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WO2000065039A2 - Identification d'elements de controle d'adn reagissant a des stimuli specifiques - Google Patents

Identification d'elements de controle d'adn reagissant a des stimuli specifiques Download PDF

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WO2000065039A2
WO2000065039A2 PCT/US2000/011460 US0011460W WO0065039A2 WO 2000065039 A2 WO2000065039 A2 WO 2000065039A2 US 0011460 W US0011460 W US 0011460W WO 0065039 A2 WO0065039 A2 WO 0065039A2
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genes
gene
mutants
sar
expression
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Klaus Maleck
Kay Ann Lawton
Robert Arthur Dietrich
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Syngenta Participations Ag
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • C12N15/8239—Externally regulated expression systems pathogen inducible
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Definitions

  • the invention generally relates to the use of gene expression profiling to identify groups of genes that show a similar pattern of expression in response to biotic and abiotic stimuli.
  • the invention more particularly relates to the use of the expression profile data thus generated to uncover gene groups that are co-regulated and to identify common DNA sequences that function to regulate gene expression in response to specific stimuli.
  • Plants are constantly challenged by a wide variety of pathogenic organisms including viruses, bacteria, fiingi, and nematodes. Crop plants are particularly vulnerable because they are usually grown as genetically-uniform monocultures; when disease strikes, losses can be severe. However, most plants have their own innate mechanisms of defense against pathogenic organisms. Natural variation for resistance to plant pathogens has been identified by plant breeders and pathologists and bred into many crop plants. These natural disease resistance genes often provide high levels of resistance to or immunity against pathogens.
  • SAR Systemic acquired resistance
  • HR hypersensitive response
  • Lesion mimic phenotypes can be caused by mutations in R genes, as seen in the Rpl mutant in maize (Hu et al., 1996) but they can also be caused by metabolic perturbations (Dangl et al, 1996), and loss-of-function mutations in putative transcription factors (e.g. Isdl; Dietrich et al., 1997). Mutants deficient for R gene- specific signal transduction provide another link between the induction of HR and SAR.
  • the mutant ndrl (non-race specific disease resistance), which was isolated in a screen for susceptibility to avirulent Pseudomonas syringae strains, is deficient for the induction of a local defense reaction induced by R genes of the LZ-NBS-LRR class (Aarts et al., 1998; Century et al., 1995). Subsequently, SAR cannot be induced in these interactions in the ndrl mutant. Similarly, in the edsl (enhanced disease susceptibility) mutant, the signaling cascade induced by R genes containing an N-terminal TIR domain is interrupted (Aarts et al, 1998; Parker et al, 1996).
  • Avr-R gene interaction edsl (enhanced disease Susceptibility to avirulent (Parker et al, 1996) susceptibility) Peronospora parasitica isolates, member of the converging TIR-NBS- LRR R gene signaling pathway ndrl (non-race-specific disease Susceptibility to avirulent (Century et al, 1995) resistance) Pseudomonas syringae strains, convergence of LZ-NBS-LRR R gene signaling
  • Isdl 6 (lesions simulating Identification of spontaneous (Dietrich et al, 1994) disease resistance) lesion formation. Wild-type alleles are involved in limiting initiation or spreading of cell death acd2 (accelerated cell death) Same as Isds (Greenberg and Ausubel, 1993) cell death cims/cpr (constitutive Marker gene overexpression (PR-1 or (Bowling et al, 1994) immunity/ PR-2); Role in SA biosynthesis or constitutive ER gene S R upregulation expression) dndl (defense, no death) Absence of HR when inoculated with (Yu et al, 1998) avirulent Pseudomonas syringae, constitutive immunity pad (phytoalexin deficient) No phytoalexin accumulation after (Glazebrook and infection by the moderate virulent Ausubel, 1994) pathogen Pseudomonas syringae pv.
  • niml/nprl/sail no Susceptibility to virulent Peronospora (Cao et ⁇ /., 1994; immunity/no PR genes/SA parasitica isolates after chemical Delaney et al, 1995; insensitive) immunization, hypersensitive to Shah et al, 1997) Pseudomonas syringae, counter selection using a SA-inducible promoter; Niml seems to be a central component of SAR
  • PR gene expression non-SAR mutants edrl enhanced disease Resistance to virulent Pseudomonas (Frye and Innes, 1998) resistance
  • syringae pathovars also resistant to Erisyphe cichoracearum SAR can be distinguished from other inducible disease resistance responses by a number of associated biochemical and physiological changes, which eventually confer an increased resistance to a secondary pathogen attack.
  • SAR markers are proteins whose expression is tightly correlated with the onset of SAR in uninfected tissue (Metraux et al, 1989; Uknes et al, 1992; Ward et al, 1991). All of the SAR markers fall in the class of PR proteins, which were originally identified as low-molecular weight, acidic proteins accumulating after TMV infection of tobacco leaves (Van Loon, 1985) or other pathological and stress-related situations ( Kombrink and Somssich, 1997). In tobacco, the set of SAR markers is encoded by at least nine gene families (Alexander et al, 1992; Ward et al, 1991).
  • PR-1 the most specific marker protein.
  • PR-2 the most specific marker protein.
  • PR-5 the most specific marker protein.
  • the Arabidopsis PR- 1 promoter has been examined in detail for responsiveness to salicylic acid (SA) and 2,6- dichloro-isonicotinic acid (INA). Only two or three active promoter domains have been identified, none of them with homology to ethylene response binding protein sites, which may reflect on a molecular level the observed overall specificity of PR-1 induction (Lebel et al, 1998).
  • SA salicylic acid
  • acetylsalicylic acid could induce disease resistance and the expression of PR genes (Van Loon and Antoniw, 1982; White, 1979).
  • PR genes Van Loon and Antoniw, 1982; White, 1979.
  • SA accumulation is not only concomitant to but also required for SAR induction.
  • SA is synthesized in plant cells from phenylalanine, which is converted to t-cinnamic acid (t-CA) by PAL, a key enzyme in the phenylpropanoid pathway.
  • t-CA is converted to SA via benzoic acid, presumably in the chloroplast (Yalpani et al., 1993).
  • Free SA is rapidly conjugated to the ⁇ -O-D glycoside (Enyedi et al, 1992) by an SA-inducible UDP-glucose:SA 3-O- glycosyltransferase (Enyedi and Raskin, 1993). Only free SA is active, but its action can be mimicked by the functional analogs BTH (Benzothiadiazol, CGA 245704) and INA (2,6- dichloro-isonicotinic acid, CGA 41396) (Friedrich et al, 1996; G ⁇ rlach et al, 1996; Lawlon et al, 1996; Vernooij et al., 1995).
  • JA wound-inducible, jasmonic acid
  • ethylene mediated defense response has primarily been studied in the context of induced resistance to insect predation in tomato and tobacco, and more recently, also in Arabidopsis (McConn et al., 1997). It is triggered by wounding and insect feeding and results in the induction of proteinase inhibitor (PI) genes (Creelman and Mullet, 1997; Ryan, 1990). The proteinase inhibitors interfere with digestion in the insect gut and discourage further feeding.
  • PI proteinase inhibitor
  • PR-3 chitinase
  • PR-4 thaumatin-like protein
  • thionins PR-12; Vignutelli et al., 1998) in Arabidopsis.
  • Defensins and thionins are small, cystein-rich peptides ( ⁇ 5 kDa) with potent in vitro activity inhibiting fungal growth (Bohlmann, 1994; Broekaert et al, 1995).
  • Structurally related peptides can be found not only in other plant species, but also in insects where they are also likely to participate in antimicrobial host defenses (Hancock et al, 1995). Overexpression of defensins or thionins in plants leads to enhanced resistance to certain pathogens that are not affected by SAR (Epple et al, 1997; Terras et al, 1995).
  • a genetic block in the wound-response signaling cascade renders plants more susceptible to necrotrophic fungal pathogens.
  • the jasmonate insensitive mutant jarl and the jasmonate-deficient triple fad3-2fad7-2fad8 mutant are both more susceptible than wild-type plants to Botrytis cinerea or Pythium irregulare but not to "classical" SAR pathogens such as Peronospora (Staswick et al, 1998; Thomma et al, 1998; Vijayan et al, 1998).
  • mutants in the ethylene perception show increased susceptibility (or increased tolerance) to avirulent and virulent pathogens (Bent et al, 1992; Knoester et al., 1998; Lund et al, 1998).
  • expression of an antisense construct of a lipoxygenase gene in tobacco results in reduced jasmonate synthesis and increased susceptibility to Rhizoctonia solani and Phytophtora parasitica (Ranee et al, 1998).
  • some components of the wound response may be involved in resistance to certain pathogens as well as in resistance to insects.
  • the pathogen spectrum might however vary from plant to plant and may overlap with the pathogen spectrum of SAR.
  • ISR Induced Systemic Resistance
  • ISR Inhibits apoptosis apoptosis apoptosis apoptosis apoptosis aposis aposis aposis aposis aposias syringae
  • ISR confers quantitative resistance to fungal (Fusarium oxysporium) and bacterial pathogens (Pseudomonas syringae) and appears to be independent of S A, but dependent on ethylene and jasmonic acid (Pieterse et al, 1998).
  • none of the typical marker genes for JA/ethylene induced resistance is expressed during ISR.
  • a screen for mutants unable to activate SAR after INA treatment was performed.
  • Six alleles of a mutant hypersensitive to Peronospora were isolated (Delaney et al, 1995).
  • the same gene was isolated, thus underlining the central importance of the NIM1/NPR1 (no immunity, no PR gene expression) gene for SAR activation downstream of SA (Cao et al, 1994).
  • the niml/nprl gene was cloned by map-based cloning.
  • the sequence has homologies to some ankyrin-containing transcription factor regulators, such as I ⁇ B ⁇ (Cao et al, 1997; Ryals et al, 1997).
  • Overexpression of the NIM1 gene results in plants that are poised to respond faster and stronger than wild-type plants after (subclinical) concentrations of chemical inducers (Cao et al, 1998; U.S. Patent No. 6,031,153).
  • NIM1 is also required for the SA- independent ISR (Pieterse et al, 1998).
  • niml/nprl does not entirely block the resistance observed in some of the cpr mutants (Bowling et al, 1997; Clarke et al, 1998).
  • Arabidopsis thaliana Is A Model System To Study SAR:
  • Tobacco is however not easily amenable to genetic studies for gaining molecular insights into the components of the SAR signaling cascade.
  • Arabidopsis is attractive as a research tool because of its diminutive stature, short generation time (6 to 8 weeks), high seed yield and its small, well-characterized genome, which makes it of great use in the dissection of other signal transduction pathways, such as the ethylene or the ABA signal transduction.
  • members of several important plant pathogens are virulent on Arabidopsis (Meyerowitz and Somerville, 1994).
  • Phylogenetically Arabidopsis belongs to the Brassicaceae family, which encompasses many crop plants, such as cabbage and mustard (Price et al, 1994).
  • Arabidopsis thaliana has a small genome (110 Mb) with a high gene density (about 1 gene per 4 kb, or an estimated 21,000 - 25,000 genes in total). Less than 10% of the genome, including centromeric and telomeric repeats, contains dispersed repetitive elements (Goodman et al, 1995; Pruitt and Meyerowitz, 1986). These characteristics make Arabidopsis an ideal plant for genetic and physical mapping projects. A large number of genetic markers (Table 2) and genetically diverse land races exist. A significant fraction of the genome has been assembled into physical contigs in high capacity cloning vectors, such as YACs, BACs and PI clones (Schmidt, 1998).
  • Table 2 Genetic Marker Types Currently Used In Arabidopsis Genetic Mapping Projects (partly derived from Rafalski et al, 1996, p.72).
  • DNA required 2 - 10 mg 10 - 25ng 50 - 100 ng 50 - 100 ng l - 2 mg 10 - lOO ng
  • N/A not applicable (per primer, several fragments are obtained, with numbers varying with the PCR conditions)
  • Functional genomics describes the combined efforts to elucidate the functions of the increasing numbers of unknown genes identified by mass-sequencing. Currently, the functions of roughly 50% of all putative genes are unknown (Bevan et al., 1998). Homology searches, expression profiling, knock-out mutant analysis, overexpression studies, and protein-protein interaction analysis might all yield clues to the biochemical, cellular, adaptive or developmental role of a given protein (Bouchez and Hofte, 1998). To match the high- throughput sequencing effort, highly paralleled technologies have been conceived, mostly to gather gene expression patterns (Table 3).
  • Differential PCR-based Leads primarily to (Diatchenko et ⁇ /., 1996) display, PCR normalization and the identification of select enrichment of differentially differentially abundant expressed genes, RNA molecules, successive yielding a cDNA expression analysis library still to be done.
  • Not high-throughput Inducible Gene Expression A principal advantage to be realized through genetic engineering of plants is the controlled expression of chimeric genes so that they are expressed only at the appropriate time, to the appropriate extent, and in some situations in particular parts of the plant. For example, the energy expended by a plant to continuously produce high levels of a foreign protein could prove detrimental to the plant, whereas if the gene were expressed only when desired, the drain on energy and therefore yield could be reduced.
  • the phenotype expressed by the chimeric gene could result in adverse effects to the plant if expressed at inappropriate times during development.
  • tissue in culture or in a bioreactor the untimely production of a desired secondary product could lead to a decrease in the growth rate of the culture, resulting in a decrease in the yield of the product.
  • specific regulation of plant gene expression by exogenous application of chemicals to increase or decrease expression of transgenes of interest could be of particularly great commercial value to both seed and crop protection businesses as well as to end users (e.g. food processors) of agricultural commodities.
  • Promoters activated by specific stimuli can be used for regulated expression of value added traits, input traits and output traits as well as for production of certain proteins (e.g. antibodies, etc).
  • PR genes are known to be induced by various internal and external factors including plant hormones, heat shock, chemicals, pathogens, lack of oxygen, and light.
  • exogenous application of SA induces SAR and expression of PR genes (Ward, et al. 1991; Uknes, et al, 1992) as well as of synthetic compounds such as 2,6- dichloroisonicotinic acid (INA) (Vernooij, et al, 1995) and benzo(l,2,3)thiadiazole-7- carbothioic acid S-methyl ester (BTH) (Friedrich, et al, 1996; Lawton, et al, 1996). Therefore, induction of PR protein genes by chemicals or pathogens provides a foundation to address the problem of controlling gene expression in plants and plant tissue.
  • INA 2,6- dichloroisonicotinic acid
  • BTH benzo(l,2,3)thiadiazole-7- carbothioic acid S-methyl ester
  • Myb-like transcrition factor (mybl) was isolated and its expression shown to be inducible by SA and tobacco mosaic virus (Yang, et al. 1996). Furthermore, it was shown to bind in vitro to a fragment of the tobacco PR- la promoter (positions -679 to -487 from the transcription start site) containing a Myb-like recognition site (positions -520 to -514). Moreover, a sequence in the tobacco PR-2d promoter (-348 to -324) was shown to bind in vitro to another protein.
  • United States Patent No. 5,614,395 describes the Arabidopsis PR-1 protein gene and its chemically inducible promoter. As described in this patent, the full-length Arabidopsis PR-1 promoter fragment was fused to the firefly luciferase (LUC) gene and ultimately cloned into plasmid pAtPRl-S, which is in turn transformed into Arabidopsis plants for chemical induction analysis.
