WO2009041805A1

WO2009041805A1 - Defense priming in plants

Info

Publication number: WO2009041805A1
Application number: PCT/NL2007/050472
Authority: WO
Inventors: Jurriaan Ton; Sjoerd Van Der Ent; Maria Helena Adriana Van Hulten; Cornelis Marinus Jozef Pieterse
Original assignee: Universiteit Utrecht Holding B.V.
Priority date: 2007-09-28
Filing date: 2007-09-28
Publication date: 2009-04-02

Abstract

The present invention relates to the field of biotic and/or abiotic stress resistance of plants, especially to methods for activating priming in plants and methods for determining the priming status of plants using priming-specific marker genes.

Description

DEFENSE PRIMING IN PLANTS

FIELD OF THE INVENTION

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. Methods for determining the priming status and/or capability of plants using markers are provided. Such methods can be used to determine the (natural or induced) variation in priming status and/or priming capability between plants and to generate, identify or select plants which have an increased priming capability and/or an augmented priming status (e.g. constitutively primed). Especially, 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).

BACKGROUND OF THE INVENTION 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).

The plant hormones salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) play important roles in these stress-inducible defense mechanisms (Glazebrook, J. (2005) Annu . Rev. Phytopathol. 43, 205-227; Mauch-Mani, B., and Mauch, F. (2005) Curr. Opin. Plant Biol. 8, 409-414; Van Loon, et al (2006) Trends Plant Sci. 11, 184-191; Seki, M., et al (2007) Opinion Plant Biol. 10, 296-302). Both constitutive and pathogen-inducible defense mechanisms contribute to the plant's basal defense barrier.

As a result of the evolutionary arms-race between plants and microbial parasites, many pathogens have evolved the ability to circumvent or suppress the basal defense layer. Consequently, plants have co-evolved additional layers of defense, allowing them to detect invaders at earlier stages of infection. The extensively-studied mechanisms of i?-gene dependent resistance is an example of such a superimposed layer of defense (McDowell, J.M., and Woffenden, BJ. (2003). Trends Biotechnol. 21, 178-183; Chisholm, S.T., et al, (2006). Cell 124, 803-814). In addition to these innate defense reactions, plants also possess the ability to acquire an enhanced level of resistance against future attack upon perception of specific environmental cues. In many cases, this so-called induced resistance is not based on a direct activation of defense mechanisms, but on a sensitization of the tissue that results in a faster and stronger expression of basal defense mechanisms once the plant is exposed to pathogen attack. This phenomenon is called "priming" (Conrath, U. et al (2006) MoI. Plant- Microbe Interact. 19, 1062-1071).

The classical form of induced resistance develops upon localized infection by a necrosis-inducing pathogen, which causes a systemic acquired resistance (SAR) that protects against a variety of different pathogens (Ryals, et al (1996) Plant Cell 8, 1808- 1819; Durrant, W.E., and Dong, X. (2004) Annu. Rev. Phytopathol. 42, 185-209). 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). In addition to SAR, 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). Unlike SAR, this so-called rhizobacteria-induced systemic resistance (ISR) functions independently of SA, but requires components of the JA- and ET-dependent response pathways (Pieterse et al., 1998, supra). Interestingly, both ISR and SAR require an intact NPRl protein, suggesting that they are controlled by partially overlapping signaling pathways. However, it is commonly assumed that the SAR and ISR pathways diverge downstream of NPRl, because ISR, unlike SAR, is not accompanied by systemic expression of PR genes (Van Wees et al. (1999) Plant MoI. Biol. 41, 537-549; Verhagen et al. (2004). Plant-Microbe Interact. 17, 895-908). Apart from biologically induced SAR and ISR, there are many chemicals that trigger similar induced resistance responses. Most of these agents activate the SAR pathway, because they trigger a similar set of PR genes, or fail to induce resistance in mutants that are impaired in the SAR pathway (Lawton, K.A. et al. (1996) Plant J. 10, 71-82; Dong, H. et al. (1999) Plant J. 20, 207-215). However, the non-protein amino- acid β-aminobutyric acid (BABA) has been shown to trigger a partially different induced resistance response than SAR or ISR. Although BABA-induced resistance against Pseudomonas syringae pv tomato DC3000 (Pst DC3000) and Botrytis cinerea resembles pathogen-induced SAR in its requirement of SA and NPRl (Zimmerli et al. (2001) Plant Physiol. 126, 517-523; Zimmerli et al. (2000) Proc. Natl. Acad. Sci. USA 97, 12920-12925), 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). Despite the differences in signal transduction pathways between SAR, ISR and

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.

Although 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.

The inventors investigated the differences and similarities in signaling pathways that control the priming during rhizobacteria-mediated ISR and BABA-IR. In addition, they examined the involvement of transcription factors (TFs) during the onset of ISR- and BABA-IR-related priming. Because many plant TFs are thought to be tightly regulated at the transcriptional level (Chen, et al. (2002) Plant Cell 14, 559-574; Lee, et al. (2006) Proc. Natl. Acad. Sci. USA 103, 6055-6060), the inventors performed a genome -wide RT-qPCR profiling of about 2.300 TF genes upon induction of the primed defense state during ISR and BABA-IR. The inventors found that priming by ISR- inducing WCS417r bacteria and BABA is marked by characteristic transcriptional changes of specific sets of TF genes.

Without limiting the scope of the invention, it is believed that priming results in an increased level of (inactive) TF proteins in the primed tissue compared to non-primed tissue. Upon subsequent exposure of the tissue to 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. constitutively) and/or for screening micro-organisms, compounds or compositions for their ability to induce priming. Although BABA is used to induce priming and disease resistance, there remains a need for 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).

The availability of sets of marker genes which are indicative of the primed defense status means that it is not necessary anymore to use host plant-specific pathogen- or other stress treatments to determine the primed defense state, which has been necessary so far and which is both laborious and time consuming because it requires disease resistance bioassays with pathogenic microorganisms.

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.

DEFINITIONS

"Priming" 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. In contrast to induced resistance that is based on a direct induction of active defense mechanisms, 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).

"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). "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. "Priming status", "priming state", or "primed defense state" 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. not primed as indicated by the TF profile) and/or quantitative measure of the priming status (partially primed referring to TFs having a weaker expression level than fully primed). "Priming capability" or "priming capacity" refers to the ability of a plant or plant part to become "primed", i.e. priming can be activated in the plant or plant part. "Priming inducer" or "priming agent" refers herein to conditions or compounds which are able to induce priming, i.e. to activate priming and induce a sensitized state.

