WO2018101824A1

WO2018101824A1 - Plants comprising pathogen effector constructs

Info

Publication number: WO2018101824A1
Application number: PCT/NL2017/050798
Authority: WO
Inventors: Xiaotang DI; Harrold Alfred VAN DEN BURG; Franciscus Lambertus Wilhelmus Takken
Original assignee: Universiteit Van Amsterdam
Priority date: 2016-11-30
Filing date: 2017-11-30
Publication date: 2018-06-07

Abstract

The present invention relates to a chimeric gene for the extracellular production in plant cells, transformed with the chimeric gene, of a pathogen effector, the chimeric gene comprising: (a) a promoter sequence which functions in plant cells; and (b) a coding sequence, operably linked to the promoter, encoding a fusion protein consisting of a pathogen effector fused in translation frame to a signal peptide for targeting said pathogen effector to the plant secretory pathway

Description

Title: Plants comprising pathogen effector constructs FIELD OF THE INVENTION

The invention is in the field of crop protection. In particular, the invention relates to plants exhibiting improved resistance to pathogens. The invention further relates to methods of ameliorating pathogen resistance in crops, and methods of inducing pathogen-induced immunity and/or cell death in plant cells.

BACKGROUND OF THE INVENTION

Plants have evolved several different defence mechanisms to prevent or overcome pathogen infection. For instance, plants possess passive defence mechanisms in the form of physical and chemical barriers. The waxy cuticle covering the epidermis and their complex cell walls containing highly crosslinked polysaccharides that surround each cell pose physical barriers. Furthermore, upon wounding chemical barriers are mounted and antimicrobial proteins and phenolic compounds are released.

Plants also rely on an innate immune system; a basal defence system present at the cell surface, where specialized cell-surface receptors detect pathogens through recognition of so-called pathogen associated molecular patterns (PAMPs). Some examples of PAMPs are conserved motifs in bacterial flagellin or bacterial lipopolysaccharides (LPS) or fungal chitin. The perception of PAMPs by pattern recognition receptors (PRRs) activates signal-transduction cascades that turn on basal defence

mechanisms referred to as pathogen-triggered immunity (ΡΊΊ), which include callose and silicone deposition, production of reactive oxygen species, salicylic acid and ethylene, and expression of defence genes, including pathogenesis-related genes (PR). PTI suffices to halt further proliferation of the majority of non-adapted microbes.

Pathogens, such as phytopathogenic bacteria, may suppress this basal defence mechanism through secretion of specialized effector proteins. In the case of bacterial pathogens these effectors are typically directly translocated into the host cell cytoplasm by the type III secretion system (T3SS), which is found in most pathogenic Pseudomortas, Ralstonia solanacearum, Xanthomonas and Erwinia species. Many effectors of filamentous pathogens, such as fungi or oomycetes, also act intracellularly but it is currently unknown how they are translocated into host cells.

Pathogen effectors can activate a third layer of defence in plants, the so-called effector-triggered immunity (ETI). Plants are able to detect the presence of effectors inside the plant cell through intracellular receptors encoded by "disease-resistance" (R) genes that encode R proteins. The majority of plant R proteins contain a nucleotide-binding site and leucine- rich repeats ( BS-LRR), and these NBS-LRR proteins mediate resistance against a large range of plant pathogens. Specific activation of such a NBS- LRR R protein due to direct or indirect recognition of its cognate pathogen effector protein (a pathogen effector, or Avirulence protein) results in a defence response that halts further pathogen ingress. The effector-triggered- immune (ΕΊΊ) response is qualitatively similar to the PTI response, but is quantitatively faster and stronger and often encompasses a programmed cell death response of the infected plant cells. Induction of cell death is not a requirement for resistance, as e.g. recognition of the coat protein of Potato Virus X by the potato NBS-LRR protein Rx results in extreme-resistance without induction of programmed cell death or lesion formation. Together these ETI responses result in containment of the pathogen and prevent its systemic spread in the host plant. NBS-LRR-mediated resistance is further associated with massive transcriptional reprogramming and production of reactive oxygen species, nitric oxide and salicylic acid, which function as direct antimicrobial agents and as signaling agents triggering Systemic Acquired Immunity.

ETI is based on the phenomenon that both inheritance of resistance in the host and the pathogen's ability to trigger immunity are controlled by a pair of matching genes: a dominant resistance R) gene in the plant, and a corresponding (or "cognate") dominant avirulence (Avr) gene or pathogen effector gene in the pathogen. Plants that produce a specific R gene product are resistant towards a pathogen that produces a matching Avr gene product. This gene-for-gene relationship is a widespread and very important aspect of plant disease resistance. Resistance conferred by a resistance gene is typically pathogen race-specific (narrow spectrum) as it is based on recognition of a single Avr determinant of the pathogen.

Avirulence effectors, however, play an important role as virulence factors in genetically susceptible hosts. Hence, there exists strong selection pressure on recognized pathogens to evade introgressed ETI in crops. ETI can be evaded by mutating or shedding the cognate Avr genes or by acquiring new effectors that suppress ETI. In this unending arms race between plants and pathogens, both the plant and its breeder are always one step behind.

Present strategies for managing disease resistance based on PTI or ΕΊΊ, therefore, only provides a short-lived solution to confer resistance to specific pathogenic strains. Likewise, pathogens can develop resistance to pesticides, making also this strategy non-durable.

Hence, there is a need for a durable strategy to develop or support plant disease resistance towards pathogens. There is also a need for methods that provide broad-spectrum resistance in plants to, preferably, many pathogens, including microbial pathogens such as fungi, oomycetes, bacteria, nematodes, mites and preferably also insects, parasitic plants and other parasites. Because activation of a resistance-conferring defence system in plants is associated with loss of both energy and metabolites that otherwise could be used for growth, it is preferred that such a system is only induced when needed, e.g. when the pathogen enters the plant and is about to cause disease resulting in yield/quality loss. There is also a need for methods to provide pathogen resistance in plants that can be applied in a wide variety of crops, e.g. ornamentals, shrubs, vegetables, etc.

The present invention aims to overcome the disadvantages of the prior art and offers tools that enable plant breeders to provide plants with pathogen resistance that is durable, inducible, and applicable to a wide variety of plants and pathogens. SUMMARY OF THE INVENTION

The present inventors have found that uptake of pathogen effector proteins by plant cells depends on the presence of a pathogen, and does not occur in the absence of a pathogen. This observation forms the basis of the present invention. The present invention provides a plant that produces an extracellular localized (e.g. in the apoplast or in the xylemsap) pathogen Avr effector that can be recognized by a matching intracellular R protein of the plant. In the absence of a pathogen the intracellular R protein does not perceive the extracellular Avr effector and plants develop normally without mounting ΕΊΊ. However, in the presence of a pathogen, uptake of the Avr effector causes activation of the R protein resulting in an effective ETI response that halts further pathogen ingress and proliferation.

The system uses a genetically encoded pathogen effector (protein with "avirulence" activity) whose uptake by the plant cell specifically triggers ETI upon its perception by its cognate intracellular immune receptor (R protein). The pathogen Avr effector can be localised in the apoplast and/or xylem sap from which plant cells can retrieve it. It is also envisioned as an embodiment that the pathogen Avr effector may be applied directly to plants, or introduced in other ways such as through the

application of plant-colonizing micro-organisms that themselves do not trigger uptake, but is preferably produced by the plants themselves, which have, e.g., been transformed to produce the pathogen effector. Alternatively, the pathogen Avr effector may be produced in the plants themselves upon expression by plant viruses in cells of the plant. The genetically encoded pathogen effector may thus also be encoded by a plant viral vector that expresses the pathogen effector in non-transgenic plants expressing the cognate intracellular R protein. In such embodiments, the invention may provide a non-transgenic plant comprising as an episome a recombinant plant viral vector expressing a fusion protein consisting of a pathogen effector fused in translation frame to a signal peptide for targeting said pathogen effector to the plant secretory pathway. The invention thus also provides a combination of a non-transgenic plant and a transformed plant viral vector for expressing the said fusion protein in said non-transgenic plant.

The present invention provides inducible broad-spectrum resistance to any pathogen that triggers pathogen effector uptake during infection. An important advantage of this invention is that resistance is durable, as it can only be overcome when the pathogen loses its ability to trigger pathogen effector uptake, which essentially means that it will also lose its pathogenicity. Another advantage is that the invention can be applied in a wide variety of plants, including ornamentals and vegetables that are susceptible to microbial pathogens that trigger pathogen effector uptake. It is an important advantage of this invention that the method provides resistance against root-, vasculature- and foliar -invading

pathogens.

In a first aspect, the present invention provides a plant comprising at least one i?-gene encoding an intracellular R protein for mounting an immune response in cells of said plant, said plant further comprising a chimeric gene, either stably integrated in the genome or present on an episome, for the extracellular production of a pathogen effector in cells of said plant that express the chimeric gene, the chimeric gene comprising:

(a) a promoter sequence which functions in plant cells;

(b) a coding sequence, operably linked to the promoter, encoding a fusion protein consisting of said pathogen effector fused in translation frame to a signal peptide for targeting said pathogen effector to the plant secretory pathway; and

(c) optionally, a 3' non-translated region which encodes a polyadenylation signal which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA;

wherein the pathogen effector is directly or indirectly recognized by the intracellular R protein.

In a preferred embodiment of a plant of the invention, the pathogen effector is heterologous with respect to the plant, the R protein, the promoter, the signal peptide, and/or the optional 3' non-translated region. In certain preferred embodiments, R protein, promoter, signal peptide, and 3' non-translated region may be encoded by endogenous plant sequences.

In another preferred embodiment of a plant of the invention, the plant is a transgenic plant, stably transformed with the chimeric gene.

In an alternative preferred embodiment of a plant of the invention, the plant is a non-transgenic plant comprising an

extrachromosomal expression vector for expression of the chimeric gene.

In a preferred embodiment of a plant of the invention, the i?-gene is endogenous, but it may be heterologous, in which case the heterologous R- gene may be located on an extrachromosomal expression vector, or may be integrated in the plant's genome.

In another aspect, the present invention provides a chimeric gene for the extracellular production of a pathogen effector in cells of a plant that express the chimeric gene, the chimeric gene comprising: (a) a promoter sequence which functions in plant cells;

(c) optionally, a 3' non-translated region which encodes a polyadenylation signal which functions in plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA.

In a preferred embodiment of a chimeric gene of the invention, the pathogen effector is heterologous with respect to the promoter, the signal peptide, and/or the optional 3' non-translated region.

In another preferred embodiment of a chimeric gene of the invention, the pathogen effector is cognate with an intracellular R protein expressed in said plant. In aspects of this invention, the effector is denoted "cognate" when it triggers a plant defense and/or immune signaling response upon direct or indirect recognition only in plants that express the aforementioned R protein. Hence, the effector and its cognate intracellular R protein are transcripts of a gene-for-gene R protein/Avr pair. Such a pair consists of an intracellular R protein and a pathogen effector that is recognized (directly or indirectly) by the intracellular R protein, which recognition causes an ETI response in said plant.

In another preferred embodiment of a chimeric gene of the invention, the promoter is adapted to cause sufficient expression of the fusion polypeptide to mount an ETI response in said plant cells upon exposure of said plant cells to a pathogen that facilitates uptake of the extracellular pathogen effector in said plant cells, preferably wherein the promoter is a constitutive promoter.

In another preferred embodiment of a chimeric gene of the invention, the pathogen effector originates from an organism selected from the group consisting of fungi, oomycetes, bacteria, nematodes, mites, insects, and parasitic plants.

In another preferred embodiment of a chimeric gene of the invention, the pathogen effector is selected from the group consisting of Avr2 (AM234063.1) of Fusarium oxysporum, Avra10, Avrkl and AvrMLal of Blumeria graminis, AvrL567 (AY510102.1), AvrM (DQ279870), AvrP1234 (EU642499) and AvrP4 (ABB96263.1) of Melampsora lini, AVR-Pia, Avr- Pik, Avr-Pita (AF207841), Avr-Pita2, and Avrl-C039 (AF463528) of

Magnaporthe grisea or Magnaporthe oryzae, AvreRpgl of Puccinia graminis f sp. Tritici, Avr3 of Bremia lactucae, ATR1 (PDB ID: 3RMR_A), ATR3, ATR13 (PDB ID:2LAI), HaRxLl- HaRxL147, HaRxLL3a- HaRxLL495c, HaRxLCRN4b of Hyahperonospora arabidopsidis, AvrB and AvrRPPlA of Peronospora parasitica, AvrRPPlB, AvrRPPlC, AvrRPP2, AvrRPP4, AvrRPP5, and AvrRPP8 of Peronospora parasitica, Avrl (DS028168), AVR2,Avr-blbl (IpiO) (DS028419), Avr-blb2 (DS028242), PiAvr2

(DS028133), Avr3a (EF587759), Avr2 (EEY61966), Avr3b, Avr10, Avrll (DQ390339), and AVR4 of Phytophthora infestans, Avrla (EF463064.1), Avr3a (EF587759.1), AVRlb-1, AVRlk, and Avr3c (FJ705360.1) of

Phytophthora sojae, AvrLml (AM084345.1), AvrLm6 (AM259336.1),

AvrLm4-7 (AM998638.1) of Leptosphaeria maculans, Avra10 (DQ679913), and Avrkl (DQ679912) of Blumeria graminis f. sp. hordei, and OEC45- OEC115 of Golovinomyces orontii, and homologs thereof, wherein PDB indicates the accession number in the Protein Data Bank, Berman et al., 2000, Nucleic Acids Research 28 (1): 235-242; and other accession numbers refer to the Genetic Sequence Data Bank of October 15, 2016, NCBI- GenBank Flat File Release 216.0).

In general, examples of pathogen effectors that are useful in the present invention are those that are (i) secreted by a pathogen in the plant's extracellular space as defined herein (the endogenous pathogen gene encoding such effectors may thus usually comprise an endogenous signal sequence), and that are (ii) recognized in the host plant cell by an ETI receptor. The classes of pathogens containing such effectors are, preferably, extracellular pathogens that have no specialized mechanism for injecting effectors. Hence, bacterial type III effectors or effectors that nematodes or lice that are directly inserted or brought into a cell, are preferably not used in aspects of this invention. In contrast, effectors of fungi, oomycetes and nematodes or other pathogens that are excreted in the extracellular space and then have to cross the plant's plasma membrane, are preferred.

In yet another aspect, the present invention provides a vector comprising a chimeric plant gene of the present invention, preferably wherein said vector is selected from a cloning vector, an expression vector, a plant transformation vector, and a plant viral expression vector.

In yet another aspect, the present invention provides a plant cell comprising a chimeric gene of the present invention or a vector according to the present invention.

In yet another aspect, the present invention provides a plant, which has been regenerated from a plant cell of the present invention, preferably wherein said plant is resistant to a pathogen that facilitates uptake of the extracellular pathogen effector in said plant cells.

In yet another aspect, the present invention provides a method for producing a pathogen-resistant plant, which comprises

(a) introducing into a plant cell a chimeric gene of the present invention; and

(b) regenerating pathogen-resistant plants from said plant cells.

In yet another aspect, the present invention provides a method for generating effector-triggered immunity in a plant, the method comprising:

(a) allowing the expression in said plant of at least one endogenous or heterologous 2?-gene encoding an intracellular immune receptor;

(b) introducing the chimeric gene of the present invention into a cell of said plant; (c) growing a plant from the plant cell produced in step b) for a time sufficient to produce and translocate the pathogen effector to the

extracellular space; and

(d) contacting said plant with a pathogen to thereby facilitate uptake in cells of said plant of the pathogen effector from the extracellular space.

In aspects of this invention, plants may be transformed with the chimeric gene or may transiently express the chimeric gene. In some embodiments, the chimeric gene encodes a fusion protein wherein the pathogen effector is fused in translation frame to a plant-derived signal peptide for targeting said pathogen effector to the plant secretory pathway (that is, for extracellular secretion). The fusion protein may also consist of the pathogen effector combined with its natural, endogenous, signal peptide. In the latter case, the signal peptide will be heterologous to the plant.

It is preferred that the pathogen effector is a pathogen effector whose uptake in plant cells is pathogen-dependent.

It is further preferred that at least one of the promoter sequence, the signal sequence and the 3' non-translated region is heterologous to the (nucleic acid) sequence encoding the pathogen effector.

The chimeric gene of the present invention may in an alternative embodiment, comprise: a) a promoter that is active in plant cells; b) a first DNA sequence, operably linked to the promoter, encoding a signal sequence; c) a second DNA sequence, operably linked to the promoter, encoding a pathogen effector; wherein the first and second DNA sequences are linked in translation frame and together encode a fusion protein comprising the signal sequence and the pathogen effector, and wherein the signal sequence is for targeting said pathogen effector to the plant secretory pathway.

In all aspects, the pathogen effector could be used in combination with its original (non-heterologous) signal peptide, in order to targeting said pathogen effector to the plant secretory pathway when the fusion peptide is expressed in the plant, as many signal peptides comprise conserved sequences and may well be recognized by the plant, thereby resulting in efficient secretion of the protein. It is thus generally preferred that signal peptides are used that are functional in plant cells and that ensure efficient secretion in the extracellular space. Their origin is not limiting in aspects of this invention. It is a preferred embodiment in aspects of this invention that the signal peptide triggers protein secretion effused protein sequences via the ER/Golgi pathway.