  • the transgenic Arabidopsis lines carrying the PR-1 promoter/LUC gene fusion are then treated by spraying with isonicotinic acid (INA). When analyzed, the transgenic lines showed significantly higher induction of luciferase activity compared to water-treated controls.
  • LOC firefly luciferase
  • INA was shown to induce expression in transformed plants of a chimeric gene comprising the full-length Arabidopsis PR-1 promoter fragment.
  • WO 98/03536 described deletion mutants of the Arabidopsis PR-1 promoter that are shorter than the full-length Arabidopsis PR-1 promoter sequence, yet still yield similar induction of gene expression upon the application of a chemical regulator.
  • the present invention addresses the aforementioned needs by providing a new method whereby the entire genome (transcriptome) is simultaneously surveyed for genes that change in expression in response to biotic and abiotic factors. By comparing gene expression changes across various treatments, groups of co-regulated genes (regulons) are identified and the genomic sequences of genes within a regulon are examined to identify common sequence motifs that are likely to act as regulatory elements. These regulatory elements are then used to make promoters that drive controlled gene expression. This approach uses experimental design based upon the biology of the study system in combination with bioinformatics to analyze the results.
  • gene expression profiling using DNA microarrays is used to study the transcriptome of a plant to identify groups of genes that show a similar pattern of expression in response to biotic and abiotic stimuli, especially biotic and abiotic inducers of SAR.
  • Expression profile data can uncover gene groups that are co-regulated (regulons), and can be used to identify common DNA sequences that function to specifically regulate gene expression in response to exogenous factors but not endogenous signals.
  • the resulting regulatory sequence elements can be cloned and used to precisely regulate genes of interest in transgenic plants.
  • the present invention is useful for identifying genes that are responsive to BTH and/or pathogens, using PCR select and microarray gene chip technology. Experiments are conducted to compare expression profiles in response to biotic and abiotic inducers of SAR and to assess the requirement for salicylic acid and the NIM1 gene for mRNA accumulation. By analysis of data generated with cDNA microarrays, sets of genes that are responsive specifically to exogenous application of BTH, a chemical that can activate the SAR response, can be identified.
  • the present invention is directed to a method for isolating a regulatory DNA sequence from a differentially expressed gene, comprising:
  • the present invention is directed to a method for isolating a common regulatory DNA sequence from a group of co-regulated genes, comprising:
  • said regulatory DNA sequence is a promoter.
  • said genome is a plant genome.
  • said expression profile is obtained using a DNA microarray.
  • said two or more different conditions comprise biotic stimuli.
  • said two or more different conditions comprise abiotic stimuli.
  • said two or more different conditions comprise biotic and abiotic stimuli.
  • said genome is a plant genome and wherein said two or more different conditions comprise at least one SAR inducing condition or at least one SAR repressing condition.
  • said least one SAR inducing condition comprises pathogen infection, SA application, BTH application, NIM1 gene expression, or a cim mutation, and preferably said least one SAR repressing condition comprises NahG expression or a niml mutation.
  • the present invention is directed to a method for isolating a regulatory DNA sequence from a differentially expressed plant gene, comprising:
  • the present invention is directed to a method for isolating a common regulatory DNA sequence from a group of co-regulated plant genes, comprising:
  • said regulatory DNA sequence is a promoter.
  • said least one SAR inducing condition comprises pathogen infection, SA application, BTH application, NIM1 gene expression, or a cim mutation
  • said least one SAR repressing condition comprises NahG expression or a niml mutation.
  • said two or more different conditions comprise BTH application, and at least one condition selected from the group consisting of pathogen infection, SA application, and a niml mutation.
  • said two or more different conditions comprise BTH application, pathogen infection, and SA application, and wherein one or more genes are identified that are inducible by BTH application but not by pathogen infection or SA application.
  • said two or more different conditions comprise BTH application and a niml mutation, and wherein one or more genes are identified that are inducible by BTH application in niml mutant plants. It is desirable in the above embodiments that said one or more genes are inducible at least 5x by BTH application.
  • PR-1 transcription may not be strictly limited to defense responses of plants to pathogen attack, and in fact PR-1 expression has been observed in other circumstances as well (Uknes et al, 1993).
  • the PR-1 peptide is neither required nor sufficient to confer complete disease resistance. Rather, it is thought that the combination of many factors (chitinases, glucanases, antimicrobial peptides) together confer the broad-spectrum disease resistance observed during SAR. Based on structural similarity to small antimicrobial peptides, PR-1 is likely to be one of the factors contributing to this resistance.
  • luciferase reporter gene To isolate new disease resistance mutants we screened for plants that constitutively expressed the PR-1 gene.
  • PR-1 gene expression we chose the luciferase reporter gene because luciferase activity can be monitored in vivo without affecting the integrity of the plant. This feature opens up the possibility to rapidly examine many plants as well as to reexamine the same tissue several times throughout the experiment (Millar et al, 1992). Also, luciferase activity can easily be reexamined in vitro providing a mean for fast confirmation and quantification of results obtained by in vivo monitoring. Two lines of evidence correlate light emission by ER-E/luciferase plants with SAR gene expression:
  • the size of the EMS mutant screen was designed to near-saturate the genome with point mutations that might lead to constitutive PR-1 gene expression.
  • the mutation rate corresponded to a 400- fold increase of the natural mutation frequency (which has been estimated to 5 x 10 "4 mutations per gene per generation; Feldmann et al, 1994; Li and Redei, 1969).
  • the Ml and for the most part the M2 populations were much smaller.
  • EMS introduces primarily G-A transitions through O 6 alkylation of guanine (Britt, 1999).
  • Other commonly used mutagens in Arabidopsis include foreign insertion sequences, such as T-DNA or heterologous transposons that have been engineered to carry selectable marker genes (for review, see Stiekema and Pereira, 1998).
  • T-DNA insertions occur randomly throughout the genome and do not need to be mobilized (Schulz et al, 1994). Although only a few mutations per genome are introduced by T-DNA mutagenesis (thus increasing the required mutant population size), more mutations result in a detectable phenotype than with EMS mutagenesis. Furthermore, T- DNA mutagenesis facilitates the cloning of the mutant gene (especially in cases where the phenotype is difficult to determine).
  • mutants Characterization of the different mutants validated the approach taken since all mutants with increased luciferase activity exhibited increased resistance to several virulent pathogens.
  • the mutants fall into two classes, Isd mutants, the predominant class, and cim mutants. Since we were interested in mutants in SAR signal transduction, and not in mutants in which SAR is induced as a consequence of spontaneous cell death, we focused the study on cim mutants. cim mutants define a diverse group of loci with different disease resistance spectra.
  • SAR signaling might be regulated by a cascade of repressors, in analogy to for example the pathway controlling apoptosis in Drosophila and C elegans (McCall and Sach and Sachr, 1997; Vaux and Strasser, 1996).
  • a complex network of repressors (bcl-2/ced-9, p35, and others) keep the cell alive and the loss of one of these factors is sufficient to induce apoptosis.
  • the SAR signaling cascade would be turned on by a loss-of-function mutation in a negative regulator, and this mutation could be dominant or appear to be dominant as a result of haplo-insufficiency (Melnick et al, 1993).
  • the cim mutations are often not expressed in 100% of the self-progeny. Incomplete penetrance has also been found in several hormone mutants (for instance impaired in the ethylene or giberellic acid pathways; Kieber, 1997; Ogas et al, 1997) and also in SAR mutants (e.g. Isd2, IsdT). To date, no genetic explanation for incomplete penetrance of mutations in isogenic backgrounds has been found. Exogenous and endogenous events, such as heat, light, and cell homeostasis can be decisive in triggering a labile switch, as known in less complex biological systems, such as the phage lambda lysis-lysogeny decision (McAdams and Shapiro, 1995; Ptashne, 1992).
  • This bistable behavior is well known in biological network theory, and occurs also in higher eukaryotic cells: Feedback regulation can result in the presence of two discrete steady-state activities, such that a small stimulus is sufficient for a cell to trigger the transition to one state and to stabilize the cell in this state (Bhalla and Iyengar, 1999). Such a labile equilibrium would be in accordance with haplo-insufficient mutations. The loss of one gene copy of a regulatory factor might still allow proper regulation until stress situations titer this factor and the mutation becomes phenotypically evident.
  • This value can be compared to up to 4 milligram salicylic acid per gram fresh weight in the cim mutants, which corresponds to a 15-fold increase to uninduced wild-type levels. It should be noted that ectopic expression of tryptophan decarboxylase in potato resulted in a severe alteration of the phenylpropanoid pathway. The plants were morphologically unchanged, though hypersusceptible to pathogens because of depletion of the chorismate pool (Yao et al, 1995).
  • Plant metabolism appears to be very flexible in reacting to exogenous manipulations, such as the inhibition of amino acid biosynthesis (Guyer et al, 1995), or the perturbation of the carbohydrate homeostasis (Stitt et al, 1990). Plants that overexpressed a yeast invertase increased the glycolysis and were able to define new "Sink” and "Source” tissues (Sonnewald et al, 1991). Similarly, it is likely that cim mutants can compensate for the increased energy flux through the SAR pathway by enhancing and deviating the metabolism, cim mutants often appear darker green, and might have a higher chlorophyll content than wild-type. It is also remarkable in this context that cim mutants show enhanced transcription of genes encoding functions in energy metabolism, photosynthesis and protein biosynthesis. Similar results in parsely cells also revealed extensive changes in metabolism during fungal infection (Batz et al, 1998).
  • cim mutants are able to develop an HR in response to an avirulent bacterial pathogen but some appear to simply bypass HR (cz ' m713, cz ' m ⁇ lO).
  • Arabidopsis mutants with a similar phenotype, called dnd (defense, no death) have recently been isolated (Yu et al, 1998). They exhibit disease resistance to two virulent pathogens and do not develop an HR when inoculated with avirulent Pseudomonas strains, although they remain fully resistant.
  • HR may be required in wild-type to potentiate an SAR-inducing signal, possibly via the release of reactive oxygen species (Alvarez et al, 1998).
  • the quantitative differences in disease resistance and biochemical markers among the cim mutants reveal a complex regulation pattern of the different signaling branches of disease resistance responses in Arabidopsis.
  • the deciphering of the Arabidopsis genome will allow the monitoring of expression of all genes, as has been done for S.
  • Metabolite profiling depends largely on the extraction protocol and on the detection method and hence detects only subsets of metabolites, for example compartmental or structural. Besides these technical limitations, it is currently unknown how many changes in secondary metabolites are induced during plant pathogen defense. In a similar study, in barley, only a few changes were detected in the biochemical cytosolic and cell wall composition during pathogen infection (von Roepenack et al, 1998). In conclusion, it is not straightforward at this time to match proteins to gene induction, and metabolites to proteins. Hence it is difficult to match traits to genes.
  • cim695 and cim.713 which exhibit an SA-independent resistance, do accumulate SA to five fold higher levels than wild type.
  • the resistance conferred by mutations cim695 and czm713 may therefore lie in a feedback loop as suggested above, triggering multiple resistance mechanisms including SA-independent resistance pathways which lead subsequently to SA accumulation.
  • SA-independent resistance has been described in the literature. For instance, a jasmonate-dependent defense response in Arabidopsis has been shown to confer resistance to a distinct set of pathogens (Thomma et al, 1998). This wound- and necrotrophic-inducible disease resistance is correlated with the expression of the PDF 1.2 gene (Penninckx et al, 1998).
  • NahG suppresses SAR gene expression in crosses to two of the SAR-constitutive cim mutants, cim ⁇ and cimll, to a baseline resembling that of NahG-expressing plants.
  • NahG expression results in a characteristic gene expression fingerprint in secondary tissue from plants inoculated in primary tissue with avirulent bacteria. This corresponds to the inability of these plants to establish SAR.
  • the corresponding primary tissues in NahG-expressing plants display changes in gene expression which compares very closely to wild-type primary, infected tissue and this sample does not cluster with other NahG samples.
  • the cluster containing EST 209E19T7 defines genes that are transcriptionally induced in NahG-expressing plants.
  • the cluster containing EST 118P18T7 defines genes that are not significantly responsive to SAR- inducing conditions like chemical and genetic induction, but do respond to avirulent bacteria and are downregulated in NahG expressing plants.
  • Phenylalanine ammonia lyase (PAL) and 20 other ESTs that cluster together are repressed by NahG expression, but are induced during the maintenance phase of SAR, for example in cim mutants or 48 hours after BTH treatment.
  • the cluster of "PRl like" genes exhibits similar induction behavior to genes in, the PAL gene cluster but these genes are only weakly suppressed in NahG-expressing plants.
  • the PRl regulon contained 25 other ESTs (17 different genes). These are prime candidates for SAR marker genes and the encoded proteins are likely to play a physiological role in SAR. The estimated 1.5 to 2-fold redundancy of our EST set is a good internal control for this analysis and we also included three replicates of the PR5 and the PerC cDNAs (as well as 28 other relevant cDNAs) on the DNA-microarray. All three copies of the two genes cluster with PRl, showing the robustness of the DNA microarray analysis.
  • cluster analysis of expression profiles provides a tool to derive physiological functions of genes. This is important for sequences with no close homologs in the databank (for example EST 134C2OT7 or EST 192 K7T7) and also for genes with structural similarity to genes with known function (such as asparagine synthetase).
  • EST 134C2OT7 or EST 192 K7T7 genes with structural similarity to genes with known function (such as asparagine synthetase).
  • gene profiling is a powerful tool in understanding signaling cascades, and their interactions.
  • NIM1INPR1 One central regulator gene of the SAR signaling cascade, NIM1INPR1 was cloned independently by two groups and seems to be part of a signal transduction cascade with homology to the mammalian I ⁇ B/NF- ⁇ B pathway (Ryals et al, 1997; Cao et al, 1997; Baeuerle and Baltimore, 1988). Interesting parallels to this conserved pathway in the innate immune response of Drosophila and mammals to the defense response of plants have been drawn (Belvin and Anderson, 1996).
  • T4 DNA polymerase and T4 DNA ligase are purchased from New England Biolabs (Boston, MA) or, if not available from this provider, from Boehringer Mannheim (Indianapolis, IN) or Stratagen (La Jolla, CA). Lysozyme, bovine serum albumin fraction IV and V, and ribonuclease A are delivered by Sigma.
  • AmpliTaq Gold® from Perkin-Elmer (Foster City, CA) or PCR beads ("Ready-to-goTM"; Pharmacia Biotech Inc., Piscataway, NJ) are used.
  • PCR Long range PCR is performed using a special enzyme mix, xTth DNA polymerase (Perkin-Elmer, XL-PCR kit).
  • DNA size ladder either the lamba DNA-Hmdm digest (1-23 kb), the phiX174 DNA-H ⁇ eDI digest (0.1 - lkb), or the 1 kb ladder (1 - 10 kb) from New England Biolabs is employed.
  • Plasmid minipreparations are prepared using Promega's wizard® miniprep kit (Promega Corp., Madison, WI). For maxipreparations, Qiagen's maxiprep kit (Qiagen Inc., Chatsworth, CA) is used.
