve high level expression of effector nucleic acid sequences through gene amplification that is heritable when stably integrated into the host chromosome and cell autonomous.

[0193] Replicase genes a re selected so that they recognize the Rep recognition elements required for release of a replicon from the proreplicon and autonomousepisomal re lication of the replicon. Exemplary rep genes include those from ssDNA plant viruses, such as Geminiviruses and Nanoviruses, as well as those from bacteria, including phyto plasmaI rep genes. For example, a Mastrevirus repgene encoding both Rep and RepA proteins can be included in a construct for expressing an effector nucleic acid sequence. In otherexamples, a Curtovirus, Topocuvirus or Begomovirus rep gene is included. In further examples, a Nanovirus rep gene encoding the master replication initiation protein (M-Rep) is included.

3.3 Otherseauences

[0194] Other sequences can be included within oradjacentto the expression cassettes or constructs described herein to promote any one or more of integration of the constructs into the plant genome, selection or screening of transgenic host cells and/or transgenic hosts.

[0195] The expression cassettes or constructs can also be introduced into a vector, such as a plasmid. They can be introduced into the same vector or different vectors. Furthermore, a vector can include two or more of a first construct or expression cassette, and/or two or more of a second construct or expression cassette, so that the vector comprises two or more copies of the a modulator nucleic acid sequence and/or two or more copies of a target nucleic acid sequence. Similarly, in embodiments where a third construct is utilised (e.g., to express a repgene), a vector can include two or more copies of the third construct. Plasmid vectors include additional DNA sequencesthat provide foreasy selection, amplification, and transformation ofthe expression construct in prokaryotic and eukaryotic cells, e.g., pUC-derived vectors, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors, or pBS-derived vectors. Additional nucleic acid sequences include origins of replication to provide forautonomous replication ofthe vector, selectable markergenes, desirably encoding antibioticor herbicide resistance, unique multiple cloning sites providing for multiple sites to insert nucleic acid sequences or genes.

[0196] Desirably, the vector contains oneormore elements that permit stable integration ofthe construct into the host cell genome or autonomous replication ofthe vector in the cell independent ofthe genome ofthe cell. In particular embodiments, the vector contains one or more elements so that the construct is stably integrated into the hostcell ge nome when the vector is introduced into a host cell. In some examples, the vector contains additional nucleicacid sequences fordirecting integration by homologous recombination into thegenome ofthe host cell, which facilitate integration ofthe construct into the host cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integrational elements should desirably contain a sufficient numberof nucleicacids, such as 100 to 1,500 nts, usually 400 to 1,500 nts and more usually 800 to 1,500 nts, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome ofthe host cell. Furthermore, the integrational elements may be non-coding orcoding nucleic acid sequences.

[0197] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.

[0198] To facilitate identification of transformants, a selectable or screenable marker gene is included adjacent to or within the constructs ofthe present invention. The actual choice of a marker is not crucial as long as it is functional in combination with the host cell of choice. The marker gene and effector nucleicacid sequence (and optionally a rep gene) do not have to be linked, since co -transformation of unlinked genes is also an efficient process in transfection or transformation, especially transformation of plants (see e.g., U.S. Pat. No.4,399,216).

[0199] Included within the terms selectable orscreenable markergenes aregenesthat encode a "secretable marker" whose secretion can be detected as a means of identifying or selecting fortransformed cells. Examples include markers that encode a secretable antigen that can be identified by antibody interaction, or secretable enzymes that can be detected by their catalyticactivity. Secretable proteins include, butare not restricted to, proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin ortobacco PR-S); small, diffusible proteins detectable, e.g. by ELISA; and small active enzymes detectable in extra cellularsolution (e.g., a-amylase, β-lactamase, phosphinothricin acetyltransferase).

[0200] Exemplary selectable markers forselection of plant transformants include, but are not limited to, a hyg gene which encodes hygromycin B resistance; a neomycin

phosphotransferase (neo) gene conferring resistance to kanamycin, paromomycin, G418 and the like; a glutathione-S-transferase gene from rat liver conferring resistance to glutathione derived herbicides; a glutamine synthetase gene conferring, upon expression, resistance to glutamine synthetase inhibitors such as phosphinothricin; an acetyl transferase gene from Streptomyces viridochromogenes conferring resistance to the selective agent phosphinothricin; a gene encoding a 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance to N- phosphonomethylglycine; a bargene conferring resistance against bialaphos; a nitrilase genesuch as bxn from Klebsiella ozaenae which confers resistance to bromoxynil; a dihydrofolate reductase (DHFR) gene conferring resistance to methotrexate; a mutant ace tola ctate synthase gene (ALS), which confers resistance to imidazolinone, sulfonylurea orother ALS-inhibiting chemicals; a mutated anthranilate synthase gene that confers resistance to 5-methyl trypto han; or a dalapon dehalogenase gene that confers resistance to the herbicide 2,2 -dichloropropionic acid.

[0201] Exemplary screenable markers include, but are not limited to, a uidA gene encoding a β-glucuronidase (GUS) enzyme forwhich various chromogenic substrates a re known; a β-galactosidase gene encoding an enzyme for which chromogenic substrates are known; an aequorin gene which may be employed in calcium-sensitive bioluminescence detection; a green fluorescent protein gene; a luciferase (luc) gene, which allows for bioluminescence detection; a β- lactamase gene, which encodesan enzyme forwhich various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); an R-locus gene, encoding a product that regulates the production of anthocyanin pigments (red colour) in plant tissues; an a-amylase gene; a tyrosinase gene, which encodes an enzyme ca able of oxidizing tyrosine to do pa and dopaquinone which in turn condenses to formthe easily detectable compound melanin; ora xylE gene, which encodes a catechol dioxygenase that can convert chromogenic catechols.

