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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • 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
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/13—Plant traits
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to the field of plant immunity and defense, especially to the sensitization of plants (e.g. seeds, whole plant seedlings or mature plants) or plant parts (e.g. leaves) to future attack by pests or pathogens (biotic stress) and/or abiotic stresses called 'priming' and thus to the generation of broad spectrum disease resistance.
• plants e.g. seeds, whole plant seedlings or mature plants
• plant parts e.g. leaves
• 'priming' abiotic stresses
• TF gene expression profiling methods transcription profiling of one or more sets of transcription factor (TF) genes are provided herein as markers for priming. Priming status and/or capability (and stress resistance capability) can, thus, be inferred from TF marker gene expression, without a need for exposing the plant to biotic or abiotic stress. Plants, seedlings or plant parts having such an altered priming status and/or capability are also an embodiment of the invention. Also provided are methods for identifying microorganisms, compounds or compositions which are able to cause (activate or induce) priming when brought into contact with plants or plant parts, or to increase the priming capability (i.e. the responsiveness of the plant or plant part to future pest or pathogen attack and/or abiotic stress exposure).
• Such biotic and/or abiotic compounds are useful in agriculture and horticulture for activating priming, whereby the seeds or plants (or plant parts) have a faster and/or stronger defense response to a broad spectrum of stresses compared to non-primed seeds, seedlings or plants (or plant parts) which rely on the "direct defense" response (which is both slower and weaker, and is thus often insufficient to fend of pathogen attack and to protect the plant from stress).
• Plants have evolved sophisticated mechanisms to defend themselves against insects and microbial pathogens. Apart from constitutive defense barriers, plants have a large spectrum of pathogen-inducible defense mechanisms at their disposal.
• Well- characterized examples of such pathogen-inducible defenses are the production of antimicrobial compounds, such as pathogenesis related (PR) proteins and phytoalexins, as well as formation of callose-rich cell wall appositions at the sides of fungal or oomycetous attack (Hammerschmidt, R. (1999), Annu. Rev. Phytopathol. 37, 285-306; Van Loon, L.C., and Van Strien, EA. (1999) Physiol. MoI. Plant Pathol. 55, 85-97; Ton, J., and Mauch-Mani, B. (2004) Plant J. 38, 119-130).
• PR pathogenesis related
• SAR systemic acquired resistance
• the signaling pathway controlling SAR depends on the plant endogenous accumulation of salicylic acid (Gaffney, T. et al. (1993) Science 261, 754-756) and an intact defense regulatory protein NPRl (Cao, H. et al. (1994) Plant Cell 6, 1583-1592).
• NPRl is an essential regulatory compound in SA-dependent basal resistance and SAR, and has been shown to control the expression of many stress-related genes, such as pathogenesis-related (PR) genes and genes involved in the secretory pathway ( Wang, D. et al. (2005). Science 308, 1036-1040).
• PR pathogenesis-related
• colonization of plant roots by specific strains of non-pathogenic, fluorescent Pseudomonas spp. also triggers systemic resistance against a wide range of pathogens (Pieterse et al. (1996) Plant Cell 8, 1225-1237; Pieterse et al. (1998) Plant Cell 10, 1571-1580; Ton et al. (2002) MoI. Plant-Microbe Interact. 15, 27-34).
• ISR rhizobacteria-induced systemic resistance
• BABA-induced resistance against the oomycetous pathogen Hyaloperonospora parasitica and the fungal pathogens Alternaria brassicicola and Plectosphaerella cucumerina is fully functional in Arabidopsis genotypes impaired in SAR signaling (Zimmerli et al., 2000, supra; Ton and Mauch-Mani, 2004, supra).
• This SA- and NPRl -independent form of BABA- induced resistance is based on priming for augmented depositions of callose-rich papillae at the sides of pathogen penetration, and depends on an intact ABA- and phosphoinositide (PI)- signaling ( Ton et al. (2005) Plant Cell 17, 987-999).
• PI phosphoinositide
• BABA-IR BABA-IR
• they are all associated with a priming of the tissue for enhanced activation of basal defense mechanisms after pathogen attack. Because priming allows the plant to adjust the efficiency of its innate immune system to the environmental conditions, it can be regarded as an adaptive defense strategy. Recently, it has been demonstrated that priming of the plant's innate immune system yields broad-spectrum resistance with minimal reductions in plant growth and seed set (see Van Hulten et al. 2006, Proc. Natl. Acad. Sci. USA 103, 5602-5607). Hence, defense priming is an important regulatory system that increases the plant's ability to cope with the immanently changing conditions in its environment.
• priming has been known for years, the current understanding of the molecular mechanisms behind this phenomenon remains rudimentary. It is, however, hypothesized that induction of priming leads to an increase in the amount of cellular components with important roles in defense signaling (Conrath et al., 2006, supra). Because there are barely any defense mechanisms that are activated directly upon induction of priming, it can be assumed that these signaling components remain inactive until the plant is exposed to pathogen attack. Upon subsequent attack by a pathogen, a specific subset of the signaling proteins becomes activated, resulting in an augmented activation of the appropriate basal defense reaction in primed cells.
• TFs transcription factors
• priming results in an increased level of (inactive) TF proteins in the primed tissue compared to non-primed tissue.
• stress e.g. pathogen or pest attack or abiotic stress
• these inactive TFs become active and regulate gene expression of defense genes, such that a faster and/or stronger defense response is mounted by primed tissue compared to unprimed tissue.
• Specific and robust sets of TFs are provided herein, whose expression levels can be used as (bio)markers for the priming status and/or capability of plants and for use in methods for generating plants having an altered priming status or wherein priming is activated (e.g.
• BABA is used to induce priming and disease resistance
• other chemical or biological compounds and compositions which can be used to induce priming in plants (such as in seeds or whole seedlings or mature plants) and/or plant parts (e.g. leaves).
• Transcription profiling of priming-specific marker genes using methods such as RT- qPCR are much faster and more sensitive and can be applied easily in a large variety of crop plants and horticultural plants.
• the sets of TFs provided herein provide, for the first time, easy, reliable markers for the state of priming and enable the breeding of plants having an altered priming status and/or capability. Similarly, it is much more efficient to screen the effect of biological and/or chemical compounds or compositions on such a set of priming-specific marker genes and to thereby identify compounds which can be used in agriculture and horticulture for inducing (or activating) broad- spectrum disease resistance.
• Primed plants or plant parts can be selected and differentiated from unprimed plants, allowing a more directed and more uniform planting of primed plants, e.g. in areas prone to biotic and/or abiotic stresses, such as salt- or drought stress or heat stress, and the like.
• Natural or induced variation in priming ability can now also be screened quickly and plants or plant parts can be selected and/or bred which have an altered priming state and/or capability, such as a constitutively active priming state. Such plants will benefit from a faster and/or stronger stress resistance response (compared e.g. to unprimed plants which must rely on a "direct defense" response) when being subsequently exposed to one or more stresses, while having a normal growth and reproductive potential.
• Primer refers herein to the sensitization of a plant or plant part so that it is able to activate defense mechanisms faster and/or stronger when exposed to one or more biotic and/or abiotic stresses compared to a non-primed control plant or plant part, which must rely on a 'direct defense' response.