  • Nucleic acids are separated on agarose gels (low EEO, Sigma).
  • low melt SeaPIaque GTG agarose is used (FMC bioproducts, Rockland, ME)
  • Metaphor gels are used (FMC) and for separation of smaller fragments, as well as for heteroduplex analysis, 10% 19:1 polyacrylamide gels (BioRad) are used.
  • nucleic acids separated on gels are photographed on Polaroid black and white print film, iso3000/36° (Cambridge, MA).
  • nucleic acids are transferred onto GeneScreen Plus membranes (NENTM Life Science Products, Boston, MA) or HybondTM-N+ membranes (Amersham, Arlington Heights, IL). Random primer DNA labeling mix is obtained from GibcoBRL Lifescience. Radioisotopes [ ⁇ - 32 P] dCTP are delivered by International Biotechnologies Inc. (New Haven, CT). Radioactive signals are visualized on Kodak X-OMAT film (Roley, NY).
  • double-autoclaved water is used for the preparation of the buffers and media. Prior to use, the solutions are autoclaved or filter-sterilized through 2 ⁇ m filters (Nalgene, Rochester, NY).
  • Media are supplemented with antibiotics, if needed: 50 mg/1 kanamycin, 50 mg/1 ampicillin, 50 mg/1 rifampicin, 15 mg/1 tetracyclin, 25 mg/1 chloramphenicol, or 30 mg/1 hygromycin.
  • RNA sample buffer 50 ml formamide/bromophenol blue (10:1)
  • Phenol is saturated with Tris-HCl pH 8.0 except for use in the Trypan Blue stain mix. 3. Biological Materials
  • Arabidopsis thaliana (Heynh.) ecotypes Wassilewskija (Ws-0); Columbia (Col-0), and Landsberg erecta (Ler) are obtained from Lehle Seeds (Round Rock, TX).
  • a hygromycin resistant NahG line in the Col-0 background is used for crosses.
  • DH10B F " mcrA ⁇ (mcrCB-bsc.SMR-mrr) endAl, recAl, gyrA96, thi-1, hsdR.17 (rk “ rnk + ), supE44, re/Al, deoR, ( ⁇ 80-i/ ⁇ c ⁇ (/ ⁇ cZ)M15)
  • A(l ⁇ cIZYA- ⁇ rgF) (GibcoBRL)
  • Saccharomyces cerevisiae strain AB1380 Mat ⁇ , psi+, ura3-52, trpl, ade2-l, canl- 100, lys2-l, his5 (Burke et al, 1987)
  • Agrobacterium tumefaciens Agrobacterium tumefaciens strain GV3101, containing the pMP90 vir plasmid (Koncz and Schell, 1986) Pseudomonas syringae strains
  • Erysiphe cichoracearum strain UCSC is provided by R. Innes (Indiana University, IN).
  • pHD-1 is identical to pBluescript but contains a polylinker cloned in the Notl site (Hofte et al, 1993).
  • YAC clones, BAC clones and BAC library filters are obtained from the ABRC stock center (Ohio State University, OH).
  • Cosmid library pOCAl 8 binary vector, bacterial selection: Tet r , plant selection:
  • the endonucleolytic cleavage of D ⁇ A by restriction enzymes is carried out according to the manufactures specifications.
  • CAPS marker development 5 ⁇ l of a 25 ⁇ l PCR reaction is used in a 20 ⁇ l digest.
  • digests are performed in 200 ⁇ l volume, using up to 3 ⁇ g DNA.
  • DNA fragments are either gel purified or, for PCR-generated fragments, purified in solution.
  • gel purification small pieces of low-melt agarose are isolated and DNA is extracted using the AdvantageTM PCR pure Kit (Clontech, Palo Alto, CA).
  • AdvantageTM PCR pure Kit Clontech, Palo Alto, CA
  • DNA in solution e.g. PCR fragments
  • the GeneClean in kit Biol 01, Inc., Vista, CA is used according to the manufactures recommendations.
  • Ligations of DNA fragments to vector DNA are performed according to standard protocols (Sambrook et al, 1989). Usually, 50 to 100 ng vector DNA are mixed with a two- to threefold excess of fragment DNA in a 30 ⁇ l reaction volume. The reactions are performed overnight at 16°C for both sticky and blunt end ligations. One to 5 units T4 ligase are used per reaction. PCR fragments are cloned by TOPO-TA cloning following the instructions of the manufacturer (Invitrogen).
  • a 100 ml LB culture is inoculated with 0.5 ml of a liquid overnight culture of E. coli DH5 ⁇ and grown with shaking at 37°C until an optical density (O. D. 6 oo) of 0.5 has been reached. Cultures are chilled on ice and cells are collected by centrifugation (5 min, 5000g). Cells are resuspended in 7.5 ml transformation buffer I (100 mM RbCl , 45 mM MgCl 2 , 35 mM potassium acetate, 10 mM CaCl 2 , 0.5 mM LiCl, 15%) glycerin, pH 5.8) and incubated for 10 min on ice.
  • transformation buffer I 100 mM RbCl , 45 mM MgCl 2 , 35 mM potassium acetate, 10 mM CaCl 2 , 0.5 mM LiCl, 15%
  • Plasmids are transformed into E. coli using a modified version of the heat-shock protocol (Dagert and Ehrlich, 1979). An aliquot of competent cells is thawed on ice and incubated for 10 min with the DNA. After a heat pulse (1 min, 42°C), the cells are again incubated on ice for 2 min. 200 ⁇ l SOC media at room temperature are added and the mixture is incubated at 37°C for an hour. Cells are plated on LB plates containing the selective antibiotics and in appropriate cases isopropylthiogalactose (IPTG) and 5-bromo-4-chloro-3- indoyl- ⁇ -D-galactose (X-Gal).
  • IPTG isopropylthiogalactose
  • X-Gal 5-bromo-4-chloro-3- indoyl- ⁇ -D-galactose
  • Epicurian Coli® ultracompetent cells E. coli XL-2 Blue; Stratagene are transformed following the manufactures instructions.
  • Agrobacterium To transform a binary vector into Agrobacterium, 40 ⁇ l electrocompetent Agrobacterium cells are thawed on ice and 2 to 10 ng plasmid DNA is added. The mixture is transferred into a prechilled 0.2 ml electroporation cuvette (BioRad) and the cells are electroporated at 2.0 Volts, 600 Ohms, 25 ⁇ Farad, 6 msec time constant using a Gene Pulser (BioRad). Immediately, 1 ml of 2 x YT medium is added and the suspension is incubated at 37°C for one hour under shaking.
  • BioRad Gene Pulser
  • Cells are collected by centrifugation, resuspended in a small volume LB medium and spread onto LB plates containing the appropriate antibiotic (Kanamycin for pCB200). Plates are inoculated 2 to 3 days at 28°C before inoculating 50 ml liquid LB cultures (supplemented with kanamycin and rifampicin) for transformation. 10 ml of this culture are used after 24 - 36 hours incubation at 28°C to inoculate 500 ml LB cultures.
  • Plasmids are isolated following a lysis in 5 M NaCl, 20% sarkosyl solution and then the protocol of the Wizard plasmid minipreparation is followed (Promega, section 2.4.7).
  • the preparation of small amount plasmid DNA is carried out following a method by Birnboim and Doly (1979). 3 ml overnight cultures are concentrated by centrifugation, and resuspended in 200 ⁇ l solution I (50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM ⁇ DTA). The solution is placed on ice and 200 ⁇ l of solution II (0.2 M NaOH, 1% SDS) are added to lyse the cells. Cell debris is precipitated with 200 ⁇ l 5 M potassium acetate and separated from the supernatant by centrifugation. DNA is precipitated from the supernatant by 1/10 volume sodium acetate and 2 volumes ethanol. The pellet is resuspended in 50 ⁇ l water.
  • the Wizard plasmid minipreparation kit (Promega) is used for 3 ml overnight cultures according to the manufactures recommendations.
  • DNA Sequencing is done according to the Sanger 2',3'-didesoxy technology (Sanger et al, 1977), using the big Dye terminator ready reaction mix (ABI/ Advanced Biotechnologies, Inc., Columbia, MD), supplemented with 2 mM MgCl 2 , 80 mM Tris-HCl, pH 8.0 buffer.
  • transposon-mediated sequencing is carried out (Kimmel et al, 1997) using the primer island transposition kit (Perkin Elmer). All sequencing reactions are carried out in Peltier Thermal cyclers (MJ Research Inc., Watertown, MA) and loaded onto 5% acrylamide long-range gels (FMC ready mix). Fluorescence is read by an ABI Prism 377 DNA sequencer (ABI) and bases are called using Phred/Frap/Consed software (University of Washington, Seattle, WA; Ewing et al, 1998; Gordon et al, 1998).
  • sequencer software For sequence assembly and comparison, and restriction site mapping, the sequencer software (Gene Codes Corp., Ann Arbor, MI; Version 4.0 for Windows) is used.
  • BLAST2 software Altschul et al, 1990 are run at NCBI against GenBank (www/ncbi.nlm.nih.gov/BLAST/) or against the Arabidopsis thaliana database (http://genome-www2.stanford.edu cgi-bin/AtDB/nph-blast2atdb).
  • A. thaliana (L.) Heynh. ecotypes Zer, Ws-0 and Col-0 are sown in 200-ml containers in an all-purpose soil mix (Germination Mix, superfine C. Fafard Inc.; Agawam, MA) that has been autoclaved twice for 70 min or once for 2 hours after 24 h hydration to allow fungal sporulation.
  • the seeds are surface-sterilized with bleach (50% v/v commercial bleach, 0.01 % v/v sodium dodecyl sulfate or another wettable agent) for 5 min and for 5 min with 80% ethanol, and washed several times in sterile distilled water before sowing.
  • Plants are grown at 20 - 24°C, 60% relative humidity, 9 hr day/15 hr night (short day, SD), 250 ⁇ E/m 2 s. Prior to germination, the flats are covered with plastic domes. For older plants, the soil surface is allowed to dry between waterings. Alternatively, plants are cultivated on GM-agarose in petri dishes in 0.1% GM top agar under sterile conditions and either SD or long day (LD; 15 hr day, 9 hr night) in high densities (up to 10 plants per 1 cm 2 ).
  • A. thaliana is performed on half-closed buds of flowers from the female parent plant. It is confirmed with the aid of a dissecting microscope that the anthers have not yet released pollen on the stigma. From the male parent plant, a dehiscing anther is removed with forceps and pollen is transferred to the stigma of the female parent.
  • Plants are genetically transformed using an adapted protocol of vacuum infiltration (described by Bechtold et al. (1993)).
  • IM infiltration media
  • section 4F binary vector
  • Infiltration is accomplished by creating and releasing a vacuum in the chamber containing the plants.
  • Plants are then cultured as described above and the seeds are harvested and subjected to selection either on GM plates containing 50 mg/1 Kanamycin (Valvekens et al, 1988) or on soil with 3 to 4 spray treatments of 160 mg/1 Basta® (glufosonate ammonium) in the first two weeks of development in order to identify the transformants (Akama et al, 1995).
  • Transfers are accomplished by spraying oospores on compatible A. thaliana cultivars that are grown under high relative humidity (95%) at 15°C in a culture chamber.
  • Peronospora oospores are isolated from infected A. thaliana leaves by vortexing the leaves in distilled water. Spores are counted in a hemacytometer and the concentration is adjusted to 10 5 - 10 6 spores per ml. The supernatant is then used directly either to spray the planosphaere (Dietrich et al, 1994) or to infiltrate the leaf apoplast of Arabidopsis thaliana with a 1 ml syringe gently pressed onto the subfacial leafside.
  • the apoplast of leaves of four weeks old cim plants, BTH-activated Col-0 (0.3 mM, 2 days prior to infection) and water-treated Col-0 control plants are injected with Pseudomonas syringae pv. maculicola ES 4326 (Schott et al, 1990) or Pseudomonas syringae pv. tomato DC3000 (Dong et al, 1991) at 2 x 10 5 cells per ml. Samples are taken at 0, 1, 3 and 5 days after injection.
  • cim mutants are selected based on in vivo expression of the ER-i/luciferase gene. For crosses of cim mutants to the NahG line, where ER-//luciferase expression is suppressed in all cases, resistance is evaluated on population level.
  • Callose depositions are detected using an aniline blue stain on 5 ⁇ m thick leaf sections (Hunt et al, 1997). Leaves are fixed in 10% formaldehyde solution (45% ethanol, 10% formaldehyde, 5% acetic acid), and embedded in paraffin blocks. Microtom leaf sections (made by Experimental Pathology Laboratory, Durham, NC) are mounted on microscope slides and deparaffinated by two successive 5 min incubations in 100% xylene, two 5 min incubations in 100% ethanol and one 5 min incubation in each of the following: 75% ethanol, 40% ethanol, and water. For callose staining, samples are incubated for 5 min in 0.15 M K HPO 4 and 0.01% aniline blue. Samples are mount in 70% glycerol, 30% aniline blue stain and visualized using ultraviolet epifluorescence (390 - 430 nm) as described in Dietrich et al. (Dietrich et al, 1994).
  • the absorbence of 5 ⁇ l of the supernatant is measured at 595 nm after the addition of 20% v/v protein assay-solution (BioRad; Hercules, CA) in a total volume of 500 ⁇ l.
  • PAL activity in ⁇ Kat/kg protein can be calculated according to: ⁇ E/h x 27.8/mg protein (Kombrink and Hahlbrock, 1986). Protein content is determined as described above (8 A) using the Bradford reagent.
  • samples are harvested in triplicates and analyzed as previously described (Enyedi et al, 1992; Uknes et al, 1993).
  • 0.3 g of frozen, ground A. thaliana leaves are extracted with 3 ml 90% methanol during 20 minutes of sonication. After centrifugation at 4000g for 20 min, the pellet is further extracted with 2 ml 100% methanol and then spun down again (4000g, 20 min), and the two supernatants are combined. Samples are split into two equal parts and dried in a speedvac.
  • the first series of samples (free SA) is suspended in 2.5 ml 5% trichloroacetic acid (TCA; 5 min sonication) and SA is extracted twice with 2.5 ml extraction buffer.
  • Each extract is dried in a speedvac, resuspended in 150 ⁇ l 20% methanol, filtered in a spincolumn (Titan-MSF nylon microsample filters, 0.2 ⁇ m; SRI Scientific resources Inc.; Eatontown, NJ) and transferred to an HPLC autosampler vial.
  • a spincolumn Tian-MSF nylon microsample filters, 0.2 ⁇ m; SRI Scientific resources Inc.; Eatontown, NJ
  • 50 ⁇ l of each extract is injected in a C-18 HPLC column (Dynamax 60, Rainin Instrument Comp.; Wobura, MA).
  • RNA Total RNA is isolated from 1 g frozen, powderized leaf tissue that is ground to a fine powder in liquid nitrogen. The samples are resuspended in 2.5 ml RNA extraction medium (50 mM Tris-HCl, pH 8.0, 4% w/v 7-amino salicylic acid, 1% w/v 1,5-naphtalene disulfonic acid (Arcos Chemicals, NJ) and 2.5 ml water-saturated phenol (Lagrimini et al, 1987). After addition of 2.5 ml chloroform, phases are separated by centrifugation (10 min at 7000g).