The promoter used in aspects of this invention is preferably adapted to cause sufficient expression of the fusion polypeptide to mount an effective plant immune response in said plant cells upon exposure of said plant cells to a pathogen that facilitates uptake of the extracellular pathogen effector in said plant cells. In principle, any suitable promoter can be used, including constitutive promoters.

In a chimeric gene of the invention the coding sequence preferably encodes a pathogen effector that is secreted by the pathogen but whose action and/or recognition occurs intracellularly. An example of such a pathogen effector protein is Avr2 from Fusarium oxysporum, which is recognised by the intracellular R protein 1-2. Other examples are disclosed herein above and in Table 1, and throughout the text.

The R gene, is aspects of this invention, and the intracellular R protein that it encodes, may be endogenous to the plant or plant cell of the present invention. Alternatively, the R gene may not be naturally present in the plant or plant cell of the present invention. In such instances, the heterologous R gene, in aspects of this invention, may be carried on the same nucleic acid construct that carries the chimeric gene of the present invention, or may be brought into the plant or plant cell in aspects of this invention by means of a separate nucleic acid construct using any suitable transformation or vector system as described herein. The R gene can also be obtained from natural (germplasm) resources and introgressed in the plant via crossing. Expression of the heterologous R gene may thus be accomplished by using the same or different expression system from that used to drive expression of the heterologous pathogen effector, and expression may be under the control of the same or a different promoter.

An aspect of this invention is also a combination of a plant and a chimeric gene or vector comprising the chimeric gene of the invention (preferably a recombinant plant viral expression vector comprising the chimeric gene of the invention). It is preferred in such a combination that the plant comprises the cognate R protein to the pathogen effector expressed from the chimeric gene.

In another aspect, the present invention relates to a use of a plant, or plant part, of the invention for the production of a foodstuff or feedstuff, such as a vegetable or fruit. The foodstuff or feedstuff may be processed for consumption. Alternatively, the invention provides a method for (downstream) processing of a plant, or plant part such as a vegetable or fruit, of the invention, comprising the step of: a) processing a plant, or plant part, of the invention for consumption. Food or feed processing techniques depend on the type of plant and its edible parts, and are generally known and available to a person skilled in the art. DESCRIPTION OF THE DRAWINGS

Figure 1 shows that Avr2 exerts its virulence function inside host cells. (A) A schematic diagram of Avr2 in which the signal peptide is boxed and the two cysteine residues and the predicted RxLR (Arg-x-Leu-Arg)-like motif are marked red (and underlined) and blue (and underlined), respectively. (B) Ten-day-old seedlings of wild type (Moneymaker), full- length Avr2-4 and ΔspAvr2-30 transgenic tomato plants were inoculated with water (mock), wild type Fusarium Fol007 or FoLΔAvr2. Three weeks after inoculation, (C) mean plant weight and (D) average disease index of 20 plants were scored. Error bar represent means ± SE. (*P<0.05; ** P<0.01; ***P<0.001). For clarity only one representative transgenic line is shown. Figure 2 shows that Avr2 accumulates in xylem sap and apoplastic fluids of Avr2 plants. (A) Western blot showing accumulation of HASBP-tagged Avr2 in total protein extracts of wildtype and transgenic tomato plants expressing either full-length Avr2 or ΔspAvr2. The top blot was probed with an antibody targeted against Avr2 while the bottom blot was developed using an HA antibody. The top band (*) corresponds to the size of HASBP-tagged Avr2 whereas the lower band (#) represents the size of a non-tagged Avr2. (B) Western blot of xylem sap and apoplastic fluid isolated from the above mentioned plants probed with an Avr2 specific antibody. Avr2 accumulates in apoplastic fluid and xylem sap of transgenic tomato plants expressing full-length Avr2-, but not in plans expressing ΔspAvr2. The molecular weight, as indicated by the precision plus protein standard (Bio-Rad), is shown on the left.

Figure 3 shows that /-2-carrying tomato plants do not trigger immune signaling upon Avr2 exposure. (A) Scions of four- week-old tomato plants expressing 1-2 grafted onto a wild-type Moneymaker, a ΔspAvr2 or an Avr2 rootstock. Representative grafts are shown four-weeks-post grafting. Note that all grafts grew normally and did not develop autoimmune symptoms (B) Western blot analysis of xylem sap harvested ±10 cm above the graft. The Avr2 protein could be readily detected in xylem sap of 1-2 scions placed on an Avr2 rootstock, but not in xylem sap isolated from scions grafted on either wild-type or a ΔspAvr2 roots stock. As a positive reference Avr2-containing xylem sap was harvested from tomato plants inoculated with Fol007. (C) Avr2-7 and ΔspAvr2-30 transformants were crossed to 1-2 tomato plants. Two weeks after germination ΔspAvr2/I-2 plants developed clear autoimmune phenotypes; i.e. necrotic lesions, reduced plant weight and stunted growth, whereas no symptoms developed on Moneymaker //- 2 or Avr2/I-2 progeny. (D) Western blot analysis shows accumulation of Avr2 in Avr2 and ΔspAvr 2 transgenic tomato plants and their ΔspAvr2/I-2 and Avr2/I-2 progenies. The blot was probed with an antibody targeted against Avr2. Lower panel shows a Ponceau S staining that serves as loading control.

Figure 4 shows that infiltration of A. tumefaciens in Avr2/I-2 tomato plants triggers cell death. (A) Four-week-old wild-type Moneymaker, 1-2 and Avr2/I-2 tomato plants infiltrated with either infiltration buffer ("-") or agrobacterium expressing GUS or ΔspAvr2. The left side of each leaf is buffer infiltrated and the right site is infiltrated with agrobacterium carrying either a GUS or ΔspAvr2 construct. Photographs were taken 4dpi. The bottom panel shows the same leaves stained with trypan blue to visualize cell death. (B) 20 leaves of wild-type Moneymaker, 1-2 and Avr2/I- 2 tomato plants were scored for their response following infiltration. The assay was repeated twice with similar results.

Figure 5 shows the presence of Avr 2 and 1-2 gene in ΔspAvr2/I-2 andAvr2/I-2 tomato plants. (A) Ethidium bromide stained agarose gel showing the PCR products obtained with either Avr2 or 1-2 specific primers using DNA extracted from the indicated plants. The GeneRuler lkb DNA Ladder (Fermentas) is shown on the left. (B) Western blot analysis shows accumulation of Avr 2 in the parental Avr 2 and two independent Δsp Avr 2 transgenic tomato plants, and in two independent ΔspAvr2/I-2 andAvr2/I- 2 progenies. The blot was probed with an antibody targeted against Avr 2. The precision plus protein standard (Bio-Rad) is shown on the left.

Figure 6 shows that Avr2/I-2 tomato plants are less susceptible to the plant pathogen Phytophthora infestans than the parental Avr2 and 1-2 tomato lines. (A) Disease symptom classes of tomato leaves inoculated with P. infestans. Class 1 and 2 show weak and strong necrosis, respectively, which is limited to the site of inoculation. Class 3 shows spreading necrosis. (B) The bar graph depicts the percentage of inoculation sites showing a particular disease class. The tested plant genotypes are indicated on the bottom, for each plant line 40 P. infestans inoculation sites were classified. DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein shall have the same meaning as is commonly understood by one skilled in the art to which the present invention belongs. Where permitted, all patents, applications, published applications, and other publications, including nucleic acid and polypeptide sequences from GenBank, SwissPro and other databases referred to in the disclosure are incorporated by reference in their entirety.

The term "plant", as used herein, refers to any type of plant. As used herein, the term "plant" includes the whole plant or any parts or derivatives thereof, preferably having the same genetic makeup as the plant from which it is obtained, such as plant organs (e.g. harvested or non- harvested carrot root), plant cells, plant protoplasts, plant cell and/or tissue cultures from which whole plants can be regenerated, plant calli, plant cell clumps, plant transplants, seedlings, hypocotyl, cotyledon, plant cells that are intact in plants, plant clones or micropropagations, or parts of plants (e.g. harvested tissues or organs), such as plant cuttings, vegetative propagations, embryos, pollen, ovules, flowers, leaves, seeds, clonally propagated plants, roots, taproots, stems, root tips, grafts, parts of any of these and the like. Also any developmental stage is included, such as seedlings, cuttings prior or after rooting, mature plants, roots or leaves. Alternatively, plant part may also include a plant seed that comprises one or two sets of chromosomes derived from the parent plant.

Below is an exemplary description of some plants that may be used in aspects of the invention. However, the list is provided for illustrative purposes only and is not limiting, as other types of plants will be known to those of skill in the art and could be used with the invention. The term preferably refers to a cultivated plant, more preferably a breeding line, still more preferably an essentially homozygous breeding line. A common class of plants exploited in agriculture are vegetable crops, including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head, leaf, romaine), bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas, parsnips, chicory, Chinese cabbage, peppers, collards, potatoes, cucumber plants (marrows, cucumbers), pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots, endive, garlic, spinach, green onions, squash, greens, beet (sugar beet and fodder beet), sweet potatoes, swiss-chard, horseradish, tomatoes, kale, turnips, and spices.

Other types of plants frequently finding commercial use include fruit and vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, quince almonds, chestnuts, filberts, pecans,

pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropical fruits, melon, mango, papaya, and lychee.

Many of the most widely grown plants are field crop plants such as evening primrose, meadow foam, corn (field, sweet, popcorn), hops, jojoba, peanuts, rice, safflower, small grains (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), fiber plants (cotton, flax, hemp, jute),

Lauraceae (cinnamon, camphor), or plants such as coffee, sugarcane, tea, and natural rubber plants.

Another economically important group of plants are ornamental plants. Examples of commonly grown ornamental plants include

Alstroemeria (e.g., Alstoemeria brasiliensis ), aster, azalea (e.g.,

Rhododendron sp.), begonias (e.g., Begonia sp.), bellflower, bouganvillea, cactus (e.g., Cactaceae schlumbergera truncata ), camellia, carnation (e.g., Dianthus caryophyllus ), chrysanthemums (e.g., Chrysanthemum sp.), clematis (e.g., Clematis sp.), cockscomb, columbine, cyclamen (e.g.,

Cyclamen sp.), daffodils (e.g., Narcissus sp.), false cypress, freesia (e.g., Freesia refracta ), geraniums, gerberas, gladiolus (e.g., Gladiolus sp.), holly, hibiscus (e.g., Hibiscus rosasanensis ), hydrangea (e.g., Macrophylla hydrangea ), juniper, lilies (e.g., Lilium sp.), magnolia, miniroses, orchids (e.g., members of the family Orchidaceae ), petunias (e.g., Petunia hybrida ), poinsettia (e.g., Euphorbia pulcherima), primroses, rhododendron, roses (e.g., Rosa sp.), snapdragons (e.g., Antirrhinum sp.), shrubs, trees such as forest (broad-leaved trees and evergreens, such as conifers) and tulips (e.g., Tulipa sp.). Plants useful in the methods of the invention include plants amenable to transformation techniques.

Whenever reference to a "plant" or "plants" (or a plurality of plants) according to the invention is made, it is understood that also plant parts (cells, tissues or organs, seeds, severed or harvested parts, leaves, seedlings, flowers, pollen, fruit, stems, roots, callus, protoplasts, etc), progeny or clonal propagations of the plants which retain the distinguishing characteristics of the parents (e.g. presence of a trans-gene), such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived therefrom are encompassed herein, unless otherwise indicated.

The term "crop plant", as used herein, refers to a plant that is harvested or provides a harvestable product. Suitable plants for use in aspects of the invention also include protected (greenhouse) crop plants.

As used herein, the term "genetically modified plant" refers to a plant whose genome has been changed using genetic modification

techniques. The term includes reference to a transgenic plant, which itself denotes a plant comprising a transgene. The term "non-genetically modified plant" in the context of the present invention refers in particular to plants that are not considered as plants that are genetically modified under Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms. Plants obtained through crossing and mutagenesis, and that are not obtained by methods that involve the use of recombinant nucleic acid molecules or genetically modified plant cells are considered non-genetically modified plants.

The term "species" includes any taxonomic group of organisms, which can interbreed, and thereby includes sub-species, varieties, accessions and cultivars.

The term "variety" or "cultivar" means a plant grouping within a single botanical taxon of the lowest known rank, which grouping,

irrespective of whether the conditions for the grant of a breeder's right are fully met, can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, distinguished from any other plant grouping by the expression of at least one of the said

characteristics and considered as a unit with regard to its suitability for being propagated unchanged. The term "cultivar" refers to a variety, strain or race of plant that has been produced by horticultural or agronomic techniques and is not normally found in wild populations.

The term "accession" when used herein is associated with sources of plants and refers to a plant or group of similar plants or group of seeds from these plants received from a single source at a single time. Accessions are generally indicated by an "accession number", which number refers to a unique identifier for each accession and is assigned when an accession is entered into a plant collection. The terms "germplasm" and "accessions" are somewhat interchangeable, and use of the term "accession" is not meant to exclude from the method of the invention use of wild accessions of plants that are not uniquely identified or part of a germplasm collection.

As used herein, the term "pathogen" refers to any plant pathogen including a plant pathogenic virus, a plant pathogenic bacterium, a plant pathogenic fungus, a plant pathogenic oomycete, a plant pathogenic nematode and a plant pathogenic arthropod. Pathogens used in aspects of this invention may include, but are not limited to plant pathogenic oomycetes, such as Phytophthora infestans, Phytophthora sojae and

Phytophthora ramorum, Albugo spp's, Bremia spp's, Pythium spps's and Hyaloperonospora ssp's, plant pathogenic bacteria such as Pseudomonas syringae, Xyella spps and Xanthomonas ssp's, plant pathogenic fungi such as Cladosporium fulvum, Alternaria species, Mycosphaerella species,

Verticiulum species, Melampsora lini, Magnaporthe oryzae, Rhizoctonia solani, Puccina species, Colletotrichum spp., Blumeria graminis, Fusarium oxysporum, Fusarium graminearum, and plant pathogenic nematodes, such as Globodera pallida and G. rostochiensis.

As used herein, the term "resistance response" includes reference to the display of a resistance phenotype following pathogenic challenge, or following pathogen effector protein contact or pathogen effector gene expression as indicated herein. Such phenotype may be, for example, the extent, rate of progress, or degree of occurrence of a necrotic reaction in the plant tissue. A necrotic reaction is not essential during a resistance response as referred to herein. A resistance response includes reference to ETI responses in which cell death is not induced but wherein the plant is resistant. Thus, for instance, cell death is potentially not triggered but the plant still exhibits resistance.

The term "nucleic acid sequence" (or nucleic acid molecule), as used herein, refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages, including a DNA or KNA molecule in single or double stranded form, and particularly a DNA encoding for a protein or protein fragment. The term also includes modified or substituted sequences comprising non- naturally occurring monomers or portions thereof. The nucleic acid sequences of the present invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. An "isolated nucleic acid sequence" refers to a nucleic acid sequence, which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.

As used herein the terms "pathogen effector" and "pathogen effector polypeptide", both of which refer to the pathogen effector gene product, are to be understood as general terms that include reference to avirulence factors, elicitors, and (Avr) effectors. The term "pathogen effector" is used here in the broadest sense and applies to any pathogen molecule that is recognized by a plant immune receptor triggering a resistance, defense, immune or ΕΊΊ response, which can comprise an HR. The term preferably does not refer to PAMPs in the case that these molecules are recognized by extracellular R proteins. Pathogen effectors that are used in aspects of this invention are secreted by the pathogen in the extracellular space (e.g. xylem, apoplast or even extrahaustorial spaces) of the plant and recognized in the plant cell through intracellular R proteins, preferably but not exclusive to NB-LRR proteins. As used herein, the terms "avirulence factor" and "elicitor" are interchangeably used, and are broadly drawn to a molecule of phytopathogenic origin that triggers a resistance- response in the plant. Without wishing to be bound by theory, in the context of the present invention, it is assumed that inheritance of both resistance in the host and the pathogen's ability to cause disease is controlled by pairs of matching genes. The first member of the gene pair is a plant gene called the resistance (R) gene. The second member of the pair is a parasite- or pathogen-derived gene called the avirulence (Avr) gene. Plants producing a specific R gene product are resistant towards a pathogen that produces the corresponding Avr gene product (also known as avirulence factor or elicitor).

The term "effector" is commonly used to indicate a potential . Avr gene for which the cognate R gene is not yet known. The terms "pathogen effector" and "pathogen effector polypeptide" refer to any and all polypeptide sequences of a pathogen effector including all pathogen-derived Avr effector polypeptides and polypeptides comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any pathogen effector polypeptide or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding a pathogen effector or capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding a pathogen effector but for the use of synonymous codons.

The terms "nucleic acid sequence encoding a pathogen effector" and "nucleic acid sequence encoding an Avr effector polypeptide", which may be used interchangeably herein, refer to any and all nucleic acid sequences encoding a pathogen effector polypeptide, including any bacterial, fungal, oomycetal, nematodal, mite, insect, parasitic plant and other parasite Avr effector polypeptide. Nucleic acid sequences encoding a pathogen effector polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the pathogen effector polypeptide sequences set forth herein; or (ii) hybridize to any pathogen effector nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.