4 Methods of producing transgenic hosts

[0202] The present invention contemplates introducing the subject nucleic acid constructs and expression system in any plant host in which it is desired to improve a growth characteristic of the host, as described for example herein, and o tional to express a nucleic acid sequence fo interest. Representative plant hosts are suitably selected from monocotyledons, dicotyledons and gymnosperms. The plant maybe an ornamental plantorcrop plant. Illustrative examples of host cells from ornamental plants include, but are not limited to, host cells from Malus spp, Crataegus spp, Rosa spp., Betula spp, Sorbus spp, Olea spp, Nerium spp, Salix spp and Populus spp. Illustrative examples of host cells fromcrop plants include host cells from plant species that are cultivated in orderto produce a harvestable product such as, but not limited to, Abelmoschus esculentus (okra), Acacia spp., Agave fourcroydes (henequen), Agave sisalana (sisal), Albizia spp., Allium fistulosum (bunching onion), Allium sativum (garlic), Allium spp. (onions), Alpinia galanga (greater ga la nga), Amaranthus caudatus, Amaranth us spp., Anacardium spp. (ca shew), Ananas comosus (pinea pple), Anethum graveolens (dill), Annona che mola (cherimoya ), Apios americana (America n pota to ea n), Arachis hypogaea (pea nut), Arctium spp. (burdock), Artemisia spp. (wormwood), Aspalathus linearis (red bush tea), Athertonia diversifolia, Atriplex nummularia (old ma n sa Itbush), Averrhoa carambola (sta rf ruit), Azadirachta indica

(neem), Backhousia spp., Bambusa spp. (ba mboo), Beta vulgaris (suga r beet), Boehmeria nivea (ra mie), bok choy, Boronia megastigma (sweet boronia), Brassica carinata (Abyssinia n musta rd), Brassica juncea (India n musta rd), Brassica napus (ra peseed), Brassica oleracea (ca bbage, broccoli), Brassica oleracea va r Alboga bra (ga i lum), Brassica parachinensis (choi sum), Brassica pekensis (Wong bok or Chinese ca bbage), Brassica spp., Burcella obovata, Cajanus cajan (pigeon pea ), Camellia sinensis (tea ), Cannabis sativa (non-d rug hemp), Capsicum spp., Carica spp.

(pa paya ), Carthamus tinctorius (safflower), Carum carvi (ca raway), Cassinia spp.,

Castanospermum australe (blackbea n), Casuarina cunninghamiana (beefwood), Ceratonia siliqua (ca rob), Chamaemelum nobile (cha momile), Chamelaucium spp. (Gera ldton wax), Chenopodium quinoa (q uinoa), Chrysanthemum (Ta nacetum), cinerariifolium (pyreth ru m), C/cer arietinum

(chickpea), Cichorium intybus (chicory), Clematis spp., Clianthus formosus (Sturt's desert pea), Cocos nucifera (coconut), Coffea spp. (coffee), Colocasia esculenta (ta ro), Coriandrum sativum (coria nder), Crambe abyssinica (cra mbe), Crocus sativus (saffron), Cucurbita foetidissima (buffa lo gourd), Cucurbita spp. (gourd), Cyamopsis tetragonoloba (gua r), Cymbopogon spp. (lemongrass), Cytisus proliferus (tagasaste), Daucus carota (ca rrot), Desmanthus spp., Dioscorea esculenta (Asiatic ya m), Dioscorea spp. (ya ms), Diospyros spp. (persimmon), Doronicum sp., Echinacea spp., Eleocharis dulcis (water chestnut), Eleusine coracana (finger millet), Emanthus arundinaceus , Eragrostis tef (tef), Erianthus arundinaceus , Eriobotrya japonica (loquat), Eucalyptus spp., Eucalyptus spp. (gil ma llee), Euclea spp., Eugenia malaccensis (jumba ), Euphorbia spp., Euphoria longana (longa n), Eutrema wasabi (wasa bi), Fagopyrum esculentum (buckw heat), Festuca arundinacea (ta ll fescue), Ficus spp. (fig), Flacourtia inermis, Flindersia grayliana (Queensland ma ple), Foeniculum olea a, Foeniculum vulgare (fennel), Garcinia mangostana (ma ngosteen), Glycine latifolia, Glycine max (soybea n), Glycine max (vegeta ble soybean), Glycyrrhiza glabra (licorice), Gossypium spp. (cottons), Grevillea spp., Grindelia spp., Guizotia abyssinica (niger), Harpagophyllum sp., Helianthus annuus (high oleic sunflowers), Helianthus annuus (monosun sunflowers), Helianthus tuberosus (Jerusa lem a rtichoke), Hibiscus cannabinus (kenaf), Hordeum bulbosum, Hordeum spp. (waxy ba rley), Hordeum vulgare (ba rley), Hordeum vulgare subsp. sponta neum, Humulus lupulus (hops), Hydrastis canadensis (golden sea l), Hymenachne spp., Hyssopus officinalis (hyssop), Indigofera spp., Inga edulis (ice crea m bea n), Inocarpus tugiter, Ipomoea batatas (sweet potato), Ipomoea sp. (ka ng kong), Lablab purpureus (w hite la bla b), Lactuca spp. (lettuce), Lathyrus spp. (vetch), Lavandula spp. (lavender), Lens spp. (lentil), Lesquerella spp. (bladderpod), Leucaena spp., Lilium spp., Limnanthes spp. (meadowfoam), Linum usitatissimum (flax), Linum usitatissimum (linseed), Linum usitatissimum (Linola.TM.), Litchi chinensis (lychee), Lotus corniculatus (birdsfoot trefoil), Lotus pedunculatus, Lotus sp., Luffa spp., Lunaria annua (honesty), Lupinus mutabilis (pea rl lupin), Lupinus spp. (lupin), Macadamia spp., Mangifera indica (ma ngo), Manihot esculenta (cassava ), Medicago spp. (lucerne), Medicago spp., Melaleuca spp. (tea tree), Melaleuca uncinata (broombush), Mentha tasmannia, Mentha spicata (spea rmint), Mentha X piperita (peppermint), Momordica charantia (bitter melon), Musa spp. (ba na na), Myrciaria cauliflora (ja botica ba), Myrothamnus flabellifolia, Nephelium lappaceum (rambutan), Nerine spp., Ocimum basilicum (basil), Oenanthejavanica (water dropwort), Oenothera biennis (evening primrose), Olea europaea (olive), Olearia sp., Origanum spp.