• Many induced resistance phenomena that are effective against a broad range of pathogens, insects and abiotic stress are, at least partly, based on priming.
• priming for defense confers a low -cost protection of the plant in terms of fitness, as quantified by growth- and reproductive capacity (see van Hulten et al. PNAS 2004, p5602-5607).
• Priming thus covers priming for all types of inducible resistance, such as SAR-related priming (priming for SA- inducible defense), BABA- induced priming (priming for SA-inducible defense and priming for cell wall defense), and ISR related priming (priming for JA-inducible defenses).
• SAR-related priming primary for SA- inducible defense
• BABA- induced priming primary for SA-inducible defense and priming for cell wall defense
• ISR related priming priming for JA-inducible defenses
• Induced resistance or “inducible resistance” refers herein to the ability of plants to increase their level of resistance against future stress/pathogen attack upon appropriate stimulation, and types of induced resistance are Systemic Acquired Resistance (SAR; activated in distal parts of a plant upon localized pathogen attack and involving Salicylic Acid (SA) and NPRl), Induced Systemic Resistance (ISR; activated for example by non-pathogenic rhizobacteria or other bacteria and involving Jasmonic Acid (JA), ethylene and NPRl) and BABA-Induced Resistance (BABA-IR).
• SAR Systemic Acquired Resistance
• ISR Induced Systemic Resistance
• JA Jasmonic Acid
• BABA-IR BABA-Induced Resistance
• Direct defense or “induced defense” refers to defense mechanisms activated in non- primed plants, such as upon a first exposure to stress or pathogen attack.
• Primer status refers to the present priming condition in which a plant or plant part resides, e.g. it can be 'not primed' (priming is not activated) or it can be 'partially primed' or 'fully primed' (priming is activated), so that it responds faster and/or more strongly to future exposure to biotic and/or abiotic stress compared to an unprimed plant or plant part, i.e. it has an enhanced stress response (see below).
• the gene expression profile of priming-specific marker genes (TFs) provided herein is used as a qualitative (primed vs.
• Primer refers herein to conditions or compounds which are able to induce priming, i.e. to activate priming and induce a sensitized state.
• Primer specific marker genes refers to genes, whose mRNA expression level or profile is indicative of the priming state and/or priming capability of the tissue.
• seed priming (which is also a form of priming but which does not target the same physiological processes as defense priming according to the invention) has a known meaning in the art and refers to a pre -treatment of seeds to improve seed germination behaviour and u niformity of seedling emergence.
• Different priming methods are known, such as osmo-priming (using liquid carriers of water), matrixpriming (using solid water carriers) or hydropriming (using pure water). The principle of all priming methods is the same: pre-treatment of seeds in order to provide water in a controlled manner.
• seed priming herein in relation to the instant invention
• a priming agent e.g. a coating comprising a priming agent
• the seedlings and plants emerging from the primed seeds become defense primed through the priming agent.
• Plant pathogens refer herein to biotic agents, which are capable of causing disease on plants, such as plant pathogenic fungi (e.g.
• biotrophic or necrotrophic species bacteria, viruses, oomycetes, mycoplasma like organisms, nematodes, insects and the like.
• all strains, races or pathovars of a pathogen species which are capable of causing disease on host tissue are included herein.
• subgroups, such as subspecies of pests and pathogens are included.
• animal species, such as insects and nematodes are referred to as "plant pests" rather than pathogens, but for simplicity the term “plant pathogens” as used herein encompasses also pests unless indicated otherwise.
• “Broad spectrum disease resistance” refers to a host plant or plant part being resistant to a broad range of pest and/or pathogen species, including subgroups of such species, e.g. pathovars, biotypes, strains, etc.
• Disease resistance refers herein to various levels of disease resistance and/or tolerance of a plant, including susceptibility, moderate resistance and high resistance or complete resistance to one or more pathogens. It can be measured and optionally quantified by comparison of pathogen-induced disease symptoms (such as frequency and/or size of lesions, etc.) as well as the extent of tissue colonization by the pathogen, relative to those seen in susceptible control plants when grown under identical disease pressure. Such disease bioassays can be carried out using known methods. Disease resistance can also be indirectly measured as higher growth and/or yield of resistant plants compared to susceptible plants when grown under disease pressure.
• Stress refers to conditions or pressures of physical, chemical or biological origin acting on a plant or plant cells which may result in yield loss and/or quality loss of a plant, but which is preferably not lethal to the plant.
• Biotic stress refers to stress caused by biotic (live) agents, such as fungi, viruses, mycoplasma like organisms, insects, bacteria, nematodes etc. (i.e. especially plant pests and pathogens).
• Stress resistance refers to various levels of stress tolerance, i.e. a plants ability to cope with biotic and/or abiotic stress. Stress resistance can be classified into susceptibility, moderate tolerance to one or more biotic and/or abiotic stresses and high tolerance or complete resistance to one or more biotic and/or abiotic stresses.
• Enhanced stress resistance and “enhanced disease resistance” and “ enhanced defence response” refers to any statistically significant increase in stress resistance / disease resistance of a plant or plant tissue compared to a suitable control, such as an un-primed plant. Especially a stronger defence res ponse and/or a faster defence response following pest or pathogen challenge and/or abiotic stress challenge are included herein.
• the defence response / disease resistance is at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, or even 100% higher than in the control plant, using appropriate bioassays and/or field assays and/or the defence response is mounted at least lhr, 2hrs, 3hrs, 4hrs, 5hrs, 6hrs or more earlier than in the control.
• the expression of defence related genes and/or the production of defence- related proteins can be used to assess the speed and/or strength of the defence response.
• nucleic acid sequence refers to a DNA or RNA molecule in single or double stranded form, particularly a mRNA encoding a protein, such as a TF according to the invention.
• isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated.
• protein or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 -dimensional structure or origin.
• isolated protein is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
• gene means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA, or RNA transcript) in a cell, operably linked to suitable regulatory regions (e.g. a promoter).
• a gene may thus comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3 'nontranslated sequence comprising a polyadenylation site.
• ortholog of a gene or protein refers herein to the homologous gene or protein found in another species, which has the same function as the gene or protein, but (usually) diverged in sequence from the time point on when the species harbouring the genes diverged (i.e.
• Orthologs of the Arabidopsis TF genes may thus be identified in other plant species based on both sequence comparisons (e.g. based on percentages sequence identity over the entire sequence or over specific domains) and/or functional analysis.
• homologous and heterologous in the context of transgenic organisms refer to the relationship between a nucleic acid or amino acid sequence and its host cell or organism.
• a homologous sequence is thus naturally found in the host species (e.g. a tomato plant transformed with a tomato gene), while a heterologous sequence is not naturally found in the host cell (e.g. a tomato plant transformed with a sequence from potato plants).
• the term “homolog” or “homologous” may alternatively refer to sequences which are descendent from a common ancestral sequence (e.g. they may be orthologs).
• “Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA molecule.
• Upregulation of gene expression refers to an amount of mRNA transcript levels of at least about 2 times the level of the reference (control) sample, preferably at least about 3x, 4x, 5x, 1Ox, 2Ox, 30x or more.