The term "intracellular" in the context of R proteins in aspects of the present invention includes reference to, but is not limited to soluble proteins, and includes reference to proteins that are (loosely) attached to intracellular membranes, such as the plasma membrane, and for which ligand perception/recognition depends fully on molecular interactions inside the cell.

By the term "substantially identical" it is meant that two polypeptide sequences preferably are at least 70% identical, and more preferably are at least 85% identical and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical. "Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical" or "essentially similar" when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685

Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program "needle" (using the global Needleman

Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for 'needle' and for 'water' and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as using the Smith Waterman algorithm, are preferred. Alternatively percentage similarity or identity may be deteimined by searching against public databases, using algorithms such as FASTA, BLAST, etc.

By "at least moderately stringent hybridization conditions" it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution.

Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the T_m, which in sodium containing buffers is a function of the sodium ion concentration and temperature (TV = 81.5°C - 16.6 (Logio [Na⁺]) + 0.41(%(G+C) - 600/1), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1°C decrease in T_m, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred

embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5 x sodium chloride /sodium citrate (SS lo x Denhardt's solution/ 1.0% SDS at T_m (based on the above equation) - 5° C, followed by a wash of 0.2 x SSC/0.1% SDS at 60° C. Moderately stringent (moderate stringency) hybridization conditions include a washing step in 3 x SSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2003, 6.3.1. - 6.4.10, and in: Green and Sambrook, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 4th edition, 2012.

"Stringent hybridisation conditions" can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. The stringency of the hybridization conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt (NaCl) concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g. 100 nt) are for example those which include at least one wash in 0.2X SSC at 63°C for 20 min, or equivalent conditions. Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g. 10Ont) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Green and

Sambrook, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 4th edition, 2012. "High stringency" conditions can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (20x SSC contains 3.0 M NaCl, 0.3 M Na-citrate, pH 7.0), 5x Denhardt's (100X

Denhardt's contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 μg/ml denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non-specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0. lx SSC, 0.1% SDS.

"Moderate stringency" refers to conditions equivalent to hybridization in the above described solution but at about 60-62° C. In that case the final wash is performed at the hybridization temperature in lx SSC, 0.1% SDS.

"Low stringency" refers to conditions equivalent to hybridization in the above described solution at about 50-52° C. In that case, the final wash is performed at the hybridization temperature in 2x SSC, 0.1% SDS. See also Green and Sambrook, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 4th edition, 2012.

As used herein, the term "gene" refers to a functional protein, polypeptide or peptide-encoding nucleic acid unit. As will be understood by those skilled in the art, this functional term includes genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been designed and/or altered.

Purified or isolated genes, nucleic acids, proteins and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated. The term "gene" also refers to a DNA sequence comprising a region

(transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable transcription regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' non-translated leader sequence (also referred to as 5' UTR, which corresponds to the transcribed mRNA sequence upstream of the translation start codon) comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3' non-translated sequence (also referred to as 3' untranslated region, or 3' UTR) comprising e.g. transcription termination sites and

polyadenylation site (such as e.g. AAUAAA or variants thereof).

The term ''chimeric" as used herein in the context of nucleic acid sequences or genes refers to at least two linked nucleic acid sequences which are not naturally linked. Chimeric nucleic acid sequences or genes include linked nucleic acid sequences or genes of different natural origins. For example a nucleic acid sequence constituting a plant promoter linked to a nucleic acid sequence encoding a pathogen-derived Avr effector (pathogen effector) is considered chimeric. Chimeric nucleic acid sequences also may comprise nucleic acid sequences of the same natural origin, provided they are not naturally linked. For example a nucleic acid sequence constituting a promoter obtained from a particular cell-type may be linked to a nucleic acid sequence encoding a polypeptide obtained from that same cell-type, but not normally linked to the nucleic acid sequence constituting the promoter. Chimeric nucleic acid sequences also include nucleic acid sequences comprising any naturally occurring nucleic acid sequence linked to any non- naturally occurring nucleic acid sequence. The term "chimeric gene" is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more sense sequences (e.g. coding sequences) or to an antisense (reverse complement of the sense strand) or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription).

The terms "expression construct", "nucleic acid construct" or "vector" are herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology and which is used to deliver exogenous DNA into a host cell. The vector backbone may for example be a binary or superbinary vector (see e.g. US 5,591,616 ,

US2002138879 and WO 95/06722 ), a co-integrate vector or a T-DNA vector, as known in the art, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence / promoter is already present, only a desired nucleic acid sequence (e.g. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence / promoter. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like. The term "expression

construct" is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. Generally, the nucleic acid encoding a gene product is under transcriptional control of a promoter.

The term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation. "Expression of a gene" refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into a RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment) or which is active itself (e.g. in posttranscriptional gene silencing or RNAi, or silencing through miRNAs). The coding sequence is preferably in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment.

The term "transient expression" in the context of expression of a gene or nucleotide sequence or "transiently expressed" refers to the expression of a gene or nucleotide sequence that is not integrated into the host chromosome but which can function either independently (e.g., by being a part of an autonomously replicating plasmid or an expression cassette) or as a part of another biological system, such as a virus, for example.

Transient expression may be achieved by "transient transformation" of a host cell, which term refers to the introduction of foreign DNA or a

nucleotide sequence of interest into the host cell (for example, by such methods as Agrobacterium-mediated transformation or biolistic

bombardment) without integration of the foreign DNA or nucleotide sequence of interest into a host cell chromosome, thereby precluding stable maintenance of the foreign DNA or nucleotide sequence of interest in the progeny of the host cell.

As used herein, the term "immune receptor" refers to the array of innate immune system receptors inside the plant cell involved in

interpreting signals from potential pathogens and potential commensals or mutualists. Immune receptors in the context of aspects of this invention include in particular the immune receptors that are localized in the cytosol, the largest group of these being nucleotide-binding oligomerization domain (NOD)-like receptors / nucleotide-binding leucine-rich repeat (NLR) (NLRs). However, the invention is not limited to these receptors as also other types of intracellular immune receptors could be used. It will be understood that the structure of the intracellular immune receptor is not relevant. It is an essential aspect of this invention that the immune receptor is localised intracellularly and is able to perceive (either directly or indirectly) the intracellular presence of an effector protein that is taken up from the extracellular space. Immune receptors not intended for use in the context of aspects of this invention are R proteins and pattern-recognition receptors for which the ligand-recognition/perception domain or binding site is localized on the outside of the plant cell membrane surface. Ligand in this context means the protein, peptide, nucleotide, metabolite, chemical compound that specifically activates intracellular signaling of any type of receptor resulting in a defense or immune response. Intracellularly localized R proteins are the particularly preferred immune receptor in aspect of this invention. The terms "immune receptor" and "R protein" may be use interchangeably herein. The term "R gene" refers to a resistance (R) gene encoding an R protein for resistance against pathogens in a plant, and includes reference to R genes that are the result of recombination events and that may have been assembled, synthesized, or otherwise produced, preferably as a result of man-made efforts, and any genes that are replicated or otherwise derived from genes in plant genomes that convey plant disease resistance against pathogens producing the matching Avr proteins. The main class of R- proteins consists of multi-domain proteins carrying a nucleotide binding domain (NB) and a leucine-rich repeat (LRR) domain. These proteins are often referred to as NB-LRR or NLR R-protein. Generally, the NB domain binds ATP/ADP, whereas the LRR domain is often involved in protein- protein interactions as well as effector recognition. NB-LRR R-proteins can be further subdivided into the Toll interleukin 1 receptor (TIR-NB-LRR or TNL) and non-TIR or coiled-coil (CC-NB-LRR or CNL) proteins. Resistance can be conveyed through a number of mechanisms including: (i) The R protein interacts directly with the Avr gene product of a pathogen (receptor- ligand model); or (ii) The R protein guards another protein that is

manipulated by the Avr protein (Guard/Decoy Hypothesis). The workings of the present invention are not limited by the mechanism of action. Once the intracellular R protein detects the presence of a pathogen, as a result of the internalization of the heterologous plant-produced pathogen effector externalized by the plant, the plant mounts a defence response targeted against the pathogen. It is possible to transfer an R gene from one plant to another and provide a plant of the present invention with an R gene that is cognate to the specific plant-produced heterologous pathogen effector.

Orthologs and homologs of R genes are also envisioned for use in this invention, as are heterologous R genes. Although the scientific term "R gene" is commonly understood to also include those genes that encode extracellular R proteins, such as cell surface-localised receptor-like proteins (RLPs), the term "R gene", as used herein, refers to genes encoding intracellular immune receptors, and not to extracellular or surface-localized proteins that perceive extracellular ligands, such as the Cf gene-encoded RLP proteins, and is thus meant as referring to an "intracellular R gene".

The terms "homologous" and "heterologous" refer to the relationship between a nucleic acid or amino acid sequence and its host cell or organism or other molecules, especially in the context of transgenic organisms. A homologous sequence is thus naturally found in the host species (e.g. a tomato plant transformed with/or introgressed with a tomato gene), while a heterologous sequence is not naturally found in the host cell (e.g. a tomato plant transformed with a sequence from potato plants). The term "heterologous" may also refer to molecules being of different origin, e.g. derived from organisms belonging to different taxa. Depending on the context, the term "homolog" or "homologous" may alternatively refer to sequences which are descendent from a common ancestral sequence (e.g. they may be orthologs). Homologs include sequences having a sequence identity of at least 70%, preferably more than 80%, still more preferably at least 90%, 95%, 98%, or 99% over the entire length of the sequence with the sequence of the nucleic acid or amino acid sequence to which it is

homologous.

The term "ortholog" of a gene or protein refers herein to the homologous gene or protein found in another species, which has the same function as the gene or protein, but (usually) diverged in sequence from the time point on when the species harbouring the genes diverged (i.e. the genes evolved from a common ancestor by speciation). Orthologs of a gene from one plant species may thus be identified in other plant species based on both sequence comparisons (e.g. based on percentages sequence identity over the entire sequence or over specific domains) and functional analysis.

The term "heterologous gene" refers to a coding sequence for a heterologous peptide or polypeptide. Due to the inherent degeneracy of the genetic code, a number of nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence may be

generated and used to clone and express a given heterologous peptide or polypeptide. Thus, for a given heterologous peptide or polypeptide encoding nucleic acid sequence, it is appreciated that as a result of the degeneracy of the genetic code, a number of coding sequences can be produced that encode the same protein amino acid sequence. Such substitutions in the coding region fall within the range of sequence variants covered by the present invention. Any and all of these sequence variants can be utilized in the same way as described herein for the exemplified heterologous peptide or polypeptide encoding nucleic acid sequence. As will be understood by those of skill in the art, in some cases it may be advantageous to use a

heterologous peptide or polypeptide encoding nucleotide sequences possessing non-naturally occurring codons. Codons preferred by a particular eukaryotic host can be selected, for example, to increase the rate of expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence. As an example, it has been shown that codons for genes expressed in rice are rich in guanine (G) or cytosine (C) in the third codon position. Changing low G + C content to a high G + C content has been found to increase the expression levels of foreign protein genes in barley grains. The DNA sequences employed in the present invention may be based on a gene codon bias found in the targeted crop/plant along with the appropriate restriction/recombination sites for gene cloning.

The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, three-dimensional structure or origin. A "fragment" or "portion" of a protein may thus still be referred to as a "protein". An "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

The term "signal sequence" or a "signal peptide", as the terms are used interchangeably herein, is an N-terminal polypeptide sequence, which is effective to localize the peptide or protein to which it is attached to a selected intracellular or extracellular location, preferably via the

endoplasmatic reticulum (ER). The type of signal sequence used is not critical, as long as the pathogen effector is targeted to the plant secretory pathway for extracellular secretion. Preferably, the signal sequence targets the attached peptide or protein to the endoplasmic reticulum (ER) for extracellular secretion.

A "3' UTR" or "3' non-translated sequence" (also often referred to as 3' untranslated region, or 3' end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof). After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added.

The term "transcription regulatory sequence", as used herein, refers to a nucleic acid sequence that is capable of regulating the (rate of) transcription of a nucleic acid sequence operably linked to the transcription regulatory sequence. A transcription regulatory sequence as herein defined will thus comprise all of the nucleic acid sequence elements necessary for initiation of transcription (promoters or promoter elements), for maintaining and for regulating transcription, including e.g. attenuators or enhancers, but also silencers. Although mostly the upstream (5') transcription

regulatory sequences of a coding sequence are referred to, regulatory sequences found downstream (3') of a coding sequence are also encompassed by this definition. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream (5') with respect to the direction of transcription of the transcription initiation site of the gene (the transcription start is referred to as position +1 of the sequence and any upstream nucleotides relative thereto are referred to using negative numbers), and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase,

transcription initiation sites and any other DNA motifs (cis acting

sequences), including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount, timing, and tissue-type of transcription from the promoter. Examples of eukaryotic cis acting sequences upstream of the transcription start (+1) include the TATA box (commonly at approximately position -20 to -30 of the transcription start), the CAAT box (commonly at approximately position -75 relative to the transcription start), 5'enhancer or silencer elements, etc.

The term "constitutive promoter", as used herein, refers to a promoter that is active in essentially all tissues and organs under most physiological and/or developmental conditions (such as the CaMV 35S promoter). More preferably, a constitutive promoter is active under essentially all physiological and developmental conditions in all major organs, such as at least the leaves, stems, roots, seeds, fruits and flowers. Most preferably, the promoter is active in all organs under most (preferably all) physiological and developmental conditions. Tissue-specific or tissue- preferred promoters can also be referred to as being "constitutively active". The promoter is thus active under most developmental and/or physiological conditions, albeit in only a specific tissue or mainly in a specific tissue. A "promoter which has constitutive activity" or which is "constitutive" in a plant or plant cell refers, therefore, to a nucleic acid sequence which confers transcription in the plant or plant cells in the specific tissue under most physiological and developmental conditions.

The term "inducible promoter", as used herein, refers to a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated.

The term "tissue specific promoter", as used herein, refers to a promoter that is only active in specific types of tissues or cells, such as trichome cells, epidermal cells or leaf mesophyll cells. The promoter activity can therefore be described by referring to the circumstances under which the promoter confers transcription of the nucleic acid sequence operably linked downstream (3') of the promoter.

A "tissue preferred" promoter is preferentially, but not exclusively, active in certain tissues or cells, such as for example in trichome cells and epidermis cells.

The term "operably finked" as used herein is synonymous to the term "operatively finked", and means that a nucleic acid is placed into a functional relationship with another nucleic acid sequence. For example, a promoter, or other expression control element, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Linking between two nucleic acid sequences in a construct may be accomplished by ligation at convenient restriction sites, synthesis of synthetic dsDNA (also known as gene synthesis) and/or DNA recombination site strategies. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Operably finked DNA sequences being finked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame so as to produce a "chimeric protein". A "chimeric protein" or "hybrid protein" is a protein composed of various protein "domains" (or motifs) which is not found as such in nature but which are joined to form a functional protein, which displays the functionality of the joined domains. A chimeric protein may also be a fusion protein of two or more proteins occurring in nature. The term

"domain" as used herein means any part(s) or domain(s) of the protein with a specific structure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain. One such a domain may be a signal sequence. A chimeric protein is encoded by a chimeric gene.

The term "apoplastic space", as used herein, is broadly drawn to everything outside the plasma membrane of the plant cell, and includes reference to the "extracellular space" and "intercellular space". Strictly speaking, the apoplast is the space outside the plasma membrane within which material can diffuse freely, following secretion from the host cell, or following secretion from a plant-invading pathogen. It is interrupted by the Casparian strip in roots, by air spaces between plant cells and by the plant cuticle. Structurally, the apoplast is formed by the continuum of cell walls of adjacent cells as well as the extracellular spaces, forming a tissue level compartment comparable to the symplast. The apoplastic route facilitates the transport of gasses, water and solutes across a tissue or organ.

The term "transgenic" refers to an organism or cell having received genetic material from a different organism or cell, resulting in the introduction of foreign DNA into said organism or cell, either naturally, or by any of a number of genetic engineering techniques.

The term "transforming" or "transformation" refers to the process of introducing DNA into a recipient plant cell, and includes both the subsequent integration into the plant cell's chromosomal DNA, as well as the transient transformation whereby the DNA is expressed from

extrachromosomal elements. A number of techniques are known in the art for transformation of plants or plant cells in general, including

Agrobacterium-mediated transformation, electroporation, microinjection, microprojectile or particle gun technology (biolistics), liposomes,

polyethylene glycol (PEG) mediated transformation, wounding, vacuum infiltration, passive infiltration or pressurized infiltration, and reagents that increase free DNA uptake. Identification of transformed cells or plants is generally accomplished by including a selectable marker in the- transfonning vector or by direct screening for the presence of the transgene. Transformation of a cell may be stable or transient. The term "transient transformation" or "transiently transformed" refers to the introduction of one or more transgenes into a cell in the absence of integration of the transgene into the host cell's genome. One possible embodiment of aspects of in the present invention comprises the transformation of plant cells with a putative R gene. This method involves infection with Agrobacterium tumefaciens and is well known to one of skill in the art. The method is for instance described in detail in Van de Hoorn et al., MPMI Vol. 13, No. 4, 2000, pp. 439-446, and Sparkes et al. (2006) Nat Protoc 1:2019-2025. In some preferred embodiments of aspects of the invention, stable

transformation is used to generate transformed plant lines. Stable

transformation may for instance be accomplished by using agroinfiltration. In addition to stable transformed plant lines, transient expression may for instance be accomplished by using plant viral expression vectors as described elsewhere herein.