(marjoram, oregano), Oryza spp. (rice), Oxalis tuberosa (oca), Ozothamnus spp. (rice flower), Pachyrrhizus ahipa (yam bean), Panax spp. (ginseng), Panicum miliaceum (common millet), Papaver spp. (poppyj, Parthenium argentatum (guayule), Passiflora sp., Paulownia tomemtosa (princess tree), Pelargonium graveolens (rose geranium), Pelargonium sp., Pennisetum americanum (bulrush or pearl millet), Persoonia spp., Petroselinum crispum (parsley), Phacelia tanacetifolia (tansy), Phalaris canariensis (canary grass), Phalaris sp., Phaseolus coccineus (scarlet runner bean), Phaseolus lunatus (lima bean), Phaseolus spp., Phaseolus vulgaris (culinary bean), Phaseolus vulgaris (navy bean), Phaseolus vulgaris (red kidney bean), Pisum sativum (field pea), Plantago ovata (psyllium), Polygonum minus, Polygonum odoratum, Prunus mume (Japanese apricot), Psidium guajava (guava), Psophocarpus tetragonolobus (winged bean), Pyrus spp.

(nashi), Raphanus satulus (long white radishor Daikon), Rhagodia spp. (saltbush), Ribes nigrum (black currant), Ricinus communis (castor bean), Rosmarinus officinalis (rosemary), Rungia klossii (rungia), Saccharum officinarum (sugarcane), Salvia officinalis (sage), Salvia sclarea (clary sage), Salvia sp., Sandersonia sp., Santalum acuminatum (sweet quandong), Santalum spp.

(sandalwood), Sclerocarya caffra (marula), Scutellaria galericulata (scullcap), Secale cereale (rye), Sesamum indicum (sesame), Setaria italica (foxtail millet), Simmondsia spp. (jojoba), Solanum spp., Sorghum almum (sorghum), Stachys betonica (wood betony), Stenanthemum scortechenii, Strychnos cocculoides (monkey orange), Stylosanthes spp. (stylo), Syzygium spp., Tasmannia lanceolata (mountain pepper), Terminalia karnbachii, Theobroma cacao (cocoa), Thymus vulgaris (thyme), Toona australis (red cedar), Trifoliium spp. (clovers), Trifolium alexandrinum (berseem clover), Trifolium resupinatum (persian clover), Triticum spp., Triticum tauschii, Tylosema esculentum (morama bean), Valeriana sp. (valerian), Vernonia spp., Vetiver zizanioides (vetiver grass), Vicia benghalensis (purple vetch), Vicia faba (faba bean), Vicia narbonensis (narbon bean), Vicia sativa, Vicia spp., Vigna aconitifolia (mothbean), Vigna angularis (adzuki bean), Vigna mungo (black gram), Vigna radiata (mung bean), Vigna spp., Vigna unguiculata (cowpea), Vitis spp. (grapes), Voandzeia subterranea (bambarra groundnut), Triticosecale (triticale), Zea mays (bicolour sweetcorn), Zea mays (maize), Zea mays (sweet corn), Zea mays subsp. mexicana (teosinte), Zieria spp., Zingiber officinale (ginger), Zizania spp. (wild rice), Ziziphus jujuba

(common jujube). In particular embodiments, the first and second constructs are introduced into Gossypium spp. (cottons), Nicotiana tabacum (tobacco), Ananas comosus (pineapple), Saccharum spp (sugarcane), Musa spp (banana), Lycopersicon esculentum (tomato) and Solanum tuberosum (potato) cell, Manihot spp. (cassava), Zea mays (maize), Triticum spp. (wheat), bean, Capsicum spp. (pepper), Lactuca sativa (lettuce), Carica papaya (papaya), Beta vulgaris (beet), Brassica oleracea co nva r. capitata (cabbage), Ipomoea batatas (sweet potato) or Fa baceae family

(legumes) host cells.

[0203] Constructs corresponding to the subject expression system may be introduced into a plant of interest or part thereof using any suitable method, and the kind of method employed will differ depending on the intended cell type, part and/or organ ism of interest. For example, four general classes of methods for delivering nucleic acid molecules into cells have been described: (1) chemical methods such as calcium phosphate precipitation, polyethylene glycol (PEG)-mediate precipitation and lipofection; (2) physical methods such as microinjection, electro poration, acceleration methods and vacuum infiltration; (3) vector based methods such as bacterial and viral vector-mediated transformation; and (4) receptor-mediated. Transformation techniques that fall within these and other classes a re well knownto workers in the art, and new techniques are continually becoming known. The particular choice of a transformation technology will be determined by its efficiency to transform certain host species as well as the experience and preference of the person practicing the invention witha pa rticular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce a synthetic construct of the invention into cells is not essential to ora limitation of the invention, provided it achieves an acceptable level of nucleic acid transfer. Thus, the constructs can be introduced into tissues or host cells by any number of routes, including viral infection, phage infection, microinjection, electroporation, orfusion of vesicles, lipofection, infection by

Agrobacterium tumefaciens or A. rhizogenes, or protoplast fusion. Jet injection may also be used for intra -muscular administration (as described for example by Furth etal., 1992, Anal Biochem 205:365-368). The synthetic constructs may be coated onto microprojectiles, and delivered into a host cell or into tissue by a particle bombardment device, or "gene gun" (see, forexample, Tang et al., 1992, Nature 356:152-154).

[0204] Guidance in the practical implementation of transformation systems for plant improvement is widely available to those skilled in the art (see e.g., Birch (1997) An. Rev. Plant Physiol. Plant Mol. Biol.48: 297-326). Non -limiting examples of methods for transformation of plants include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria) , viral-mediated nucleicacid delivery, silicon carbide or nucleicacid whisker mediated nucleicacid delivery, liposome mediated nucleicacid delivery, microinjection, microparticle bombardment, calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation,

electroporation, nanoparticle-mediated transformation,, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any otherelectrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki etal. ("Procedures for Introducing Foreign DNA into Plants"in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds.(CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska {Cell. Mol. Biol. Lett.7:849-858 (2002)).