• Downregulation of gene expression refers to an amount of mRNA transcript levels of at least about 2 times lower than the level of the reference (control) sample, preferably at least about 3x, 4x, 5x, 10x, 15x lower.
• Constant refers to an essentially equivalent mRNA transcript level as in the reference sample.
• housekeeping genes such as glyceraldehydes-3 -phosphate dehydrogenase, albumin, actins, tubulins, 18S or 28S rRNA
• constant transcript level e.g., glyceraldehydes-3 -phosphate dehydrogenase, albumin, actins, tubulins, 18S or 28S rRNA
• “Relative” mRNA expression levels refer to the change in expression level of one or more genes relative to that in another sample, preferably compared after "normalization” of the expression levels using e.g. housekeeping genes.
• the fold change (upregulation or downregulation) can be measured using for example quantitative real-time PCR.
• the fold change can be calculated by determining the ratio of an mRNA in one sample relative to the other.
• Mathematical methods such as the 2(- Delta Delta C(T)) method (Livak and Schmittgen, Method 2001, 25: 402-408) or other mathematical methods, such as described in Pfaffl (2001, Nucleic Acid Research 29: 2002-2007) or Peirson et al. (2003, Nucleic Acid Research 31: 2-7) may be used.
• “Absolute” mRNA expression levels refer to the absolute quantity of mRNA in a sample, which requires an internal or external calibration curve and is generally more time consuming to establish than relative quantification approaches.
• substantially identical means that two peptide or two nucleotide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default parameters, share at least a certain percent sequence identity.
• RNA sequences are said to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
• Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA.
• EmbossWIN version 2.10.0
• the program "needle” using the same GAP parameters as described above.
• local alignment algorithms such as the Smith Waterman algorithm (Smith TF, Waterman MS (1981) J. MoI. Biol 147(l);195-7), used e.g. in the EmbossWIN program "water”.
• Default parameters are gap opening penalty 10.0 and gap extension penalty 0.5, using Blosum62 for proteins and DNAFULL matrices for nucleic acids.
• “Stringent hybridization conditions” can also be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances.
• stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH.
• Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60 0 C. Lowering the salt concentration and/or increasing the temperature increases stringency.
• Stringent conditions for RNA-DNA hybridizations are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.
• Stringent conditions for DNA-DNA hybridization are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50 0 C, usually about 55°C, for 20 min, or equivalent conditions.
• the term "comprising” is to be interpreted as specifying the presence of the stated parts, steps or components, but does not exclude the presence of one or more additional parts, steps or components.
• indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
• the indefinite article “a” or “an” thus usually means “at least one”.
• plant refers to any organism of which the cells or some of the cells contain chloroplasts. It may refer to the whole plant (e.g. the whole seedling) or to parts of a plant, such as cells, tissue or organs (e.g. pollen, seeds, gametes, roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainable from the plant, as well as derivatives of any of these and progeny derived from such a plant by selfing and/or crossing. The term encompasses plants and/or plant parts of any developmental stage, unless indicated otherwise. "Plant cell(s)” include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., either in isolation or within a tissue, organ or organism.
• a “crop plant” refers herein to a plant species which is cultivated and bred by humans and excludes weeds such as Arabidopsis thaliana.
• a crop plant may be cultivated for food purposes (e.g. field crops), or for ornamental purposes (e.g. production of flowers for cutting, grasses for lawns, etc.).
• a crop plant as defined herein also includes plants from which non-food products are harvested, such as oil for fuel, plastic polymers, pharmaceutical products, cork and the like.
• PCR primers include both degenerate primers and non-degenerate primers (i.e. of identical nucleic acid sequence as the target sequence to which they hybridize).
• Oligonucleotides refer to nucleic acid fragments suitable for use as PCR primers or hybridization probes, e.g. coupled to a carrier in a nucleic acid microarray.
• DNA Microarray or “DNA chip” is a series of known DNA sequences (oligonucleotides or oligonucleotide probes) attached in a regular pattern on a solid surface, such as a glass slide, and to which a composition consisting of or comprising target sequences are hybridized for identification and/or quantification.
• TF transcripts and proteins are produced in the tissue upon priming and remain inactive until a subsequent stress challenge occurs.
• the defense response (and stress resistance) of the primed tissue is, thereby, faster and/or stronger compared to a direct response of an un-primed plant.
• the TF proteins present in the primed tissue thus, lead to a faster and/or augmented transcription of defense genes following exposure to biotic and/or abiotic stress compared to non -primed tissue.
• TF genes mark the priming response, and play a key role in the establishment of the priming. They identified specific sets of TF genes (referred herein to a "priming specific TF genes" or “TF marker genes” or “priming markers”, whose expression profile can be used for various purposes, e.g.
• TF genes for use as according to the invention are provided in Tables 1, 2 and 3 of the Examples. The genes listed are mRNA/cDNA sequences found in the weed Arabidopsis thaliana. Figure 6 and Tables 1 and 2 show that specific sets of TF genes were either significantly upregulated or significantly downregulated (transcription was induced or repressed by more than 2 fold compared to the base level of transcription) following priming of wild type plants. From all TF genes that were differentially expressed between primed and unprimed leaves upon treatment with Pseudomonas fluorescens WCS417r and/or BABA, a set of 37 genes (Table 3 and SEQ ID NO: 1-37) was identified as being priming specific marker genes (see Examples).
• TF genes were selected that showed differential expression (2-fold up or down) under the following conditions: only differentially expressed in wild- type plants after BABA treatment (10 TF genes); differentially expressed in both wild-type and nprl-1 plan ts after BABA treatment (6 TF genes); - differentially expressed in wild-type plants after BABA treatment, and differentially expressed in wild- type plants after treatment with WCS417r bacteria (4 TF genes); differentially expressed in both wild-type and nprl-1 plants after treatment with BABA, and differentially expressed in wild-type plants after treatment with WCS5417r bacteria (9 TF genes); or only differentially expressed in wild- type plants after treatment with WCS417r bacteria (8 TF genes).
• the selected TF genes were validated in 3 independent biological samples from replicate experiments.
• priming specific marker gene nucleic acid sequences comprise or consists of one or more of SEQ ID NO: 1-37, one or more of the cDNA/mRNA sequences provided by The Arabidopsis Information Resource (TAIR) Accession numbers listed in Tables 1, 2 and 3, and "variants" of any of these nucleic acid sequence, wherein variants comprise at least 70%, 80%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid sequence identity to any of these sequences, preferably when aligned pairwise over the entire length (using e.g. the program "needle", which uses the Needleman & Wunsch algorithm, with a gap opening penalty of 10.0 and gap extension penalty 0.5, and the DNAFULL matrix).
• TAIR Arabidopsis Information Resource
• cDNA mRNA
• Variants of the above marker genes include for example homologs or orthologs of these genes from other species, such as agricultural species and/or horticultural species.
• orthologs of the Arabidopsis TF sequences include sequences from Solanaceae (tomato, potato, tobacco, etc.), monocotyledonous plants, dicotyledonous plants, cereals, such as maize, wheat, rice, Brassicaceae, etc.