The term "selectable marker" is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. Selectable marker gene products confer, for example, antibiotic resistance, such as the nptll gene and the like, or more preferably, herbicide resistance or another selectable trait such as a phenotypic trait (e.g. a change in pigmentation) or a nutritional requirement. The term "reporter" is mainly used to refer to visible markers, such as green fluorescent protein (GFP), eGFP, luciferase, bioluminescence, GUS and the like.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". It is further understood that, when referring to "sequences" herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to. Description of preferred embodiments

A major limitation associated with current genetic-based disease control practices, whether based on GMO of non-GMO practice, is that they are either crop-specific or pathogen-specific. Current non-GMO strategies that aim for disease resistant plants are typically based on introgression of dominant resistance genes encoding immune receptors recognizing specific effector proteins into crops (i.e. classic plant breeding). These receptors confer race-specific resistance, which is typically narrow spectrum and often non-durable as the pathogen can evade recognition upon mutation or loss of a single effector gene as described herein. Alternatively, breeders use recessive susceptibility genes that disturb compatibility between pathogen and host. The latter type of resistance is often non-race specific and potentially more durable, but since the genes involved often play key roles in plant growth or development, pleiotropic effects often limit their usefulness (van Schie and Takken, Annual Review Phytopathol., 2014). In both cases the applicability of the use of Resistance and Susceptibility genes is restricted by their availability in crossable plant species and the presence of S gene homologues that remain functional. Introgression of genes in crops from wild relatives or landraces is typically a tedious and lengthy process. The present invention overcomes these limitations, as the method is based on the introduction of a transgene encoding an extracellular effector in a plant that has a matching immune receptor localized in the cytosol.

Many GMO based strategies have been proposed in the literature, but none of them are based on activation of the plant immune system following pathogen-induced uptake of a transgenically produced

extracellular localized pathogen effector protein. Exposure of the plant immune system to a transgenically encoded pathogen effector protein as a means to control plant disease has been described in US 5,866,776. This patent publication describes the controlled production of a pathogen effector inside a plant that contains the matching immune receptor. In US 5,866,776 expression of the pathogen effector gene is driven by a pathogen-inducible promoter. Upon infection the pathogen effector gene is expressed, resulting in production of the Avr effector protein and subsequent induction of plant immunity by the corresponding R protein. Induction of the promoter to drive effector gene expression requires an initial recognition of the pathogen's presence by the host.

The present invention differs from the prior art in that the strategy is distinct. For instance, in aspects of this invention, the method may be based on the constitutive expression of the pathogen effector, which can be either systemic or tissue specific in case leaf- or root-specific resistance is preferable. Another difference between the present invention and the prior art is that in the present invention the pathogen effector is targeted for secretion and its recognition and activation of immunity is preferably mediated by pathogen-induced re-uptake of the pathogen effector rather than pathogen-induced production thereof in the plant.

US 5,866,776 describes expression of the Avr effector by a pathogen-inducible promoter after which the effector is perceived by the matching immune receptor. A major obstacle for application of the invention described in US 5,866,776 is that identification of a plant promoter that is only and specifically induced by pathogens has so far been unsuccessful, prohibiting the use of this technology in crops.

A variation of this idea is patented in WO02/02787 (Al) in which a pathogen-inducible promoter drives expression of a transcription factor that activates plant immunity. The main difference between the prior art and the present invention is, besides the type of promoters used to drive effector gene expression, the mechanism on which perception of the effector is based. In the present invention the pathogen effector (extracellular) is physically and spatially separated from the immune receptor (intracellular) and co-localization is only facilitated by the presence of a pathogen while in the US 5,866,776 patent both the receptor and pathogen effector co-localize upon expression of the latter and activation depends on the induction of pathogen effector gene expression. The methods described in WO02/02787 differ also by the type of promoter used and the mechanism involved in activating immune signaling.

Resistance conferred by the present invention is essentially broad spectrum, e.g. to all pathogens that trigger pathogen effector uptake, and durable as it is not based on recognition of a single trait (i.e. effector) that can be shed/mutated by the pathogen. In theory the patent is applicable to all plant species that can be transformed and for which an effector-immune receptor pair can be identified of which effector uptake is triggered by the pathogen.

In the Examples below, the present inventors describe the use of a secreted Avr2 effector from Fusarium oxysporum f.sp. lycopersici that is recognized by the 1-2 R protein from tomato. However, it will be understood by one of skill in the art that the methods of this invention can potentially be used for any pathogen effector/ immune receptor gene pair of which uptake of the pathogen effector by the host cell is triggered by the pathogen. Pathogen effector

The "pathogen effector", also referred to herein as "pathogenic effector" or "Avr effector protein" is a pathogen-derived protein, that, in aspects of this invention, has a corresponding or cognate immune receptor (such as the cognate "R protein") inside a plant cell. The nucleic acid sequences encoding a pathogen effector that may be used in accordance with aspects of this invention may be any nucleic acid sequence encoding a pathogen effector polypeptide. The pathogen effector is chosen such that presence of both the pathogen effector and its cognate R protein in a plant cell triggers an effective ETI, immune or defence response that halts further ingress and/or proliferation of the pathogen. Hence, in aspects of this invention, reference may also be made to a specific pathogen

effector/immune receptor gene pair in the context of nucleic acid sequences encoding the pathogen effector protein/immune receptor protein pair.

A number of useful pathogen effector/immune receptor gene pairs, that be used in aspects of this invention is displayed in Table 1. The effectors and R-genes listed therein can also be used individually.

In the same manner, other useful pathogen effector/immune receptor gene pairs are a pair of nucleic acid sequences encoding (i) effector protein NSm, or an immunogenic or immune response-inducing part thereof, from tomato spotted wilt virus (TSWV) (Genbank Acc. No.

ATL64765.1; i.a. described in Kormelink et al., Virology, 200(l):56-65

(1994), and Kormelink et al., J Gen Virol, 73(Pt 11):2795-S04 (1992)), and intracellular R protein Sw-Sb (Genbank Acc. No. AY007366.1, tospovirus resistance protein B), the latter originating from Solatium lycopersicum

(tomato); (ii) effector protein Six8 (Genbank Acc. No. ACN69118.1) from

Fusarium oxysporum f.sp. lycopersici, and intracellular R protein SNC1 (Genbank Acc. No. 023530.5), the latter originating from Arabidopsis thaliana; (iii) effector protein Avr3a from P. Infestans (indicated by Genbank Acc. No. BE776395.1), and intracellular R protein R3a (Genbank Acc. No. AY849382.1); and (iv) effector protein mutant Avr2^T145E (encoded by

Genbank Acc. No. AM234063.1, with a T-→E substitution mutation on position 145), and intracellular R protein 1-2 (Genbank Acc. No.

AF118127.1). A plant of the invention may thus contain a pair of nucleic acid sequences as indicated above. Preferably, for the pair indicated under (i), the plant is a tomato plant, preferably Solarium lycopersicum, or a root(stock) that allows for grafting and growth of tomato, potato and/or eggplant. Such a plant preferably provides resistance to Tospoviruses such as TSWV. Preferably, for the pair indicated under (ii), the plant is

Arabidopsis thaliana. Preferably, for the pair indicated under (iii), the plant is a potato plant, preferably S. tuberosum. Preferably, for the pair indicated under (iv), the plant is a tomato plant, preferably Solanum lycopersicum, or a root(stock) that allows for grafting and growth of tomato, potato and eggplant. It is again noted that these effectors and R-genes can also be used individually and/or in different plants.

An example of such a suitable pathogen effector is Avr2 from Fusarium oxysporum, which is recognised by the intracellular NLR protein 1-2 from tomato. Other examples from secreted effectors that are recognised by intracellular immune receptors are Avr2 (AM234063.1) of Fusarium oxysporum, Avra10, Avrkl and AvrMlal of Blumeria graminis, AvrL567 (AY510102.1), AvrM (DQ279870), AvrP1234 (EU642499) and AvrP4

(ABB96263.1) of Melampsora lini, AVR-Pia, Avr-Pik, Avr-Pita (AF207841), Avr-Pita2, and Avrl-C039 (AF463528) of Magnaporthe grisea or

Magnaporthe oryzae, AvreRpgl of Puccinia graminis f sp. Tritici, Avr3 of Bremia lactucae, ATR1 (PDB ID: 3RMR_A), ATR3, ATR13 (PDB ID:2LAI), HaRxLl- HaRxL147, HaRxLL3a- HaRxLL495c, HaRxLCRN4b of

Hyaloperonosporaarabidopsidis, AvrB and AvrRPPlA of Peronospora parasitica, AvrRPPlB, AvrRPPIC, AvrRPP2, AvrRPP4, AvrRPP5, and AvrRPP8 of Peronospora parasitica, Avrl (DS028168), AVR2,Avr-blbl (IpiO) (DS028419), Avr-blb2 (DS028242), PiAvr2 (DS028133), Avr3a (EF587759), Avr2 (EEY61966), Avr3b, Avr10, Avrll (DQ390339), and AVR4 of

Phytophthora infestans, Avrla (EF463064.1), Avr3a (EF587759.1), AVRlb- 1, AVRlk, and Avr3c (FJ705360.1) of Phytophthora sojae, AvrLml

(AM084345.1), AvrLm6 (AM259336.1), AvrLm4-7 (AM998638.1) of

Leptosphaeria maculans, Avra10 (DQ679913), and Avrkl (DQ679912) of Blumeria graminis f. sp. hordei, and OEC45-OEC115 of Golovinomyces orontii, and homologs thereof.

It is a feature of the above mentioned effectors is that they are secreted by the pathogen into the apoplastic/extracellular spaces of the plant cell, which typically implies that they carry a signal peptide. The effectors can directly be injected in the apoplastic spaces using a stylet, as for instance done by aphids, nematodes and other sucking and piercing insects. Alternatively, for pathogens that colonise the apoplastic spaces the effectors can be secreted directly into the apoplastic space. For pathogens that form specialised feeding structures, such as haustoria, effector secretion will mostly occur in the extrahaustorial matrix. This

extahaustorial matrix is on one side restricted by the plasmamembrane from the pathogen and on the other side by the plasmamembrane from the host plant. A neckband closes this space from the rest of the apoplastic spaces in the host.

Other pathogens, such as Magnaporthe oryzae, form invasive hyphae that are enclosed in host-derived extrainvasive hyphal membrane. The effectors are secreted into the invasive hyphae, that like the haustorial matrix forms a sealed and distinct apoplastic compartment that is separated from both the symplast and the bulk apoplast. In all above cases the effector is secreted into the apoplastic space on the outside of the host cell

membrane. This in contrast to effector proteins that are secreted directly inside the host cell, by for instance, but not exclusively, a type 3 secretion system (also known as TTSS, type III secretion, or Hrp-dependent secretion) as used by many bacterial pathogens. The latter class of effectors is excluded for use in this invention as they are not taken up by the host, but injected into the host by the action of the pathogens. For an overview of suitable extracellular effectors useful in aspects of this invention, reference is made to Giraldo & Valent, 2013 (Nature Reviews Microbiology 11, 800- 814). Suitable effectors for use in this invention may also be identified experimentally, for instance by using a gene gun or a biolistic particle delivery system for delivering expression constructs with and without signal peptide sequence to a resistant plant cell. Expression of the effector construct in a resistance plant will not result in an immune response when the effector protein is in the apoplast, but an immune response will be mounted when it is produced in the cell, as will be the case for the construct without signal peptide. Alternatively, an experiment can be designed wherein infiltration of the effector protein in a leaf will not result in an immune response, whereas expression in the cell, for example by means of Agrobacterium tumefaciens-mediated transient transformation of the plant cells using a construct for expression of the effector without signal peptide will result in an immune response.

Table 1: Effector/R protein pairs of secreted effector proteins that are recognised by intracellular immune receptors.

For the R proteins that are not cloned their putative location is prediced based on for instance their map position.

The nucleic acid sequence encoding the pathogen effector may be altered, to improve expression levels for example, by optimizing the nucleic acid sequence in accordance with the preferred codon usage for a particular plant cell type which is selected for the expression of the pathogen effector polypeptide. Comparison of the codon usage of the nucleic acid sequence encoding the pathogen effector polypeptide with the codon usage of the plant cell type will enable the identification of codons that may be changed.

Construction of synthetic genes by altering the codon usage is well known in the art.

Use may also be made of pathogen effector orthologs.

Immune receptors (R proteins)

The type of immune receptor responsible for interacting intracellularly with the pathogen effector is not critical for the present invention, as long as it is an intracellular immune receptor that is cognate to the pathogen effector and is capable of mounting an effector-triggered immunity (ΕΊΊ) response. It is envisioned that in preferred embodiments, the plants own endogenous intracellular immune receptors can be used to mount the required immune response against the pathogen. The plant cell genome contains numerous genes encoding intracellular immune receptors that can mount ETI. The Arabidopsis genome contains about 200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Hence, the immune receptor cognate to the pathogen effector used in aspects of this invention may suitably be endogenous. Alternatively, or in case a suitable pathogen effector protein/immune receptor protein pair cannot be composed, use may be made of an immune receptor of heterologous origin. The gene for such a heterologous immune receptor may suitably be integrated in a gene construct, as a separately encoded nucleic acid sequence.

Numerous R genes, belonging to different classes, have been cloned from many plant species (Dangl and Jones, 2001; Hulbert et al., 2001, as referenced by Meyers et al., Plant Cell. 2003 Apr; 15(4): 809-834.). The largest class of known R proteins includes those that contain a nucleotide binding site and leucine-rich repeat domains (NBS-LRR proteins). NBS-LRR proteins may recognize the presence of the pathogen directly or indirectly. In theory, specific recognition of multiple pathogens could necessitate the activity of numerous R genes.

It is preferred that in aspects of this invention, use is made of an R protein that mediates intracellular effector recognition. Effector perception by the R protein could be either direct, or indirect via a host protein that is targeted by the effector protein. In either case, perception of the effector triggers activation of the R protein resulting in the induction of ETI halting further pathogen ingress and/or proliferation. To examine whether an R protein recognizes an effector intracellular the effector protein could be produced inside the cells by means of transient expression and by monitoring the induction of ETI responses. When immune responses, such a cell death or induction of defence marker genes and/or ROS or electrolyte leakage are observable it implies that recognition is intracellular. To ascertain that the effector is not internalized by a host autonomous process in the absence of the pathogen the effector protein can be infiltrated inside the apoplastic spaces of the plant and/or expressed from the plant cell for extracellular secretion. For a suitable effector/R protein pair the presence of the Avr effector protein in the apoplastic space will not trigger ETI, while its intracellular production does.

A prerequisite for suitable R proteins in this invention is that they localize inside the host cell, i.e. they do not carry a signal peptide that targets them to the extracellular membrane.

One of skill in the art will be able to identify additional suitable pathogen effector /immune receptor pairs for use in generating resistance in transgenic plants according to the present invention.

Alternatively, it is not essential to aspects of this invention that the immune receptor is known. One of skill in the art will be able to determine if a certain pathogen effector is capable of mounting a localized immune response in plant cells of a plant species, cultivar, variety or accession of interest at the site of infection by a pathogen or triggered by recognition of a heterologously produced pathogen effector by any immune receptor present in said plant cell. Suitable methods are exemplified in the Examples described herein.

In short, to identify and select a suitable pathogen effector capable of mounting a localized immune response in a plant of interest against a pathogen of interest, transgenic plants of the plant of interest carrying the cognate R protein may be produced that produce cytosolic or apoplastic localized effector proteins. For a suitable pair only the cytosolic localized effectors triggers ETI, resistance or immunity. Alternatively, purified effector protein can be administered to a plant carrying the cognate R protein. If the effector is suitable no ETI, resistance or immune response will be induced in the absence of a pathogen. Signal sequence for secretion into apoplastic space

Secretion of the pathogen effector protein can suitably, and preferably, be mediated by inclusion of a signal peptide for protein secretion via the ER/Golgi pathway. Hence, aspects of the invention, such as the fusion proteins of the invention, further comprise a signal peptide functional for targeting said fusion protein to the apoplast. This may be achieved with a signal peptide that targets the fusion protein into the endoplasmatic reticulum (ER) and subsequently into the (Golgi) secretory pathway. All signal peptides of proteins known to be secreted or targeted to the apoplast may be used for the purposes of the invention. Preferred examples are signal peptides like that from the PRla protein of tomato. Further examples are the signal peptides of pectin methylesterase or of Nicotiana tabacum tyrosine and lysine rich protein (NtTLRP). A further example is the signal peptide of apoplastic isoperoxidase from zucchini (Cucurbita pepo) (APRX) (Carpin etal., 2001 , The Plant Cell, 13, 511-520). The signal peptide has to be comprised in said fusion protein such that it is functional for said targeting. The signal peptide is thereto positioned at the N-terminus of the fusion protein for functional targeting of the fusion protein to the apoplast.

