Detection of NPR1 for the assessment of plant defense mechanism activation

Abstract

The present invention relates to anti-NPR1 antibodies specifically directed against the monomeric form of the NPR1 protein. The present invention also relates to a method for detecting the activation of plant defenses by quantifying the monomeric form of the NPR1 protein in a plant sample using an anti-NPR1 antibody according to the present invention.

Description

Title of the invention: Detection of NPR1 for the assessment of the activation of plant defense mechanisms technical field

[0001] The invention relates to antibodies specifically directed against the monomeric form of the NPR1 protein, as well as their use in methods for detecting the activation of plant defenses by detecting or quantifying the monomeric form of the NPR1 protein. Previous technique

[0002] The reduction in the use of conventional plant protection products, as planned in the Ecophyto 2025 plan, is leading to the development of new strategies for more sustainable and environmentally friendly agricultural practices. In this context, new varieties less dependent on inputs are expected to emerge, and new practices associated with these varieties need to be developed. The development of plant defense stimulants (PDS) is a strategy that is attracting increasing attention. PDS can be of natural or synthetic origin, but in both cases are subject to the regulations in force concerning the placing on the market of plant protection products (Regulation (EC) No 107 / 2009).

[0003] However, although they represent a promising strategy for more sustainable agriculture, the effectiveness of plant protection products (PPPs) in the field remains unpredictable, and there are few or no tools to assess their activation capacity. Thus, many PPP product projects developed in the laboratory fail to obtain marketing authorizations due to a lack of analytical resources and / or because of unusable results from experiments conducted under natural conditions. In practice, PPP treatments are generally applied preventively and repeated frequently because their duration of action is limited. Plants may also be unresponsive at the time of treatment (variety, growth stage, etc.). Furthermore, beyond a certain pest pressure, a supplementary chemical treatment is recommended (either in a mixture or separately).Several tests under semi-controlled conditions have refuted the results of laboratory studies, and consequently, several products and molecules have been banned. This discrepancy between efficacy results obtained under more or less controlled conditions (phytotron vs. greenhouse) is even more pronounced when trials are conducted under natural conditions (open field). Indeed, climatic hazards are inevitable, and pest pressure is sometimes highly variable. The results of these trials are therefore unpredictable, difficult to interpret, and... Their reproducibility is low. Some products have nevertheless succeeded in proving their effectiveness, but there is a significant disparity in the methods used to evaluate the products.

[0004] Identifying effective SDP molecules and highly receptive varieties requires reliable, efficient, and routinely usable screening tools. Developing a reliable method for detecting the activation of plant defense mechanisms is crucial for evaluating these types of products in the field under optimal conditions. This tool is essential at several levels, both for testing plant receptivity at selected experimental sites and for monitoring product efficacy over time.

[0005] Currently, there are various methods for detecting the activation of plant defenses by SDPs:

[0006] An optical transmitter / receiver, developed by the company Force A and called Multiplex®, allows for the "quantification" of molecules such as anthocyanins, flavonols, etc. This portable tool enables the rapid acquisition of easily usable data. Furthermore, the non-destructive measurements allow for multiple measurements to be taken on the same organ, thus providing information on the temporal evolution of responses. However, the measured markers are not very specific. They are secondary metabolic compounds specific to each plant variety, which play very diverse roles in the physiology and development of these plants.

[0007] There is also a molecular diagnostic tool developed by INRA Angers, called "qPFD" (Quantitative Low Density Chip: quantitative RT-PCR in microplate / low density DNA chip) which makes it possible to evaluate target genes whose expression, alone or in combination, provides information on the state of stimulation of the plants' natural defenses (WO 2011 / 161388).