  • 2.5 RNA extraction medium 50 mM Tris-HCl, pH 8.0, 4% w/v 7-amino salicylic acid, 1% w/v 1,5-naphtalene disulfonic acid (Arcos Chemicals, NJ) and 2.5 ml water-saturated phenol (Lagrimini et al, 1987). After addition of 2.5 ml
  • aqueous phase is transferred to a new tube and nucleic acids are precipitated with the addition of 1/10 volume 3 M sodium acetate, pH 5.2 and 2 volumes ethanol at -20°C for 30 min.
  • Precipitates are spun down (10 min at 7000g) and the dried pellets are resuspended in 2 ml double-distilled water.
  • RNA is precipitated overnight at 4°C with the addition of 1.25 ml 8 M LiCl.
  • the precipitate is pelleted by centrifugation (10 min at 7000g) and the pellet is rinsed with 80% ethanol.
  • RNA pellets are resuspended in 100 ⁇ l water and the absorbence at 260 nm and 280 nm are measured in a spectrophotometer (UV-160 visible recording spectrophotometer, Shimadzu; Columbia, SC) to determine the amount and the purity of the RNA.
  • a spectrophotometer UV-160 visible recording spectrophotometer, Shimadzu; Columbia, SC
  • PolyA RNA is enriched using a poly dT magnetic bead technique (Promega). One mg total RNA is hybridized to the poly dT-biotin nucleotides according to the manufactures recommendations. Avidin-coated magnetic beads allowed the separation of polyA RNA from non poly-adenylated RNA. The RNA is released from the poly dT probes in low salt buffers and concentrated by precipitation with ethanol. 10% of the obtained polyA RNA is used for spectrometric analysis.
  • Plant DNA is extracted using the CTAB-method described by Rogers and Bendich (1988). 1 - 2 leaves per sample are ground in liquid nitrogen with a Polytron (Brinkmann Instruments Inc. Westbury, NY), then vortexed with 200 ⁇ l 2 x CTAB buffer. After heating at 65°C for 15 min, 200 ⁇ l chloroform are added and the well-mixed extraction is centrifuged for 2 min (10,000g). DNA is precipitated from the resulting supernatant with 3 volumes of ethanol at -20°C. The precipitate is spun down (10,000g, 15 min) and the pellet is rinsed with 70% ethanol. After drying, the pellet is resuspended in 30 ⁇ l 10 mM Tris-HCl, pH 8.5. D. Plant DNA extraction for Southern blot analysis and pooled progeny analysis (F3 populations)
  • 1 to 2 g ground tissue are mixed on ice with 12 ml extraction buffer (0.1 M Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0, 0.5 mM NaCl, 10 mM ⁇ -mercaptoethanol; Dellaporta et al, 1983). After adding 0.8 ml 20% SDS, the extract is incubated at 65°C for 10 min. Cell debris is precipitated with 4 ml 5 M potassium acetate during a 20 min incubation at 4°C and separated from the supernatant by centrifugation at 4°C (10 min, 8000g).
  • the supernatant is filtered through prewetted Miracloth and DNA is precipitated with 8 ml isopropanol at -80°C (30 min). DNA is pelleted, dried and resuspended in 4 ml TE and again precipitated with sodium acetate and ethanol. After resuspension in 400 ⁇ l TE, an RNase A digest is performed for 10 min at 37°C (final concentration RNase A: 50 ⁇ g/ml). The samples are extracted once with a 1:1 mixture of phenol : chloroform, once with chloroform, precipitated by addition of sodium acetate and ethanol and resuspended in 50 ⁇ l water. For less tissue (15 - 500 mg), the protocol is scaled down 20-fold and the RNase treatment is omitted. 10 ⁇ l is used in restriction digests for Southern blot analysis.
  • BAC DNA minipreparations are done according to a protocol by Colltt et al. (1998), using a modified alkaline lysis method.
  • 3 ml LB overnight cultures containing either 50 ?g/ml kanamycin (IGF BACs) or 12.5 ⁇ g/ml chloramphenicol (TAMU BACs) are pelleted and resuspended in 100 ⁇ l of chilled resuspension solution (25 mM Tris-HCl, pH 8.0, 50 mM glucose, 10 mM EDTA, pH 8.0).
  • Cells are lysed by adding 200 ⁇ l lysis buffer (0.2 NNaOH, 1% SDS).
  • Cell debris and chromosomal DNA are precipitated by 150 ⁇ l 5 M potassium acetate, pH 4.8. After a 5 min centrifugation at maximal speed in a tabletop centrifuge, the supernatant is transferred to a new tube and the crude DNA is precipitated by adding 2 volumes of ethanol. The DNA is pelleted for 5 min as before, washed in 70% ethanol and resuspended in 100 ⁇ l of TE buffer containing 0.1% SDS and 100 ⁇ g/ml proteinase K, followed by a one-hour incubation at 37°C. The reaction is extracted with 100 ⁇ l phenol : chloroform (1 :1), then with 100 ⁇ l chloroform. The DNA is precipitated with 2 volumes ethanol and washed as before, then resuspended in 50 ⁇ l water. F. BAC DNA maxipreparations
  • BAC DNA a protocol provided by Choi et al. (1995) is used in a modified version. 2 liter overnight bacterial cultures (LB plus antibiotic) are harvested by centrifugation. The pellet is resuspended in a lysozyme solution (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0, 5 ⁇ g/ml lysozyme) and incubated on ice for 5 min. 40 ml of an alkaline lysis solution (0.2 NNaOH, 1% SDS) are added and after 5 min incubation at 4°C, 30 ml of ice-cold potassium acetate solution (5 M, pH 4.8) are added.
  • a lysozyme solution 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0, 5 ⁇ g/ml lysozyme
  • 40 ml of an alkaline lysis solution 0.2 NNaOH
  • DNA is precipitated by 0.6 volumes isopropanol at -80°C. DNA is pelleted (30 min, 15000g), dissolved in TE and subjected to RNase A digest (20 ⁇ g/ml, 45 min at 37°C). DNA is extracted with an equal volume phenol : chloroform (1:1), and with chloroform, then precipitated with sodium acetate and ethanol. DNA is taken up in 200 ⁇ l water.
  • the purification followed the Qiagen maxipreparation protocol for very- low-copy cosmids (Qiagen Inc., Valencia, CA).
  • the DNA is taken up in 20 ⁇ l of 10 mM
  • the pellet is resuspended in 0.4 ml TE and the DNA is precipitated with 10 ⁇ l ammonium acetate and 1 ml ethanol. DNA is resuspended in 50 ⁇ l TE. 10. Analysis Of Membrane-Bound Macromolecules
  • RNA blot analysis 10 ⁇ g of purified RNA (0.25 ⁇ g poly A RNA) in 15 ⁇ l water is heated for 15 min at 65°C after addition of 34 ⁇ l RNA sample buffer (65% v/v formamide:bromophenol blue (1 :10), 21.5% v/v formaldehyde (37%), 13% MSE, 0.5% v/v ethidium bromide (10 mg/1)).
  • the samples are loaded on a denaturing agarose-gel (1.2% w/v agarose, 1 x MSE, 3% v/v formaldehyde) and run at 12 V/cm for 1 hour (Sambrook et al, 1989).
  • RNA is linked to the membrane by UV-crosslinking (Stratalinker®, Stratagene; La Jolla, CA) at 1200 ⁇ J.
  • hybridization-buffer 500 mM NaPO 4 , pH 7.0, 1 mM EDTA, 7% v/v SDS, 1% w/v BSA (fraction V); Church and Gilbert, 1984
  • 100 ⁇ l of an [ ⁇ - 32 P] dCTP labeled probe (random priming, Feinberg and Vogelstein, 1983) is added and the membrane is incubated overnight; Church and Gilbert, 1984).
  • the membrane is washed twice with washing buffer (40 mM NaPO 4 buffer, pH 7.0, ImM EDTA, 1% v/v SDS) containing 5 g/1 bovine serum albumin (BSA) (each 20 min, 65 °C) and once without BSA (15 min, 65°C).
  • BSA bovine serum albumin
  • the blot is exposed for at least half an hour to a Phosphorlmager® screen (Molecular Dynamics; Sunnyvale, CA) and, depending upon the observed intensity, exposed for several hours to days on XAR-5 scientific imaging film at - 80°C in the presence of an intensifier screen.
  • the experiments are repeated at least twice for every probe.
  • Table 4 Clones Used As Probes For The Characterization Of Gene Expression In cim Mutants In Northern Blot Analysis And DNA Microarrays.
  • DNA is digested overnight in 200 ⁇ l reaction volume.
  • DNA is precipitated by sodium acetate and ethanol and resuspended in 20 ⁇ l water.
  • the DNA is loaded in presence of 4 ⁇ l loading dye onto a 0.9% TBE agarose gel (run either overnight at 1 V/cm, or 3 to 4 hours at 4 V/cm).
  • the gel is soaked in 0.25 NHC1 for 20 min, then for 30 min in denaturation solution (1.5 M ⁇ aCl, 0.5 M ⁇ aOH) and twice for 20 min each in neutralization solution (3M ⁇ aCl, 0.5 M Tris-HCl, pH 7.5), as described by Ausubel et al. (1987).
  • the D ⁇ A is transferred overnight onto a nitrocellulose GeneScreen Plus® membrane (Du Pont- ⁇ ew England Nuclear) in 10 x SSC, as described above for Northern blots (section 10A). Hybridization and washing are performed as for Northern blot analysis.
  • Dot blots are performed using a Bio-Dot apparatus (Bio-Rad) as described by G ⁇ rlach et al (1995). Two ⁇ g of total RNA are denatured in 6 x SSPE (20 x SSPE: 20 mM EDTA, pH 7.4, 3 M NaCl, 0.2 M sodium phosphate, pH 7.4) containing 20% deionized formaldehyde for 15 min at 55°C and then chilled on ice. Two volumes of ice-cold 15 x SSPE are added and the samples are applied to a GeneScreen Plus® membrane which is pretreated with 12 x SSPE. After crosslinking, prehybridization and hybridization are performed as described for Northern blot analysis (section 10 A).
  • BAC filters are hybridized according to the TAMU BAC filter manual (Version 2, http://tamu.edu:8000/ ⁇ creel/bacman2.html). New filters are prehybridized at 65°C twice for 8 hours in prehybridization buffer (0.5 M NaHPO 4 , pH 7.2, 7% SDS, 1% BSA, (fraction V), 1 mM EDTA, 10 mg/ml sheared salmon sperm DNA), used filters only once. After adding the probe, hybridization took place in the same buffer for 18 to 36 hours at 65°C. Filters are washed with 0.5 x SSC, 0.1% SDS 3 times for 20 min at 65 °C and exposed on a Phosphorlmager screen (MolecularDynamics) as described above.
  • prehybridization buffer 0.5 M NaHPO 4 , pH 7.2, 7% SDS, 1% BSA, (fraction V), 1 mM EDTA, 10 mg/ml sheared salmon sperm DNA
  • proteins from 0.25 g pulverized tissue are extracted in 500 ⁇ l extraction buffer (0.25 mM Tris-HCl, pH 6.8, 4.5 M Urea, 2% SDS, 5% ⁇ -mercaptoethanol). Equal amount of protein (determined by Bradford reagent, section 2.8.1) are loaded onto a 10% Tris-glycine gel (Novex, San Diego, CA) and run at 10 V/cm for 1 to 2 hours in 1 x running buffer (25 mM Tris-HCl, pH 8.3, 250 mM glycine, 0.1% SDS).
  • the gel is soaked for one hour in equilibration buffer (20 mM Tris-HCl, pH 8.0, 150 mM glycine, 20% methanol) and proteins are transferred by electroblotting onto a nitrocellulose membrane (100 V, 1 hour constant current, Novex X-Cell II Blot module).
  • the membrane is washed for 10 minutes in wash buffer (1 x PBS, 0.1% Tween-20), then incubated for one hour in blocking buffer (1 x PBS, 0.1% Tween-20, 5% milk powder).
  • the PR-1 specific antibody is bound at 4°C overnight in incubation buffer (1 x PBS, 0.1% Tween- 20, 1% milk powder) and unspecifically bound antibodies are removed by washing the membrane four times in wash buffer.
  • the second antibody (antilgG rabbit conjugated with alkaline phosphatase) is bound for 3 hours at room temperature in incubation buffer and the membrane is washed as before.
  • the protein is detected using the NBT (p-nitroblue tetrazolium) method as described by Harlow and Lane (1988).
  • PCR Polymerase chain reactions
  • SSLP microsatellite amplification
  • Each reaction contained 5 ⁇ l of 10 fold diluted DNA from the DNA minipreparation (see above, section 2.9.3), 2.5 ⁇ l PCR 10 x reaction buffer (Perkin Elmer), 2 ⁇ l of a 10 mM dNTP stock solution, 1 ⁇ l of forward and reverse primers (20 ?M primer stocks), 0.3 ⁇ l AmpliTaq® gold DNA polymerase (5 U/ml, Perkin Elmer) and water qs. 25 ⁇ l.
  • a typical reaction temperature cycle is: 10 min at 94°C, 40 cycles of 15 sec 94°C, 15 sec 55°C and 30 sec 72°C, then a last polymerization step at 72°C for 10 min.
  • the PCR conditions are more variable depending on the length of the expected fragment size (2 min for 2 kb) and the melting temperature of the primers.
  • a typical thermocycle program is: 10 min at 94°C, 35 cycles of 30 sec 94°C, 30 sec 56°C and 2 min 72°C, then a last polymerization step at 72°C for 10 min (Konieczny and Ausubel, 1993). If PCR beads are used (Pharmacia Biotech), only 2 min at 94°C are used prior to thermo cycling. Pooled PCR samples are used for restriction digest of fragments. For sequenced PCR fragments, restriction fragment polymorphisms are identified using the dCAPS software (Neff et al, 1998) rather than by random trial.
  • PCR fragments are pooled from at least two PCR reactions to minimize sequence differences generated during PCR, purified in solution (section 2.4.2) and diluted to a concentration of 100 ⁇ g/ml.
  • PCR fragments are TA-cloned into pCR2.1- TOPO following the instructions given by the manufacturer (Invitrogen).
  • a set of 30 SSLP and CAPS primer pairs (see Appendix) is used on segregating F2 populations to establish an initial map position. Genetic map distances are determined using MAPMAKER 3.0 b (Lander et al, 1987; Lincoln et al, 1992) run on a Sun SPARC workstation. Recombination frequencies are calculated using the MAPMAKER F2 algorithm and converted to map distances in centiMorgans (cM) using the Kosambi function (Kosambi, 1944). D. Long range PCR
  • the lower phase (40 ⁇ l) contained final concentrations of 1 x buffer (Perkin-Elmer), 200 ⁇ M of each dNTP, 1.25 mM MgOAc, and 1.5 ⁇ M of each primer. It is covered with a wax bead and heated at 80°C for 5 min, then chilled to 20°C.
  • the upper phase (60 ⁇ l), containing 450 pM DNA, 4 Units rTth polymerase (Perkin Elmer) and 1 x buffer, is added on top of the lower phase.