The signal peptide may be of plant origin, or, alternatively , of pathogen origin, or any heterologous origin but functional for protein secretion via the ER in the plant. In preferred embodiments of the present invention, the chimeric nucleic acid sequence comprises a "signal sequence". A signal sequence (also referred to herein as "signal peptide^*') is a short (5- 35 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. These proteins include those that reside either inside certain organelles (the ER, Golgi or endosomes), secreted from the cell, or inserted into most cellular membranes.

The signal sequence, in aspects of this invention, may include any amino acid sequence capable of directing the Avr effector polypeptide, when expressed, to a desired location within the plant cell that results in its secretion in the extracellular space. Suitably, in order to ensure secretion in the extracellular space, the heterologous Avr effector is targeted to the ER. In order to achieve targeting of the Avr effector to the ER, the nucleic acid sequence encoding the Avr effector polypeptide is linked or fused to the signal sequence that causes the Avr effector to be exported to the ER. In a preferred embodiment, the nucleic acid sequence encoding the Avr effector polypeptide is expressed in such a manner that the Avr effector polypeptide does not accumulate in the cytoplasm. It is an aspect of this invention that the nucleic acid sequence (RNA) encoding the pathogen effector polypeptide is translated by the ribosome at the ER membrane surface to ensure direct translocation of the protein into the ER lumen during peptide synthesis from which it is then exported via the Golgi system to the extracellular spaces (e.g. apoplast or xylem), in order to avoid contact between the pathogen effector polypeptide and the cytosolic immune receptor prior to the re-internalization of the pathogen effector polypeptide by pathogen presence. Hereby it is preferred that pathogen presence refers to presence of (a part of) the pathogen on the cell that expresses the chimeric gene of the present invention.

Methods of achieving protein secretion in plant cells through the use of signal sequences are well known in the art. Use may for instance be made of the signal peptide of the pathogenesis-related protein lb (sPRl), a protein secreted in tobacco mosaic virus-infected tobacco leaves, and/or of the prepropeptide of cecropin B (sCEC), a peptide secreted into the hemolymph of the insect Hyalophora cecropia as described elsewhere (Denecke, J., et aL (1990). Plant Cell 2, 51-59). Other exemplary signal peptides that may be used in aspects of this invention include the tobacco pathogenesis related protein (PR-S) signal sequence (Sijmons et al., 1990, Bio /technology, 8:217-221), lectin signal sequence (Boehn et al., 2000, Transgenic Res, 9(6):477-S6), signal sequence from the hydroxyprohne-rich glycoprotein from Phaseolus vulgaris (Yan et al., 1997, Plant Phyiol.

115(3):915-24 and Corbin et al., 1987, Mol CeU Biol 7(12) .4337-44), potato patatin signal sequence (Iturriaga, G et al., 1989, Plant Cell 1:381-390 and Bevan et al., 1986, Nuc. Acids Res. 41:4625-4638.) Such targeting signals are in vivo cleaved off from the Avr effector polypeptide during translocation to the ER, which for example is typically the case when an apoplast targeting signal, such as the tobacco pathogenesis related protein- S (PR-S) signal sequence (Sijmons et al., 1990, Bio /technology, 8:217-221) is used. Other signal peptides can be predicted using the SignalP World Wide Web server (http://www.cbs.dtu.dk/services/SignalP/) which predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms. It is preferred that the signal sequence and the sequence encoding the pathogen effector polypeptide are linked through a cleavable sequence or cleavage site for the release of pathogen effector polypeptide in the lumen of the ER. Once in the lumen of the ER, all further information for specific transport is preferably present in domains of the protein remaining after cleavage of the signal peptide and other post- translational modifications.

The signal sequence, in aspects of this invention, may include a signal sequence that is not heterologous with respect to the pathogen effector.

Polyadenylation sequence

Posttranscriptional cleavage of mRNA precursor is an essential step in mRNA maturation. Following cleavage, most eukaryotic mRNAs, with the exception of replication-dependent histone transcripts in some organisms, acquire a poly(A) tract at their 3' ends. The process of 3'-end formation promotes transcription termination and transport of the mRNA from the nucleus, as explained in detail in e.g. Zhao J. et al. Microbiol Mol Biol Rev. 1999 Jun; 63(2): 405-445). To this end, the coding sequence for the heterologous Avr effector protein is preferably fused to a 3' non-translated region (UTR) which encodes a polyadenylation signal which functions in plant cells to cause the addition of poly adenylate nucleotides to the 3' end of the mRNA. Suitable such 3' non-translated regions for expression in plants are described in Loke J.C. et al, (Plant Physiol. 2005 Jul; 138(3): 1457- 1468), and include those of pea rbcS, CaMV, figwort mosaic virus, rice tungro bacilliform virus (RTBV), nos, ocs, and maize 27-kD protein gene (all references for these are provided in Loke J.C. et al, supra). In general, plant poly(A) signals suitable for use in this invention may be one of three major groups: far upstream elements (FUE), near upstream elements (NUE; an AAUAAA-like element), and cleavage sites (CSs) as described e.g. in Loke J.C. et al, supra. The composition of plant consensus signals, such as CSs, is a YA (CA or UA) dinucleotide situated within a U-rich region. The NUE region is A rich and spans about 6 to 10 nucleotides (nt) located between 13 and 30 nt upstream of the CS. FUE, the control or enhancing element, is a combination of rather ambiguous UG motifs and/or the sequence ULTGUAA Promoters

In accordance herewith the nucleic acid sequence encoding a pathogen effector is linked to a nucleic acid sequence capable of controlling expression of the pathogen effector polypeptide in a plant cell. Accordingly, the present invention also provides a nucleic acid sequence encoding a pathogen effector linked to a promoter capable of controlling expression in a plant cell. Nucleic acid sequences capable of controlling expression in plant cells that may be used herein include any plant derived promoter capable of controlling expression of polypeptides in plants. Generally, promoters obtained from dicotyledonous plant species will be used when a

dicotyledonous plant is selected in accordance herewith, while a

monocotyledonous plant promoter will be used when a monocotyledonous plant species is selected. In one embodiment, a promoter is used which results in the expression of the Avr effector polypeptide in the entire plant. Constitutive promoters that may be used include, for example, the 35S cauliflower mosaic virus (CaMV) promoter (Rothstein et al., 1987, Gene 53: 153-161), the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171; US Patent 6,429,357), a ubiquitin promoter, such as the corn ubiquitin promoter (US Patents 5,879,903; 5,273,894), ubiquitin-10 promoter from Arabidopsis tkaliana (Greffen et al., Plant J., 2010, 64:355-65), RPS5A promoter of Arabidopsis thalUma (Tsutsui H and Higashiyama, 2016, Plant Cell Physiology), the parsley ubiquitin promoter (Kawalleck, P. et al., 1993, Plant Mol. Biol. 21:673-684). the Arabidopssis thaliana hydroperoxide lyase promoter (proHPL; Schafer et al. 2002), the tobacco cryptic promoter

(p_roENTCUP2, Malik et al 2002) and the Triticum aestivum lipid transfer protein promoter (Simmonds et al. 2001) fused to intron 6 of the alcohol dehydrogenase 1 gene from Zea mays (p_roTAPADH, J. Simmonds,

unpublished data). Certain genetic elements capable of enhancing

expression of the Avr effector polypeptide may also be used herein. These elements include the untranslated leader sequences from certain viruses, such as the AMV leader sequence (Jobling and Gehrke, 1987, Nature, 325: 622-625) and the intron associated with the maize ubiquitin promoter (US Patent 5,504,200).

Generally the chimeric nucleic acid sequence will be prepared so that genetic elements capable of enhancing expression will be located 5' to the nucleic acid sequence encoding the pathogen effector polypeptide.

Aspects of the present invention may, in preferred embodiments, be based on the constitutive expression of an extracellular pathogen effector in a plant expressing a cognate intracellular immune receptor. The expression of the pathogen effector can be specifically targeted towards certain tissue-types to confer resistance to pathogens infecting these specific tissues. As a consequence, depending on the purpose, a constitutive plant promoter may be chosen to drive expression of the pathogen effector gene, or, in an alternative embodiment, a tissue-specific-promoter may be selected to drive expression of the pathogen derived effector gene.

Marker genes

Pursuant to the present invention the expression vector may further contain a marker gene. Marker genes that may be used in

accordance with the present invention include all genes that allow the distinction of transformed cells from non-transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin (US Patent 6,174,724), G418, bleomycin, hygromycin which allows selection of a trait by chemical means or a tolerance marker against a chemical agent, such as the normally phytotoxic sugar mannose (Negrotto et al., 2000, Plant Cell Rep. 19: 798-803). Other convenient markers that may be used herein include markers capable of conveying resistance against herbicides such as glyphosate (US Patents 4,940,935; 5,188,642),

phosphinothricin (US Patent 5,879,903) or sulphonyl ureas (US Patent 5,633,437). Resistance markers, when linked in close proximity to nucleic acid sequence encoding the Avr effector polypeptide, may be used to maintain selection pressure on a population of plant cells or plants that have not lost the nucleic acid sequence encoding the Avr effector

polypeptide, as may screenable markers that may be employed to identify transform ants through visual inspection. Expression vectors

In accordance with the present invention the chimeric nucleic acid sequences comprising a promoter capable of controlling expression in plant and linked to a nucleic acid sequence encoding a pathogen effector

polypeptide can be integrated into a recombinant expression vector which ensures good expression in the plant cell. The vector may be a plant viral expression vector, or a transformation vector for transient or stable transformation of the plant cells. Accordingly, the present invention includes a recombinant expression vector comprising in the 5' to 3' direction of transcription as operably linked components: (i) a nucleic acid sequence capable of controlling expression in plant cells; and (ii) a nucleic acid sequence encoding a pathogen effector polypeptide; wherein the expression vector is suitable for expression in a plant cell. The term "suitable for expression in a plant cell" means that the recombinant expression vector comprises the chimeric nucleic acid sequence of the present invention linked to genetic elements required to achieve expression in a plant cell, such as promoters functional in plant cells. Genetic elements that may be included in the expression vector in this regard further include a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication and the like. In preferred

embodiments, the expression vector further comprises genetic elements required for the integration of the vector or a portion thereof in the plant cell's nuclear genome, for example the T-DNA left and right border sequences which facilitate the integration into the plant's nuclear genome in embodiments of the invention in which plant cells are transformed using Agrobacterium tumefaciens (aka Rhizobium radiobacter). The recombinant expression vector generally comprises a transcriptional terminator which besides serving as a signal for transcription termination further may serve as a protective element capable of extending the mRNA half life. The transcriptional terminator may generally have a length from about 200 nucleotides to about 1000 nucleotides and the expression vector is prepared so that the transcriptional terminator is located 3' of the nucleic acid sequence encoding the pathogen effector. Termination sequences that may be used herein include, for example, the nopaline termination region (Bevan et al., 1983, Nucl. Acids. Res., 11: 369-385), the phaseolin terminator (van der Geest et al., 1994, Plant J. 6: 413-423), the arcelin terminator (Jaeger GD, et al., 2002, Nat. Biotechnol . 20:1265-8), the terminator for the octopine synthase genes of A. tumefaciens or other similarly functioning elements.

Recombinant vectors suitable for the introduction of nucleic acid sequences into plants include Agrobacterium and Rhizobium based vectors, such as the Ti and Ri plasmids, including for example pBIN19 (Bevan, Nucl. Acid. Res., 1984, 22: 8711-8721), pGKB5 (Bouchez et al., 1993, C R Acad. Sci. Paris, Life Sciences, 316:1188-1193), the pCGN series of binary vectors (McBride and Summerfelt, 1990, Plant Mol. Biol., 14:269-276) and other binary vectors (e.g. US Patent 4,940,838). The recombinant expression vectors of the present invention may be prepared in accordance with methodologies well known to those skilled in the art of molecular biology. Such preparation will typically involve the bacterial species Escherichia coli as an intermediairy cloning host. The preparation of the E. coli vectors as well as the plant transformation vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gelectrophoresis, DNA sequencing, the Polymerase Chain Reaction (PGR) and other methodologies. A wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E. coli, are vectors such as pBR322, the pTJC series of vectors, the M13mp series of vectors, pBluescript etc. Typically, these cloning vectors contain a marker allowing selection of transformed cells. Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in E. coli grown in an appropriate medium. Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells.

Further, general guidance with respect to the preparation of recombinant vectors may be found in, for example, Green and Sambrook, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 4th edition, 2012. In accordance with the present invention the chimeric nucleic acid sequence is introduced into a plant cell and the cells are grown into mature plants, wherein the plant expresses the Avr effector polypeptide. In accordance herewith any plant species or plant cell may be selected.

Methodologies to introduce plant recombinant expression vectors into a plant cell, also referred to herein as "transformation", are well known to the art and typically vary depending on the plant cell that is selected. General techniques to introduce recombinant expression vectors in cells include the use of liposomes, electroporation, chemically mediated free DNA uptake techniques, for example CaCl2 mediated nucleic acid uptake, free DNA delivery via microprojectile bombardment (biolistics), and

transformation using naturally infective nucleic acid sequences, for example virally derived nucleic acid sequences, or Agrobacterium or Rhizobium derived sequences, polyethylene glycol (PEG) mediated nucleic acid uptake, microinjection and the use of silicone carbide whiskers. A chimeric plant gene containing a coding sequence of a heterologous Avr effector of the present invention may for instance be inserted into the genome of a plant by plant transformation vectors including those derived from a Ti plasmid of A tumefaciens, as well as those disclosed, e.g., in EP 0 120 516.

In preferred embodiments, a transformation methodology is selected which will allow the integration of the chimeric nucleic acid sequence in the plant cell's genome, and preferably the plant cell's nuclear genome. The use of such a methodology in the production of transgenic plants according to this invention is preferred as it will result in the transfer of the chimeric nucleic acid sequence to progeny plants upon sexual reproduction. Especially preferred transformation methods that may be used in this regard include biolistics and Agrobacterium mediated methods.

Transformation methodologies for dicotyledenous plant species are well known. Generally, Agrobacterium mediated transformation is used because of its high efficiency, as well as the general susceptibility by many, if not all, dicotyledenous plant species. Agrobacterium transformation generally involves the transfer of a binary vector, such as one of the hereinbefore mentioned binary vectors, representing plant transformation vectors, comprising the chimeric nucleic acid sequence of the present disclosure from E. coli to a suitable Agrobacterium tumefaciens strain (e.g. EHA101 and LBA4404) by, for example, tri-parental mating with an E. coli strain carrying the recombinant binary vector and an E. coli strain carrying a helper plasmid capable of mobilizing the binary vector to the target Agrobacterium strain, or by direct DNA transformation to the

Agrobacterium strain (Hofgen et al., Nucl. Acids. Res., 1988, 16:9877). Other techniques that may be used to transform dicotyledenous plant cells include biolistics (Sanford, 1988, Trends in Biotechn. 6:299-302); electroporation (Fromm et al., 1985, Proc. Natl. Acad. Sci. USA., 82:5824-5828); PEG mediated DNA uptake (Potrykus et al., 1985, Mol. Gen. Genetics, 199:169- 177); microinjection (Reich et al., 1986, Bio/Techn. 4:1001-1004); and silicone carbide whiskers (Kaeppler et al., 1990, Plant Cell Rep., 9:415-418) or in planta transformation using, for example, a flower dipping

methodology (Clough and Bent, 1998, Plant J., 16:735-743).

Monocotyledonous plant species may be transformed using a variety of methodologies including particle bombardment (Christou et al., 1991, Biotechn. 9:957-962; Weeks et al., 1993, Plant Physiol. 102:1077-1084; Gordon-Kamm et al., 1990, Plant Cell. 2:5603-618); PEG mediated DNA uptake (European Patents 0292 435; 0392 225) or Agrobacterium mediated transformation (Goto-Fumiyuki et al., 1999, Nature-Biotech. 17:282-286).

Also, the present invention provides recombinant plant viral expression vectors comprising the chimeric gene of the present invention.

Plant viral vectors

In aspects of this invention, the pathogen effector produced in the plant and targeted to the plant secretory pathway for subsequent

extracellular secretion, may also be encoded by a plant viral vector. Such a plant viral vector is then used to express the pathogen effector in non- transgenic plants expressing the cognate intracellular immune receptor.

In such embodiments, the invention may provide a non-transgenic plant comprising (e.g. as an episome) a transformed plant viral vector or a transgenic or recombinant virus, expressing a fusion protein as described herein consisting of a pathogen effector fused in translation frame to a signal peptide for targeting said pathogen effector to the plant secretory pathway.

Suitable plant viral vectors include polynucleotides carried by a virus for transfection into a host cell. In particular, viral vectors that are capable of autonomous replication in a host cell into which they are introduced may be used and will not be integrated into the host plant cell genome. Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain aspects of this invention are directed to (non- transgenic) plants having as an episome a transformed plant viral vector. In such aspects, the vectors are capable of directing the expression of genes to which they are operatively-hnked, and they may also be referred to herein as "viral expression vectors."