"Priming 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. The term "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. This will initiate early stages of germination, but does not permit radicle protrusion. After priming the seeds are dried again. Later on, after sowing and exposure to water these primed seeds germinate faster, and seedling emergence is synchronized. Thus, when referring to "seed priming" herein in relation to the instant invention "defense priming" of the seeds (e.g. by treatment of the seeds with a priming agent, e.g. a coating comprising a priming agent) is referred to, unless indicated otherwise. 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. Generally all strains, races or pathovars of a pathogen species which are capable of causing disease on host tissue are included herein. Also subgroups, such as subspecies of pests and pathogens are included. Generally 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).

"Abiotic stress" refers to stress caused by abiotic (non-living) agents, such as temperature stress (cold/freezing, heat), salinity (salt), wind, metals, day-length (photoperiod), water-stress (such as too little or too much water availability, i.e. drought, dehydration, water-logging, etc.), wounding, radiation, nutrient deprivation, etc. "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. For example, 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 (such as LOX2 and pathogenesis-related proteins like PR-I, PR-2, etc.) can be used to assess the speed and/or strength of the defence response. The term "nucleic acid sequence" (or nucleic acid molecule) 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. An "isolated nucleic acid sequence" refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated.

The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 -dimensional structure or origin. An "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.

The term "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. The term "ortholog" of a gene or protein refers herein to the homologous gene or protein found in another species, which has the same function as the gene or protein, but (usually) diverged in sequence from the time point on when the species harbouring the genes diverged (i.e. the genes evolved from a common ancestor by speciation). Orthologs of 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.

The terms "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). Depending on the context, the term "homolog" or "homologous" may alternatively refer to sequences which are descendent from a common ancestral sequence (e.g. they may be orthologs). "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. Generally, housekeeping genes (such as glyceraldehydes-3 -phosphate dehydrogenase, albumin, actins, tubulins, 18S or 28S rRNA) have a constant transcript level.

"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.

The term "substantially identical", "substantial identity" or "essentially similar" or "essential similarity" 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. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). It is clear that when 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. or using in EmbossWIN (version 2.10.0) the program "needle", using the same GAP parameters as described above. For comparing sequence identity between sequences of dissimilar lengths, it is preferred that local alignment algorithms are used, 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. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60⁰C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridizations (Northern blots using a probe of e.g. lOOnt) 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 (Southern blots using a probe of e.g. lOOnt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50⁰C, 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.

In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The term "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.

DETAILED DESCRIPTION

It was found that the phenomenon known as "priming" causes a change in the expression of TF genes in sensitized tissue (upregulation or downregulation), without directly inducing defense-related marker genes (such as PR genes) to a level that is indicative of active expression of defense mechanisms. Without limiting the scope of the invention, it is believed that 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. For example, the production of defense proteins such as those involved in SA-induced defense, JA-induced defense and callose deposition, is faster and/or stronger in the primed plant upon challenge. For example, BABA-primed plants already expressed relatively high levels of PR-I at 8 hrs after pathogen challenge, whereas non-primed plants did not reach this level of PR-I expression until 24 hours after pathogen challenge (see Examples). The inventors thus found that (specific sets of) 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. (1) as biomarkers for the priming state and/or priming capability of plants and plant parts, (2) for selecting or generating plants or plant parts having a modified priming state or ability, and (3) for the easy and large scale screening of compounds or compositions which are able to alter the priming status of plants or plant parts. These embodiments will be described in more detail herein below, following the description of the TF genes and sets of TF genes suitable for transcription profiling and for use in the above methods.

TF genes for use as according to the invention The TF genes for use 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). Thus, based on the first whole-genome screen, 37 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 Thus in one embodiment of the invention priming specific TF marker genes 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).

Also encompassed are fragments of any one of SEQ ID NO: 1-37, and of the nucleic acid sequences listed by Accession number in Tables 1, 2 or 3, and fragments of any of these or of any of the variants, wherein fragments comprise nucleic acid sequences with at least 5, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 50, 100, 150, 200 or more contiguous nucleotides. Fragments can for example be used as primers or probes, to detect the mRNA (cDNA) transcript level of the TF genes or to isolate / amplify orthologs from other plant species.

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. For example, 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. computer programs such as FASTA, BLAST, etc.). 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.

Thus, in one embodiment of the invention, 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. 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 Blossum62 matrix). 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. For example 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). 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). Likewise, nucleic acid fragments of such sequences can be used as hybridization probes. 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. Examples of 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:

1. At least 5, 10, 15, 20, 25, or more, e.g. at least 30, 35 or 37 or more of priming up- or down-regulated TF genes, such as selected from SEQ ID NO: 1-37 and/ or from variants thereof. 2. At least 5, 10, 15, 20, 25, or more, such as at least 30, 35, 37, 40, 50, 60, 70, 80 or more of priming upregulated TF genes, such as selected from Table IA and/or

Table IB and/or Table 1C, or variants thereof.

3. At least 5, 10, 15, 20, 25, or more, such as at least 30, 35, 37, 40, 50, 60, 70, 80 or more of priming downregulated TF genes, such as selected from Table 2 A and/or Table 2B and/or Table 2C, or variants thereof.

4. At least 5, 10, 15, 20, 25, or more, such as at least 30, 35, 37, 40, 50, 60, 70, 80 priming upregulated (such as selected from Table IA and/or IB and/or 1C) and at least 5, 10, 15, 20, 25, or more, such as at least 30, 35, 37, 40, 50, 60, 70, 80 priming downregulated TF genes (such as selected from Table 2A and/or 2B and/or 2C).

As mentioned, 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. Thus, homologs or orthologs of the TF genes can be identified and detected in the following species:

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).

Methods of using TF genes as (bio)markers for the priming state and/or priming capability of plants and plant parts

In one embodiment, 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.) or plant part, such as a leaf, is provided. The method comprises the steps of: (a) obtaining a nucleic acid sample from said plant or plant part; and

(b) determining the (relative or absolute) mRNA expression level of priming-specific marker genes in said sample; and (c) inferring from the expression profile and/or expression level the priming state and/or priming capability of said plant or plant part, wherein said marker genes are selected from the those listed in Table 1, 2 and/or 3, or genes comprising at least 70%, at least 80%, at least 90%, at least 95%, 98%, 99% or more nucleic acid identity to those marker genes.