• Such genes can be identified using various methods in the art, such as nucleic acid based hybridization methods (e.g. Southern blot or Northern blot), protein based methods (e.g. Western blot), PCR based methods, sequencing, in silico analysis (using e.g.
• Variants can be identified in any agricultural or horticultural species, wherein priming is to be analyzed and/or modified. Once the variant nucleic acid is identified, its expression level within the tissue or cells of the plant or plant part can be analyzed using methods known in the art, such as PCR based methods. Variants are preferably functional variants, i.e. they also encode TFs having the same or similar biological function as the proteins encoded by the Arabidopsis genes.
• Variants of the marker nucleic acid sequences include also nucleic acid sequences encoding the TF proteins depicted in SEQ ID NO: 123 - 159, or variants thereof (such as orthologues from other species), or of the TF proteins encoded by the nucleic acid sequences listed in Tables 1, 2 and/or 3. Due to the degeneracy of the genetic code, several different nucleic acid sequences may encode the same amino acid sequence.
• the TF marker genes can be characterized by the amino acid sequence of the TF protein and/or by the activity of the protein as transcription factor.
• TF marker proteins comprise proteins depicted in SEQ ID NO: 123-159, proteins encoded by the nucleic acid sequences of the Accession numbers of Tables 1, 2 and 3 and variants of any of these.
• a variant protein is a protein comprising at least 50%, 60%, 65%, 70%, 75%, 80%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to any of these sequences, preferably when aligned pairwise over the entire length (using e.g.
• Fragments of such proteins are polypeptides of less then the full length of the protein, such as peptides comprising at least 5, 10, 20, 30, 40, 50, or more contiguous amino acids of the TF proteins or variants.
• Biological function of such proteins or peptides can be tested by the ability of the protein or peptide to regulate (induce or repress) transcription of target genes in a plant cell.
• the protein or peptide can be expressed in a transgenic host cell or organism (e.g. in a plant) and the ability of the TF to regulate target gene transcription can be analyzed.
• the invention makes in one embodiment use of PCR primers and/or probes and kits for detecting TF nucleic acid sequences, especially TF transcripts (cDNA or mRNA).
• TF nucleic acid sequences especially TF transcripts (cDNA or mRNA).
• Degenerate or specific PCR primer pairs to amplify TF nucleic acids (or parts thereof or variants thereof) from samples can be synthesized based on SEQ ID NO's 1-37 or sequences of Table 1, 2 and 3, or variants thereof, as known in the art (see Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany).
• a "priming" detection kit may comprise either TF marker gene specific primers and/or TF marker gene specific probes, and an associated protocol to use the primers or probe to detect TF transcripts in a sample.
• primer pairs capable of amplifying and detecting TF transcripts are provided in SEQ ID NO: 49-122, which depicts primer pairs capable of amplifying the TF marker gene transcripts of SEQ ID NO: 1-37.
• Preferred sets of TF marker genes whose expression is indicative of the priming status and/or priming capability are the following:
• the TF marker genes detected in the methods according to the invention are those endogenous to the plant which is to be used in the method.
• the primers or probes should thus be capable of detecting the endogenous mRNA transcripts of the TF genes (which can also be quantified).
• Detection and optionally quantification of TF transcripts in the following plant species is possible, preferably by first determining the nucleic acid sequences of the TF genomic sequences and/or mRNA (cDNA) sequences using methods known in the art and by then using these nucleic acid sequences to design primers and/or probes for transcript detection and/or quantification.
• cDNA mRNA
• Monocotyledonous plants or dicotyledonous plants for example maize/corn (Zea species, e.g. Z. mays, Z. diploperennis (chapule), Zea luxurians (Guatemalan teosinte), Zea mays subsp. huehuetenangensis (San Antonio Huista teosinte), Z. mays subsp. mexicana (Mexican teosinte), Z. mays subsp. parviglumis (Balsas teosinte), Z. perennis (perennial teosinte) and Z. ramosa), wheat (Triticum species), barley (e.g.
• Hordeum vulgare oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypium species, e.g. G. hirsutum, G. barbadense), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B. rapa, etc), sunflower (Helianthus annus), tobacco (Nicotiana species), alfalfa (Medicago sativa), rice (Oryza species, e.g. O.
• sativa indica cultivar-group or japonica cultivar-group forage grasses, pearl millet (Pennisetum spp. e.g. P. glaucum), tree species (e.g. fruit trees such as apple, pear, etc.), vegetable species, such as Solanum ssp e.g.
• tomato (Lycopersicon esculentum now reclassified as Solanum lycopersicon), potato (Solanum tuberosum, other Solanum species), eggplant (Solanum melongena), peppers (Capsicum annuum, Capsicum frutescens), pea, Cucurbitaceae (such as cucumber), melons, carrot, onion, leek, bean (e.g. Phaseolus species), soybean, fleshy fruit (grapes, peaches, plums, strawberry, mango) ornamental species (e.g. Rose, Petunia, Chrysanthemum, Lily, Gerbera species), woody trees (e.g. species of Populus, Salix, Quercus, Eucalyptus), fibre species e.g. flax (Linum usitatissimum) and hemp (Cannabis sativa).
• Cucurbitaceae such as cucumber
• melons such as cucumber
• carrot onion, leek, bean (e.
• a method for determining the priming state and/or the priming capability of a plant e.g. a seed or germinated seed, a whole seedling, etc.
• the method comprises the steps of: (a) obtaining a nucleic acid sample from said plant or plant part; and
• the nucleic acid sample may be obtained from a part of the plant (e.g. a leaf, root, etc.), or whole plant (e.g. seed or seedling), or a plurality of plants or plant parts, such as from a batch of seedlings or leaves.
• leaf aerial tissue most preferably leaf tissue is used.
• tissue samples are taken and optionally pooled prior to nucleic acid isolation or detection.
• the nucleic acid sample is preferably an mRNA or total RNA or cDNA sample.
• the nucleic acid sample is preferably extracted from the cells, e.g.
• nucleic acid sample encompasses plant tissue samples, such as unprocessed or partially processed tissue samples (e.g. freezing in liquid nitrogen and/or grinding).
• the plant or plant part may be of any species, as priming is likely a common defense strategy throughout the plant kingdom.
• the plant is preferably an agricultural or horticultural plant species, such as any of the species listed above. It may be a cultivated plant or a wild accession.
• nucleic acid samples from several plants and/or plant parts are analyzed, in order to account for variation in gene expression between plants and/or sampling.
• Control nucleic acid samples are included, depending on the aim of the marker analysis. For example, control samples (or reference samples) may be from unprimed plants or plant parts, whereby the TF expression level of which is then compared to samples of primed plants or plant parts.
• a step (a') may precede step (a) and comprise contacting plants or plant parts with one or more priming agents or potential priming agents (see further below).
• samples obtained from suitable controls would be samples from primed and/or unprimed plants.
• unprimed and primed samples are preferably compared for their TF marker gene expression before (or with) and after (or without) priming.
• primed plants or plant parts can be included and compared, for example BABA may be used as priming agent (e.g.
• priming agents in different concentrations to determine the sensitivity of the tissue for BABA-priming
• other known priming agents may be contacted with the plants or plant parts (e.g. bacterial strains as shown in the Examples).