  • Thermocycling is as follows: 94°C, 1 min, 16 cycles (94°C 30 sec, 68°C 10 min), and 14 cycles (94°C 30 sec, 68°C 10 min with 15 sec extension every cycle), 72°C, 10 min. 10 ⁇ l of the reactions are analyzed on 0.9% agarose gels.
  • genomic fragments (200 bp - 1 kb) are amplified from two A. thaliana ecotypes using PCR primers with inco ⁇ orated T7 (5'taatacgactcactataggg - SEQ ID NO:l) and SP6 (5'atttaggtgacactatagga - SEQ ID NO:2) promoters.
  • T7 5'taatacgactcactataggg - SEQ ID NO:l
  • SP6 5'atttaggtgacactatagga - SEQ ID NO:2 promoters.
  • both sense and antisense RNA probes are made, according to the manufacturers instructions of the MisMatch DetectTM II kit (Ambion, Inc., Austin, TX).
  • Equal volumes of SP6 transcripts are mixed to T7 transcripts of the other ecotype, heated at 95 °C for three min and cooled to room temperature. Different RNase digestions of the homo- and hetero-RNA duplices are performed as recommended. Di
  • Ligase is heat inactivated at 70°C (15 min), DNA is precipitated by ethanol, and circularized DNA is cleaved with Evwl (T7 end) or EsrBI (Sp6 end) in a 10 ⁇ l volume reaction.
  • Evwl T7 end
  • EsrBI EsrBI
  • pBelo flanking DNA standard PCR is performed with 56°C annealing temperature and 2 min extension time at 72°C, using for the T7 end the primers: 5'ttcccaacagttgcgcagc (S ⁇ Q ID NO:3) and 5'tcttcgctattacgccagct (S ⁇ Q ID NO:4), and for the Sp6 flanking DNA, the primers: 5'tcacacaggaaacagctat (S ⁇ Q ID NO:5) and 5'acacaacatacgagccggaa (S ⁇ Q ID NO:6).
  • PCR fragments are purified as described in section 2.4.2
  • thermal asymmetric interlaced PCR (TAIL PCR) is used as described by Liu et al. (1995).
  • One out of six low stringency primers is used in successive PCR with three nested high stringency primers on either the right border, or the left border of the T-DNA.
  • Reactions are performed on 5-fold diluted CTAB DNA minipreparations.
  • the products of the second and third PCR are analyzed on agarose gels. If a small size difference between the second and the third PCR fragment is detected, the product of the third PCR is either purified for direct sequencing or cloned for sequencing by TA cloning into the vector pCR2.1. 12.
  • Est stock cultures are duplicated from liquid cultures by transferring cells into 96 well flat bottom culture plates (Falcon), containing LB freezing buffer (LB supplemented with 36 mM K 2 HPO 4 , 13.2 mM KH 2 PO 4 , 1.7 mM sodium citrate, 0.4 mM MgSO 4 , 6.8 mM (NH 4 ) 2 SO 4 , 4.4%o (v/v) glycerol) with a disposable 96 needle inoculation tool. After growth overnight at 37°C, the cultures in microtiter plates are sealed using self-adhesive plastic seals (USA Scientific Plastics) and stored at -80°C.
  • LB freezing buffer LB supplemented with 36 mM K 2 HPO 4 , 13.2 mM KH 2 PO 4 , 1.7 mM sodium citrate, 0.4 mM MgSO 4 , 6.8 mM (NH 4 ) 2 SO 4 , 4.4%o (v/v) glycerol
  • PCR For PCR, cultures are diluted 1 :100 in 10 mM Tris-HCl, pH 8.5 and 10 ⁇ l of the dilution per 50 ⁇ l PCR reaction is used.
  • a QfiU2 machine (Genetix, Wales, GB) is used.
  • a Qpix robot (Genetix) is used.
  • Membranes are put onto an agarose plate and colonies grew overnight. Cells are lysed on the membrane, and DNA is fixed on the support as described by Nizetic et al. (1991).
  • IEF Isoelectric focusing
  • the tube gels are sealed to the top of stacking gels which are on top of 10% acrylamide slab gels (0.75 mm thick) and SDS slab gel electrophoresis is carried out for about 4 hrs at 12.5 mA.
  • SDS slab gel electrophoresis is carried out for about 4 hrs at 12.5 mA.
  • the slab gel is fixed in a solution of 10% acetic acid/ 50%) methanol overnight.
  • phosphorylase A 94,000
  • the polyacrylamide gel is soaked in the staining solution (0.1% w/v Coomassie Blue, 16% v/v acetic acid, 42% v/v methanol) for several hours.
  • the gel is destained in aqueous acetic acid (12% v/v isopropanol, 16% v/v acetic acid.
  • the gel is fixed at room temperature successively in 20% (w/v) trichloroacetic acid for one hour, twice for 30 min in 40% (v/v) ethanol, 10% (v/v) actic acid and twice in water for 20 min.
  • the gel is soaked for 30 min in a 10% (w/v) glutaraldehyde solution, followed by 3 washes in water (20 min each).
  • the proteins are stained for 30 min in silver diamine solution (freshly made up 0.26% (w/v) NaOH, 1.8% (w/v) ammonia, 3% (w/v) silver nitrate), washed three times in water and developed for 10 min in developing solution (0.05% (w/v) citric acid, 0.02% (v/v) formaldehyde) then transferred into stop solution (40% (v/v) ethanol, 10% (v/v) acetic acid).
  • the stained gels are dried between sheets of cellophane.
  • a PCR on colonies is performed. Bacteria are subcultured in 96 well format plates overnight in LB freezing media (supplemented with Amp). Aliquots of the cultures are diluted 1 : 100 in 10 mM Tris-HCl, pH 8.5 and 10 ⁇ l of the dilutions are used per 50 ⁇ l PCR.
  • the PCR mix contained per 50 ⁇ l reaction 5 ⁇ l AmpliTaq buffer (Perkin Elmer), 10 ⁇ l dNTP mix (20 mM each), 2 times 5 ⁇ l modified primers (M13 forward: 5'amino tgtaaaacgacggccagt - SEQ ID NO:7, M13 reverse: 5'amino ggaaacagctatgaccat - SEQ ID NO:8, 10 ⁇ M each), 1 ⁇ l AmpliTaq Gold (Perkin Elmer).
  • PCR thermocycling is performed in a Perkin Elmer 9700 PCR machine as follows: 10 min at 95°C, 40 cycles of 30 sec at 95°C, 30 sec at 51°C, 2 min extension at 72°C, followed by a 10 min extension at 72°C. 5 ⁇ l of all PCR reactions are run on a 1.2% agarose gel (1 x TBE, 6 V/cm). After further purification through QIAquick-96 microfiltration columns (Qiagen) and lyophilization, PCR products are resuspended in 10 ⁇ l of 3 x SSC and spotted onto silane-coated glass slides (Synteny, Inc., Fremont, CA). The DNA is rendered single stranded by heat or alkali treatment.
  • single strand reverse transcription from an oligo-dT primer is performed in presence of Cy3-dCTP or Cy5-dCTP (Amersham, Arlington Heights, IL) using 600 ng polyA RNA per sample.
  • the reverse transcription reaction is performed in a 25 ⁇ l volume with 2 ⁇ g oligo(dT) 21 -mer, 500 ⁇ M each of dATP, dGTP and dTTP, 280 ⁇ M dCTP, 40 ⁇ M of Cy3 dCTP or Cy5 dCTP, 40 units RNAsin (Promega) and 200 units Superscript II reverse transcriptase (Life Technologies) in 1 x Superscript first strand buffer.
  • RNAs from non-coding yeast genomic DNA are added into the reverse transcription reaction at 0.006 ng, 0.06 ng and 0.6 ng, respectively (ratios of the control RNA to polyA RNA are 1:100,000, 1:10,000, and 1:1000 (w/w), respectively).
  • ratios of the control RNA to polyA RNA are 1:100,000, 1:10,000, and 1:1000 (w/w), respectively.
  • the reactions of two samples are combined and treated with 5 ⁇ l of 0.5 M sodium hydroxide and 5 ⁇ l of 10 mM EDTA for 10 min at 65°C to stop the reaction and degrade the RNA.
  • Probes are purified using two successive Chroma Spin 30 gel filtration spin columns (Clontech) and lyophilized. Probes are resuspended in 20 ⁇ l hybridization buffer (5 x SSC, 0.2% SDS) and applied to the microarray (Schena et al, 1996). Hybridization is carried out at 60°C for at least 12 hours (Synteni). The slide is rinsed for 5 min each in 5 x SSC, 0.1% SDS and in 0.2 x SSC, 1% SDS at room temperature. Two-channel, confocal laser microscopes are used to scan the fluorescence emission after excitation at 532 nm and 633 nm (Shalon et al, 1996).
  • tomato (Pst) DC3000 avrRpml at 106 cfu/ml harvested 44 hours after inoculation tissue harvested after 4 hours (12) or 48 hours (13) after treatment with 0.3 mM BTH; tissue harvested 48 hours after inoculation with a suspension of 105 spores/ ml of the compatible isolate of Peronospora parasitica pv. Emwa (14) and of infected primary (15) and systemic secondary (16) tissue of wild-type plants harvested 44 hours after inoculation with Pst DC3000.
  • PR-1 The expression of the PR-1 gene is the most reliable marker for the onset of SAR in Arabidopsis (Uknes et al, 1992).
  • the ER-7 gene encodes a small (preprotein: 17677 Da, 161 aa; cleaved: 14880 Da, 135 aa), acidic (pl of approximately 4.0) apoplastic protein.
  • PR-1 Although the function of the PR-1 protein remains unknown, several studies have shown that PR-1 might play a direct role in conferring resistance to fungal pathogens. PR-1 has an antimicrobial activity in vitro and confers resistance to oomycetes when overexpressed in planta (Alexander et al, 1993).
  • PR-1 protein homologs were found not only in di- and monocotyledonous plants, but also in mammals and insects: PR-1 belongs to a family of cystein-rich secretory proteins (CRISPs) that groups mammalian SCP/TPX1 (sperm coating glycoprotein/testis specific protein) insect AG3/AG5 (venom allergen), fungal SC7/SC14 (Schizophyllum commune), and plant PR-1 proteins together.
  • CRISPs cystein-rich secretory proteins
  • the sequence identities of the homologs range between 30% and 80%). While the function of the human PR-1 homolog is less well understood, in insects, PR-1 -like proteins make up a major venom allergen.
  • PR-1 protein The close sequence homologies of the PR-1 protein to secreted cystein-rich proteins suggest a possible function of PR-1 in defense similar to other small antimicrobial peptides, such as plant defensins or thionins. This finding is consistent with the previously observed effects of PR-1 on fungal pathogens.
  • ER-7/luciferase plants were sprayed with three chemical activators of SAR: SA (5 mM), INA (375 ⁇ M) or BTH (375 ⁇ M or 5 mM) and luciferase activity was determined every 24 hours during a period of four days. For each measurement, six samples consisting of six leaves each were harvested. INA and BTH treatment at the standard concentration of 375 ⁇ M caused an induction of luciferase activity of more than 2000 fold within 48 hours and this level was maintained for at least two more days.
  • SA 5 mM
  • INA 375 ⁇ M
  • BTH 375 ⁇ M or 5 mM
  • ER-7/luciferase plants were sprayed at 24 and 12 hours before the pathogen treatment with 7.5 mM luciferin to inactivate luciferase (Millar et al, 1992) and to reduce background induction. At 0 hours, approximately 50% of the area of fully developed leaves were infiltrated with either water or with a spore solution of E. parasitica Emwa (Table 5). The incompatible interaction triggered a more than 150 fold systemic induction of the luciferase activity within three days. This induction could also be followed in vivo.
  • Table 5 Induction of the ER-7/luciferase transgene after treatment with the avirulent pathogen E. parasitica Emwa ( ⁇ mwa). Luciferase activity was inactivated 24 and 12 hours before the experiment by luciferin treatment. Values represent relative inductions of luciferase in vitro activity compared to water treated plants (set to 1). Each time point consists of 20 leaves infiltrated with either water or a spore solution of P. parasitica Emwa (10 5 spores per ml) derived from 10 different plants.
  • mutants that constitutively express the ER-7/luciferase gene
  • the 6 ⁇ line was submitted to EMS mutagenesis.
  • Mutant screens in A. thaliana are usually performed in the M2 generation.
  • the mutations are heterozygous and the plants are chimeric, since in A. thaliana seeds, at the time of the chemical mutagenesis, 12 cells represent the origin of the vegetative parts of the Ml plants.
  • the size of the Ml population is dictated by the cost of the mutagenesis on the one hand and the size of the genome, and the mutation rate of the mutagen (mutations per genome and generation) on the other hand.
  • GECN genetically effective cell number.
  • the required size of the M2 population can be derived from the size of the Ml to maximally exploit the genetic potential of the Ml .
  • the frequency of putative mutants did however not vary with the conditions, and was approximately 2.4 x 10 " .
  • 160 Ml pools contained at least one plant that constitutively expressed the ER-7/luciferase gene. In total, 603 putative mutants were identified in this in vivo screen. Almost all of them were confirmed by the in vitro luciferase analysis in the M2 or their progeny. The phenotypes were therefore considered to be caused by genetic mutations. We expected a multigenic regulation of ER-7 gene expression and, thus, a high number of mutants.
  • mutants were further increased by the redundancy of mutant identifications in the M2, which provided several fold coverage of the Ml gene pool (M2 saturation of the Ml) and the fact that several independent mutations per gene (alleles) might have been identified (Ml saturation of the genome). To find out how many independent genes are actually involved in the SAR cascade, all 603 mutants would have to be mapped or crossed to each other.
  • T-DNA insertion mutagenesis might also yield mutants with constitutive ER- 7/luciferase expression, within a reasonable population size.
  • T-DNA insertions to mutagenize the genome has the disadvantage of being labor-intensive and having a low mutation rate (1-2 inserts per genome, compared to over 80 point mutations per genome by EMS), but has the potential advantage of easy cloning of the mutated, "tagged' gene, by plasmid rescue or other techniques.
  • Tl population primary, hemizygous transformants
  • ER-7 luciferase expression 10,000 Tl lines were screened for in vivo PR-1 /luciferase activity, and 80 of them were retested in the T2 generation. Strong luciferase activity was confirmed in 7 T2 populations (see section 4 ⁇ , table 15). This low rate of confirmation in the T2 generation was caused by the low cut-off of luciferase activity that was used in the Tl as a criteria for selection in order to also find codominant mutants. Therefore, more false positive plants were retained than in the EMS screen.
  • the goal of this study was to identify SAR activated mutants that do not show spontaneous cell death, according to the definition of cim mutants.
  • the 603 mutants were therefore subjected to a Trypan Blue lesion staining in the M2 and the M3 generation and examined both macroscopically and microscopically for cell death. More than 90 mutants did not show macroscopic patches of cell death, but only 16 did not have any cell death under our growth conditions, as revealed by microscopy after staining. Most of the other mutants developed necrotic lesions at some stage of their life cycle, mostly in the leaf tips.