[0205] Thus, in some pa rticular embodiments, the introduction of a construct into a plant host is via bacterial-mediated transformation, particle bombardment transformation, calcium- phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus-mediated nucleicacid delivery, whisker-mediated nucleicacid delivery, microinjection, sonication, infiltration, polyethylene glycol-mediated transformation, any other electrical, chemical, physical and/or biological mechanism that results in the introduction of nucleic acid into the plant host.

[0206] Agrobacterium-med\ated transformation is a commonly used method for transforming plants, in particular, dicot plants, because of its high efficiency of transformation and becauseof its broad utility with many different species. Agrobacterium -mediated transformation typically involves transfer of the binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain that maydepend on the complement of vir genes carried bythe host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (Uknes etal. (1993) Plant Cell 5:159-169). The transferof the recombinant binary vector to Agrobacterium can be accomplished bya tripa rental mating procedure using Escherichia coli carrying the recombinant binary vector, a helperf. coli strain that carries a plasmid that is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation (Hofgen & Willmitzer (1988) Nucleic Acids Res.16:9877). Transformation of a plant by recombinant/Agrodacierii/m usually involves co- cultivation of the Agrobacterium with explants fromthe plant and follows methods well known in the art. Transformed tissue is regenerated on selection medium carrying an antibioticor herbicide resistance marker between the binary plasmid T-DNA borders.

[0207] Another method for transforming plant hosts involves propelling inert or biologically active particles at planttissuesand cells. See, e.g., U.S. Patent Nos.4,945,050;

5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the nucleicacid of interest. Alternatively, a cell orcells can be surrounded by the vectorso thatthe vector is carried into the cell by the wake of the particle. Biologically active particles {e.g., a dried yeast cell, a dried bacterium ora bacteriophage, each containing one or more nucleic acids sought to be introduced) also can be propelled into plant tissue. Thus, in particular embodiments of the present invention, a plant cell can be transformed by any method known in the art and as described herein and intact plants can be regenerated fromthese transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture and/or cultured protoplasts is described, forexample, in Evans etal. (Handbook of Plant Cell Cultures ,Vol.1, MacMilan

Publishing Co. New York (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)). Methods ofselecting for transformed, transgenic plants, plant cells and/or plant tissue culture are routine in the art and can be employed in the methods of the invention provided herein. Likewise, the genetic properties engineered into the transgenic seeds and plants, plant parts, and/or plant cells of the present invention described above can be passedon by sexual reproduction orvegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling . A nucleotide sequence therefore can be introduced into the plant, plant part and/or plant cell in any numberof ways that are well known in the art. The methods of the invention do not depend on a particular method for introducing oneor more nucleotide sequences into a plant, only that they gain access to the interior of at least one cell of the plant. Where more than one nucleotide sequence is to be introduced, the respective nucleotide sequences can be assembled as part of a single nucleicacid construct/molecule, or as separate nucleicacid constructs/molecules, and can be located on the same ordifferent nucleicacid

constructs/molecules. Accordingly, the nucleotide sequences can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol. In some embodiments of this invention, the introduced nucleic acid molecule may be maintained in the plant cell stably if it is incorporated into a non- chromosomal autonomous replicon or integrated into the plant chromosome(s). Alternatively, the introduced construct may be present on an extra -chromosomal no n -rep Heating vector and be transiently expressed or transiently active. Whether present in an extra -chromosomal non-replicating vectorora vectorthat is integrated into a chromosome, the nucleicacid molecule can be present in a plant expression construct.

[0208] To confirm the presence of the constructs in the regenerating plants, a varietyof assays may be performed. In specific embodiments, expression of a heterologous or reporter gene in tissues, developing seeds, young seedlings and mature plants may be monitored, according to some embodiments, by immunological, histochemical, m NA expression or activity assays. Choice of expression assay forthe expression cassette may depend upon the nature of the heterologous coding sequence. For example, RNA gel blot analysis may be used to assess transcription if appropriate nucleotide probes are available. If antibodies to the polypeptide encoded by the heterologous gene (e.g., coding sequence)are available, western analysisand

immunohistochemical localization may be used to assessthe production and localization of the polypeptide. Depending uponthe heterologous gene, appropriate biochemical assays may be used.

5 Isolation of gene products

[0209] The present disclosure further relates to methods for isolating and/or purifying an expression product (e.g., a nucleic acid and/or a protein) of a nucleic acid sequence of interest from a plant. According to some embodiments, these methods may comprise producing a transgenic protein in a plant, extracting juice containing the transgenic protein from the plant, cleaning the juice to remove particulate matter, and/ortransmitting the juice through at least one membrane to produce two fractions, one of the fractions containing the transgenic protein. In some embodiments, a transgenic protein may comprise a lectin, an enzyme, a vaccine, a bacterial lytic peptide, a bacterial lytic protein, an antimicrobial peptide, an antimicrobial peptide protein, an antiviral peptide, an antiviral protein, an insecticidal peptide, an insecticidal protein, a therapeutic peptide, and a therapeutic protein.

[0210] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by wayofthe following non-limiting examples.

EXPERIMENTAL

[0211] To investigate the biological and evolutionary ramifications of the insertion in RDR1 in N. benthamiana, the present inventors compared their laboratory isolate (LAB), and a well-known European laboratory line (16c), with accessions of the species taken from the extremities of its natural distribution. The inventors' LAB isolate and the internationally widespread laboratory lines come from the Granites site in the Northern Territory of Australia (Figure la and notes on provenance). The wild accessions are representative of theirfive geographic habitat zones, that are separated by deserts or mountain ranges⁹ (Figure la), and they were named to reflectthe State orTerritor of theirorigin: Western Australia (W A), north Western Australia

(NWA); Northern Territory (NT), South Australia (SA) and Queensland (QLD). Within an accession there is no obvious morphological variability but differences in plant form, leaf shape, flower and seed size between the accessions are clearly apparent (Figure lb, Table 1). PC R analysis revealed thatthe laboratory strains andone wild accession, from SA, each contain a 72-nt insertion in their RDRl (Figure lc,d) but the other wild accessions do not.