• priming states or priming capability of different types of priming such as BABA-IR or ISR.
• samples may be analyzed, such as nucleic acid samples from primed- , unprimed- , differently primed plant tissue, from different plant species and accession, or from different plant parts (roots, leaves, aerial parts of seedlings, seeds - preferably post germination, etc.).
• step (b) the mRNA expression level (or corresponding cDNA level) of priming- specific marker genes in said sample is determined.
• quantitative PCR methods preferably quantitative RT-PCR, or nucleic acid hybridization based methods (for example microarray hybridization).
• Quantitative PCR may be carried out by conventional techniques and equipment, well known to the skilled person, described for instance in S. A. Bustin (Ed.), et al., A-Z of Quantitative PCR, IUL Biotechnology series, no 5, 2005.
• a preferred method is Reverse Transcription quantitative PCR (RT-qPCR) (see Czechowski et al., 2004, Plant J.
• the step (b) involves the detection of relative or absolute expression level of the marker gene mRNA transcript(s) in the sample(s).
• the method used is preferably very sensitive and able to detect very few mRNA molecules per sample.
• RT-qPCR can, for example detect one transcript molecule per 1000 cells.
• TF levels in Arabidopsis ranged from of 0.001 - 100 copies transcript per cell (Czechowski et al., 2004, supra).
• primers or probes are used which are capable of hybridizing to the selected TF transcripts, such as primers or probes (oligonucleotides) which are essentially similar or identical to part of the endogenous TF transcripts of the plant species being analyzed.
• RNA arrays where the cDNAs or oligonucleotides of the cDNAs are placed on carrier, such as a chip (e.g. an Affymetrix chip) and contacted with the nucleic acid sample of the tissue.
• carrier such as a chip (e.g. an Affymetrix chip)
• Suitable methods for microarray detection and quantification are well described in the art and may for instance be found in: Applications of DNA Microarrays in Biology. R.B. Stoughton (2005) Annu.Rev.Biochem. 74:53-82, or in David Bowtell and Joseph Sambrook, DNA Microarrays: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, 2003 ISBN 0-870969-625-7.
• nucleic acid molecules e.g. single stranded oligonucleotides according to the invention
• the arrayed nucleic acid molecules are complementary to the nucleotide sequences according to the invention, and the location of each nucleic acid on the chip is known.
• DNA chips or microarrays have been generally described in the art, for example, in US 5,143,854, US 5,445,934, US 5,744,305, US 5,677,195, US 6,040,193, US 5,424,186, US 6,329,143, and US 6,309,831 and Fodor et al.
• Step (c) involves analyzing the expression data of the marker genes determined in the different samples and inferring from the expression level the priming state and/or priming capability of said plant or plant part. The comparison to the suitable control samples allows such interferences to be made. For example, comparison of the expression profile of the test samples to a "primed" sample and/or an "unprimed” sample, allows one to conclude that the test sample is from either a primed or unprimed plant or plant part (or bulk).
• the priming state can also be verified by analyzing the speed and/or strength of the defense response of the plant material using for example a bioassay.
• Candidate plant genotypes and/or priming agents may also be further tested for growth and (seed) yield performance, or effects on growth and (seed) yield performance, respectively, under varying degrees of biotic and/or abiotic stress.
• Various steps of the method may be repeated one or more times. Selected plants may be selfed and/or crossed one or more times and e.g. progeny of such sellings or crosses may be tested for an altered priming state (see further below).
• This procedure may lead to the generation of a novel generation of crops with enhanced performance under one or more stressful conditions, or crop protection agents that enhance the performance of crops under one or more stressful conditions.
• consitutively primed plants or plants which continuously have a (significantly) higher priming state than the reference plants can be identified and selected.
• Further uses include the identification of over-represented DNA motifs in the promoter regions of priming-related TF genes, which can be used as a bait to identify key regulatory TFs for priming that are not transcriptionally regulated.
• the priming capability of a collection of plants or plant part scan be analysed using the TF marker gene expression.
• Plants or plant parts in which a marker gene expression profile indicative of a primed state cannot be induced in any way have no priming capability, while those in which it can be induced (or in which it is induced constitutively or to a higher level) have a good priming capability and can be selected for further use, such as breeding plants with higher or constitutive priming levels or priming capability (e.g. a stronger responsiveness to one or more priming agents).
• the marker genes are selected from the group consisting of: (a) nucleic acid sequences comprising or consisting of SEQ ID NO: 1-37,
• nucleic acid sequences comprising at least 70%, preferably at least 80%, 90%, 95%, 99% or more nucleotide sequence identity to SEQ ID NO: 1-37.
• Other preferred sets of marker genes are described elsewhere (see above).
• primer pairs which are TF transcript specific can be designed for each marker gene and can be used in the method.
• Czechowski et al. 2004, supra
• Such primer pairs can be used (see Supplmentary Material Table Sl of the Czechowski et al. 2004 paper), or primer pairs can be designed using known methods.
• SEQ ID NO: 49-122 provide primer pairs for amplifying the TF marker genes of SEQ ID NO: 1-37, respectively.
• primer pairs may be designed, such as fragments comprising 14-30 contiguous nucleotides of the cDNAs of SEQ ID NO: 1-37 (or complement strands thereof) and/or of marker genes of Table 1, 2 or 3.
• primer pairs can be designed for amplifying homologs or orthologs of any of the TF marker genes of SEQ ID NO: 1-37 and/or of Table 1, 2 or 3 from nucleic acid samples of other plant species, such nucleic acid sequences comprising at least 70%, preferably at least 80%, 90%, 95%, 99% or more nucleotide sequence identity to the sequences of Table 1, 2 or 3.
• labeled primers or oligonucleotides are used to quantify the amount of reaction product.
• a convenient system for quantification is the immunolabeling of the primers, followed by an immuno -lateral flow system (NALFIA) on a pre-made strip (references: Kozwich et al., 2000, Applied and Environmental Microbiology 66, 2711-2717; Koets et al., 2003, In: Proceedings EURO FOOD CHEM XII - Strategies for Safe Food, 24-26 September 2003, Brugge, Belgium, pages 121 -124; and van Amerongen et al., 2005 In: Rapid methods for biological and chemical contaminants in food and feed. Eds. A. van Amerongen, D. Barug and M. Lauwaars, Wageningen Academic Publishers, Wageningen, The Netherlands, ISBN: 9076998531, pages 105-126).
• RNA isolation As a positive control for the RNA isolation, reverse transcriptase reaction, amplification reaction and detection step, amplification and detection of a constitutively expressed housekeeping gene may be included in the assay, such as ribosomal (18S or 25S) rRNA's, actin, tubulin, ubiquitin or GAPDH (see SEQ ID NO: 38-48, which provide control cDNAs, whose expression can be detected).
• ribosomal (18S or 25S) rRNA's such as ribosomal (18S or 25S) rRNA's, actin, tubulin, ubiquitin or GAPDH (see SEQ ID NO: 38-48, which provide control cDNAs, whose expression can be detected).