  • mutant 779 One of the mutants with spontaneous cell death, designated mutant 779, was included in all the following experiments as a control. Mutant 779 displayed patches of autofluorescence and callose that normally accompany HR-like cell death. No callose was detected in the 16 cim mutants. Although free of lesions, pleiotropic phenotypic alterations in the 16 cim mutants were not separated from the mutation that caused constitutive ER gene expression by three backcrosses. In general, cim mutants have a prolonged life cycle, a delayed flowering time (one to four weeks later than wild-ty ⁇ e Col-0) and they set fewer seeds (approximately one third of Col-0). Some mutants also showed reduced germination.
  • Leaf morphology varied from long, often curly leaves (c/m205, c/m716), to extremely small, round leaves (cim677, c/m810). Mutant cim677 showed a bright green leaf pigmentation, other cim mutants (c/m713, c/m810) had dark-green leaves. However, normal leaf morphology was also found, albeit mostly in the weaker mutants, c/m328 and c/m658 (weakness based on ER-7 gene expression and SA content, see below) as well as in the mutant cim713 that differed from wild-type only in size.
  • All 16 mutants originated from different seed pools and were therefore considered independent mutations. All mutants were backcrossed at least three times to the ER- 7/luciferase parental line.
  • Table 6 Genetic characteristics of the 16 cim mutants identified in the ER-7/luciferase EMS mutant screen.
  • cim ⁇ 1 originated from the same Ml seed lot as cim , they may be identical
  • F2 populations of backcrosses containing 20 to 100 plants were screened for constitutive luciferase activity and the resulting data were subjected to ⁇ 2 analysis (Table 6).
  • the expression of the reporter gene in the FI confirmed in random samples by Northern blot analysis for endogenous ER-7 expression, indicated that in all but two cases (ciml, cim677) the mutant phenotype was dominant.
  • the analysis of the F2 segregation ratios suggested that many of these mutations were not fully penetrant.
  • cim phenotype Populations of usually 50 to 80 F2 plants (mutant crossed to ecotype Ler) with preselected phenotype were used to look for linkage between the cim phenotype and genetic markers. About 30 SSLP and CAPS markers that were evenly distributed throughout the genome, were chosen to find a primary linkage. If available, closer markers were chosen to further define this map position.
  • c/m713 was placed on the genetic map of Arabidopsis thaliana on chromosome 1 between markers mi291a and markers nga280 (see below, section 4B).
  • cim205 is also located on chromosome 1, between markers nga280 (20 recombinants in 116 analyzes meiosis) and ml85 (19 recombinants in 148 meiosis).
  • c/m8 is located on chromosome 2, between markers ve017 (16 recombinants in 148 meiosis) and ngal68 (9 recombinant in 122 meiosis).
  • cim695 lies on chromosome 5 between markers DFR (22 recombinants in 106 meiosis and LFY (17 recombinant in 110 meiosis).
  • the map positions of the mutations on chromosome 1 and 2 do not match the map position of known mutations in genes encoding functions in disease resistance and or SAR.
  • Mutant cim695 is in a region of chromosome 5 termed MRC-J, which contains a number of R gene homologs (Botella et al, 1997; Holub and Beynon, 1997). c/m205 and ctm713 map close to but distinct from cpr ⁇ 5 (Clarke et al, 1998).
  • SAR SAR-degrading NahG lines.
  • a control treatment with a virulent Erysiphe cichoracearum pathogen caused a 7-fold increase in total SA content after three days of infection.
  • Table 7 Salicylic acid content is increased in most cim mutants. Total salicylic acid content was determined by HPLC separation of an organic tissue extraction. Results (in ng SA per mg tissue fresh weight) are mean values and standard deviations of three independent measurements. As a comparison, SA content was measured in wild-type tissue infected with Erysiphe cichoracearum, harvested 3 days after inoculation.
  • mutant total SA (ng/mg fresh weight) wild-type 296 +/- 25 wild-type + Erysiphe 2030 +/- 890 cim% 1958 +/- 835 cim.205 1657 +/- 436 cim32S 899 +/- 16 cim658 294 +/- 21 cim ⁇ 77 4154 +/- 211 cim695 2256 +/- 223 cim7 ⁇ 3 1500 +/- 78 cim7 ⁇ 6 1350 +/- 267 cimSl 3415 +/- 331 cimS24 2190 +/- 491
  • lesion mimics have higher levels of S A than cim mutants, but the two distributions are overlapping: cim mutants can have more SA than lesion mimic mutants, cim mutants are hence not simply a weak subclass of lesion mimic mutants, but ought to be considered as a distinct class of mutants.
  • Free SA content was about tenfold less than total SA and was always correlated to the total SA content thus excluding from our collection mutations in the regulation of this equilibrium or in the degradation/conjugation of SA. Based on SA content (and ER gene expression, see below), mutants can be classified into strong cim mutants (e.g.
  • HI. cim mutants can accumulate low levels of camalexin Plants under pathogen attack accumulate antimicrobial molecules, called phytoalexins.
  • the major phytoalexin in Arabidopsis is camalexin, derived from a tryptophan precursor.
  • Camalexin can be visualized under UV light after TLC separation of methanol plant extracts. Both a synthetic standard and tissue harvested after Pseudomonas infection were used as controls.
  • the Rf for synthetic camalexin was a bit lower (0.77) than the R f for camalexin in complex mixtures (0.78).
  • Mutants c/m328, cim677, cim.716, 779 and cimSlO accumulated between 1 ⁇ g camalexin/cm (based on the synthetic camalexin standard) and the amount that accumulated in Esew_7omo « ⁇ s-infected tissue after 3 days (literature value: 1.2 ⁇ g carnal exin crn ; Glazebrook and Ausubel, 1994).
  • Mutant cimS24 accumulated less than 1 ⁇ g/cm 2 , and c/m8, czm658, cim695 and ciml 13 did not show substantial amounts of camalexin.
  • D. cim mutants exhibit resistance to pathogenic microorganisms In order to show that SAR (or LAR) is constitutively activated in cim mutants, resistance to SAR pathogens must be shown.
  • cim.32 , cz ' m ⁇ lO are completely resistant to E. cichoracearum, and others completely susceptible.
  • the resistance did not correlate with the strength of PR-1 gene expression or SA content.
  • the two strongest mutants cim677 and c/m810 were resistant, but c/m328, with low ER-7 gene expression and SA accumulation, also displayed an almost complete resistance (disease rating 1.01).
  • Some cim mutants exhibit a good resistance to virulent Pseudomonas syringae pathovars
  • mutants cim677, cim695, cim713, and c/m810 exhibited a bacterial proliferation reduced more than 10-fold compared to wild-type (Table 8).
  • mutants c/m8 and c/m824 are both in the class of "strong" mutants, they were at least as susceptible to this P. syringae isolate as wild-type (Table 8).
  • the response to avirulent Pseudomonas strains also differed among the cim mutants.
  • cim mutants c/m205, c/m328, cim716, cimS24
  • Other cim mutants did not show the typical HR symptoms or HR was delayed.
  • cim677 the HR occurred 2 to 3 hours earlier than in wild-type, in which the HR was visible 8 to 9 hours after inoculation.
  • This mutant showed increased sensitivty to virulent Pseudomonas strains, although the injection of MgCl 2 did not have any visible effect on the plant.
  • Table 8 Resistance of cim mutants to Pseudomonas syringae pv. ES4326. Bacterial colony forming units (cfu) were counted at three and five days after infiltration from four independent experiments, each containing four leaf punches per mutant per time point. While in most cims bacterial growth is significantly limited, in some mutants (cim8, cim824) proliferation is not reduced.
  • Bacterial viable count expressed as cfu (colony forming units per 4 leaf discs), calculated from four independent repetitions, dpi: days after inoculation
  • mutants at molecular level gene expression for known marker genes of various pathways was analyzed. The results may provide insight into the signaling pathways that are turned on in cim mutants and allow a further understanding of the maintenance phase of SAR.
  • ROS reactive oxygen species
  • ROS are degraded in the plant cell by superoxide dismutases (SOD), and successively in the Halliwell-Asada (Ascorbate-Glutathione) cycle, reducing H 2 O 2 to H O under use of NADPH (Asada, 1994; Halliwell and Gutteridge, 1989).
  • SOD superoxide dismutases
  • Halliwell-Asada Ascorbate-Glutathione
  • catalases may dismutate cellular H 2 O 2 .
  • three genes encode for catalases, that are 70 to 72% identical at the nucleotide level (Frugoli et al, 1996). The specific functions of catalase isozymes are still not well understood. The expression of the catalase 2 and 3 genes is not significantly altered in the cim mutants, or by pathogen treatment. Peroxidases also degrade H O by oxidizing specific substrates. They are responsible for lipid peroxidation, and cell wall cross-linking.
  • Expression of the two other Arabidopsis SAR genes Uknes et al, 1992), PR-2 (data not shown) and PR-5 was also elevated in most of the cim mutants, though not to the same degree as ER-7. It has been previously shown that the regulation of these genes does not always correlate with ER-7 gene expression (Reuber et al, 1998). The PR-5 gene expression was, however, induced in all mutants, with the exception of the weakest mutant, ctm658.
  • Thionins and defensins are genes that are induced by pathogen attack. They are not induced during SAR but are regulated by an SA-independent, JA-dependent signaling pathway ( ⁇ pple et al, 1998; Penninckx et al, 1996). Both an antagonistic interaction and a concomitant induction of PDF1.2 and ER-7 expression have been described in the literature (see Maleck and Dietrich, 1999 for review). Interestingly, thionin2.1 was induced in some cim mutants (c/m658, cim677, cim716 and mutant 779). PDF1.2 is known to be induced in several lesion mimics, and was also induced in mutant 799.
  • LTP lipid transfer proteins
  • the NIM1 gene is modestly SA- inducible (Cao et al, 1997; Ryals et al, 1997) and is induced in the cim mutants with the highest SA accumulation (cim205, cim677, cim695, cimSlQ, cimS24).
  • the NDRl gene that is induced in incompatible plant-pathogen interactions reflects these weak changes.
  • the previously described induction of the NDRl gene by pathogens was not observed in the pathogen-infected control (tissue harvested eight days after inoculation with E. parasitica Noco2). This finding is consistent with the observation that NDRl function is not required for resistance to Noco2 (Century et al, 1997).
  • PAL genes which encode the putative rate-limiting enzyme in the general phenylpropanoid pathway leading to SA biosynthesis (Bate et al, 1994) and which are inducible by many biotic and abiotic factors (Wanner et al, 1995) were induced in some mutants (e.g. in cim713, cim716) and by BTH treatment, but not in others (cim695, mutant 779).
  • PATl phosphoribosyl-anthranilate synthase
  • Arabidopsis vegetative storage protein acid phosphatase gene (AtVSP), a marker for jasmonic acid induced gene expression (Berger et al, 1995) was either weakly or not induced.
  • PR-4 expression is ethylene-inducible. Its expression in the cim mutants was about 20- fold weaker than that observed in an ethylene treated control plant and not induced above wild-type level.
  • Rabl8 gene an example of an ABA-inducible gene (Merlot and Giraudat, 1997). Rabl8 gene expression was induced in c/m328, cim695 and mutant 779.
  • Table 10 ⁇ lement-to-element variability on the DNA microanay. cDNA clones were spotted three times on the array and expression values were compared in three independent hybridization experiments, using as probes RNA from cim713, cim205 and c/m328, and wild- type as comparison. Raw data (expression) and mean and standard deviation (mean+/-std) for 11 genes with significant inductions are shown.
  • a pair of probes was prepared. Single strand cDNA was labeled during synthesis with the fluorochromes cyanine cy3 (red) or cy5 (green). These dyes differ only in one double bond and it is likely that they are incorporated with identical efficiency in the first strand cDNA synthesis.
  • the cyanines have very high, but similar molar absorptivity ( ⁇ > 50000 cm _1 M " ) and large fluorescence enhancements upon binding to nucleic acids. Cy3 and cy5 have distinct and narrow emission peaks at 532 nm and 633 nm, respectively.
  • a third validation is a direct comparison between differential gene expression quantified by the DNA microarray technology and by Northern blot analysis (Table 11). With few exceptions, such as the ER-7 gene expression in cim2Q5 and c/m713, the DNA microarray gives higher absolute values of gene induction than obtained in Northern blot analysis. This is not surprising because the values are normalized on a scale from +100 to -100 which appears to extend the scale (compare section 5B, where the same phenomenon was observed). Apparently, the yeast RNA that was spiked for normalization purposes in the plant samples did not match exactly the abundance of plant mRNA in the cell. Induced expression levels between 3-fold and 6-fold are reported by both technologies to similar levels (e.g. PR-5, PAL).
  • Table 11 Comparison between gene expression quantification by the DNA microarray and by Northern (RNA) blot analysis.
  • the distribution of the FSI shows that most mRNAs fall into the class of low- to medium-abundant mRNAs (1 - 50 mRNA per cell; 1000 - 10,000 FSI). Only a few have intensities above 11,000 FSI, which corresponds to highly abundant transcripts (100 - 500 transcripts per cell, based on an estimated total number of 100,000 transcripts per cell; Kamalay and Goldberg, 1980). The largest changes in abundance were observed for low copy mRNAs that are usually more than 10-fold induced. The highly expressed housekeeping genes do not change the transcription rate in the cim mutants.
  • Table 12 Counts of elements displaying altered gene expression in three cim mutants, in plants treated with BTH and NahG plants. The total number of elements giving valid signals is shown, along with the number of elements with at least a 2-fold, 2.5-fold, 3-fold, or 4-fold difference in signal in the mutant (or treatment) compared to untreated wild-type.
  • the five elements in cim32S that displayed a more than 4 fold change in expression are three elements for ER-7, one element for PR-5 and est 203C22T7 (extensin).
  • mutants were created: Genes that changed significantly in one mutant (more than 2.5 fold) were grouped together and their gene expression data in the other mutants were obtained and plotted in profiles.
  • the expression of 86 genes with differential expression in mutant cim205 was compared to their expression in cim713, cim328 and in the BTH control experiment.
  • a characteristic of cim205 is the group of repressed genes around the est 246D2T7 and est 246B12T7 (both derived from genes encoding a senescence associated proteins).
  • est 212B17T7 is also strongly suppressed. This reduction in expression is not found in the other cim mutants, but is seen in the BTH treated tissue.
  • est 203C22T7 BLAST similarity: extensin
  • mutant cim713 This is not the case in mutant cim713. Most of the 153 selected genes with altered expression (cut-off 2.5-fold) in this mutant are upregulated and only a few are downregulated. It is evident that many more elements display significant alterations in gene expression in c/m713 compared to cim2 5. Although the spectrum of gene inductions in the other two mutants looks very similar to the spectrum of cz ' m713, those gene inductions are usually weaker and not necessarily significant. Several elements with the most dramatic changes in expression are annotated and the results of BLAST similarity searches are indicated in table 13.
  • SAR genes include the known SAR genes, and genes encoding for cell-wall modifying proteins, such as extensins (est 118N4T7 and 203C22T7) and xyloglucan endotransglycosylase related proteins (est E12G2T7 and 92121T7), as well as genes whose induction was not expected, such as the genes encoding squalene monooxygenase and a cytochrome P450, and also genes with unknown protein function (est 156F15T).