[0212] Inoculating the plants with a spectrum of viruses from 5 different families ¹⁰ (Virgaviridae, Potyviridae, Bromoviridae, and Geminiviridae, and a native Australian sobemo virus) showed that almost irrespective of the virus type, the SA, LAB and 16C plants, gave severe symptoms, often including dwarfing ordeath (Figures 2b and 5), whereas the WA, NT, QLD, and to a lesser extent NWA isolates, produced milder symptoms and the plants were able to flower and set seed. Infection with fluorescence-tagged viruses showed that the responses are due to differences in virus spread and replication (Figure 2f,g). To verify that the dichotomy in defense performance is due to differences in RDRl integrity, progeny plants from a self -fertilized parent, generated from a LAB x QLD cross, were tested for their response to TMV-U1 (Figure 2a, c). All of the progeny homozygous for the insertion-mutation soon died, those heterozygous developed some necrotic leaves but survived to flowering, and those homozygous for the insert -free RDRl were taller, healthier, flowered profusely and set seed (Figure 2a). Furthermore, down-regulating RDR1 expression, using an artificial microRNA, in the most robustly virus -resistant wild N.

benthamiana accession (WA) rendered it hype rs usee ptible to TMV-Ul (Figure 2d,e).

[0213] RDR1 may be an important player in the RNA interference (RNAi) mechanism defending plants against viruses¹¹, so the present inventors investigated whether there are other defective genes in this pathway contributing to the viral hypersensitivity in SA, LAB, and 16c. The transcriptomes of the wild accessions were sequenced, and assembled, and their core 27 RNAi genes compared with those ofthe LABtranscriptome and genome¹² _' ¹³ _' ¹⁴ (www.benthgenome.com, www.benthgenome.qut.edu.au). With the exception of RDR1, the transcripts ofthe RNAi genes from the wild accessions have comparable expression levels to those of LAB and similarly full length ORFs (www.benthgenome.com, Figure 6). Combining this with the results ofthe complementation and silencing experiments demonstrates that the major cause of hypersensitivity to viral infection in laboratory strains of N. benthamiana is, indeed, the dysfunctionality of its RDR1.

[0214] Searching the non-redundant nucleotide database at NCBI for sequences similar to the 72 bp that disrupt the RDR1 gene in LAB and SA returned onlya single BLASThit, against RDRl in N. benthamiana and none for all the other 46 species examined (Figure 7). However, in the N. benthamiana LAB genome assembly (www.benthgenome.com) the present inventors identified the sequence not only in the RDRl gene on scaffold Nbv0.5scaffold3298, as expected, but also within Nbv0.5scaffold954, with a 71/72 nt match (Figure 8a-c). This lattersequence is in a 100 kb untranscribed, non-coding region and seems the likely origin of the DNA inserted into RDRl . To identify when this debilitating RDRl insertion occurred in the antecedent of LAB and SA, the RDRl sequences and three other genes routinely used in phylogeny studies, (glutamine synthetase (Gs/), maturase K (MatK) and alcohol dehydrogenase C (Adh ), were obtained from DNA amplification of different Australian Nicotiana {Suaveolentes) family members, including our six N. benthamiana accessions, and subjected to phylogeneticand mo lecularclockanalyses. Using the sequences with or without RDRl sequences gave non-conflicting trees that placed QLD, NT, and WA into one subclade and LAB with SA into another. Two different molecular clock assessments gave estimations of sub-clade divergence to ~520 Kya, (95% credibility interval: 183 Kya - 1.23 Mya) and a polartree ofthe relationships between N. benthamiana isolates, nine other Australian Nicotianas and 31 non-Australian Nicotianas (Fig.3a). More refined timing ofthe RDRl insertion event, by cladisticanalysis using 21 canonical single -copy¹⁵ N. benthamiana gene sequences (Table 2), confirmed the close relationship between WA, QLD and NT with estimated divergence points of 240 and 430 Kya. It placed NWA within this clade at a d ivergence point of 810 Kya (Figure 3b, c) and LABand SA into a sub-clade thatdiverged fromthe insert-free lineages about 880 Kya. As SA and LAB split from each other 710Kya, this dates the insertion event between 880 and 710 Kya.

[0215] The regions where N. benthamiana plants grow today have experienced dramatic climatic changes in the past. The first progenitors ofthe Australian Nicotiana family probably arrived, by Pacific island hopping, about 20 Mya, when the conditions were warmand wet^16,17. This was followed by a long period of intense aridification (10-6 Mya) pursued by cycles of warm-and-wet/cool-and-dry periods. About 1 Mya, when the populations of N. benthamiana represented by our accessions started diversifying, the arid landforms of central and northwestern Australia had developed and during the last 0.8 My large mobile dune sands provided further gene- flow barriers¹⁷. Super-imposing the unrooted phylogenetic distance tree ofthe N. benthamiana accessions ontothe geographic map of theirdistribution (Figure 3d) shows a remarkable concordance. The degree of inter-relationship between SA, LAB, NT and QLD correlates closely with the distances of their physical separation, suggestive of linear diversification during range expansion along an east/west axis. NWA is a clear outlier in both phylogenetic and geographic terms. Curiously, WA forms a clade with NT and QLD, despite the geographic separation, and may be the result of long-range dispersal by wind, birds or humans.