• Primers may be labeled with direct labels such as FITC (fluorescein), SYBR® Green, Texas Red, Rhodamine and others or with tags such as biotin, lexA or digoxigenin which may be visualized by a secondary reaction with a labeled streptavidin molecule (for instance with carbon or a fluorescent label) or a labeled antibody (labeled with fluorescent molecules, enzymes, carbon, heavy metals, radioactive isotopes or with any other label).
• direct labels such as FITC (fluorescein), SYBR® Green, Texas Red, Rhodamine and others or with tags such as biotin, lexA or digoxigenin which may be visualized by a secondary reaction with a labeled streptavidin molecule (for instance with carbon or a fluorescent label) or a labeled antibody (labeled with fluorescent molecules, enzymes, carbon, heavy metals, radioactive isotopes or with any other label).
• the plant or plant part from which the nucleic acid sample is obtained is preferably an agricultural crop plant or a horticultural plant. Plant species are listed further above. It is understood that the marker genes detected in plant species other than Arabidopsis are homologs or orthologs of the Arabidopsis genes of Tables 1, 2 and/or 3. The primer pairs are thus designed to amplify the homologous or orthologous transcripts.
• the primer pairs designed for Arabidopsis TF genes may also work for other plant species, such as Brassicaceae species, e.g. Brassica species. However, the homologous or orthologous sequences can easily be identified and isolated and primers capable of detecting these can be designed using undue experimentation.
• the method may optionally further comprise the step
• One of the advantages of the method is that disease or stress resistance assays are not necessary in order to determine the resistance level, as the level is correlated to the marker gene expression profile (i.e. with the priming status).
• the priming state and/or capability can be modified by identifying plants having such a modified state or capability, either naturally (making use of natural variation) or artificially (by inducing variation, e.g. mutagenizing plants or plant parts using one or more mutagens).
• a method for generating and/or selecting a plant or plant part having an enhanced priming state (and thus enhanced biotic and/or abiotic stress resistance) and/or enhanced priming capability comprising the steps of:
• priming state of a plurality of plants or plant parts (either natural plants or plant parts or optionally mutagenized plant or plant parts) by analyzing the mRNA expression level of priming-specific marker genes selected from the group consisting of the genes of Table 1, 2 and 3, or genes comprising at least 70% nucleic acid identity to those marker genes and of suitable control plants or plant parts;
• Steps (a) and (b) and/or (c) and/or (d) may be repeated one or more times.
• the plants identified in (d) may be crossed or selfed or treated otherwise and retested according to the method.
• the starting plant material may optionally be mutagenized, i.e. treated with one or more mutagens (mutagenic agents).
• “Mutagenesis” refers to the process in which plant cells (e.g., a seed or tissues, such as pollen, etc.) are contacted one or more times to a mutagenic agent, such as with a chemical mutagen, fast neutron mutagenesis, gamma irradiation, or a combination of the foregoing.
• a mutagenic agent such as with a chemical mutagen, fast neutron mutagenesis, gamma irradiation, or a combination of the foregoing.
• the desired mutagenesis may be accomplished by use of chemical means such as by contact with ethylmethylsulfonate (EMS), ethylnitrosourea, etc., by the use of physical means such as x-ray, etc, or by gamma radiation, such as that supplied by a Caesium 137 source.
• EMS ethylmethylsulfonate
• gamma radiation such as that supplied by a Caes
• the plant or plant part selected in step (d) is in one embodiment constitutively primed, in the absence of biotic and/or abiotic stress and/or priming agents.
• a natural plant variant , or natural mutant or induced mutant, plant is selected which has a significantly enhanced priming state, preferably a constitutive priming state, so that it need not be treated with priming agents prior to planting.
• Such plants preferably have no negative agronomical characteristics associated with them as a result of the altered priming state. For example, yield should be comparable to the normal plant. Alternatively, plants which respond to lower amounts of priming inducer can also be identified.
• Plants or plant parts generated or identified and selected by this method are also encompassed herein.
• priming agents do already exist, there is a need for new priming agents and reliable, easy methods for screening a large number of biological or chemical compounds or compositions for their use as priming agents.
• the invention provides a method for identifying biological or chemical compounds, or compositions comprising biological or chemical compounds, which are capable of activating or inducing or enhancing priming in a plant or plant part, comprising the steps of:
• marker genes are selected from the group consisting of the genes of Table 1, 2 and 3, or genes comprising at least 70% nucleic acid identity to those marker genes.
• the steps may be repeated once, two or more times.
• any biological or chemical compound may be contacted with the plants or plant parts.
• a plurality of different compounds can be contacted in parallel with plants or plant parts.
• each test compound is brought into physical contact with one or more individual plants.
• Contact can also be attained by various means, such as spraying, spotting, brushing, applying solutions or solids to the soil, to the gaseous phase around the plants or plant parts, dipping, etc.
• the test compounds may be solid, liquid, semi-solid or gaseous.
• test compounds can be artificially synthesized compounds or natural compounds, such as proteins, protein fragments, volatile organic compounds, plant or animal or microorganism extracts, metabolites, sugars, fats or oils, microorganisms such as viruses, bacteria, fungi, etc.
• the biolo gical compound comprises or consists of one or more microorganisms, or one or more plant extracts or volatiles (e.g. plant headspace compositions).
• the microorganisms are preferably selected from the group consisting of: bacteria, fungi, mycorrhizae, nematodes and/or viruses . It is especially preferred that the microorganisms are non-pathogenic to plants, or at least to the plant species used in the method. Especially preferred are bacteria which are non-pathogenic root colonizing bacteria and/or fungi, such as Mycorrhizae. Examples include Pseudomonas fluorescens strains, such as P.
• fluorescens WCS417r Pseudomonas putida strains, such as P. putida WCS358, and various Glomus species. Obviously other strains and species may be used. Mixtures of two, tree or more compounds may also be applied to start with, and a mixture which shows an effect on priming can then be separated into components which are retested in the method. Using mixtures, also synergistically acting compounds can be identified, i.e. compounds which provide a stronger priming effect together than the sum of their individual priming effect.
• a compound or composition identified using the method is also encompassed herein.
• the compound identified may be used to make a priming agent or inducer, i.e. to make a composition comprising or consisting of suitable amount of the compound in a suitable formulation, as known in the art.
• a priming agent or inducer i.e. to make a composition comprising or consisting of suitable amount of the compound in a suitable formulation, as known in the art.
• compositions are liquid or solid (e.g. powders) and can be applied to the soil, seeds or seedlings or to the aerial parts of the plant.
• the DNA motif 5'TAG[TA]CT 3' was identified as being present in a large number of transcription regulatory elements of priming-specific marker genes according to the invention.
• the fourth nucleotide may be T or A, and is therefore bracketed.
• the motif may thus either be 5'TAGTCT 3' (3'ATCAGA 5') or 5' TAGACT 3'(3'ATCTGA 5').
• a method for identifying other priming-specific marker genes comprising: a. identifying plant genes which comprise the motif TAG[TA]CT in their transcription regulatory region (as a c ⁇ -acting element), for example in silico, and b. testing whether the identified gene, or a homologue or ortholog of the gene, is upregulated following priming of the plant in which the gene (or homologue or orthologue) occurs in nature, compared to suitable control treatments, in order to verify that the gene is a priming specific marker gene and c. using said gene, or a variant thereof (such as a homologue or orthologue), or a fragment of any of these, in a method according to the invention, as described.