  • cell-wall modifying proteins such as extensins (est 118N4T7 and 203C22T7) and xyloglucan endotransglycosylase related proteins (est E12G2T7 and 92121T7)
  • genes whose induction was not expected such as the genes encoding squalene monooxygenase and a cytochrome P450, and also genes with unknown protein function (est 156F15T).
  • mutant cim328 only 47 genes show more than 2.5 fold alterations in expression compared to wild-type. 35 genes are found only in the cim205 gene group (40%), and 107 genes are unique to the cim713 gene group (70%). In c/m328, only 13 genes were found that did not also have altered expression in one of the other mutants. More than half of the genes induced in c/m328 showed also altered expression in one or both of the other mutants: 11 elements induced in cim328 were also induced genes in mutant cim205, 6 are induced in mutant ciml 13 and 17 genes are common to all three gene groups. These 17 elements are listed in table 13.
  • 3 est 118N4T7 is likely to be a chimeric clone
  • the microarray can thus be a powerful tool in dissecting pathways.
  • gene expression profiles describe more precisely the characteristics than single marker genes do.
  • the fingerprints for cim mutants show many similarities (the "SAR profile"), but also distinct features that might eventually help to explain phenotypic differences of the mutants.
  • EPSPS is not part of the phenylpropanoid pathway, but of the shikimate pathway, furnishing the phenylpropanoid precursor phenylalanine.
  • cDNAs were not full-length clones, but only fragments as described in Table 4.
  • Two- dimensional (2D) polyacrylamide gel electrophoresis can resolve between 2500 and 10,000 proteins but the low loading capacity, unprecise pH gradients and difficult identification (either by comparison or by microsequencing) limit the actual use of this technology (Pennington et al, 1997).
  • 2D gel electrophoresis has been used before to characterize Arabidopsis mutants (Santoni et al, 1994). We attempted to compare results obtained with the DNA microarray to changes in protein patterns. Total protein extracts of c* ' m328, czm713, cim677, NahG, c/m713xNahG, and BTH-treated wild-type plants were separated on 2D gel electrophoresis.
  • the crossing partner A. thaliana ecotype Ler does not contain the ER-7/luciferase reporter gene, therefore one quarter of the F2 will not be usable for phenotyping.
  • Plants containing the ER-7/luciferase transgene can be identified by selection on kanamycin or by PCR screening for the T-DNA. Although growth on GM plates under selection does not induce the ER-7 gene expression, the phenotype changes slightly and phenotyping was never solely based on results obtained from those experiments. Therefore, a PCR for the luciferase gene was also established and run on all F2 plants and on at least 6 F3 progeny, to allow plants homozygous or heterozygous for the reporter gene to be distinguished.
  • ER-7 can be induced by various stresses. Although the levels of induction are usually small (with the exception of cell death-inducing events), the high sensitivity of the ER-7/luciferase system might make it difficult in some cases to distinguish between stress induction and genetic induction.
  • RNA analysis The detection of the ER-7 marker gene expression by dot blot (RNA) analysis is feasible in the F2, and was used to confirm in random samples the phenotype determination by luciferase activity but does not help to improve the reliability of the phenotype determination.
  • the rate of phenotype miscalls was the same as when F2 plants were scored for luciferase activity.
  • the Cim phenotype is only expressed in a small percentage of the F2 plants, the penetrance is incomplete (15% vs. 75% expected).
  • luciferase in vivo assay would allow the screening of 10,000 F3 plants in a reasonable amount of time. Because of the penetrance problem encountered in the Eer ecotype, about 5000 F2 plants were screened for constitutive luciferase expression. 855 F2 plants, for which the phenotype could be called without doubt, were used for mapping purposes. Usually, they were homozygous for the cim mutation. For the 855 F2 plants, the presence of the luciferase gene was confirmed by PCR. The F3 populations were scored on both kanamycin selection and without selection.
  • SSLP and CAPS markers are polymorphic between the A. thaliana ecotypes Col-0 and Eer. To get a rough map- position, about 30 markers were used on a F2 population of cim.713 of 65 individuals. Linkage to the phenotype was found with the SSLP marker nga280 (at 81.4 cM on the Lister and Dean RI map, 2 recombinants in 124 meiosis). The next closest SSLP markers, nga248 (at 40.0 cM) and ngal 11 (at 111.4 cM) showed less linkage. In this genomic region, no PCR- based genetic marker was available.
  • RFLP markers were present in the interval, but these are difficult to use on individual F2 Arabidopsis plants because of the amount of genomic DNA required.
  • To convert RFLP markers into PCR-based markers several RFLP probe clones were sequenced and sequence-specific primers were designed. PCR fragments from both parental ecotypes were digested using 48 to 80 different restriction endonucleases to find a cleaved amplified polymorphic sequence (CAPS). Polymorphisms were detected as differences of fragment sizes after electrophoretic separation. This procedure was successful for the RFLP markers mi209, mi304 and mi291a, and for the gene NIA2. No polymorphism was detected for RFLP marker mi 106.
  • PAP240 was identified as an expressed sequence tag hybridizing to a YAC clone in this region.
  • the clone obtained from M. Raynal, INRA, Perpignan, France
  • the marker PAP240 divided the genetic distance between the markers mi291a and mi209, and narrowed the interval containing the cim mutation to roughly 2 cM.
  • the Arabidopsis genome is well represented in several large-capacity vectors. Three YAC and two BAC libraries exist that are partly assembled into contigs. From the physical map, sequence information can be derived to design new genetic markers and eventually to construct a high-resolution genetic map. Sequence information can be obtained from publicly available BAC end sequences (http://genome.bio.upenn.edu), YAC end rescue, cloning of BAC (random or end-) fragments, or by the identification of ests that hybridize to BAC clones. With the rapid progress of the Arabidopsis sequencing project, the chance of finding sequence information of entire BAC clones is also increasing.
  • APK100 and LOX were used as anchor points on the physical map. They were hybridized to BAC filters containing subsets of the two available BAC clone libraries, IGF and TAMU (ABRC stock center). APK100 hybridized to BAC F16J8, F15I10, F22G10 and F8H4. The LOX probe hybridized to BAC T7N22, T3A10, and to the BAC clones F19C6, F9I9, F26H12 and F5P9. From these starting points, a physical contig was constructed, using both experimental and non-experimental data. J. ⁇ cker (Univ. Pennsylvania, PA), and T.
  • Altmann's laboratory (Max-Planck-Institut fur molekulare Dephysiologie, Golm, Germany) provide hybridization data of BAC clones to BAC end fragments and construct BAC contigs (http://www.mpimp- golm.mpg.de/101/mpi_mp_map/bac.html). The considered region was however not yet contiged.
  • Washington University (St. Louis, MO) provides H dm fingerprints of BAC clones (http://genome.wustl.edu/gsc/cgi-bin/arab/atdatabase.shtml). Based on similarity in restriction fragment patterns, different BAC clones can be aligned with certain probabilities.
  • Simple sequence repeats like polydT, or polydCA, were identified on the BAC F20D21 and flanking primers were designed to identify length polymorphisms (SSLP). Usually, these repeats are meiotically unstable and vary in size between evolutionarily distinct ecotypes.
  • BAC F20D21 As a last, most accurate (and costly) solution, systematic sequencing of genomic DNA from both parents corresponding to the insert cloned in BAC F20D21 was conducted. PCR primers were designed every 1 kb, the slightly overlapping fragments purified and sequenced with the same PCR primer pair. Of the 100 kb of BAC F20D21 that were sequenced, only 7 single nucleotide polymorphisms between the ecotypes Col-0 and Eer were detected, 5 of them were restriction fragment polymorphisms and 4 were converted into CAPS markers (orf5, 20D21-2, orf52, cf2-12). The fifth polymorphism was already used as the RFLP marker 20D21-13. This is an extremely low rate of genetic polymorphism and explains the difficulties encountered in marker development by random trials.
  • BAC clone T22H22 which overlaps with the right (SP6) end of the BAC F20D21 was, similar to F20D21, partly sequenced by the Arabidopsis genome initiative in the course of this work (AC005388). PCR fragments on BAC T22H22 were generated of both parental lines and sequenced.
  • the locus T26 (at 26 kb from the BAC end F20D21) was polymo ⁇ hic and the recombinant F2 plant number 1006 had a recombination event between this marker and the mutation, thus limiting the physical interval on the right side (table 15).
  • BAC end sequences between APK100 and F20D21-2 were used in Southern blot analysis to find restriction length polymo ⁇ hisms (the BAC end sequences were too short to develop CAPS markers), but no such polymo ⁇ hism was detected. Therefore other mismatch detection methods, used primarily in mammalian mapping projects and routine identification of known mutations, were tried.
  • Heteroduplex analysis detects changes in confirmation of DNA duplexes caused by single base pair mismatches. PCR fragments in the corresponding genomic region, are denatured, mixed and hybridized with PCR products of the second parental ecotype. Any point mutation results in the formation of two heteroduplexes as well as two homoduplexes. The heteroduplexes have altered confirmations, which can be detected by the altered migration in a polyacrylamide gel. Hauser et al. found in Arabidopsis 50% of 36 loci (230 bp to 1000 bp PCR fragments) to be polymo ⁇ hic (Hauser et al, 1998).
  • sequencing seemed to be the last solution to identify the rare nucleotide polymo ⁇ hisms in this genomic region between the two ecotypes Col-0 and Eer.
  • long (26-mer) PCR primers from several BAC end sequences in the direction of BAC F20D21
  • a long range PCR fragment between BAC end F17M20 and the left (T7) end of BAC F20D21 was amplified and cloned into the vector pCR2.1.
  • These additional 9.1 kb were sequenced by random transposon integration. Marker development based on this sequence is in progress that will hopefully limit the cosegregating interval on the left side of BAC F20D21.
  • BAC F20D21 and T22H22 were identified using consensus prediction programs (Genscan, http://ccr.081.mit.edu/Genscan.html).
  • BAC F20D21 contained 28 putative genes. For some, the prediction was confirmed by the presence of ests in the databank or by Northern blot analysis. ORFs were sequenced in the Col-0 wild-type and cim713 mutant. In cases where the sequenced fragments did not overlap with the next fragment, Northern blot analysis was performed to detect potential point mutations in promoter elements, leading to changes in gene expression.
  • the number of inserts was also estimated by probing genomic Southern blots from each of the mutants with T-DNA probes (using pBluescript and the RB). This method usually gave a higher estimate of T-DNA inserts than segregation analysis. There are two possible reasons for this discrepancy. Multiple T-DNAs may have inserted at a single genetic locus, or partial T-DNAs, lacking the BAR gene but containing pBluescript and RB segments may have been inserted.
  • Table 16 Genetic analysis of cim mutants generated by T-DNA insertion. Out of 10,000 primary transformants, 7 lines were retained that showed reproducible ER-7/luciferase activity in the next generations. Those were submitted to segregation analysis on selective media (Basta) and Southern blot analysis to identify T-DNAs that cosegregate with the phenotype. Plasmid rescue, or TAIL PCR was performed to clone flanking genomic DNA. line luciferase number luciferase number TAIL (T)/ activity ' inserts segregation in inserts plasmid rescue
  • flanking genomic fragments were sequenced and the inserts compared by BLAST similarity search to sequences in the Arabidopsis genome or elsewhere.
  • Two flanking sequences were derived from sequenced parts of the A. thaliana genome, two had homology to Arabidopsis ests and two had no significant homology.
  • One plasmid contained only the T- DNA cloning vector, probably integrated into the genome by inefficient cleavage of the T- DNA at the border sequence during the transformation.
  • TAIL PCR was performed to get LB- and RB-flanking genomic DNA.
  • TAIL PCR was successful and several fragments per line were either subcloned or directly submitted for sequencing. Some fragments contained only the T-DNA sequence, possibly because of tail-to-head or head-to-head cointegrations of several T-DNAs into the genome.
  • known genes or sequenced genomic regions were cloned. The fragments were used in Southern blot analysis to confirm the insertions and to identify the gene knock-out that cosegregated with the phenotype. These genes will be transformed into the mutants to establish wild-type phenotypes and to genetically prove the cloning of cim genes.
  • the Cim phenotype is not expressed in 100% of the progeny, making genetic analysis in F2 populations difficult, especially if no genetic marker linked to the mutation can be followed.
  • mutant cim713 expressed elevated PDF 1.2 mRNA levels that were decreased in a NahG background, but resistance to E. parasitica was retained in both mutants.
  • transcription of neither SAR genes nor PDF 1.2 is increased in cim713 when crossed to NahG, revealing a novel, unrelated mechanism for resistance to E. parasitica.
  • cim205 x NahG expresses more than 30 genes 5- to 10-fold stronger than wild-type that are not differentially expressed in NahG or ciml 13 x NahG. About 20 genes that are reduced between 5- to 10-fold in NahG are either not as strongly repressed in the double mutants or are not repressed at all. Thus, there are differences between the two double mutants and NahG.
  • Table 17 ests that revealed differential gene expression in cim205 x NahG and cim713 x NahG on the DNA microarray (“chip”), and the values obtained in Northern blot analysis ("blot"). est/clone homology BTH NahG c//n205xNahG cwii7 3xNahG blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blot chip blo
  • Hybridizations were done by Synteni, Inc., Fremont, CA as described by Ruan, et al, 1998.
  • We also compared more than 40 DNA microanay data points to Northern blot results and found a linear conelation of r 0.83.
  • the EST "Unigene" set was obtained from the Arabidopsis Biological Resource Center, (Ohio State University, Columbus, OH). 10,000 of the 14,000 clones were amplified by PCR using the M13 universal reverse and -21 forward primer, with modified 5' amino end for spotting onto a glass slide. A fraction of the ESTs, including all mentioned in this manuscript, were resequenced at our facility. Roughly 30 % of the EST sequences did not conespond to the original sequence as represented in the AATDB. Based on our own BLAST search and on estimations made by others (Delseny et al, 1997), we extrapolated the redundancy of the "Unigene” set to 1.5 to 2-fold.
  • Table 18 Diversity of conditions used to describe the transcriptome of Arabidopsis thaliana during SAR.
  • NahG suppresses SAR gene expression in crosses to two of the SAR-constitutive cim mutants, cim ⁇ and cimll, to a baseline resembling that of NahG-expressing plants.
  • NahG expression results in a characteristic gene expression fmge ⁇ rint in secondary tissue from plants inoculated in primary tissue with avirulent bacteria. This conesponds to the inability of these plants to establish SAR.
  • the conesponding primary tissues in NahG-expressing plants display changes in gene expression which compares very closely to wild-type primary, infected tissue and this sample does not cluster with other NahG samples.
  • the cluster containing EST 209E19T7 defines genes that are transcriptionally induced in NahG-expressing plants.
  • the cluster containing EST 118P18T7 defines genes that are not significantly responsive to SAR- inducing conditions like chemical and genetic induction, but do respond to avirulent bacteria and are downregulated in NahG expressing plants.
  • Phenylalanine ammonia lyase (PAL) and 20 other ESTs that cluster together are repressed by NahG expression, but are induced during the maintenance phase of SAR, for example in cim mutants or 48 hours after BTH treatment.