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. In a preferred embodiment leaf aerial tissue, most preferably leaf tissue is used. When seeds are used, preferably TF expression levels and/or profiles are analyzed post germination, e.g. in the seedlings. Thus, in a first step, 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. using standard nucleic acid extraction methods. Crude extracts or crude tissue samples, such as homogenized plant tissue, may also be used as nucleic acid sample in which the marker transcripts are detected and/or quantified. Thus, when referring to "a nucleic acid sample" in the method, this 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 (or plurality of plants or plant parts) 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. Preferably, 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. Thus, 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). In order for determining the priming state of a plurality of "test" plants (having an unknown priming state), samples obtained from suitable controls would be samples from primed and/or unprimed plants. In order for determining the priming capability of a plant species or of cultivars or accessions of a plant species (or of several plant species), it is understood that unprimed and primed samples are preferably compared for their TF marker gene expression before (or with) and after (or without) priming. Also differently primed plants or plant parts can be included and compared, for example BABA may be used as priming agent (e.g. in different concentrations to determine the sensitivity of the tissue for BABA-priming) or other known priming agents may be contacted with the plants or plant parts (e.g. bacterial strains as shown in the Examples). By using different sets of TF marker genes one can also differentiate between priming states or priming capability of different types of priming, such as BABA-IR or ISR.

Thus, several 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.).

In step (b) the mRNA expression level (or corresponding cDNA level) of priming- specific marker genes in said sample is determined. This can be done using various methods, such as (but not limited to) quantitative PCR methods, preferably quantitative RT-PCR, or nucleic acid hybridization based methods (for example microarray hybridization). Quantitative PCR (qPCR) 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. 38, 366-379; Czechowski et al. 2005, Plant Physiol. 139, 5-17). The step (b) involves the detection of relative or absolute expression level of the marker gene mRNA transcript(s) in the sample(s). As the expression level of transcription factor genes is very low, 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). Preferably 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.

Alternatively methods for determining the transcript level include for example DNA 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. 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. To construct a DNA microarray, nucleic acid molecules (e.g. single stranded oligonucleotides according to the invention) are attached to a solid support at known locations or "addresses". 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. Such 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. (1991) Science 251: 767- 77, each incorporated by reference. See also technology providers, such as Affymetrix Inc. (www.affymetrix.com). These arrays may, for example, be produced using mechanical synthesis methods or light-directed synthesis methods that incorporate a combination of photolithographic methods and solid phase synthesis methods. Also methods for generating labelled polynucleotide and for hybridizing them to DNA microarrays are well known in the art. See, for example, US 2002 /0144307 and Ausubel et al., eds. (1994) Current Protocols in Molecular Biology, Current Protocols (Greene Publishing Associates, Inc., and John Wiley & Sons, Inc., New York; 1994 Supplement). However, DNA arrays are less preferred herein, as the detection limit is much lower than for PCR based methods.

The TF proteins of SEQ ID NO: 123-159 and/or variants thereof or fragments of any of these may also be detected and optionally quantified in samples. Methods using antibody based detection are known in the art (e.g. ELISA). 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). One can then select the primed or unprimed plant(s) or plant parts for further use or further treatment and discard the undesired plant(s) or plant parts. Optionally 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. For example 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.

Similarly, 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).

In one specific embodiment the marker genes are selected from the group consisting of: (a) nucleic acid sequences comprising or consisting of SEQ ID NO: 1-37,

(b) 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).

Thus, 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) for example developed 1465 PCR primer pairs for quantifying transcripts for the majority of TF genes in Arabidopsis by real-time RT-PCR. 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. Obviously, other 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. Similarly, 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.

Preferably, labeled primers or oligonucleotides are used to quantify the amount of reaction product. Other techniques capable of quantifying relative and absolute amounts of mRNA in a sample, such as NASBA (Nucleic Acid Sequence Based Amplification), may also be suitably applied. 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).

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). 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).

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

(d) inferring the biotic and/or abiotic stress resistance level of said plant or plant part from the priming status without carrying out a biotic and/or abiotic stress resistance assay.

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). Methods for selecting or generating plants or plant parts having a modified priming state or ability

As there is natural variability in the priming status and capability of plants, 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).

Therefore, a method is provided 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:

(a) determining the 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;

(b) identifying one or more plants or plant parts having a priming-specific mRNA expression profile indicative of said plant or plant part being in a primed state (or in an enhanced priming state) in the absence of exposure to biotic and/or abiotic stress and/or in the absence of a priming inducer and/or in response to lower amounts of priming inducer; and

(c) optionally testing the biotic and/or abiotic stress resistance level of said selected plants of (b); and

(d) selecting those plants or plant parts identified in (b) for breeding, such as selling or crossing or clonal propagation.

Steps (a) and (b) and/or (c) and/or (d) may be repeated one or more times. For example the plants identified in (d) may be crossed or selfed or treated otherwise and retested according to the method. Thus, the steps are similar to those described further above, which embodiments are therefore also applicable here. The starting plant material may optionally be mutagenized, i.e. treated with one or more mutagens (mutagenic agents). "Mutagenesis", as used herein, 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. 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.

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. Thus, 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.

Methods for the easy and large scale screening of compounds or compositions which are able to alter the priming status of plants or plant parts Although 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:

(a) contacting a plant or plant part with one or more biological or chemical compounds, or compositions comprising one or more biological or chemical compounds; and (b) obtaining a nucleic acid sample from said treated plant or plant part and optionally from a control plant or plant part;

(c) determining the mRNA expression level of priming-specific marker genes in said sample; and (d) identifying those compounds or compositions for which the mRNA expression profile is indicative of the plant being in a primed state of defense and/or an enhanced priming state, wherein said 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.

Again, the above embodiments also apply to this embodiment. The steps may be repeated once, two or more times.

In step (a) 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. Preferably 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.

The 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.

In a preferred embodiment 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. Preferably 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.

Methods for identifying other priming-specific marker genes and/or for identifying key regulator proteins (e.g. transcription factors) which induces transcription of priming- specific marker genes

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').

Therefore a method for identifying other priming-specific marker genes is provided herein, comprising: a. identifying plant genes which comprise the motif TAG[TA]CT in their transcription regulatory region (as a cώ-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. Thus, the use of the nucleic acid motif TAG[TA]CT (or of nucleic acid sequences comprising this motif) for the identification of priming specific marker genes is provided herein. 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.

Also, 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.

Thus, the use of the nucleic acid motif for identifying and preferably isolating key priming regulator proteins is provided herein.

In other words, a method for using the motif TAG[TA]CT for identifying transcription factor proteins that regulate priming-specific transcription of marker genes.

The method uses known molecular biology and bioinformatics methods. For example, step a) may involve the use of fluorometric DNA-protein binding assays, electrophoretic assays (e.g. gel mobility shift assays), etc. Uses according to the invention

Use of one or more 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 according to the invention

In another embodiment kits for analyzing the priming state and/or capability of plants or plant parts is provided (see also elsewhere herein). Such kits 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.