[0216] The placement of LAB and SAtogetherin a sub-clade distinct from the other isolates, on the basis of sequence similarities, is not only reflected in differences in virus susceptibility and presence or absence ofthe 72-bp insertion, butalso in a numberof other characteristics including the aridity of their habitat, and the size of their flowers (Figure la,b, Table 1). This raised the possibility that some of these attributes a re associated with the insert -derived viral hypersusceptibility. The present inventors reasoned that for the RDRl mutation to be retained for such a long period, it must confer a survival advantage, or the re is a barrier preventing its loss. All of theiraccessions are self-compatible, manually inter-crossable (Table 1), and N. benthamiana may be insect pollinated. Therefore, the present inventors examined their internal flower structures. LAB, 16C and SA flowers have smallercorolla diameters and tube lengths than WA, NWA, NT and QLD (Table 1) and the anthers and stigmas ofthe Iattergroup are spatia liy separated (herkogamy), favoring outcrossing (Figure 4a, Table 1 and Figure 10) whereas in LAB, 16C and SA they are in close-proximity, strongly favouring self-fertilization (Table 1 and Figure 9). This explains how the homozygous RDRl -mutation has been maintained, bythe prevention of outcrossing with individuals possessing a functional RDRl . It is also in accord with efficient self- fertilization conferring a survival advantage in central Australia, where the brief annual rainfall dictates that plants have a short lifecycle and a consequently brief opportunity for insect-mediated cross-pollination. In contrast, the WA, QLD, NWA and NT accessions, from less arid locations, have the external flower structures well suited for insect attraction and internal arrangements for enhancing outbreeding and gene flow. The abundance of insect pollinators, in less arid conditions, not only provides the means forthe functional, insert -free, RDRl allele to spread between and among populations, but also represents an increased presence of viral vectors and virus -reservoir plants applying selection pressure forthe acquisition or maintenance ofthe conferred virus resistance.

[0217] The fitness costs associated with a character determine its equilibrium value¹⁸, and the cost of resistance against pests and pathogens is a central feature in most models forthe evolution of plant defence¹⁸ _' ¹⁹. Both constitutive and induced defense costs can be high in terms of reduced growth and seed set but, under disease pressure, they provide substantial fitness 18.19,20,21 _ The present inventors noticed thatthe sizeof the seed produced fromthe wild accessions, except SA, was smallerthan from LAB or 16c, when grown underthe same conditions (Table 1). This raised the possibility that dysfunctional RDR1 in LAB, 16cand SA, by impairing viral defense, gives a metabolic economy that promotes early vigor.

[0218] To investigate this, the most effective RDR1 -targeted amiR line, #5WA, was examined alongside its no n -transgenic counterpart, WA, and LAB. Measurement showed that LAB seed is on average 39% largerthan seed from WA, and when RDR1 is silenced in WAto give #5WA, the seed size increases by 56 % and doubles in weight. (Figure 4c). The germination rate, seed vigor, and seedling growth of #5WA were a Iso dramatically increased (Figure 4c,d and Figure 10). A similar increase in seed size and weight was also observed when the Rdrl gene in QLD was silenced by an amiRNA (Table 4). This provides strong evidence that the loss of RDR1 function in LAB and SA enhances their early vigor and that the harsh conditions in central Australia have provided selection pressures that are stronger for early vigor than for defense.

METHODS PLANTS, VIRUSES AND INFECTION ASSAYS

[0219] N. benthamiana wild isolates were collected from remote geographicareas of the plant natural distribution as shown in Figure l.a. The Granites isolate has been maintained at CSIRO for more than 50 years by Nancy Burbidge and Katie Helms. The 16C line is described in Ruiz et al.²². Seed sources for each of the other Nicotiana species used in this study a re described in Marks et al.²³. Sobemovirus : Velvet tobacco mottle virus (VtMoV), Potyvirus: Turnip mosaic virus (TuMV) and Potato virus Y (PVY-D), Cucumovirus: Cucumber mosaic virus (CMV-sat), Begomovirus: Tomato yellow leaf curl virus (TYLCV), and Tobamoviruses: TMV-U1, TMV-U6, were obtained from J. Randies (the University of Adelaide, Waite campus). TMV-GFP and PVX-GFP were used as previously described²⁴.

GENERATION OF INTRASPECIFIC HYBRIDS AND BACK-CROSSES

[0220] Twelve intraspecific hybrids between LAB and QLD N. benthamiana were generated by conventional crossing and allowed to self-pollinate. The seed fromone line was collected, grown and the plants used for all further ana lysis. The plants were genotyped using the primers designed to amplifya region spanning theRDRl insertion (Table 3).

ARTIFICIAL MICRORNA DESIGNS AN N. BENTHAMIANA STABLE TRANSFORMATION.

[0221] TheRDRl target sequence (GTCGCAAAATATGCCGCCAGA) was incorporated into the design a pBlueGreen amiRNA construct²⁵, using primers pAMIR-RDRl-F and pAMIR-RDRl-R, which was electroporated into Agrobacterium tumefaciens (GV31101) and transformed into N. benthamiana WAas described previously².

TRANSCRIPTOME ASSEMBLIES

[0222] Total RNA was prepared fromthe wild isolates (SA, NT, NWA, QLD, WA), the libraries made and deep sequenced on the Illumina HiSeq 2000 sequencer at AGRF, the raw reads filtered and trimmed, and the de novo assemblies generated essentially as described previously¹².

IDENTIFYING RNAI GENE TRANSCRIPTS AND COPY NUMBER IN WILD- TYPE ASSEMBLIES

[0223] The NCBI-BLAST+ package (v2.2.26) was used to generate the transcriptomes of each wild isolate. The sequencewith the longestCDSand highest percent identity to the LAB sequence for each RNAi gene was deemed the counterpart ortholog. Copy numbers were estimated using BWA vO.7.10²⁶ and Bowtie2 v2.2.4²⁷ to map reads to the assemblies and visualization by IGV. Transcripts Per Million (TPM) values from RNA-Seq data were calculated using RSEM²⁸.

NUCLEIC ACID SEQUENCING, PHYLOGENY AND MOLECULAR CLOCK ANALYSIS.

[0224] Total RNA was isolated using TRIzol. Total genomic DNA was extracted from leaf samples as previously described¹³. Primers forgenotyping RDR1 in Nicotiana species and amplifying RDR1, Csl, AdhC and MatK genes are described in Table S5. DNA and RNAseq libraries were prepared as per manufacturer's instructions and sequenced bythe Australian Genome Research Facility, Melbourne, Australia. Raw sequences wereedited and assembled using

Geneious™ Pro, and IGV²⁹, aligned using (MUSCLE) 3.8.31³⁰, analysed using BEAST (Bayesian Evolutionary Analysis Sampling Trees) 1.7.5 and MrBayes³¹'³² and phylogenetic trees created by Figtree vl.4.0. Rate variation was modelled with an uncorrelated lognormal relaxed clock³³.