  • the cluster of "PRl like" genes exhibits similar induction behavior to genes in, the PAL gene cluster but these genes are only weakly suppressed in NahG-expressing plants.
  • the PRl regulon contained 25 other ESTs (17 different genes). These are prime candidates for SAR marker genes and the encoded proteins are likely to play a physiological role in SAR. The estimated 1.5 to 2-fold redundancy of our EST set is a good internal control for this analysis and we also included three replicates of the PR5 and the PerC cDNAs (as well as 28 other relevant cDNAs) on the DNA-microa ⁇ ay. All three copies of the two genes cluster with PRl, showing the robustness of the DNA microanay analysis.
  • cluster analysis of expression profiles provides a tool to derive physiological functions of genes. This is important for sequences with no close homologs in the databank (for example EST 134C2OT7 or EST 192 K7T7) and also for genes with structural similarity to genes with known function (such as asparagine synthetase).
  • EST 134C2OT7 or EST 192 K7T7 genes with structural similarity to genes with known function (such as asparagine synthetase).
  • genes were identified that are responsive to BTH and/or pathogens. Furthermore, we compared expression profiles in response to biotic and abiotic inducers of SAR and assessed the requirement for salicylic acid and the NIMl gene for mRNA accumulation. By analysis of data generated with cDNA microarrays, sets of genes that are responsive specifically to exogenous application of BTH were identified (see Table 20 below). The discovery of a set of BTH- inducible genes that are not responsive to SA or pathogens was especially su ⁇ rising given the expectation from previous studies of the SAR signal transduction pathway that BTH would always act as a functional analog of SA and would also activate the SAR response in the same manner as pathogen infection. The regulatory regions from these differentially expressed genes can be isolated using conventional cloning techniques and used as specifically inducible promoters, such as BTH-specific promoters.
  • Table 20 Induced genes based on >5x BTH induction in a wild-type Arabidopsis line at 4, 24, and 48 hours post-BTH and in the niml -4 mutant Arabidopsis line at 4 and 24 hours post-BTH. These are candidate genes for BTH-specific promoters that are induced by BTH but not SA or pathogens. Genes below the double line are induced by BTH in the niml -4 mutant, but are not induced by SA or pathogen. Gene names (EST IDs) in bold are induced more than 5x by BTH and are also induced in the niml-4 mutant line by BTH (NIMl independent genes), x: not in cluster analysis; # indicates cluster analysis result. Numbers indicate fold-induction relative to control treatment. If no number is present, fold-induction is less than 2.5.
  • Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity.
  • RPS2 of Arabidopsis thaliana a leucine-rich repeat class of plant disease resistance genes. Science 265, 1856-1860. Berger, S., Bell, E., Sadka, A., and Mullet, J. E. (1995).
  • Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB, genes encoding vegetative storage protein acid phosphatases, and is regulated similarly by methyl jasmonate, wounding, sugars, light and phosphate. Plant Mol. Biol 27, 933 - 942. Bevan, M., and The European Arabidopsis genome project (1998). Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391, 485 -
  • Plant defensins novel antimicrobial peptides as components of the host defense system. Plant
  • XLl-Blue a high efficiency plasmid transforming RecA Escherichia coli strain with beta galactosidase selection.
  • NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats.
  • NDRl a pathogen-induced component required for Arabidopsis disease resistance. Science 278, 1963 - 1965. Century, K. S., Holub, E. B., and Staskawicz, B. J. (1995). NDRl, a locus of Arabidopsis thaliana that is required for disease to both a bacterial and a fungal pathogen. Proc. Natl.
  • NER7 and bacterial resistance Characterization of the dominant Arabidopsis cpr6-l mutant. Plant Cell 10, 557-569. Conrath, U., Silva, H., and Klessig, D. F. (1997). Protein dephosphorylation mediates salicylic acid-induced expression of PR-1 genes in tobacco. Plant J. 11, 747-757. Creelman, R. A., and Mullet, J. ⁇ . (1997). Biosynthesis and action of jasmonates in plants.
  • Lukyanov S., Lukyanov, , Gurskaya, N., Sverdlov, E. D., and Siebert, P. D.
  • Glazebrook, J., Rogers, E. E., and Ausubel, F. M. (1997). Use of Arabidopsis for genetic dissection of plant defense responses. Annu. Rev. Genetics 31, 547 - 569. Glazebrook, J., Zook, M., Mert, F., Kagan, I., Rogeers, E. E., Crute, I. R., Holub, E. B.,
  • Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics 146, 381 - 392. Goodman, H. M., Ecker, J. R., and Dean, C. (1995). The genome of Arabidopsis thaliana.
  • Benzothiadiazole a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat. E/ ⁇ «t Cell 8, 629 - 643. Gosti, F., Berauche, N., Vartanian, N., and Giraudat, J. (1995). Abscisic acid-dependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana. Mol. Gen Genet. 246, 10 - 18. Grant, M. R., Godiard, L., Straube, E., Ashfield, T., Lewald, J., Sattler, A., Innes, R.
  • Plant J. 9, 701 - 713 Keller, H., Blein, J-P., Bonnet, P., and Ricci, P. (1996). Physiological and molecular characteristics of elicitin-induced systemic acquired resistance in tobacco. Plant Physiol.
  • TL-DNA gene 5 controls the tissue- specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383 - 396.
  • MAPMAKER An interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1, 174- 181.
  • Arabidopsis Information Service 26 CI - 72. Melan, M., A. thoroughly Dong, X., Endara, M. E., Davis, K R., Ausubel, F. M., and Peterman, T.
  • dCAPS a simple technique for the genetic analysis of single nucleotide polymo ⁇ hisms: experimental applications in Arabidopsis thaliana genetics. Plant J. 14, 387-392. Newman, T., de Bruijn, F. J., Green, P., Keegstra, , Kende, H., Mclntosh, L.,
  • Proteome analysis from protein characterization to biological function. Trends Cell
  • Arabidopsis a molecular and mo ⁇ hological perspective. In Arabidopsis, E. M.

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Abstract

Pour identifier les gènes dont les protéines interviennent dans la régulation de la résistance systémique acquise en aval de la mort cellulaire, on a utilisé un crible visant à isoler les mutants qui expriment cette résistance de manière constitutive. Le promoteur PR-1, qui est le marqueur le plus fiable pour une apparition de ladite résistance, a été cloné devant le gène rapporteur de luciférase de luciole (Photinus pyralis) et transformé en Arabidopsis. Une ligne transgénique à forme d'expression de luciférase parallèle au gène PR-1 endogène a été identifiée et soumise à une mutagenèse EMS pour isoler les mutants qui expriment de manière constitutive le gène de luciférase PR-1. Des techniques biochimiques, cytologiques, pathologiques et génétiques ont été employées pour caractériser plus avant les mutants et pour mettre en évidence l'isolation des mutants de la résistance systémique acquise en aval de la mort cellulaire. Cette caractérisation permet de faire la distinction entre les classes de mutants cim et de décrire les modifications physiologiques intervenant durant la phase de maintenance de la résistance systémique acquise. En outre, l'utilisation de puces à jeu ordonné de microéchantillons a permis d'examiner simultanément l'ensemble du génome végétal pour déceler les gènes dont l'expression se modifie en réponse à des facteurs biotiques ou abiotiques. La comparaison des modifications de l'expression génique sur différents traitements a permis d'identifier des groupes de gènes corégulés (régulons), et on a examiné les séquences génomiques de gènes dans un régulon pour identifier des motifs de séquences communes pouvant vraisemblablement tenir lieu d'éléments régulateurs. L'approche utilisée fait appel à une conception expérimentale fondée sur la biologie du système à l'étude, en combinaison avec la bioinformatique visant elle à analyser les résultats.
PCT/US2000/011460 1999-04-28 2000-04-28 Identification d'elements de controle d'adn reagissant a des stimuli specifiques WO2000065039A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027321A2 (fr) * 1999-10-13 2001-04-19 Plant Research International B.V. Procede pour detecter les differences dans l'expression genique de plantes de type sauvage et de plantes genetiquement modifiees
WO2002072789A2 (fr) * 2001-03-12 2002-09-19 Irm, Llc. Essais biologiques cellulaires grande vitesse fondes sur la genomique, elaboration de ces essais, et collections de rapporteurs cellulaires
EP1386009A2 (fr) * 2001-05-07 2004-02-04 Amersham Biosciences Corp. Conception de genes artificiels a utiliser en tant que temoins dans des systemes d'analyse d'expression genique
US7005255B2 (en) 2000-04-14 2006-02-28 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7329489B2 (en) 2000-04-14 2008-02-12 Matabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US8849577B2 (en) 2006-09-15 2014-09-30 Metabolon, Inc. Methods of identifying biochemical pathways
CN109234284A (zh) * 2018-09-14 2019-01-18 昆明理工大学 一种三七类甜蛋白基因PnTLP5及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851766A (en) * 1995-05-31 1998-12-22 Novartis Finance Corporation Process for isolating chemically regulatable DNA sequences
DE19860313A1 (de) * 1998-12-24 2000-06-29 Friedrich Felsenstein Verfahren zur Erkennung und Charakterisierung von Wirkstoffen gegen Pflanzen-Pathogene

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851766A (en) * 1995-05-31 1998-12-22 Novartis Finance Corporation Process for isolating chemically regulatable DNA sequences
DE19860313A1 (de) * 1998-12-24 2000-06-29 Friedrich Felsenstein Verfahren zur Erkennung und Charakterisierung von Wirkstoffen gegen Pflanzen-Pathogene

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
DESPREZ THIERRY ET AL: "Differential gene expression in Arabidopsis monitored using cDNA arrays." PLANT JOURNAL, vol. 14, no. 5, June 1998 (1998-06), pages 643-652, XP000960485 ISSN: 0960-7412 cited in the application *
MOLINA ANTONIO ET AL: "Impaired fungicide activity in plants blocked in disease resistance signal transduction." PLANT CELL, vol. 10, no. 11, November 1998 (1998-11), pages 1903-1914, XP000885732 ISSN: 1040-4651 *
RAFALSKI J ANTONI ET AL: "New experimental and computational approaches to the analysis of gene expression." ACTA BIOCHIMICA POLONICA, vol. 45, no. 4, 1998, pages 929-934, XP000952158 ISSN: 0001-527X *
RAMONELL K M ET AL: "Microarray dissection of systemic acquired resistance in Arabidopsis plants infected with powdery mildew." PHYTOPATHOLOGY, vol. 90, no. 6 Supplement, June 2000 (2000-06), page S63 XP000964562 Annual Meeting of the American Phytopathological Society;New Orleans, Louisiana, USA; August 12-16, 2000 ISSN: 0031-949X *
RUAN YIJUN ET AL: "Towards Arabidopsis genome analysis: Monitoring expression profiles of 1400 genes using cDNA microarrays." PLANT JOURNAL, vol. 15, no. 6, 1998, pages 821-833, XP000960486 ISSN: 0960-7412 cited in the application *
SCHENA M ET AL: "QUANTITATIVE MONITORING OF GENE EXPRESSION PATTERNS WITH A COMPLEMENTARY DNA MICROARRAY" SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 270, no. 5235, 20 October 1995 (1995-10-20), pages 467-470, XP000644675 ISSN: 0036-8075 cited in the application *
SCHENK P M ET AL: "Coordinated plant defense responses in Arabidopsis revealed by microarray analysis" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (10 OCT 2000) VOL. 97, NO. 21, PP. 11655-11660. PUBLISHER: NATL ACAD SCIENCES, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418. ISSN: 0027-8424., XP002153163 *
SOMERVILLE, S. C. ET AL: "Alternate methods of gene discovery - the candidate EST approach and DNA microarrays" CELLULAR INTEGRATION OF SIGNALLING PATHWAYS IN PLANT DEVELOPMENT. PROCEEDINGS OF THE NATO ADVANCED STUDY INSTITUTE, ACQUA FREDDA DI MARATEA, ITALY, 20-30 MAY, 1997, (1998) PP. 297-309. NATO ASI SERIES. SERIES H: CELL BIOLOGY VOL. 104., XP000964578 Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305, USA *
SPELLMAN ET AL: "COMPREHENSIVE IDENTIFICATION OF CELL CYCLE-REGULATED GENES OF THE YEAST SACCHAROMYCES CEREVISIAE BY MICROARRAY HYBRIDIZATION" MOLECULAR BIOLOGY OF THE CELL,US,BETHESDA, MD, vol. 9, December 1998 (1998-12), pages 3273-3297, XP002143737 ISSN: 1059-1524 *
WARD E R ET AL: "COORDINATE GENE ACTIVITY IN RESPONSE TO AGENTS THAT INDUCE SYSTEMIC ACQUIRED RESISTANCE" PLANT CELL, vol. 3, no. 10, 1991, pages 1085-1094, XP002134008 ISSN: 1040-4651 cited in the application *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027321A2 (fr) * 1999-10-13 2001-04-19 Plant Research International B.V. Procede pour detecter les differences dans l'expression genique de plantes de type sauvage et de plantes genetiquement modifiees
WO2001027321A3 (fr) * 1999-10-13 2001-12-27 Plant Res Int Bv Procede pour detecter les differences dans l'expression genique de plantes de type sauvage et de plantes genetiquement modifiees
US7682784B2 (en) 2000-04-14 2010-03-23 Cornell Research Foundation, Inc. Methods for drug discovery disease treatment, and diagnosis using metabolomics
US7550260B2 (en) 2000-04-14 2009-06-23 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7947453B2 (en) 2000-04-14 2011-05-24 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7910301B2 (en) 2000-04-14 2011-03-22 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7005255B2 (en) 2000-04-14 2006-02-28 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7329489B2 (en) 2000-04-14 2008-02-12 Matabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7550258B2 (en) 2000-04-14 2009-06-23 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7682783B2 (en) 2000-04-14 2010-03-23 Cornell Research Foundation, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7553616B2 (en) 2000-04-14 2009-06-30 Metabolon, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
US7635556B2 (en) 2000-04-14 2009-12-22 Cornell Research Foundation, Inc. Methods for drug discovery, disease treatment, and diagnosis using metabolomics
WO2002072789A2 (fr) * 2001-03-12 2002-09-19 Irm, Llc. Essais biologiques cellulaires grande vitesse fondes sur la genomique, elaboration de ces essais, et collections de rapporteurs cellulaires
WO2002072789A3 (fr) * 2001-03-12 2003-07-03 Irm Llc Essais biologiques cellulaires grande vitesse fondes sur la genomique, elaboration de ces essais, et collections de rapporteurs cellulaires
EP1386009A4 (fr) * 2001-05-07 2005-04-27 Amersham Biosciences Corp Conception de genes artificiels a utiliser en tant que temoins dans des systemes d'analyse d'expression genique
EP1386009A2 (fr) * 2001-05-07 2004-02-04 Amersham Biosciences Corp. Conception de genes artificiels a utiliser en tant que temoins dans des systemes d'analyse d'expression genique
US8849577B2 (en) 2006-09-15 2014-09-30 Metabolon, Inc. Methods of identifying biochemical pathways
CN109234284A (zh) * 2018-09-14 2019-01-18 昆明理工大学 一种三七类甜蛋白基因PnTLP5及应用

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