Also provided is a qPCR well-plate (i.e. 385-wells plate or more) comprising a multitude of the selected primer sets of the TFs and optionally reference genes for use in the methods according to the invention. By adding a cDNA sample and a real-time PCR mix to wells in the plate, one can quickly analyze a multitude of plant samples for a transcriptional priming profile.

SEQUENCES

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,

EBF2, GAPDH, UBI-10, Atlgl3320 and Atlg62930, respectively.

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.

FIGURE LEGENDS

Figure 1. Priming for enhanced transcription of defense-related genes in Arabidopsis.

(A) BABA-induced priming for enhanced transcription of the PR-I gene upon treatment with the SA analogue benzothiadiazole (BTH). Five -week-old plants (CoI-O) were soil-drenched with 250 μM 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 (5xlO⁷ cfu.g^"1). Shoots of 5-week-old plants were dipped in a solution containing 50 μM MeJA. Plant material was harvested at different time points after MeJA treatment.

Figure 2. Priming for cell wall defense during expression of ISR and BABA-IR against H. parasitica in CoI-O and nprl-1.

(A) Quantification of ISR and BABA-IR against H. parasitica at 8 days after inoculation. ISR was triggered by transferring 2-week-old seedlings to potting soil containing P. fluorescens WCS417r bacteria. BABA was applied to 3 -week-old plants by soil-drenching to a final concentration of 80 μM BABA. One day after soil-drench treatments, plants were challenged with H. parasitica by spraying a suspension of 5 x 10⁴ spores/mL onto the leaves. Colonization by the pathogen was visualized by lactophenol/trypan-blue staining and light microscope. 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). Asterisks indicate statistically significant different distributions of disease severity classes compared to the water control (χ² test; α = 0.05).

(B) Quantification of callose deposition at 48 h after challenge inoculation with H. parasitica. Leaves were stained with calcofiuor/aniline-blue and analyzed by epifluorecence microscopy (UV). Callose deposition was quantified by determining the percentage of callose-inducing spores in the epidermal cell layer. Inset shows a representative example of a germinating H. parasitica spore triggering callose deposition in an epidermal cell. The data presented are from a representative experiment that was repeated with similar results.

Figure 3. Priming for cell wall defense during expression of BABA-IR against H. parasitica in Col-0, nprl-1, and myb72-l. (A) Quantification of BABA-IR against H. parasitica. For induction treatments and challenge incollation, see legend to Figure 2.

(B) Quantification of callose deposition at 44 h after challenge inoculation with H. parasitica. For experimental details, see legend of Figure 2.

Figure 4. Priming for cell wall defense during expression of ISR against H. parasitica in CoI-O, ibs2, and ibs3.

(A) Quantification of ISR against H. parasitica. For experimental details see legend of Figure 2. (B) Quantification of callose deposition at 44 h after challenge inoculation with H. parasitica. For experimental details, see legend of Figure 2.

Figure 5. ISR and BABA-IR against Pseudomonas syringae pv. tomato DC3000 CoI-O, nprl, ibs2 and ibs3. (A) Quantification of ISR. Two-week-old plants were transferred to potting soil containing P. βuorescens WCS417r bacteria and three weeks later inoculated with a bacterial suspension of P. syringae pv. tomato DC3000 at 1.25xlO⁷ cfu.mL^"1. Plants were scored at 4 days after challenge inoculation. Data presented are means of the average percentage of diseased leaves per plant (± SD). Asterisks indicate statistically significant differences compared to non- induced control plants (Student's t test; α = 0.05; n = 20-25).

(B) Quantification of BABA-IR. Five- to 6-week-old plants were soil-drenched with BABA to a final concentration of 250 μM, and two days later challenge- inoculated with P. syringae pv. tomato DC3000. Inoculation and disease scoring were performed as described above.

Figure 6. RT-qPCR profiles of WCS417r- and BABA-responsive transcription factors (TF) genes in CoI-O and nprl-1. ISR and BABA-IR were triggered as described in the legend of Figure 2. 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.

Occurrences of G-box (GACGTG), PLGT-I -box (GAAAAA), and W-box (TTGACC) motifs were quantified in the promoter regions 1000 bp upstream of the TATA box, using POBO bootstrapping analysis (Kankainen and Holm, 2004). The WCS417r- and BABA-inducible TF genes in CoI-O (blue) and nprl (yellow) were compared to randomly selected promoter sequences (red) from the Arabidopsis genome.

Figure 9. Identification of a novel promoter element in the promoter regions of BABA-inducible, NPRl -dependent WRKY genes

(A) Fold inductions of 72 Arabidopsis JFi?AT TF -genes in response to BABA in CoI-O and nprl-1.

(B) Occurrences of the TAG[TA]CT motif in promoter regions of BABA-response WRKY genes (blue), BABA-nonrespnosive WRKY genes (yellow), and random Arabidopsis promoters (red).

Figure 10. Differences and similarities between WCS417r- and BABA-induced defense priming during ISR and BABA-IR against P. syringαe pv. tomato DC3000 and H. parasitica.

EXAMPLES EXAMPLE 1 - MATERIALS AND METHODS

1.1 Cultivation of plants

Seeds of Arabidopsis thaliana accession CoI-O and of the mutants nprl-1 (Cao, H., et al. 1994, Plant Cell 6, 1583-1592), myb72-l (Van der Ent et al, submitted), ibs2 (At; 5'-UTR At5g66020) and ibs3 (abal-5, npql-2) (Ton et al., 2005, supra) were sown in quartz sand. Ten days after germination, seedlings were transferred to 60-mL pots containing a sand/potting soil mixture that was autoclaved twice for 20 min with a 24-h interval. Plants were cultivated in a growth chamber with a 8-h day (200 μEm^.sec^"1 at 24°C) and 16-h night (20⁰C) 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 μM Sequestreen (CIBA-Geigy, Basel, Switzerland) once a week.

1.2 Cultivation of microorganisms

For treatment of the roots with I SR- triggering rhizobacteria, 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 MgSO₄ to a density of 10⁹ 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 MgSO₄ to a final density of 2.5xlO⁷ CFU/ml. H. parasitica WAC09 was obtained from the Plant Research Institute,

Wageningen, The Netherlands. The oomycete was maintained on susceptible CoI-O plants as described by Koch and Slusarenko (Koch, E., and Slusarenko, A. 1990 Plant Cell 2, 437-445). Sporangia were obtained by washing heavy sporulating CoI-O leaves in 10 mM MgSO₄, collected by centrifugation, and resuspended in 10 mM MgSO₄ to a final density of 5 x 10⁴ spores per ml.