EXAMPLE 1

RDR1 SILENCING IN/V. TABACUM SIGNIFICANTLY INCREASES AVERAGE SEED SIZE AND WEIGHT

[0225] The RDR1 target sequence (GTCGCAAAATATGCCGCCAGA) was incorporated into the pBlueGreen a miRNA construct, using primers pAMIR-RDRl-F and pAMIR-RDRl-R. The pBlueGreen amiRdrl vector was electro porated into Agrobacterium tumefaciens cells (GV31101) and transformed into N. tabacum.

[0226] Briefly, four- to six-inch leaves from 1- to 2-months old plants were harvested and sterilized. The excised explants were placed adaxial side up onto pre-culture medium (MSN + BAP + NAA) for 24 hr. At the same time a 25 - 50 mL culture of amiR-transformed A. tumefaciens GV3101in LB supplemented with the appropriate antibiotics was initiated. After overnight growth, cells were pelleted by centrifugation at 4000 rpmfor 10 min and the pellet was resuspended in infiltration bufferto a final OD600 of 0.5 - 1.0. The pre-cultured explants were transferred to the agro-inoculumand incubated at RTfor 30 min. The explants were then briefly blotted on sterile filter pa per and transferred to solid cocultivation medium for 3 days. At day 5, they were transferred to regeneration medium, and thenceforth transferred to fresh regeneration medium every two weeks. Growth of explants was strictly monitored, with any calli removed, any shoots excised and transferred to rooting medium in time. Plantlets with a well-established root system were transferred to soil.

[0227] Following transformation seven transformed lines were regenerated, and grown under controlled condition until seeds setting. Mature seed pods / seeds and seed lings of at least 3 of the transformed lines where analysed fortheirsize, weightand numberand compared to seeds/ seed podsand seedlings of N. tabacum control plants (not transformed with the amiRNA).

[0228] The results presented in Figures 11 and 12 clearly demonstrate that RDR1 silencing leads to plants with a substantial increase in average seed size and weight relative to control plants. TABLES

TABLE 1: Morphologic characters assessment. Morphological characters variationsamong N. benthamiana lines, assessed fora minimum of 50 plants per isolate sawn and grown in the same conditions of temperature and light.

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TABLE 2: Concatenated 21 sequences used for N. benthamiana species phylogeny analysis and Rdrl insertion tinning.

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-dhCjWSSS fi-glJl C«Ti3CCTGT^ATCAATCI

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siSiC_*S5iS_Saa;2J5. AAACCTTCCCCTTTCAeCAT

CA TATOTATCATT GATAACPCIC^'

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PP2A f*j &ACCCTG&TGrroATGT GCT

GAGSGATTTGAAaAGAi TTTC

AQAAOCAAAATGAAGOCO

i i-sgi Rev GAICGAGTCCGCAAATOSAG

TTGGTGercCTCOCA CTAT

S¾g2 Rev TCCCTGAAATTTGGAAGTCG

AG OGAGCCATKXiTAGGTG

;*· Re AAACCGCATTCACCAGAAAC

!nKNA ΒΒ8.Ϊ saiiSUKI-F TATATGCTCTTCGAGAGGGTCC^A^^

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aaii SRl -R TATATXXCT C^SATGGTCGCAAAAT^^

TABLE 4: Seed size and weight of N. benthamiana LAB, WA, QLD and their counterparts, transformed with an a miRNA targeting the Rdrl gene: WA#5 and QLD #3, and #7, were measured fortheirsize and weight. The average numberfor 3 replicates of 50 seeds is shown.

Seed size (μιτι) Seed weigh (μς)

LAB 798.774 0.0771

WA 570.7 0.0412

WA#5 891.304 0.0838

QLD 582.016 0.0516

QLD #3 654.295 0.0776

QLD #7 655.090 0.0766 TABLE 5: Plant defense pathways and associated PDR genes and encoded protein products

TABLE 6: Genomic information for PDR genes and encoded protein products listed in TABLE 4.

TABLE 7: Nucleotide sequences for various genes listed in TABLE 5.

[0229] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0230] The citation of any reference herein should not be construed as an admission that such reference is available as"Prior Art" to the instant application.

[0231] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to anyone embodiment orspecific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

BIBLIOGRAPHY

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Claims

WHAT IS CLAIMED IS:

1. A method for improving a growth characteristic of a plant, as compared to a control plant, the method comprising, consisting or consisting essentially of inhibiting expression in the plant of at least one plant defense-related (PDR) gene and/or inhibiting activity of an expression product (e.g., RNA or polypeptide) of the at least one PDR gene, wherein the at least one PDR gene comprises RDR1.

2. The method of claim 1, comprising introducing a genetic modification in the genome of the plant, which results in partial or complete loss of function of the at least one PDR gene.

3. The method of claim 2, wherein the genetic modification is introduced by site- directed mutagenesis, randomchemical mutagenesis, transposon mutagenesis, T-DNA insertion, homologous recombination, targeted induced local lesions in genomes (TILLING) or genome editing, orcombinations thereof.

4. The method of claim 2, wherein the genetic modification comprises introducing into the genome of the plant a modulator nucleic acid sequence encoding an expression product that inhibits expression of the at least one PDR gene, or that inhibits activity of an expression product of the at least one PDR gene.

5. The method of claim 4, wherein the expression product is a functional nucleic acid (e.g., siRNA, shRNA, mi RNA, nucleicacid aptamers, ribozymes, riboswitches, Ul adaptors, molecular beacons, transcriptional factor-binding regions, ere.) that inhibits expression of the PDR gene.

6. The method of claim 4, wherein the expression product is a PDR-inhibiting protein (e.g., an antibody orantibodyfragmentthat bindsto and inhibits the activity of the PDR).

7. The method of any one of claims 1 to 6, comprising inhibiting activity of an expression product of the at least one PDR gene.