• the first step can for example be carried out by using bioinformatics methods to identify nucleic acid sequences of plant derived nucleic acids comprising the motif using routine methods.
• a method for identifying one or more master regulators of priming i.e. proteins such as TFs which are capable of activating transcription of genes comprising the motif TAG[TA]CT, is provided herein.
• the method comprises the steps of: a) identifying one or more proteins which bind to the motif or are capable of binding to the motif in a nucleic acid - protein binding assay, b) isolating or purifying the protein and determining its amino acid sequence, and c) optionally determining the nucleic acid sequence of the gene encoding the protein, and d) using the information obtained above for various further uses, such as using the identified gene or variant or fragment thereof as a priming specific marker as described herein and/or for identifying compounds and/or compositions which activate expression of the gene as described, etc.
• nucleic acid motif for identifying and preferably isolating key priming regulator proteins is provided herein.
• step a) may involve the use of fluorometric DNA-protein binding assays, electrophoretic assays (e.g. gel mobility shift assays), etc.
• electrophoretic assays e.g. gel mobility shift assays
• transcription factor genes selected from selected from the group consisting of the genes of Table 1, 2 and/or 3, or genes comprising at least 70% nucleic acid identity to those genes, as biomarkers for determining the priming state and/or priming capability of plants or plant parts.
• kits for analyzing the priming state and/or capability of plants or plant parts comprise for example one or more primers or probes which are capable of detecting the TFs according to the invention, optionally control samples and/or microtitre plates, Eppendorf tubes, instructions for use, qPCR well plates as described e.g. below, etc.
• a qPCR well-plate i.e. 385-wells plate or more
• a qPCR well-plate comprising a multitude of the selected primer sets of the TFs and optionally reference genes for use in the methods according to the invention.
• SEQ ID NO 1-37 subset of priming-specific marker genes of A. thaliana
• SEQ ID NO 38-48 cDNA sequences of PR-I, PR-5, RAB18, PDF1.2, Lox2, VSP2,
• SEQ ID NO 49-122 PCR primer pairs for amplifying cDNAs of SEQ ID NO: 1-37.
• SEQ ID NO 123-159 Amino acid sequences of the TF proteins encoded by SEQ ID NO: 1-37, respectively.
• A BABA-induced priming for enhanced transcription of the PR-I gene upon treatment with the SA analogue benzothiadiazole (BTH).
• BTH the SA analogue benzothiadiazole
• Five -week-old plants (CoI-O) were soil-drenched with 250 ⁇ M BABA and 1 day later treated with BTH by spraying the indicated concentrations onto the leaves.
• Leaf material for RNA blot analysis was collected at 6 and 24 h after treatment with BTH.
• B ISR-related priming for enhanced transcription of the LOX2 gene. ISR was triggered by transferring 2-week-old seedlings (CoI-O) to soil containing P. fluorescens WCS417r bacteria (5xlO 7 cfu.g "1 ).
• Plant material was harvested at different time points after MeJA treatment.
• Disease rating is expressed as the percentages of leaves in disease classes I (no sporulation), II (trailing necrosis), III ( ⁇ 50% of the leaf area covered by sporangia), and IV (heavily covered with sporangia, with additional chlorosis and leaf collapse).
• FIG. 1 ISR and BABA-IR against Pseudomonas syringae pv. tomato DC3000 CoI-O, nprl, ibs2 and ibs3.
• B Quantification of BABA-IR. Five- to 6-week-old plants were soil-drenched with BABA to a final concentration of 250 ⁇ M, and two days later challenge- inoculated with P. syringae pv. tomato DC3000. Inoculation and disease scoring were performed as described above.
• RNA for RT-qPCR analysis was extracted from shoot material at 32 h after soil-drench treatments.
• A Number of TF genes showing > 2-fold induction or repression in the leaves upon treatment with WCS417r or BABA.
• B VEN-diagrams showing differences and similarities in transcriptional profiles upon treatment with WCS417r and BABA in CoI-O and nprl-1.
• Figure 8 Occurrence of cis-acting elements in the promoter regions of WCS417r- and BABA-inducible TF genes in CoI-O and nprl.
• Plants were cultivated in a growth chamber with a 8-h day (200 ⁇ Em ⁇ .sec "1 at 24°C) and 16-h night (20 0 C) cycle at 70% relative humidity for another 11 days. Plants were watered every other day and received half-strength Hoagland nutrient solution (Hoagland, D.R., and Arnon, D.I. (1938) Calif. Agric. Exp. Stn. Bull. 347, 36-39) containing 10 ⁇ M Sequestreen (CIBA-Geigy, Basel, Switzerland) once a week.
• Hoagland nutrient solution Hoagland, D.R., and Arnon, D.I. (1938) Calif. Agric. Exp. Stn. Bull. 347, 36-39
• 10 ⁇ M Sequestreen CIBA-Geigy, Basel, Switzerland
• the rifampicin-resistant P. fluorescens strain WCS417r (Pieterse et al., 1996, supra) was grown on King's medium B agar plates (King, E.O., et al. 1954, J. Lab. Clin. Med. 44, 301-307) for 24 h at 28°C. Bacterial cells were collected and resuspended in 10 mM MgSO 4 to a density of 10 9 CFU per ml.
• the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Whalen, M. C, et al. 1991, Plant Cell 3, 49-59), used for challenge inoculations, was cultured overnight in liquid King's medium B at 28°C, collected by centrifugation, and resuspended in 10 mM MgSO 4 to a final density of 2.5xlO 7 CFU/ml.
• H. parasitica WAC09 was obtained from the Plant Research Institute,
• ISR was activated by transplanting 2-week-old seedlings to a sand/potting soil mixture containing 5 ⁇ 10 7 CFU/g WCS417r bacteria. Control soil was supplemented with an equal volume of 10 mM MgSO 4 .
• BABA-IR was triggered by applying BABA (Sigma- Aldrich Chemie BV, Zwijndrecht, the Netherlands) as a soil-drench to the indicated concentrations.
• Disease rating was expressed as intensity of disease symptoms and pathogen sporulation on each leaf: I, no symptoms; II, trailing necrosis; III, sporangia; IV, heavily covered with sporangia, with additional chlorosis and leaf collapse.
• I no symptoms
• II trailing necrosis
• III sporangia
• IV heavily covered with sporangia, with additional chlorosis and leaf collapse.
• RNA-free total RNA was converted into cDNA using oligo-dT2o primers (Invitrogen, Breda, the Netherlands), 10 mM dNTPs, and SuperscriptTM III Reverse Transcriptase (Invitrogen, Breda, the Netherlands) according to the manufacturer's instructions.
• PCR reactions were performed with an ABI PRISM ® 7900 HT sequence detection system, using SYBR ® Green to monitor the synthesis of double stranded DNA.
• 1 ⁇ l of cDNA was mixed with 5 ⁇ l 2x SYBR ® Green Master Mix reagent (Applied Biosystems) and 200 nM of a TF-specific primer pair (Czechowski et al., 2004, supra; Czechowski, et al. 2005, Plant Physiol. 139, 5-17), in a total volume of 10 ⁇ l.