1.3 Induction treatments

ISR was activated by transplanting 2-week-old seedlings to a sand/potting soil mixture containing 5^χ10⁷ CFU/g WCS417r bacteria. Control soil was supplemented with an equal volume of 10 mM MgSO₄. BABA-IR was triggered by applying BABA (Sigma- Aldrich Chemie BV, Zwijndrecht, the Netherlands) as a soil-drench to the indicated concentrations.

1.4 Pst DC3000 bioassavs One day before inoculation with Pst DC3000, 5 weeks-old-plants were placed in 100% relative humidity. Plants were inoculated by dipping the leaves in a suspension of virulent Pst DC3000 bacteria, containing 2.5xlO⁷ CFU/ml in 10 mM MgSO₄, 0.015% (vol/vol) Silwet L-77 (Van Meeuwen Chemicals, Weesp, The Netherlands). Four days after challenge inoculation, the percentage of leaves with symptoms was determined per plant (n = 20). Leaves showing necrotic or water-soaked lesions surrounded by chlorosis were scored as diseased.

1.5 H. parasitica bioassays

Three-weeks-old plants were misted with a H. parasitica WACO9 spore suspension containing 5x10⁴ sporangia per ml. Inoculated plants were maintained at 17 ⁰C and 100% relative humidity for 24 h. Subsequently, humidity was lowered to 70% to reduce direct effects on plant development and to reduce the chance of secondary infections by opportunistic pathogens. Seven days after challenge inoculation humidity once again was raised to 100% to induce sporulation. Disease symptoms were scored for -250 leaves per treatment at nine days after inoculation. 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. To visualize trailing necrosis, infected leaves were stained with lactophenol trypanblue and examined microscopically at 5 days after inoculation as described by Koch and Slusarenko (1990, supra).

1.6 Callose quantification

Quantification of callose deposition was performed as described by Ton and Mauch- Mani (2004, supra). In short, leaves were collected at 2 d after inoculation and incubated overnight in 96% ethanol. Destained leaves were washed in 0.07 M phosphate buffer, pH 9, incubated for 15 min in 0.07 M phosphate buffer containing 0.005% calcofiuor (fluorescent brightener; Sigma-Aldrich Chemie BV, Zwijndrecht, the Netherlands) and 0.01% aniline-blue (water blue; Merck, Darmstadt, Germany), and subsequently washed in 0.07 M phosphate buffer containing only 0.01% aniline- blue to discard excess amounts of the calcofiuor. Observations were performed with an fluorescence-microscope with UV filter (bandpass 340 to 380 nm, long-path 425 nm). Callose depositions were quantified by determining the percentage of callose-inducing spores per infected leaf. 1.7 Transcription profiling

Q-PCR analysis was basically performed as described by Czechowski et al. (Czechowski, T., et al. 2004, Plant J. 38, 366-379). For the profiling of all putative Arabidopsis transcription factors, 200 μg of RNA was digested with Turbo DNA- free™ (Ambion, Huntingdon, United Kingdom) according to the manufacturer's instructions. To check for genomic DNA contamination, a PCR with primers designed on ΛtE/Z2 (At5g21120; EIL2_Y and EIL2_R) was carried out. Subsequently, DNA-free total RNA was converted into cDNA using oligo-dT2o primers (Invitrogen, Breda, the Netherlands), 10 mM dNTPs, and Superscript™ III Reverse Transcriptase (Invitrogen, Breda, the Netherlands) according to the manufacturer's instructions. Efficiency of cDNA synthesis was assessed by Q-PCR, using primers of the constitutively expressed gene AtUBIlO (At4g05320; UBIl 0_F and UBIl 0_R) and of both the 5' and 3' terminus of GlycerAldehyde-3 -Phosphate DeHydrogenase activity AtGAPDH (Atlgl3440; GAPDHy Y and GAPDH5\; GAPDHT_Y and GAPDH3\). Based on the results, cDNA of all samples was diluted to concentrations leading to UBIlO C _T (threshold cycle) values of 18 ± 0.5 for comparing TF transcript levels.

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 μl of cDNA was mixed with 5 μl 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 μl. 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. The following standard thermal profile was used for all PCR reactions: 50⁰C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60⁰C for 1 min. Amplicon dissociation curves, i.e. melting curves, were recorded after cycle 40 by heating from 60⁰C to 95°C with a ramp speed of 1.9°C min^"1. Data was analyzed using the SDS 2.2.1 software (Applied Biosystems). To generate a baseline-subtracted plot of the logarithmic increase in fluorescence signal (ΔR_n) versus cycle number, baseline data was collected between cycles 3 and 15. All amplification plots were analyzed with an R_n threshold of 0.1 to obtain C_T values. PCR efficiency (E) was estimated from the data obtained from the exponential phase of each individual amplification plot and the equation (1 + E) = 10^slope (Ramakers et al., 2003 Neurosci. Lett. 339, 62-66). To determine normalized TF expression levels (ΔC_T) the C_T of the constitutively transcribed genes At4G05320 (UBIlO), At2G28390 (SAND family), At5G46630 (Clathrin adaptor complex subunit) and At5G55840 (PPR protein) (Czechowski et al., 2005, supra) were individually subtracted from that of the TF of interest, resulting in four different values. Expression ratios are presented as (1 + E)^ΔΔCT, where ΔΔC_T = ΔCT Treat - ΔC T CM. TFs were only appointed as induced or repressed when ΔΔCT, normalized to all four constitutive genes, was Ξ 2 or # 0.5, respectively.

To confirm the results from the transcription profiling, 5 μg of 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 MylQ™ 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 μl, containing cDNA, 0.5 μL of each of the two gene-specific primers (10 pmol.μL^"1), and 3.5 μL of 2x IQ SYBR^® Green Supermix reagent. The following PCR program was used for all PCR reactions: 95 ⁰C for 3 min; 40 cycles of 95 ⁰C for 30 sec, 59.5 ⁰C for 30 sec, and 72 ⁰C for 30 sec. C_T (threshold cycle) values were calculated using Optical System Software, version 1.0 for MylQ™ (Bio -Rad, Veenendaal, the Netherlands). Subsequently, C_T values were normalized for differences in dsDNA synthesis using those of the constitutively expressed reference gene AtI gl 3320 (Czechowski et al., 2005, supra). Melting curves, were recorded after cycle 40 by heating from 55°C to 95°C with a ramp speed of 1.9°C min^"1. Again, expression ratios are presented as (1 + E)^ΔΔCT.