8. The method of any one of claims 1 to 7, wherein the improved growth characteristic is selected from any one or more of increased vigor, increased yield, increased seed size, increased leaf size, increased flower size, increased trichomes, increased plant biomass, and increased growth rate relative to the control plant.

9. The method of claim 8, wherein the increased vigor includes increased early vigor.

10. The method of claim 8, wherein the increased seed size includes an increased surface area, volume, or weight of seed.

11. The method of claim 8, wherein the increased plant biomass includes any one or more of increased plantsize, increased stalk size, increased fruit size, increased root size and increased leaf size.

12. The method of claim 8, wherein the increased growth rate includes increased growth rate of a plant at one or more stages of plant development, including embryonic stage, seedling stage, vegetative stage, juvenile stage, reproductive stage, and ripening stage or plant development.

13. The method of any one of claims 1 to 12, further comprising selecting a plant having an improved growth characteristic relative to the control plant.

14. A construct comprising a control sequence that is operable in a plant cell and that is operably connected to a modulator nucleicacid sequence encoding an expression product that inhibits expression of at least one PDR gene, or that inhibits activity of an expression product of the at least one PDR gene, wherein the at leastone PDR gene comprises RDR1.

15. The construct of claim 14, wherein the control sequence is capable of facilitating constitutive expression of the modulator nucleic acid sequence.

16. The construct of claim 14, wherein the promoter is capable of facilitating organ- specific or tissue -specific expression of the modulator nucleicacid sequence.

17. The construct of claim 14, wherein the promoter is capable of facilitating development-specific expression of the modulator nucleicacid sequence.

18. A plant cell comprising a genetic modification, which results in partial or complete loss of function of at least one endogenous PDR gene, wherein the at least one endogenous PDR gene comprises RDR1.

19. The plant cell of claim 18, further comprising a nucleic acid construct comprising a control sequence that is operable in the plant cells and that is operably connected to a nucleotide sequence of interest.

20. A plant comprising a genetic modification, which results in partial or complete loss of function of at least one endogenous PDR gene, wherein the at least one endogenous PDR gene comprises RDR1.

21. The plant of claim 20, wherein the plant includes cells that comprise a nucleic acid construct comprising a control sequence that is operable in the plant cells and that is operably connected to a nucleotide sequence of interest.

22. The plant of claim 20 or claim 21, wherein the plant is selected froman embryo, a seed, a seedling, a juvenile plant or a mature plant.

23. A harvestable part ora progeny of the plant of claim 20 or claim 21, wherein the harvestable partorthe progeny comprises the genetic modification.

24. The harvestable part of claim 23, wherein the harvestable part is selected from a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, spore, cutting, nut, or root.

25. A seed comprising a genetic modification, which results in partial orcomplete loss of function at least one endogenous PDR gene, wherein the at least one endogenous PDR gene comprises RDR1.

26. The seed of claim 25, further comprising a nucleic acid construct comprising a control sequence that is operable in a plant cell and that is operably connected to a nucleotide sequence of interest.

27. A method of producing a plant with an improved growth characteristic, the method comprising introducing a genetic modification in a plant cell, which results in partial or complete loss of function of at least one endogenous PDR gene, and regenerating a plant with a partial orcomplete loss of function of the at least one endogenous PDR gene from the plant cell, wherein the at least one endogenous PDR gene comprises RDR1.

28. An expression system for expressing a nucleicacid sequence of interest in a plant cell, the system comprising a first expression system component (e.g., comprising one or more expression cassettes or constructs) and a second expression system component {e.g., comprising one or more expression cassettes or constructs), wherein the target nucleic acid sequence is expressible fromthe first expression system component, and wherein a modulator nucleic acid sequence is expressible fromthe second expression system component, wherein the modulator nucleic acid sequence encodes an expression product that inhibits expression of a PDR gene, orthat inhibits activity of an expression product of the PDR gene, wherein the at least one endogenous PDR gene comprises RDR1.

29. A plant cell comprising the expression systemof claim 28.

30. A plant comprising the plant cell of claim 29.

31. The plant of claim 30, wherein the plant is selected from an embryo, a seed, a seedling, a juvenile plantora mature plant.

32. A harvestable part ora progeny of the plant of claim 30 or claim 31, wherein the harvestable partorthe progeny comprises the expression system of claim 25.

33. The harvestable part of claim 32, wherein the harvestable part is selected from a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, spore, cutting, nut, or root.

34. A method for expressing a nucleic acid sequence of interest in a plant, the method comprising, consisting or consisting essentially of co-expressing the nucleic acid sequence of interest and modulator nucleic acid sequence in cells of the plant, wherein expression of the modulator nucleic acid sequence produces an expression product that inhibits expression of a PDR gene orthat inhibits activity of an expression productof a PDR gene, wherein the at least one endogenous PDR gene comprises RDR1.

35. The method of claim 34, wherein the plant is is selected from an embryo, a seed, a seedling, a juvenile plant ora mature plant.

36. The method of claim 34 orclaim 35, further comprising exposing the plantto one or more stimuli that stimulate orenhance expression of the nucleicacid sequence of interest, the modulator nucleicacid sequence or both the nucleicacid sequence of interest and the modulator nucleicacid sequence.

37. The method ofanyone ofclaims 34 to 36, wherein the nucleicacid sequence of interest encodes a polypeptide of interest.

38. The method of any one of claims 34 to 37, further comprising harvesting, isolating or purifying the polypeptide of interest fromthe plant or plant part.

39. A method for monitoring a population of plants for exposure to a stress condition (e.g., biotic or abiotic stress condition) or combination of stress conditions, the method comprising, consisting or consisting essentially of introducing into the population of plants a sentinel plantthat comprises, consists orconsists essentially of a genetic modification, which results in partial orcomplete loss of function of at least one PDRgene, and examining the sentinel plant for susceptibility to the stress condition or combination of stress conditions, which is indicative of exposure of the population of plants to the stress condition or combination of stress conditions, wherein the at least one endogenous PDR gene comprises RDR1.

40. The method of claim 39, wherein the sentinel plant is located at or nearthe edge of the plant population.

41. The method of claim 39, wherein the sentinel plant is located at or nearthe centerof the plant population.