The use of plant viruses as vehicles to introduce and express nonviral genes in plants is well known to those of skill in the art. Infection of plants with modified viruses is a simple and quick method when compared to the regeneration of stably transformed plants. Plant viruses are small (3000-10,000 nucleotides), easy to manipulate, have the inherent ability to enter the plant cell, lead to the immediate expression of the heterologous gene and will multiply to produce a high copy number of the gene of interest. Thus, viral expression vectors are suitably used in aspects of this invention when engineered for delivery of the chimeric gene for the extracellular production of a pathogen effector in plant cells according to this invention. US 5,316,931 and US 5,811,653 provide examples for RNA virus vectors. In general, these plant viral expression systems will be transient expression systems as the viral expression vectors are not integrated into the genome of the host. However, depending on which virus is used, virus multiplication and gene expression can persist for long periods (up to several weeks or months).

Methods of the present invention for producing a pathogen- resistant plant or plant cell or for generating effector-triggered immunity in a plant are effected by introducing into the plant at least one plant viral expression vector encoding at least one chimeric gene according to the present invention as described herein.

The plant viral expression vector, in aspects of this invention, may, in addition to the chimeric gene according to the present invention, further encode at least one a nuclear localization signal.

The plant viral expression vector may include, but does not need to be limited to, Tobacco mosaic virus (TMV), Potato virus X (PVX), Tobacco rattle virus (TRV), Cowpea mosaic virus (CPMV), Bean Yellow dwarf virus (BeYDV), Beet soil-borne mosaic virus (BSbMV) and Potato mop top virus (PMTV).

In producing a recombinant plant viral expression vector for use in aspects of this invention, the viral nucleic acid, for instance derived from a plus sense, single stranded RNA plant virus, comprising a nucleic acid sequence that codes for a plant viral coat protein the transcription of which is under the control of, and operably linked to, a first (subgenomic) plant viral promoter, is provided with a second plant viral (subgenomic) promoter operably linked to the chimeric gene of the present invention. The first viral subgenomic promoter is preferably heterologous to the second viral promoter enabling the recombinant plant viral nucleic acid to systemically transcribe the second nucleic acid in the host plant. Alternatively, the invention may provide a combination of a non- transgenic plant and a transformed plant viral expression vector for expressing the said fusion protein in said non-transgenic plant.

Recombinant plant viral expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant viral expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. "Operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Genome editing tools

Genome editing with site-specific nucleases allows targeted transgene integration in an efficient and precise manner. Zinc-finger nucleases (ZFNs; Kim et al., 1996. Proc Natl Acad Sci U.S.A, 93:1156-1160) and transcription activator-like effector nucleases (TALENs; Christian et al., 2010. Genetics, 186;757-761) may for instance be used in aspects of this invention. In addition to the use of ZFNs and TALENs use may also be made of RNA-guided engineered nucleases, the most widely used system thereof being the type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 (CRISPR-associated) system from Streptococcus pyogenes (Jinek et al., 2012, Science, 337:816-821).

The simplicity of the type II CRISPR-Cas system from

Streptococcus pyogenes is based on the fact that it relies on only one protein, the nuclease Cas9, and two noncoding RNAs, crRNA and tracrRNA, to target a specific DNA sequence. These two noncoding RNAs can further be fused into one single guide RNA (sgRNA). The Cas9/sgRNA complex binds double-stranded DNA sequences that contain a sequence match to the first 17-20 nucleotides of the sgRNA if the target sequence is followed by a protospacer adjacent motif (PAM).

Although the CRISPR/Cas system will also allow for preparing transgenic plants according to the present invention, genome editing tools are aimed at targeting gene constructs to a specific location in the genome. Although useful in aspects of this invention, the specific genomic targeting of the chimeric gene construct of this invention, it is not limiting. Transgenic plants

Preferably, a plant of the invention is a transgenic plant. The exact plant transformation methodology for producing transgenic plants may vary somewhat depending on the plant species and the plant cell type (e.g. seedling derived cell types such as hypocotyls and cotyledons or embryonic tissue) that is selected as the cell target for transformation.

Transformation techniques may involve any one described above, including, but not limited to protoplast transfection, A twnefaciens or A rhizogenes mediated transformation, and may also include the use of genome editing tools.

Following transformation, the plant cells are grown and upon the emergence of differentiating tissue, such as shoots and roots, mature plants are regenerated. Typically a plurality of plants is regenerated.

Methodologies to regenerate plants are generally plant species and cell type dependent and will be known to those skilled in the art. Further guidance with respect to plant tissue culture may be found in, for example: Plant. Cell and Tissue Culture, 1994, Vasil and Thorpe Eds., Kluwer Academic

Publishers; and in: Plant Cell Culture Protocols (Methods in Molecular Biology 111), 1999, Hall Eds, Humana Press.

It will generally be desirable to ensure homozygosity in the transformed plants to ensure continued inheritance of the recombinant polypeptide. Methods for selecting homozygous plants are well known to those skilled in the art. Besides self-pollination and selection of the progeny alternative methods for obtaining homozygous plants that may be used include the preparation and transformation of haploid cells or tissues followed by the regeneration of haploid plantlets and subsequent conversion to diploid plants for example by the treatment with colchine or other microtubule disrupting agents. Plants may be grown in accordance with otherwise conventional agricultural practices.

Further foreseen are grafted plants, comprising a (root)stock preferably expressing in cells of said (root)stock a chimeric gene as described herein, and a scion preferably comprising at least one R-gene encoding an intracellular R protein for mounting an immune response as described herein.

As described herein, the present invention relates to methods for the production of transgenic plants that secrete a pathogen effector in the extracellular space, including the apoplast and xylem. The present inventors have surprisingly found that the presence of pathogen effectors in a plant's apoplast and xylem and their subsequent internalization triggered by the presence of a pathogen, results in a hypersensitive response in case the corresponding R protein accumulates inside the plant cell, i.e. localized to the cytoplasm and/or nucleus.

Accordingly, pursuant to the present invention a method for the expression of a nucleic acid sequence encoding a heterologous pathogen effector in plants is provided in which the method comprises: (a) providing a chimeric nucleic acid construct comprising in the 5' to 3' direction of transcription as operably linked components: (i) a nucleic acid sequence capable of controlling expression in plant cells; and (ii) a nucleic acid sequence encoding a heterologous Avr effector polypeptide (b) introducing the chimeric nucleic acid construct into a plant cell; and (c) growing the plant cell into a mature plant expressing the heterologous Avr effector. For the purpose of clarity and a concise description features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include

embodiments having combinations of all or some of the features described. In view of this passage it is evident to the skilled reader that the variants of claim 1 as filed may be combined with other features described in the application as filed, in particular with features disclosed in the dependent claims, such claims usually relating to the most preferred embodiments of an invention.

EXAMPLES

In these Examples, the inventors demonstrate that uptake in tomato plants that produce an extracellularly localised Avr2 effector is pathogen-dependent. In fact, it is shown that Uptake of the Fusarium effector Avr2 by tomato is not a cell autonomous event. The inventors demonstrate the feasibility of the invention for two pathogens, Fusarium oxysporum f.sp. lycopersici and Agrobacterium tumefaciens. Infiltration of the bacterial pathogen A. tumefaciens in tomato leaves in which both the intracellular 1-2 immune receptor and the extracellular Avr2 effector are present was found to trigger an immune response. Induction of plant immunity was visible by the induction of a localised cell death response at the infiltrated area. Cell death is a hallmark of a hypersensitive response and is typically used as a proxy for the induction of plant immunity by activated immune receptors.

Introduction

Microbe-secreted effector proteins enable pathogens to suppress or evade plant immunity responses, a prerequisite for successful infections. Most bacterial plant pathogens employ a type-Ill secretion system to directly inject type-Ill effector (T3E) proteins into the plant cytoplasm (Panstruga and Dodds, 2009). Fungi and oomycetes do not inject their effectors into plant cells, but secrete them into the extracellular spaces. Some fungal pathogens, such as Cladosporium fulvum, secrete effectors from invasive hyphae into the plant apoplast (Stergiopoulos and de Wit, 2016). Others, like Magnaporthe grisea and Phytophthora infestans, have specialized feeding structures protruding into the plant cells from which effectors are secreted (Panstruga and Dodds, 2009; Dodds and Rathjen, 2010). In either case, the effectors accumulate outside the host's plasma membrane and it is unknown how they are taken up by plant cells (Bozkurt, et al., 2012; Rafiqi et al., 2012). As many effectors have been shown to act inside the host cell (Dodds, et al., 2004; Armstrong et al., 2005; Catanzariti et al., 2006), there must be a mechanism by which effector proteins enter. Whether this process is a host autonomous mechanism or requires the presence of the pathogen is currently an unresolved question. To address this question its desirable to use a pathosystem in which effector secretion and action are spatially separated. The present inventors have studied the fungal pathogen Fusarium oxysporum, which secretes its effector proteins inside the xylem sap of infected plants (Houterman, et al., 2007). The effectors are transported with the sap stream to exert their action in various places in the plant.

F. oxysporum is a soil-borne and highly destructive pathogen causing vascular wilt disease on a wide range of plants. The F. oxysporum species complex comprises different formae speciales (f.sp.), which

collectively infect more than 100 different hosts, provoking severe losses in crops such as melon, tomato, cotton and banana, among others (Michielse, 2009). The process of infection by F. oxysporum can be divided into several steps: root recognition, root surface attachment and penetration,

colonization of the root cortex and, in the case of wilt-inducing formae speciales, hyphal proliferation within the xylem vessels (Pietro et al., 2003). Characteristic disease symptoms include vascular browning, leaf epinasty, stunting, progressive wilting, defoliation and eventually plant death.

In the past decades, the interaction between tomato and F.

oxysporum f.sp. lycopersici (Fol) has evolved into an excellent model to study the molecular mechanisms underlying disease and resistance (Takken and Rep, 2010). Over 14 putative effector proteins have been isolated from the xylem sap of infected tomato plants and are called SIX (for secreted in xylem) proteins (Houterman et al., 2007). For some of them, like Sixl, Six3, Six5 and Six6 a virulence function has been determined, making them effectors in sensu stricto (Rep et al., 2005; Houterman et al., 2009; Gawehns et al., 2014; Ma et al., 2015). Besides a virulence function some effectors have been found to act as avirulence determinants, triggering immune responses in resistant hosts. The relationship between Fol and tomato cultivars follows the 'gene-for-gene' hypothesis (Flor, 1971). According to this hypothesis disease resistance conferred by resistance (R) genes requires 'matching' avirulence (Avr) genes in the pathogen. Three R genes against Fol have been introgressed into cultivated tomato (Solarium lycopersicum): the I and 1-2 genes from S. pimpinellifolium, which confer resistance against Fol races 1 and 2, respectively, and the 1-3 gene from 5. pennellii, which confers resistance to Fol race 3. The three Fol effector proteins Avrl (Six4), Avr2 (Six3) and Avr3 (Six4), which are recognized by 1, 1-2 and 1-3, respectively, have all been cloned (Rep et al., 2004; Houterman et al., 2008; Houterman et al., 2009). They are secreted into the xylem sap during infection. Avr3 is expressed when the fungus is in contact with living plant cells (van der Does et al., 2008) while Avr2 is predominantly expressed in xylem -colonizing hyphae (Ma et al., 2013). Both Avr3 and Avr2 are important for pathogenicity (Rep et al., 2005; Houterman et al., 2009).

Notably, Avrl does not enhance virulence on a susceptible plant, but suppresses 1-2 and /-3-mediated resistance allowing the fungus to overcome the gene-for-gene resistance (Houterman et al., 2008).

Avr2 encodes a mature 15.7 kDa protein preceded by an N- terminal signal peptide. Avr2 contains two cysteine residues that might form a disulfide bond (Houterman, 2007). The protein appears in various positions in 2D gels of xylem sap from FoZ-infected tomato plants,

corresponding to apparent sizes from 11-14 kD, probably as a result of proteolytic processing from the N-terminus (Houterman et al., 2007). Race 3 strains carry point mutations in Avr2, resulting in single amino acid changes that do not affect its virulence function but allow the protein to evade /-2-mediated recognition (Houterman et al., 2009). Although Avr2 is secreted into xylem sap, the Avr2 protein is recognized intracellularly in the plant nucleus by 1-2 (Ma et al., 2013), implying uptake by host cells. Herein, the present inventors describe the generation of transgenic tomato plants expressing either full-length Avr2 or a truncated version lacking the signal peptide encoding sequence ( ΔspAvr2. Bioassays and grafting studies using these plants revealed that revealed that Avr2, besides its avirulence function, also exert its virulence function inside host cells. Having an extracellular effector that is secreted in the xylem sap, but exerts its functions inside the host cell, makes this a perfect model to study effector uptake and to reveal whether uptake is a host autonomous process or requires the presence of the pathogen.

Materials and methods

Plant material and fungal and bacterial strains

Tomato (Solarium lycopersicum) cultivar Moneymaker, which is susceptible to Fol race 2 Fol007, and a resistant cultivar, 90E341F, which contains the 1-2 resistance locus were used (Stall and Walter, 1965; Kroon and Elgersma, 1993). Tomato plants were germinated and grown on soil with 16/8 h light/dark cycles, at 22/16°C day/night and 70% relative humidity in a temperature-controlled green house. FoLΔAvr2 carrying a deletion of the Avr2 gene in the FolOO 7 background was generated as described (Houterman et al., 2009).

Construction of binary vectors

Full length Avr2 was PCR-amplified with primers FP2524 (5'- CGCTCTAGAATGCGTTTCCTTCTGCTTAT-3') and FP2274 (5'- GCGGGATCCTCCATCCTCTGAGATAGTAAG-3') using CTAPi::Avr 5 as template (Houterman et al., 2009). The obtained products were cloned into the vector pSLDB3104 (Tameling et al., 2010) between the Xbal and-BamHI restriction sites to generate SLDB3104: :Avr2. SLDB3104:: ΔspAvr2 has been described before (Ma et al., 2013). All PCR primers were purchased from MWG (http://www.mwg-biotech.com) and sequences of all plasmids were confirmed by sequence analysis. Avr2 and ΔspAvr2 were cloned behind the cauliflower mosaic virus 35S promoter for constitutive expression and fused to a C-terminal hemagglutinin (HA) and streptavidin-binding peptide (SBP) tag. The resulting vector was introduced by electroporation into LBA4404 (Hoekema et al., 1983) for tomato transformation.

Plant transformation

Moneymaker was transformed with the construct described above using Agrobacterium -mediated transformation in tomato as described before (Cortina and Culianez-Macia, 2004). Briefly, surface-sterilized seeds were sown on Murashige and Skoog (MS) agar supplemented with sucrose (15g/l). The seeds were incubated in the dark in a growth chamber at 25°C for 2 days, and subsequently exposed to light. After 10 days, the base and the tip of the cotyledons was removed and the cotyledons were placed upside up in Petri dishes containing co-cultivation medium (MS agar supplemented with 30g/l sucrose, 0.5g/l 2-(N-orpholino) ethanesulfonic acid (MES) [Duchefa] and 0.2mM Acetosyringone, pH 5.75). The plates were incubated for 24 hours at 25°C in dark. Transgenic A. tumefaciens carrying the construct of interest was grown in 30ml LBman at 28°C overnight (max 16-18 hours). After harvesting, the bacteria were resuspended in 30ml LM2 medium

(4.4g/l MS, 30g/l sucrose, 0.5g/l MES [Duchefa] and 0.2mM Acetosyringone, pH 5.75). Subsequently, the explants were incubated in the bacterial suspension for maximal 1 minute, briefly dried on sterile filter paper and placed on co-cultivation plates. The plates were incubated in the dark for 48 hours at 25°C after which the explants were transferred to selection plates (MS agar supplemented with 30g/l sucrose, 0.5g/l MES, 0.5mg/l zeatin riboside, 0.5mg/L indole-3-acetic acid (IAA), 250mg/l carbenicilline, 10Omg/1 vancomycin, and 40mg/l kanamycin, pH 5.75). Explants were transferred to fresh selection plate every two weeks. When callus appeared, it was transferred to new selection plates until shoots appeared. Upon shoot development, the shoots were harvested and transferred to root-inducing medium (MS agar supplemented with 15g/L sucrose, 0.5g/l MES, 4g/l gelrite, 50mg/l kanamycin, pH 5.75). Once roots developed, the plantlets were potted in soil and transferred to the greenhouse where they were grown under standard greenhouse with conditions of a 16h photoperiod and 70% relative humidity at 25°C.

First-generation transformants of ΔspAvr2 andAvr2 were selected on 1/2 MS medium containing kanamycin (40mg/L). For the ΔspAvr2

transgenic line, 25 seeds of nine Tl progeny were analyzed for segregation by scoring the ratio of kanamycin-resistant to kanamycin-sensitive seedlings. Six lines segregated roughly 3:1 for green versus yellowing seedlings. Subsequently the kanamycin-resistant plants were transferred to soil and self -fertilized. Homozygous single insertion lines were selected from the kanamycin resistant T2 plants according to their segregation pattern. Of each independent T2 line 25 plants were checked by PCR with primer pairs FP962 (5'-TGAGCGGGCTGGCAATTC-3') and FP963 (5'- CAATCCTCTGAGATAGTAAG-3') detecting a 273-bp fragment of

the Avr2 gene. Two lines were homozygous for the Avr2 transgene (ΔspAvr2- 3 and ΔspAvr-30). Homozygous Avr2 transgenic lines were screened using the same procedure. Eventually three of 23 Avr2 plants (Avr2-1, Avr2-4 and Avr2-7) were kept for further study.