1.8 Statistical analysis of expression data Cluster analysis (Euclidean distance) and correspondence analysis (COA) of the transcriptional patterns were performed using TIGR Multiexperiment Viewer (TMEV) software (Saeed, et al. 2003 Biotechniques 34, 374-378). The TMEV analyses of the three biological replicas were based on the Ln-transformed values of the fold inductions of each gene expression value relative to the mean of the control samples of CoI-O plants.

1.9 Promoter analysis The POBO bootstrapping program was used to define overrepresented czs-acting elements in sequences of promoters specific for the ISR- or BABA-treatment compared to sequences of random promoters, which was used as a background (Kankainen and Holm, 2004 Nucl. Acid Res 32, 222-229). The analyses was based on 1000 pseudo- clusters, each containing as much 1 kB promoter regions as were present in the smallest group of those that were to be compared, χ distribution analysis was performed to determine statistical significance of the differences between the treatments (α = 0.05).

EXAMPLE 2 - RESULTS

2.1 BABA and WCS417r prime for defense-related gene expression

Expression of 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. At 6 hours after treatment with either 50 or 200 mg/L BTH, BABA-treated clearly show an augmented induction of the PR-I gene in comparison to non-primed water-treated plants (Fig. IA). In a separate experiment, a similar pattern was found at 24 h after treatment with 150 and 300 mg/L BTH (Fig. IA). Hence, priming by BABA enhances the sensitivity to SA. To investigate whether priming by ISR-inducing P. fluorescens WCS417r bacteria is based on increased sensitivity to JA, control, and WCS417r- treated plants were tested for expression of the JA-inducible LOX2 gene at different hours after treatment of the leaves with 50 μM JA. As shown in Figure IB, WCS417r- treated plants show an accelerated LOX2 induction in comparison to non -primed control plants. Hence, ISR-related priming is associated with an increase in the responsiveness to JA, which supports our earlier finding that ISR is predominantly effective against pathogens that are resisted through JA-dependent defense mechanisms (Ton et al., 2002, supra). 2.2 ISR and BABA-IR against H. parasitica are both associated with priming for callose-rich papillae

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. parastica, we quantified the number of spores that induce callose depositions in the epidermal cell layer in ISR and BABA-IR expressing plants. As shown in Figure 2, both WCS417r bacteria and BABA increased the number callose depositions in CoI-O plants at 48 h after inoculation with H. parasitica, which was proportional to the level of induced resistance. Hence, both WCS417r and BABA prime for augmented formation of callose-rich papillae. Interestingly, WCS417r bacteria failed to trigger priming for papillae in nprl-1 plants, which correlated with a lack of ISR. On the other hand, BABA- induced priming for papillae, as well as BABA- IR against H. parasitica, 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.

2.3 Both WCS417r- and BABA-induced priming for papillae require SAClb/IBS2 and ABAl /IB S3

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). To test whether these pathways are also involved in the WCS417r- induced priming for papillae, we quantified the relative number of papillae-inducing spores in the epidermal cell layer of Col-0, the ibs2-2 mutant with a T-DNA in the 5 '-untranslated region of the SAClb/IBS2 gene, and the npq2-l mutant with an EMS-induced mutation in the ABA1/IBS3/NPQ2 gene (Ton et al., 2005, supra). At 48 h after inoculation, 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). This demonstrates that the WCS417r-induced priming for papillae depends on intact SAClb/AtIBS2 and ABA1/IBS3/NPQ2 genes. Interestingly, two mutants in the ISR signaling pathway, nprl-1 and myb72-l (Pieterse et al., 1998 supra, Van der Ent et al., submitted) were not affected in the BABA-induced priming for papillae (Figures 2 and 4). It can thus be concluded that the priming for papillae by WCS417r and BABA is controlled by partially overlapping signaling pathways.

2.4 WCS417r-mediated ISR and BABA-IR against Pst DCSOOO function both independently oϊSAClb/IBS2 and ABA1/IBS3/NPQ2 To test whether the common requirement for SAClb/AtIBS2 and ABA 1 /IB S 3 /NP Q2 in the priming for cell wall defense against H. parasitica also applies for the induced resistance against Pst DC3000, we compared ISR and BABA-IR against this pathogen in CoI-O, ibs2-2, npq2-l and nprl-1 plants. In agreement with earlier findings (Zimmerli et al., 2000, supra; Ton et al., 2005, supra), soil-drench treatment with 250 μM BABA resulted in a statistically significant reduction of disease in wild-type, ibs2- 2, npq2-l, but not in nprl-1 (Fig. 5A). Similarly, treatment with WCS417r bacteria resulted in disease suppression in Col-0, ibs2-2, and npq2-l, but not nprl-1 (Fig. 5B) showed a statistically significant reduction in disease. Hence, BABA-IR and ISR against Pst DC3000 do not involve phosphoinositide- and ABA-dependent signaling, but require an intact NPRl -protein. This also indicates that 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.

2.5 Treatment of the roots with WCS417r and BABA directly trigger changes in the expression of TF genes in the leaves of Arabidopsis

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. In order to distinguish between NPRl -dependent and NPRl -independent priming effects by BABA, we also included samples from water- and BABA-treated nprl-1 plants at 32 h after soil-drench treatment. In wild-type CoI-O plants, root colonization by WCS417r bacteria caused a >2-fold induction of 90 TF genes in the leaves (Table 1), whereas 31 TF genes were >2-fold repressed (Fig. 6A; Table 2). Upon soil-drench treatment with BABA, 186 TF genes in the leaves of CoI-O plants were >2-fold induced (Table 1), whereas 44 TF genes were >2-fold repressed (Table 2). A similar treatment of nprl-1 plants resulted in enhanced transcription of 135 TF genes (Table 1), and a repression of 140 TF genes (Fig. 6A; Table 2).

Of all BABA- inducible TF genes in CoI-O and nprl-1 plants, only 32 (10%) were inducible in both CoI-O and nprl-1 plants (Fig. 6B). This suggests a proportionally big impact of the nprl-1 mutation on BABA-induced expression of TF genes. In wild-type plants, only 27 of a total of 277 TF genes (9.7%) were inducible by both WCS417r and BABA. Hence, both priming agents induce largely different sets of TF genes (Fig. 6B).