• cDNA Prior to dispensing into individual wells, cDNA was mixed with SYBR ® Green Master Mix reagent, to ensure that all reactions contained an equal amount of template.
• ⁇ C T normalized TF expression levels
• At4G05320 UBIlO
• At2G28390 SAND family
• At5G46630 Cethrin adaptor complex subunit
• At5G55840 PPR protein
• RNA from independent experiments was used for DNase treatment and subsequent cDNA synthesis as described above.
• PCR reactions were done in optical 96 -well plates with an MylQTM Single Color Real-Time PCR Detection System (Bio-Rad, Veenendaal, the Netherlands) in combination with SYBR ® Green. Reactions were performed in a total volume of 15 ⁇ l, containing cDNA, 0.5 ⁇ L of each of the two gene-specific primers (10 pmol. ⁇ L "1 ), and 3.5 ⁇ L of 2x IQ SYBR ® Green Supermix reagent.
• TMEV TIGR Multiexperiment Viewer
• BABA-IR is marked by enhanced expression of S A -inducible gene expression upon pathogen infection (Zimmerli et al., 2000, supra), whereas expression of P. fluorescens WC S417r -mediated ISR is accompanied by a faster and stronger expression of JA- inducible genes upon pathogen infection (Van Wees, et al. 1999, supra; Verhagen, et al. 2004, MoI. Plant-Microbe Interact. 17, 895-908).
• To assess whether the priming by BABA acts through an increase in sensitivity to SA water- and BABA-treated plants were sprayed with increasing concentrations of the SA analogue BTH and subsequently tested for expression of the SA-inducible PR-I gene.
• WCS417r-mediated ISR is moderately effective against infection by the oomycete H. parasitica (Ton et al, 2002, supra). However, H. parasitica is not resisted through JA- dependent defense mechanisms (Thomrna, et al 1998, Proc. Natl. Acad. Sci. USA 95, 15107-15111). This indicates that the ISR-mediated protection against //, parasitica is based on different mechanisms than priming for JA-dependent defenses. To examine whether WCS417r-mediated ISR against H. parasticia is based on a similar priming mechanism as BABA-IR against H.
• BABA- induced priming for papillae was still intact in nprl-1 plants. This indicates that the signaling pathways controlling WCS417r- and BABA-induced priming for papillae differ in their requirement of the NPRl protein.
• BABA-induced priming for papillae in Arabidopsis depends on the SAClb/AtIBS2 and ABAl /IBSS genes, which suggests involvement a phosphoinositide- and ABA- dependent signaling pathway (Ton et al., 2005, supra).
• WCS417r-treated ibs2-2 and npq2-l plants failed to show an augmented induction of callose-rich papillae, whereas WCS417r-treated wild-type plants reacted with a statistically enhanced number of papillae compared to the corresponding controls (Fig. 3).
• BABA-IR and ISR against Pst DC3000 do not involve phosphoinositide- and ABA-dependent signaling, but require an intact NPRl -protein.
• WCS417r- and BABA- induced defense priming against Pst DC3000 is regulated by a different signaling pathway than WCS417r- and BABA-induced priming for papillae upon H. parasitica infection.
• ISR and BABA-IR are both characterized by priming for enhanced transcription of defense-related genes (Fig. 1). This prompted us to test whether this enhanced transcriptional activity is based on enhanced expression of transcription factors in response to WCS417r bacteria or BABA. To this end, the transcription of -2.300 TF genes was quantified using RT-qPCR, since this technique is significantly more sensitive for the detection of small differences in TF gene expression than DNA array technology (Czechowski et al., 2004, supra). RNA was extracted from water-, WCS417r-, or BABA-treated CoI-O plants at 32 h after soil-drench treatment.
• the profiles corresponding to 3 replicate samples from WC S417r -treated plants formed a separate cluster in comparison to the profiles from control-treated plants and plants treated with the ISR non-inducing WCS374r strain.
• the profiles of 3 replicate samples from BABA-treated Col plants formed a distinctive cluster compared to the profiles of the treatments with water and AABA (Fig. 7). This indicates that the same set of TF genes can also be used to specifically mark the BABA-induced priming response.
• promoter elements could be identified that were significantly over-represented in promoters of WCS417r-inducible TF genes of CoI-O, BABA-inducible TF genes of CoI-O, or BABA-inducible TF genes in nprl-1. All three classes of priming-inducible TF genes were enriched in PLGTl box and G- box elements that are related to responses to pathogen infection and salt-stress (Droge- Laser et al., 1997, EMBO J.
• the promoter regions of the BABA-responsive TF genes of CoI-O showed a statistically significant enrichment of W-box elements, which was absent in the promoter regions of the groups of WCS417r -responsive TF genes and BABA- inducible TF genes in nprl-1 (Fig. 8). This suggests that the NPRl -dependent induction of TF genes by BABA requires binding of WRKY transcription factors.
• WCS417r bacteria induce systemic expression of TF genes related to the regulation of JA- and ET -dependent defense reactions (Table Sl).
• the group of WCS417r-inducible TF genes contained 17 AP2/EREBPs (APETALA2/ETYLENE RESPONSIVE ELEMENTS BINDING PROTEINs) genes, amongst which the ERFl (ETHYLENE RESPONSIVE FACTOR!) gene ( At3g23240) encoding a key regulatory factor in the integration of JA- and ET-dependent signaling pathways (Lorenzo et al., 2003, Plant Cell 15, 165- 178).
• BABA primes for enhanced induction of SA-dependent defense mechanisms, which determines the level of BABA-induced protection against P. syringae and B. cinerea (Zimmerli et al., 2000; 2001; Ton et al., 2005, all supra).
• BABA induces a relatively large set of different TF genes, of which the majority was no longer inducible by BABA in the nprl-1 mutant (Figs. 6 and 7).
• NPRl is important for the BABA-induced expression of many TF genes, suggesting an important role of NPRl in the onset of priming for SA-dependent defense mechanisms.
• NPRl -dependent, BABA- inducible TF genes included 21 members of the WRKY family of TFs. Many of these genes, such as ATWRKY18, ATWRKY38, ATWRKY58, ATWRKY59, and ATWRKY70, have been reported to play an important role in the fine-tuning of SA-inducible defenses (Eulgem, 2005, Trends Plant Sci. 10, 71-78), and were recently identified as direct targets of NPRl (Wang et al, 2006, supra).
• TF gene expression point to specific signaling pathways that regulate the onset of priming through enhanced expression of defense-related TFs.
• the priming-related TF genes described in this study are controlled by other TFs that may not be regulated on the transcriptional level. Such "early-acting" TFs in the priming pathway could serve as important key regulators in the onset of priming.
• the promoter regions of both WCS417r- and BABA- inducible TF genes were significantly enriched in PLGTl- and G-boxes (Fig. 8). Both these elements have been related to transcriptional responses to pathogen infection, salt-stress, JA, and ABA (Droge-Laser et al., 1997; Faktor et al., 1997; Boter et al., 2004; Park et al., 2004, all mentioned supra).
• the promoter regions of BABA- inducible genes in the nprl-1 mutant displayed a much stronger enrichment in G-box elements than those in CoI-O (Fig. 7).

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