Primer pairs FP962 and FP963, and FP484

(AAAGCGTGGTATTGCGTTTC) and FP 165

(TTCCGGATGTCCCATAGGATCC) were used to amplify Avr2 and 1-2 from genomic DNA of Avr2/I-2 and ΔspAvr2/I-2 plants, respectively.

Protein extraction and western blotting

Protein extraction was done as described previously (Ma et al., 2015). To verify presence of Avr2 in transgenic tomato plants, leaves were harvested and snap-frozen in liquid nitrogen. After grinding the tissue with a mortar and a pestle, the powder was allowed to thaw in 2 ml protein extraction buffer per gram of tissue (25mM Tris pH 8, ImM EDTA, 150mM NaCl, 5mM DTT, 0.1% NP-40, 1 Roche complete protease inhibitor cocktail (http://www.roche.com) and 2% PVPP). Extracts were centrifuged at 12,000 g, 4°C for 10min, and the supernatant was passed over four layers of

Miracloth

(http://www.mercl-imllipore.com/NL/en/product/Miradoth,EMD_BIO- 475855) to obtain a "total" protein lysate. 20 microliter samples were mixed with Laemmli sample buffer and were run on 13% SDS-PAGE gels and blotted on PVDF membranes using semi-dry blotting. Skimmed milk powder (5%) was used as a blocking agent. The membranes were subjected to immunoblotting using anti-Avr2 antibody (1:10,000 diluted) (Ma et al., 2015). The secondary antibody goat-anti-rabbit (P31470, Pierce) was used at a 1:5000 dilution. The luminescent signal was visualized by ECL using BioMax MR film

(http://www.sigmaaldrich.com/catalog/substance/carestreamkodakbiomaxmr film 1234598765?lang=en&region=NL). Isolation ofapoplastic fluid from tomato leaf tissue

Apoplastic fluid of tomato plants was isolated as described (Joosten, 2012). Four-week-old fully stretched tomato leaves or leaflets were harvested and placed in a beaker with sterile water. The beaker was placed in a vacuum desiccator and a mild vacuum was employed using a vacuum pump. While slowly releasing the vacuum by opening the vent on the desiccator jar, the leaf tissue became water-soaked and dark in color. The infiltrated leaves were gently dried using tissue papers and then rolled up and placed in a 20ml syringe hanging in a 50ml tube. Apoplastic fluid was isolated by centrifuging at l,000g for 10 min at 4°C. For electrophoresis 20ul of collected apoplastic fluid was mixed with Laemmli sample buffer and separated on a 13% sodium dodecyl sulfate (SDS) polyacrylamide gel.

Xylem sap collection from tomato

Xylem sap was collected as described (Rep et al., 2002; Krasikov et al., 2011). Briefly, stems of six- week-old tomato plants were cut below the second true leaf and the plant was placed in a horizontal position. Then, for minimal 6h sap bleeding from the cut surface was collected in tubes placed on ice. For electrophoresis 20 microliter of collected xylem sap was mixed with Laemmli sample buffer and after heating separated on a 13% sodium dodecyl sulfate (SDS) polyacrylamide gel.

Fusarium inoculation assay

Fol was grown in minimal medium (10OmM KN03, 3% sucrose and 0.17% Yeast Nitrogen Base without amino acids or ammonia) and spores were harvested after 3-5 days of cultivation at 25°C with shaking. After washing with sterilized water the spores were diluted to 10⁷spore/ml. For bioassay, ten-day-old tomato seedlings were uprooted from the soil. The seedlings were placed for 5 rnin in the Fol spore suspension (10⁷spores/ml) and potted. Disease progression was evaluated after three weeks. Plant weight and disease index (Gawehns et al., 2014) were scored for 20 plants/treatment. Using PRISM 5.0 (GraphPad, http://www.graphpad.com) a pairwise comparison for plant weight was done using the Student's t-test and disease index data was analyzed using a nonparametrical Mann- Whitney U-test.

Phytophthora infestans inoculation assay:

Sporangia from P. infestans strain R168 were kept at 4 degree for 3 hours to facilitate hatching of zoospores. 10 μl of water containing 2.5 x 10⁴ sporangia was pipetted on the adaxial side of detached tomato compound leaves. The third compound leaf from 5-week old tomato plants was used, and 2 droplets of sporangia solution were added to each leaflet. Subsequently leafs were kept in a growth chamber at a 16 hour light/ 8 hour dark regime at 15 degree and 80 % relative humidity. After seven days the symptom development for each inoculation site was recorded. The symptom were assigned to following classes: Class 0 = no visible symptoms; class 1 = weak necrosis; class 2 = strong but contained necrosis; class 3 = spreading necrosis. Agrobacterium-mediated transient transformation in tomato leaves

The binary ctapi::GUS and ctapi::ΔspAvr2 constructs (Houterman et al., 2009) were transformed into A. tumefaciens ID 1249 (Wroblewski et al., 2005). Agrobacterium-mediated transient transformation was performed as described (Ma et al., 2012). Briefly, the agrobacteria were grown to an absorbance of 0.8 at 600nm in LB-mannitol medium (10g/l tryptone,

5g/l yeast extract, 2.5g/L NaCl, 10g/l mannitol) supplemented with 20 micromolar acetosyringone and 10mM MES pH 5.6. Cells were pelleted by centrifugation at 4000g at 20°C for 10 min and then suspended in

infiltration medium at an absorbance of 0.5. (1*MS salts, 10mM MES pH 5.6, 2% sucrose, 200 micromolar acetosyringone). Infiltration was done in four-week-old tomato leaves.

Trypan blue staining

Leaves were boiled for 5 min in a 1:1 mixture of 96% ethanol and staining solution (100ml lactic acid, 100ml phenol, 100ml glycerol, 100ml H2O and 10Omg Trypan bule). The leaves were destained in 2.5g/ml chloral hydrate in water (Ma et al., 2012).

Grafting

Four- week-old rootstocks and scions represent the best stage for grafting

(http://horticulture.ucdavis.edu/main/Deverables/Kleinhenz/tomato....graftin

g_guide.pdf). A similar diameter of the stem of the rootstock and scion increases the likelihood that their vasculatures align after grafting. The rootstock plant was cut between the cotyledons and first true leaf. The scion plant was cut at the same position at the main stem. Leaves from the scion were trimmed to reduce water loss. The stump of the scion seedling was cut to fit the shape of a two-sided wedge. Approximately one-third of each side was removed at a roughly 45° angle. The stump of the scion seedling was trimmed on both sides, creating a wedge with angled sides of approximately 45°. The wedge-shaped scion stump was inserted into the cut of the bisected rootstock stump. Parafilm was used to fix the rootstock and scions and to secure the graft. Grafted plants were placed for 5 days in a growth chamber with high humidity to reduce dehydration stress and increase the survival rate.

Results

Avr2 exerts its virulence /unction inside host cells

Avr2 was originally identified in the xylem sap of FoZ-infected tomato plants (Houterman et al., 2007), although a nuclear localization of Avr2 is required to trigger I-2-mediated resistance (Ma et al., 2013). As yet it is unknown where in the host the protein exerts it virulence function. To identify whether Avr2 acts inside or outside host cells, transgenic tomato plants stably expressing full-length Avr2 were generated. The expressed protein carries it's endogenous signal peptide (Figure 1A) for translocation into the endoplasmatic reticulum and subsequent secretion. Plant-produced Avr2 is, therefore expected to be secreted into the apoplastic spaces, allowing us to test whether it exerts its function extracellularly. In addition, stable transgenic plants were made expressing a truncated Avr2 (ΔspAvr2, Δsp for "deletion of signal peptide") encoding the mature protein without signal peptide (Ma et al., 2013). In these plants the protein is predicted to be present exclusively in the cytosol. Expression of both full-length Avr2 and ΔspAvr2 was driven by the strong and constitutive CaMV 35S promoter. Both genes were fused to sequences encoding a C-terminal hemagglutinin (HA) and streptavidin-binding peptide (SBP) tag to facilitate detection of the recombinant proteins.

From kanamycin resistant Tl plants three independent transformants containing a single copy of the Avr2 construct (35S::Avr2-l, 35S::Avr2-4 and 35S::Avr2-7) and two lines containing a single copy ΔspAvr2 lines (35S::ΔspAvr2-3 and 35S::ΔspAvr2-30) were identified based on their 3:1 segregation pattern. From these plants homozygous lines were produced, which were used in the subsequent assays. None of the transgenic lines exhibited morphological aberrations, or showed a phenotype distinct from non-transformed Moneymaker tomato plants when grown under standard greenhouse conditions. To determine whether the distinctively localized Avr2 effector proteins do complement the virulence defect of a FoLΔAvr2 (a FolAvr2 knockout; Houterman et al., 2009) strain 10-day-old seedlings of wild-type, ΔspAvr2 and full-length Avr2 transgenic tomato plants were inoculated with water (mock), wild-type Fusarium (Fol007) or the FoLΔAvr2 strain. Three weeks after inoculation, mean plant weight and average disease index of 20 plants was scored. The disease index was scored on a 0-4 scale, in which 0 means that no disease symptoms developed and 4 that plants are either dead or extremely small and wilted (Gawehns et al., 2014). Moneymaker plants inoculated with Fol007 showed severe disease

symptoms such as wilting and stunting (Figure IB shows a representative example of lines ΔspAvr2-30 and Avr2-4). As observed before (Houterman et al., 2007), the FoLΔAvr2 strain is reduced in virulence as shown by the increased vigor of the plants along with higher weights and lower disease indexes as compared to FolOO 7 inoculation (Figure 1C and D). Interestingly, the present inventors found that disease symptoms of FoLΔAvr2-infected Αυτ2 plants were at least as severe as tomato plants infected with wild-type Fol. The regain of full pathogenicity for the Fol Avr2 knockout strain on the Avr2 lines shows that plant-produced Avr2 effectively complements fungal virulence. Notably, also ΔspAvr2 tomato plants infected with FoLΔAvr2 developed severe disease symptom and showed decreased plant weight and a higher disease index. Since the latter protein does not carry a signal peptide, this strongly suggests that Avr2 exerts its virulence functions inside the host cell. The experiment was performed twice using all five transgenic lines, with similar results.

In Avr2 transgenic plants Avr2 is secreted into the xylem sap and the apoplast

To assess accumulation of Avr2 in the transgenic tomato plants, the 35S::Avr2 and 35S::ΔspAvr2 lines were subjected to immunoblot analysis using either an Avr2 specific antibody (Ma et al., 2015) or an HA antibody (Figure 2A). When probed with Avr2-specific antibody, a band with the predicted size forΔsp Avr2-HASBP (23kDa) was detected in total protein extracts from Avr2 and ΔspAvr2 transformants, but not in the

untransformed Moneymaker control plants confirming the specificity of the antibody. In the Avr2 transformants also one additional band of a smaller size (15kDa) was observed. The appearance of this smaller sized band suggests that Avr2 is secreted into the apoplastic spaces, after which the HA tag is cleaved by extracellular proteases (van Esse et al., 2006). The apparent weight fits the predicted size of the non-tagged Avr2 protein. When probed with HA antibody only the larger 23kDa band was detected, which indicates that the 15kDa band indeed contains the non-taggedΔspAvr2 protein from which the tag has been removed. To determine the in planta location of theΔsp Avr2 and full-length Avr2 proteins apoplastic fluid and xylem sap were isolated from the transgenic plants. Western blot analysis of these fluids revealed that Avr2 is present in the apoplastic fluid and xylem sap of Avr2 plants (Figure 2B). Their presence in the sap shows that i) the 35S promoter is active in the mesophyll and xylem-adjacent cells and ii) the signal peptide ofFol is functional and iii) the protein is secreted. No Avr2 protein was detected in the extracellular fluids of ΔspAvr2 plants, which shows that Avr2 is not secreted and hence must fulfill its virulence function inside the cell. Therefore, complementation of the compromised virulence of the FoLΔAvr2 strain in Avr2 plants is either due to re-uptake of secreted Avr2, or due to the activity of a cytoplasmic Avr2 pool that evaded signal peptide-mediated secretion from the plant cell.

I-2-expressing xylem-adjacent cells do not take up Avr2 host-autonomously

To determine whether tomato cells can take up Avr2 via a host- autonomous process, grafting studies were performed in which scions of tomato plants expressing 1-2 were grafted onto wild-type Moneymaker, ΔspAvr2 or Avr2 rootstocks. Since Avr2 is present in the xylem sap of Avr2 plants, through which water and nutrients are transported from roots to shoot and leaves, the effector is predicted to be transported from the Avr2 rootstock into the 1-2 scion. If the /-2-expressing cells autonomously take up the effector protein from the xylem sap then 1-2 -mediated immune responses will be induced. As predicted, and shown in Figure 3A, no difference in growth was observed when an 1-2 scion was placed on either a wild type or a ΔspAvr2 rootstock. The lack of growth retardation or necrosis in the chimaeras grafted on a ΔspAvr2 rootstock is consistent with the observation (Figure 3A) that Δsp Avr2 is not secreted and hence cannot be translocated through the plant to 1-2 expressing tissues. Importantly, also no 1-2 immune symptoms appeared in 1-2 scions grafted on an Avr2

rootstock (Figure 3A). Per genotype combination at least ten independent grafts were made and in none of them an autoimmune response was observed. The lack of 1-2 activation suggests that either Avr2 is not transported to the upper part of the plant, or that it is not taken up from the xylem sap. To distinguish between these options western blot analysis on xylem sap was done. Xylem sap was harvested from stems cut at ±10 cm above the graft to exclude possible contamination of the sap with Avr2 leaking out of damaged Avr2 expressing cells. As can been seen in Figure 3B the Avr2 protein could readily be detected in the xylem sap of 1-2 scions placed on an Avr2 rootstock, but not in xylem sap isolated from scions grafted on either wild type of an ΔspAvr2 root stocks.

These results show that Avr2 is transported from the Αυτ2 rootstock into 1-2 scions and that the absence of 1-2 -mediated immune elicitation is either due to inability of the plant to autonomously take up Avr2 from the xylem sap, or that the effector concentration is too low to trigger an I-2-mediated response. To examine both options non-transgenic Moneymaker and ΔspAvr2-30 and Avr2- 7 transgenic tomato plants were crossed with 1-2 tomato plants. Combining the resistance and avirulence gene into one plant ensures a systemic presence of both proteins and a high effector abundance inside the plant. From all three crosses Fl seeds were obtained and 15 seeds per cross were analyzed for their ability to germinate. No differences in germination frequency or timing were observed. The seedlings of the different progeny were indistinguishable from each other during the first two weeks following germination. However, whereas

Moneymaker/7-2 plantlets continued to grow normally and developed into mature plants bearing fruits, the ΔspAvr2/I-2 progeny developed a clear auto-immune phenotype; necrotic lesions emerged on the leaves and the plants showed reduced weight and stunted growth (Figure 3C). Although the ΔspAvr2/I-2 plants continued to grow and even flowered, they never developed fruits. The autoimmune phenotype of the ΔspAvr2/I-2 plant is consistent with the intracellular recognition of the protein by the 1-2 immune receptor (Ma et al., 2013) and confirms that tomato leaf cells are capable of showing an 1-2 response upon exposure to cytoplasmically localized Avr2. Therefore it was interesting to observe that no necrotic lesions developed on the Avr2/I-2 progeny. The lack of 1-2 activation in these plants implies that secreted Avr2 is not taken up from the

extracellular spaces, either the xylem or apoplast, to trigger an 1-2 response. The presence of the Avr2 and 1-2 genes in ΔspAvr2/I-2 and Avr2/I-2 progenies was verified by Avr2 and 1-2 specific primers on genomic DNA (Figure 5A). Additionally, western blot analysis of the leaves in parental lines and Fl progeny of ΔspAvr2/ 1-2 and Avr2/I-2 revealed that Avr2 is not only present in the ΔspAvr2 and Avr2 transgenic parental plants but also in their ΔspAvr2/I-2 andAvr2/I-2 progeny (Figure 3D and 5B). Taken all together, the lack of Avr2-mediated 1-2 activation in Avr2/I-2 plants suggests that /-2-expressing cells cannot autonomously take up the Avr2 effector protein from the extracellular spaces.

Infiltration of Agrobacterium in Avr2/I-2 tomato leaves triggers HR

Previously it was reported that agro-infiltration of either an Avr2- or a ΔspAvr2-encoding construct triggers /-2-dependent HR in N.

benthamiana (Houterman et al., 2009). Since the signal peptide of Avr2 is functional in planta (Fig 2), this finding suggests uptake of the secreted Avr2 protein by the plant cells in the presence of A tumefaciens. The

Avr2/I-2 plants allow us to test this hypothesis. The expectation is that upon A tumefaciens infiltration cell death will be triggered in the transgenic plants, but not in wild-type tomato. The A. tumefaciens ID 1249 strain was used to infiltrate tomato as, unlike most laboratory strains, it does not trigger necrosis in the leaf (Wroblewski et al., 2005). Four- week-old wild- type Moneymaker, 1-2 and Avr2/I-2 tomato plants were infiltrated with A tumefaciens ID 1249 delivering either GUS, which serves as a negative control, or ΔspAvr2 acting as positive control for 1-2 mediated cell death. To be able to distinguish specific responses from non-specific ones also a mock infiltration was done using buffer without A tumefaciens. To better visualize the occurrence of cell death the leaves were stained with trypan blue. At 4 days post infiltration (dpi) the majority (80%) of mock infiltrated leaves were symptomless, although some cell death directly beneath the infiltration sites was found in the other leaves (Figure 4A and B).