Table 1 - TF genes induced > 2-fold (A) by bacterium WCS417r (90 TF genes), or (B) by BABA (in wild type CoI-O; 186 TF genes) or (C) by BABA in nprl-1 mutant (135 TF genes)

Table 1-A

Table 1-B

Table 1 - C

Table 2 - genes repressed > 2-fold by (A) bacterium WCS417r, or (B) BABA (wild type Col-0) or (C) BABA (in nprl-1 mutant)

TABLE 2-A

TABLE 2 - B

TABLE 2 - C

2.6 Expression profiles of a selected set of priming-related TF as markers for priming during ISR or BABA-IR

To confirm the priming-related expression of the TF genes, we quantified the expression of a selected set of 37 WCS417r- and/or BABA- inducible TF genes in replicate biological samples. The 37 genes were selected based on the criteria described on page 14.

In addition to priming treatment by WCS417r and BABA, we also included the ISR non-inducing strain Pseudomonas fluorescens WCS374r and the inactive BABA isomere α-aminobutyric acid (AABA) as extra negative control treatments (Van Wees et al, 1997 MoI. Plant-Microbe Interact. 10, 716-724; Jakab et al, 2001, Eur. J. Plant Pathol. 107, 29-37). The resulting expression profiles were subjected to cluster analysis and correspondence analysis (COA). As shown in Figure 7, 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. This demonstrates that the expression profile of the 37 TF genes is sufficiently robust to specifically mark the onset of ISR-related priming. In a separate experiment, 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. Finally, we compared the profiles of 3 replicate samples from CoI-O and nprl-1 upon treatment with either water or BABA. The resulting expression profiles could clearly differentiate between the effects by BABA in CoI-O and nprl-1 plants (Fig. 7). These results not only confirm our findings from the genome -wide transcription profiling (Fig. 6), but they also demonstrate that the profiles of the selected set of 37 TF genes can be used as a robust marker for the establishment of priming during ISR or BABA-IR.

TABLE 3 - Dedicated set of 37 selected TF marker genes showing (A) Experiment 1, TF gene expression in response to P. fluorescens WCS417r (and P. βuorescens WCS374 negative control); and (B) Experiment 2, TF gene expression in response to BABA in COL-O (wt) and nprl-1 mutant Arabidopsis plants; and (C) Experiment 3, TF gene expression in response to BAB and AABA. Values indicate the fold induction relative to the average of the control treatments. Control treatments 1, 2 and 3 are plants grown in the absence of rhizobacteria WCS417r or WCS374r.

TABLE 3 -A

TABLE 3 -B

TABLE 3 - C

2.7 Promoter analysis of WCS417r- and BABA-inducible TF genes The transcriptional behavior of many plant genes is tightly linked to the occurrence of conserved DNA motifs in their promoter regions, called czs-acting elements (Singh et al., 2002, Curr. Opin. Plant Biol. 5, 430-436). To identify czs-acting elements involved in the priming-related induction of TF genes, we compared 1 kB promoter regions of BABA- and WCS417r-responsive using POBO software (Kankainen and Holm, 2004, supra). As shown in Figure 8, three distinct 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. 16, 726-738.; Faktor et al., 1997, Differential utilization of regulatory cis-elements for stress-induced and tissue-specific activity of a French bean chalcone synthase promoter 124, 175-182; Boter et al., 2004, Gene. Dev. 18, 1577- 1591; Park et al., 2004, Plant Phys. 135, 2150-2161). Interestingly, the promoter regions of the BABA-responsive TF genes of nprl-1 displayed a stronger enrichment in G-box elements than those of CoI-O plants (Fig. 8), indicating that this element is mostly abundant in promoters of BABA-inducible, NPRl -independent TF genes. On the other hand, 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.

2.8 Identification of a novel promoter element in BABA-inducible WRKY genes. The group of 187 BABA-inducible TFs in CoI-O contained 21 WRKY genes. Many of these WRKYs, such as WRKYl 8, WRKY38, WRKY58, WRKY59, and WRKY70, have frequently been associated with SA-dependent defenses and were recently identified as direct targets of NPRl (Wang et al, 2006, Plos Pathogens 2, 1042-1050). In agreement with this, we found that the induction of all 21 BABA-responsive WRKYs was blocked or strongly reduced in the nprl-1 mutant (Fig. 9). To further investigate the regulation of this NPRl -dependent induction of WRKY genes, we analyzed the priming-related WRKY genes for common promoter motifs. In comparison to the promoters of 51 BABA -non-responsive WRKYs and a random set of Arabidopsis promoters, the 21 BABA-inducible WRKY genes were substantially enriched in an unknown TAG[TA]CT motif (Figure 9). The fact that this motif is strongly over-represented in BABA-inducible WRKY promoters, but not in BABA- non-responsive WRKY promoters, points to the existence of a key regulatory factor in BABA-induced priming for SA- and NPRl -dependent defense mechanisms.

2.9 Discussion

Because TF activity is largely controlled at the transcriptional level in Arabidopsis (Chen et al., 2002, supra; Lee et al., 2006, supra), we screened the transcriptional response of -2.300 Arabidopsis TF genes to activation of WCS417r-mediated ISR and BABA-IR. Treatment of the roots with either WCS417r or BABA, induced major transcriptional changes in TF gene expression in the leaves (Fig. 6A). Interestingly, there was relatively little overlap between WCS417r- and BABA-targeted TF genes (Fig. 6B). Hence, priming by WCS417r and BABA is associated with transcriptional responses of largely distinct sets of TF genes. In combination with our previous finding that WCS417r and BABA prime for different sets of defense-related genes (Fig. 1, Verhagen et al., 2004; Ton et al., 2005, both supra), we propose that WCS417r-induced changes in TF gene expression contribute to the priming of JA-inducible genes, whereas the BABA-targeted TF genes contribute to the priming of SA-inducible genes (Fig. 10). WCS417r-mediated ISR in Arabidopsis is predominantly effective against pathogens that are resisted by JA- and ET -dependent basal resistance mechanisms (Ton et al., 2002, supra). In agreement with this, we found that WCS417r bacteria induce systemic expression of TF genes related to the regulation of JA- and ET -dependent defense reactions (Table Sl). For instance, 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). Furthermore, ATERFl -dependent activation of defense-related genes has been reported to be counteracted by an ABA/JA-inducible signaling pathway, which requires an intact ATMYC2 protein (Lorenzo et al., 2004, Plant Cell 16, 1938-1950). Interestingly, the AtMyC2 gene (Atlg32640) was also weakly, yet consistently, induced in the leaves after treatment with WCS417r (Table Sl; Fig. 7). Based on these results, it is tempting to speculate that ISR-inducing WCS417r bacteria sensitize the leaves for JA-dependent defense signals by altering the balance in defense-related regulatory TFs, such as AtERFl and AtM YC2.

In a previous study on the transcriptome of ISR, no direct effects by WCS417r could be detected in the leaves of Arabidopsis (Verhagen et al., 2004, supra). This contradicts our present study, in which we found consistent effects on TF gene expression in the leaves upon treatment of the roots with WCS417r (Figs. 6 and 7). This discrepancy is probably due to the difference the detection methods. Whereas Verhagen et al. (2004, supra) used micro -arrays to quantify gene expression, the transcriptional profiling in this study was based on RT-qPCR. This technique is substantially more sensitive and reliable for the detection of low abundant mRNA levels, which are characteristic for the expression of TF genes (Czechowski et al., 2004, supra).

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). Here, we showed that 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). Hence, 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. This conclusion is strengthened by the fact that the group of 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).

The direct effects of BABA and WCS417r on TF gene expression point to specific signaling pathways that regulate the onset of priming through enhanced expression of defense-related TFs. Like other genes, 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. In a first step to identify such factors, we analyzed the promoter regions of WC S417r- inducible and BABA- inducible TFs for common exacting elements. 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). Interestingly, 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). Apparently, disruption of the NPRl -dependent signaling pathway results in enhanced induction of G-box-containing TF genes by BABA. This suggests that NPRl suppresses BABA-induced expression of G-box-containing TF genes. Whether this suppression is based on similar mechanisms as NPRl -dependent crosstalk between SA- and JA-dependent defense pathways (Spoel et al., 2003, Plant Cell 15, 760-770), remains to be investigated. In addition, we found a statistically significant over-representation of W-boxes in the promoter regions of BABA- inducible genes in wild-type plants, but not in nprl-1 plants. This points to an involvement of WRKYs in the NPRl -dependent induction of TF genes by BABA, which is supported by our finding that 21 WRKY genes were >2-fold induced by BABA. Further analysis of the promoter regions of these 21 NPRl -dependent WRKY genes revealed a very significant over-representation of a yet unknown promoter element in comparison to BABA non-responsive WRKY genes (Fig. 9). We hypothesize that this TAG[TA]CT element functions as an important cώ-acting element in the NPRl -dependent activation of TF genes by BABA. Future studies will focus on the identification of TF proteins that bind to the TAG[TA]CT element, which could lead to the identification of novel key regulators in the priming for SA-dependent defense mechanisms.

Claims

1. A method for determining the priming state and/or the priming capability of a plant or plant part, such as a leaf, comprising the steps of:

(a) obtaining a nucleic acid sample from said plant or plant part; and

(b) determining the mRNA expression level of priming-specific marker genes in said sample; and

(c) inferring from the expression level the priming state and/or priming capability of said plant or plant part, wherein said 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.

2. The method according to claim 1, wherein said marker genes are selected from the group consisting of:

(a) nucleic acid sequences comprising or consisting of SEQ ID NO: 1-37,

(b) nucleic acid sequences comprising at least 70% nucleotide sequence identity to SEQ ID NO: 1-37.

3. The method according to claim 1 or 2, wherein the plant or plant part is an agricultural crop plant or a horticultural plant.

4. The method according to any one of claims 1-3, wherein the mRNA expression level is determined using a Polymerase Chain Reaction (PCR) based technique, such as RT- qPCR, whereby PCR primers are used which are fragments of SEQ ID NO: 1-37, or of the complementary sequence of these, or which are fragments of nucleic acid sequences comprising at least 70% nucleotide sequence identity to SEQ ID NO: 1-37, or of the complementary sequence these.

5. The method according to any one of claims 1-4, further comprising step

6. A method for generating or selecting a plant or plant part having an enhanced priming state and enhanced biotic and/or abiotic stress resistance, comprising the steps of: (a) determining the priming state of a plurality of plants 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; (b) identifying one or more plants or plant parts having a priming -specific mRNA expression profile indicative of said plant or plant part being in a primed state in the absence of exposure to biotic and/or abiotic stress; and

(d) selecting those plants or plant parts identified in (b) for breeding, such as selfing or crossing.

7. The method according to claim 6, wherein said marker genes are selected from the group consisting of:

(a) nucleic acid sequences comprising or consisting of SEQ ID NO: 1-37, (b) nucleic acid sequences comprising at least 70% nucleotide sequence identity to SEQ ID NO: 1-37.

8. The method according to claim 6 or 7, wherein the plant or plant part selected in step (d) is constitutively primed, in the absence of biotic and/or abiotic stress.

9. The method according to any one of the preceding claims, wherein the priming- specific marker gene comprises the nucleic acid motif TAG[TA]CT in its transcription regulatory region upstream of the coding region.

10. A plant or plant part selected according to any one of claims 6 - 9.

11. A method for identifying biological or chemical compounds, or compositions comprising biological or chemical compounds, which are capable of activating or inducing priming in a plant or plant part, comprising the steps of:

(a) contacting a plant or plant part with one or more biological or chemical compounds, or compositions comprising one or more biological or chemical compounds; and

(b) obtaining a nucleic acid sample from said treated plant or plant part and optionally from a control plant or plant part;

(c) determining the mRNA expression level of priming-specific marker genes in said sample; and (d) identifying those compounds or compositions for which the mRNA expression profile is indicative of the plant being in a primed state, wherein said 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.

12. The method according to claim 11, wherein said marker genes are selected from the group consisting of:

(a) nucleic acid sequences comprising or consisting of SEQ ID NO: 1-37,

13. The method according to claim 11 or 12, wherein the biological compound comprises or consists of one or more microorganisms, or one or more plant extracts.

14. The method according to claim 13, wherein said one or more microorganisms are selected from the group consisting of: bacteria, fungi, mycorrhizae, nematodes and viruses.

15. The method according to claim 14, wherein said bacteria are non-pathogenic root colonizing bacteria and/or fungi, such as mycorrhizae.

16. A compound or composition identified according to any one of claims 11-15.

17. Use of one or more transcription factor 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 genes, as biomarkers for determining the priming state and/or priming capability of plants or plant parts.

18. Use of a nucleic acid comprising or consisting of the motif TAG[TA]CT for identifying priming- specific marker genes and/or for identifying proteins which are capable of binding to said motif.

19. A method for identifying priming-specific marker genes comprising: a) identifying plant genes which comprise the motif TAG[TA]CT in their transcription regulatory region, using for example in silico analysis, and b) testing whether the identified gene, or a homologue or ortholog of said 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 optionally c) using said gene, or homologue or orthologue thereof, or a fragment of any of these, in a method according to any one of claims 1 - 9 or 11 -15.

20. A method for using the motif TAG[TA]CT for identify ing transcriptio n factor proteins that regulate priming-specific transcription of marker genes.