Infiltration of Agrobacterium delivering either GUS or ΔspAvr2 in wild-type plants also only showed cell death at the infiltration sites itself, and not in the sector around it, which can be attributed to mechanic damage. In contrast to this, agro-infiltration of ΔspAvr2, but not GUS, triggered cell death in a sector around the infiltration points in 20% of the infiltrated 1-2 tomato leaves. The induction of /-2-dependent cell death following transient expression of Δsp Am 2 is consistent with the former observations in N.

benthamiana, and shows that ID 1294 can be used for transient

transformation of tomato and that tomato leaves are capable of mounting an 1-2 specific response upon Avr2 perception. In contrast to the 1-2 plants, which only responded to an A

tumefaciens strain carrying ΔspAvr2, the majority of Avr2/I-2 leaves exhibited a strong cell death response of the infiltrated sector following agro-infiltration of either strain. In respectively 70% and 80% of the Avr2/I- 2 leaves cell death was induced after infiltration of either GUS or the

ΔspAvr2 construct. The cell death is independent of the construct, but requires A. tumefaciens as necrosis was not induced in the mock infiltrated sectors. Together these data shows that A tumefaciens infiltration

specifically triggers cell death in Avr2/I-2 tomato plants, likely by

facilitating the uptake of Avr2 by the plant cells.

Avr2/I-2 plants show reduced disease symptoms when challenged with Phytophthora infestans To test if the immune response in tomato plants co-expressing Avr2 and 1-2 reduces their susceptibility to pathogens, we inoculated them and the respective parental lines with the oomycete pathogen Phytophthora infestans (P. infestans). In a detached leaf assay, symptom development of each inoculation sites was observed 7 days after inoculation with P.

infestans zoospores. The symptoms were assigned to 3 different classes: weak necrosis (class 1), strong but contained necrosis (class 2) and

spreading necrosis (class 3). Inoculation sites showing no detectable symptoms were assigned class 0. For each plant line the percentage of inoculation sites showing either of these classes was recorded (Figure 6). 1-2 expressing plants showed the highest percentage of inoculation sites with class 3 symptoms, whereas Avr2 plants showed an increased percentage of inoculation sites with class 2 and class 3 symptoms compared to Avr2/I-2 co- expressing plants. In conclusion the Avr2/I-2 plants showed the lowest susceptibility to P. infestans, indicating the pathogen induced uptake of Avr2, thereby triggering an immune response that reduced the pathogen's aggressiveness.

Discussion

The present inventors herein show that expression of either full length Avr2 or ΔspAvr2 in tomato complements the compromised virulence of a FoLΔAvr2 strain. Hence, Avr2 not only exerts its avirulence function intracellularly (Ma et al., 2013), but also its virulence activity. How the protein exerts its virulence function is unknown. The observation that extracellular plant-produced Avr2 fully complements the FoLΔAvr2 strain implies that the protein is either able to evade signal peptide-mediated secretion or is taken up by the plant cells. Since the present inventors detected Avr2 in xylem sap and apoplastic fluid of Avr2 transgenic plants the signal peptide must be functional lending support to the second hypothesis. In agreement, it has been reported that Avr2 is secreted into tomato xylem vessels by Fol (Houterman et al., 2007), while it

intracellularly activates 1-2 (Houterman et al., 2009; Ma et al., 2013) further indicating that Avr2 is taken up by plant cells in the presence of Fol.

How host cells take up effectors is unknown and monitoring effector movement from the pathogen to the host cell is technically challenging (Petre and Kamoun, 2014). Also our attempts to directly visualize uptake using Fol strains producing GFP tagged effector proteins were unsuccessful as the tags were cleaved off by extracellular proteases, like they are in this study (Fig 2A). Mostly two types of assays are used to monitor the ability of effector proteins to enter host cells; "the cell re-entry" and the "protein uptake" method (Catanzariti et al., 2006; Dou et al., 2008a; Kale and Tyler, 2011). However, both assays have their limitations (Petre and Kamoun, 2014; Lo Presti et al., 2015). The first has the drawback that it is unclear whether effectors had indeed been secreted into the apoplast prior to re-internalization, and it is therefore not possible to exclude that effectors might have escaped the secretory pathway and thus remained in the cytoplasm (Oh et al., 2009). In the second assay protein is infiltrated in leaves, or added to cell suspension in which cells are stressed or wounded, that might trigger non-specific protein internalization complicating the interpretation of the data (Yaeno et al., 2011; Wawra et al., 2013).

The present inventors have overcome the limitation of these former assays by using an unique functional assay in which effector production is spatially separated from its action and in which no wounding is involved. Our grafting experiment shows that although Avr2 is present in the xylem sap of the 1-2 graft, and hence is translocated from the Avr2 rootstock, it is unable to trigger /-2-mediated immune responses in the 1-2 scion. These data suggest that /-2-expressing cells do not autonomously take up the effector protein from the xylem sap in the absence of Fol. Crosses between either Avr2 or ΔspAvr2 plants with 1-2 tomato substantiate this conclusion, as an autoimmune response was only observed in the ΔspAvr2/I- 2 crosses. Furthermore, the lack of an immune response in the Avr2/I-2 plants implies that secretion of Avr2 is a very efficient process. So although Avr2 is abundantly present in the extracellular spaces of Avr2/I-2 progeny, the secreted Avr2 is not perceived by intracellular 1-2 again implying that plant cells do not autonomously take up the Avr2 effector.

Notably, in the presence of Fol the secreted Avr2 protein is able to enter the host cell as it complements the virulence defect of a FoLΔAvr2 strain. This observation implies that during infection a factor is produced that is required for Avr2 uptake by the host cell. The identity of this factor is unknown, but since infiltration of A tumefaciens also stimulated effector uptake, the property to generate this a factor seems to be shared by other plant pathogens. Agro-infiltration of either an Avr2- or a ΔspAvr2-encoding construct triggers 7-2-dependent HR in N. benthamiana, suggesting uptake of secreted Avr2 in the presence of the bacterium (Houterman et al., 2009). In line with this finding, the present inventors herein show that agroinfiltration of Avr2/ 1-2 leaves triggers cell death irrespectively of the construct carried by the bacterium. The ID 1294 strain containing the

ΔspAvr2-encoding construct triggers a relative weak cell death response in I- 2 tomato, which is in line with the reported low transient transformation efficiency of this strain (Wroblewski et al., 2005). In agroinfiltrated leaves of Avr2/I-2 plants slightly more cell death was induced by the ΔspAvr2

carrying A tumefaciens strain than by the GUS control strain. This difference is likely attributable to a higher cytosolic Avr2 concentration due simultaneous uptake and production of Avr2 in the Avr2/I-2 cells.

Nevertheless, these results show that the mere presence of the bacterium in Avr2/I-2 plants is sufficient to trigger cell death. These findings are especially relevant for studies in which pathogen-independent uptake was suggested based on assays in which the effectors were expressed using A. tumefaciens (Rafiqi et al., 2010; KLoppholz et al., 2011).

Uptake of effectors by plants has been described before (Dou et al., 2008b; Kale et al., 2010), but it is currently unclear which properties determine whether an effector can be taken up by the host. For the AvrM and AvrL567 effectors from the flax rust fungus Melampsora lini it was shown that their N-termini are required for translocation into host cells when transiently expressed using A tumefaciens transformation (Ve et al., 2013). For ToxA from Pyrenophora tritici-repentis the C-terminal RGD motif is involved in internalization (Manning et al., 2008). The Avr2 protein does not show sequence homology with the flax rust effector proteins, nor does it contain a clearly distinguishable RGD motif. However, it has been proposed that Avr2 contains an RxLR (Arg-x-Leu-Arg)-like motif (Figure 1A) that might be involved in its uptake (Kale et al., 2010). The RxLR and DEER motifs (Δsp-Glu-Glu-Arg) are frequently found in oomycete effectors

(Bhattacharjee et al., 2006; Tyler et al., 2006; Jiang et al., 2008) and have been shown to function as a host-targeting signal allowing the protein to be translocated into host cells (Whisson et al., 2007; Dou et al., 2008a). Tyler and co-workers (2010) reported that the RxLR motif allows effectors to bind phosphatidylinositol-3-phosphate (PI3P) present on the outer surface of the plant plasma membrane enabling vesicle-mediated endocytosis. However, whether the RxLR-like motif in Avr2 is functional and required for uptake remains unclear as mutations in the motif rendered the protein unstable when transiently expressed in the plant prohibiting functional analysis (Ma, 2012).

Samuel and co-workers (2015) proposed that effectors could be transferred by extracellular vesicles (EVs). Proteins lacking secretion signals could be packaged into EVs for passage through the plasma membrane whilst proteins containing a secretion signal could be secreted into the matrix of the cell wall and then bind to EVs via a lipid binding motif. The protein then transits the cell wall as a passenger on the outer leaflet of the vesicle (Samuel et al., 2015). Whether such a mechanism applies to Avr2 is unclear. An indication that the effector might associate with vesicles is the observation that RFP-tagged Avr2 expressed from Fol during colonization of the xylem vessel forms red-fluorescent punctate spots alongside the mycelium where it touches the plant cells (Ma, 2012). A local high concentration of the RFP -labeled effector is consistent with the protein being sequestered on a specific location from where it could be internalized into vesicles. How an effector protein subsequently dissociates from a vesicle and is released in the host cytoplasm remains unclear; possibly the unknown factor produced during infection plays a determining role in this process. The nature of the factor triggering effector uptake is unknown. It might be another secreted effector protein from the pathogen, a microbial metabolite or a plant signal induced upon pathogen-inflicted damage. Since both A tumefaciens and Fol trigger uptake, and without wishing to be bound by any theory, the present inventors favor the latter option, also because the "protein uptake" method shows effector uptake by in vitro grown cultured plant cells in the absence of a microbe (Dou et al., 2008a; Kale and Tyler, 2011).

The present inventors herein describe a series of functional assays demonstrating that tomato cells do not take up the Fol Avr2 effector protein in the absence of a plant pathogen. However, effector uptake was shown in the presence of both Fol and A. tumefaciens. The Avr2/I-2 tomato plants generated in this study provide an excellent starting point to investigate whether other plant pathogens also have the ability to trigger effector uptake, a novel means of introducing disease resistance in plants. Conclusion

Pathogens secrete effector proteins to manipulate the host for their own proliferation. Currently it is unclear whether the uptake of effector proteins from extracellular spaces is a host autonomous process. The present inventors have studied this process using the Avr2 effector protein from Fusarium oxysporum f.sp. lycopersici (Fol). Avr2 is an important virulence factor that is secreted into the xylem sap of tomato following infection. Besides that, it is also an avirulence factor triggering immune responses in plants carrying the 1-2 resistance gene. Recognition of Avr2 by 1-2 occurs inside the plant nucleus. Herein, the present inventors show that pathogenicity of an Avr2 knockout Fusarium ( FoLΔAvr2) strain is fully complemented on transgenic tomato lines that express either a secreted (Avr2) or cytosolic Avr2 ( ΔspAvr2) protein, indicating that Avr2 exerts its virulence functions inside the host cells. Furthermore, our data imply that secreted Avr2 is taken up from the extracellular spaces. Grafting studies were performed in which scions of 1-2 tomato plants were grafted onto either aΔsp Avr2 or on an Avr2 rootstock. Although the Avr2 protein could readily be detected in the xylem sap of the grafted plant tissues, no I- 2-mediated immune responses were induced suggesting that 1-2 -expressing tomato cells cannot autonomously take up the effector protein from the xylem sap. Additionally,Δsp Avr2 and Avr2 plants were crossed with 1-2 plants. WhereaΔssp Avr2/I-2 Fl plants showed a constitutive immune response, immunity was not triggered in the Avr2/I-2 plants confirming that Avr2 is not autonomously taken up from the extracellular spaces to trigger 1-2. Intriguingly, infiltration of Agrobacterium tumefaciens in leaves of Avr2/I-2 plants triggered 1-2 mediated cell death, which indicates that Agrobacterium triggers effector uptake.

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Claims

1. A plant comprising at least one R-gene encoding an intracellular R protein for mounting an immune response in cells of said plant, said plant further comprising a chimeric gene for the extracellular production of a pathogen effector in cells of said plant that express the chimeric gene, the chimeric gene comprising:

(a) a promoter sequence which functions in plant cells;

wherein the pathogen effector is cognate with the intracellular R protein.

2. The plant according to claim 1, wherein the pathogen effector is heterologous with respect to the plant, the R protein, the promoter, the signal peptide, and/or the optional 3' non-translated region.

3. The plant according to claim 1 or 2, wherein the plant is a transgenic plant, stably transformed with the chimeric gene.

4. The plant according to claim 1 or 2, wherein the plant is a non- transgenic plant comprising an extrachromosomal expression vector for expression of the chimeric gene.

5. The plant according to any one of clams 1-4, wherein the i?-gene is located on an extrachromosomal expression vector.

6. A chimeric gene for the extracellular production of a pathogen effector in cells of a plant that express the chimeric gene, the chimeric gene comprising:

(a) a promoter sequence which functions in plant cells; (b) a coding sequence, operably linked to the promoter, encoding a fusion protein consisting of said pathogen effector fused in translation frame to a signal peptide for targeting said pathogen effector to the plant secretory pathway; and

7. The chimeric gene according of claim 6, wherein the pathogen effector is heterologous with respect to the promoter, the signal peptide, and/or the optional 3' non-translated region.

8. The chimeric gene of claim 6 or 7, wherein the pathogen effector is cognate with an intracellular R protein expressed in said plant.

9. The chimeric gene of any one of claims 6-8, wherein the promoter is adapted to cause sufficient expression of the fusion polypeptide to mount a immune response in said plant cells upon exposure of said plant cells to a pathogen that facilitates uptake of the extracellular pathogen effector in said plant cells, preferably wherein the promoter is a constitutive promoter.

10. The chimeric gene of any one of claims 6-9, wherein the pathogen effector originates from an organism selected from the group consisting of fungi, oomycetes, bacteria, nematodes, mites, insects, and parasitic plants.

11. The chimeric gene of any one of claims 6- 10, wherein the pathogen effector is selected from the group consisting of Avr2

(AM234063.1) of Fusarium oocysporum, Avra10, Avrkl and AvrMLal of Blumeriagraminis, AvrL567 (AY510102.1), AvrM (DQ279870), AvrP1234 (EU642499) and AvrP4 (ABB96263.1) of Melampsora lini, AVR-Pia, Avr- Pik, Avr-Pita (AF207841), Avr-Pita2, and Avrl-C039 (AF463528) of

Magnaporthe grisea or Magnaporthe oryzae, AvreRpgl of Puccinia graminis f sp. Tntici, Avr3 of Bremia lactucae, ATR1 (PDB ID: 3RMR_A), ATR3, ATR13 (PDB ID:2LAI), HaRxLl- HaRxL147, HaRxLL3a- HaRxLL495c, HaRxLCRN4b of HycUoperonospora arabidopsidis, AvrB and AvrRPP 1A of Peronospora parasitica, AvrRPPlB, AvrRPPIC, AvrRPP2, AvrRPP4, AvrRPP5, and AvrRPP8 of Peronospora parasitica, Avrl (DS028168), AVR2,Avr-blbl apiO) (DS028419), Avr-blb2 (DS028242), PiAvr2

(DS028133), Avr3a (EF587759), Avr2 (EEY61966), Avr3b, Avr10, Avrll (DQ390339), and AVR4 of Phytophthora infestans, Avr la (EF463064.1), Avr3a (EF587759.1), AVRlb-1, AVR lk, and Avr3c (FJ705360.1) of

Phytophthora sojae, AvrLml (AM084345.1), AvrLm6 (AM259336.1), AvrLm4-7 (AM998638.1) of Leptosphaeria maculans, Avra10 (DQ679913), and Avrkl (DQ679912) of Blumeria graminis f. sp. hordei, and OEC45- OEC 115 of Golovinomyces orontii, and homologs thereof.

12. A vector comprising a chimeric plant gene of any one of claims 6- 11, preferably wherein said vector is selected from a cloning vector, an expression vector, a plant transformation vector, and a plant viral expression vector.

13. A plant cell comprising a chimeric gene of any one of claims 6-11 or a vector according to claim 12.

14. A plant which has been regenerated from a plant cell of claim 13, preferably wherein said plant is resistant to a pathogen that facilitates uptake of the extracellular pathogen effector in said plant cells.

15. A method for producing a pathogen-resistant plant which comprises

(a) introducing into a plant cell a chimeric gene of any one of claims 6-11; and

(b) regenerating pathogen-resistant plants from said plant cells.

16. Method for generating effector-triggered immunity in a plant, the method comprising:

(a) allowing the expression in said plant of at least one endogenous or heterologous JZ-gene encoding an intracellular immune receptor;

(b) introducing the chimeric gene of any one of claims 6-11 into a cell of said plant;

(c) growing a plant from the plant cell produced in step b) for a time sufficient to produce and translocate the pathogen effector to the

extracellular space; and