[0008] A screening method for potentially eliciting products (GUSTAVE for GUS Technology for Analysis and Validate plant Elicitor) has also been developed and is marketed by the Applicant. The principle of this test is to use plants expressing a reporter gene encoding a β-glucuronidase, the GUS gene. This gene has been fused to the promoter of genes known to be markers of the two main defense pathways. Transgenic Arabidopsis plants possessing a promoter-marker gene:GUS construct are therefore used. When the substrate X-Gluc (5-bromo-4-chloro-3-indolyl-[3-D-glucuronic acid, cyclohexylammonium) is introduced to the plant, the GUS enzyme will cleave this substrate and produce an insoluble blue precipitate. A blue coloration then appears if the marker gene is expressed, thus allowing spatial localization of the establishment of defenses.The use of this test allows for a rapid screening of SDPs that are capable of activating defenses by easily visualizing the expression of marker genes representing one of them. or the other of the two main known defense mechanisms. This makes it easy to determine if the product is capable of activating plant defenses and also to identify which type of defense is activated.

[0009] Protection tests are also offered by numerous companies. These tests allow observation, under controlled conditions or in the field, of the effectiveness of a protective agent against pathogens. These tests therefore demonstrate the effectiveness of a protective agent but do not in any way demonstrate the activation of defenses by this product.

[0010] It appears from the literature that the NPR1 protein is a key protein in plant immunity. It is necessary for the establishment of systemic acquired resistance (SAR) involving the phytohormone salicylic acid (SA) and systemic induced resistance (ISR) involving the phytohormone pair jasmonic acid / ethylene (JA / Et), which are forms of immunity that can last up to several weeks or even several months.

[0011] The NPR1 protein is found in monomeric or oligomeric form in the plant cell. The literature indicates that the concentration level of the monomer is correlated with its activity.

[0012] Patent application WO9806748 describes acquired resistance polypeptides such as NPR1, capable of conferring resistance to a phytopathogenic agent in a plant expressing said polypeptide. The phenotypes of NPR1 mutants have demonstrated the biological importance of the NPR1 gene of Arabidopsis thaliana in controlling the defense response against a broad spectrum of pathogens.

[0013] An anti-NPR1 antibody is marketed by AGRISERA (Agrisera No. AS12 1854). However, this antibody requires prior treatment with salicylic acid and cannot detect NPR1 in plants other than Arabidopsis thaliana. Furthermore, this antibody is not specific and detects NPR1 indiscriminately. It detects both the inactivated and activated forms of the NPR1 protein without distinction. Technical problem

[0014] Thus, to date, no simple, specific, rapid and inexpensive immunological detection and quantification technique for "field" experimentation is available in view of the scientific literature and / or known to experimentation professionals.

[0015] It is therefore necessary to develop a method for detecting, quantifying and evaluating, in a simple, rapid and moderately costly manner, the activation of plant defense mechanisms, in order, in particular, to screen potentially SDP molecules, to demonstrate and / or verify the activation of plant defenses following the application of a substance and to select highly receptive varieties to the SDP. Description of the invention

[0016] The present invention therefore relates to anti-NPR1 antibodies specifically directed against the monomeric form of the NPR1 protein.

[0017] The inventors have indeed developed and used an immunization strategy allowing the obtaining of antibodies specific to an active form of NPR1, i.e., the monomeric form of the NPRL protein. This approach has the advantage of showing the activation of plant defenses regardless of the hormonal pathway implemented, and this on different plants.

[0018] Thus, and in the sense of the present invention, NPR1 has been chosen as an immunity marker enabling the multi-specific and broad-spectrum detection of plant defense activation.

[0019] The present invention also relates to a method for detecting the activation of plant defenses by detecting or quantifying the monomeric form of the NPR1 protein in a plant sample using an anti-NPR1 antibody according to the present invention.

[0020] Advantageously, this method will allow the screening of molecules, potentially SDP, for the emergence of new active substances with SDP activity, to demonstrate the activation of plant defenses following the application of a product, to verify in situ the activation of defenses following the application of SDP, to select varieties highly receptive to SDP, to measure the activation of plant defense mechanisms before treatment, and to select a plant exhibiting a state of stimulation of natural defenses likely to confer improved resistance to at least one biotic or abiotic stress.

[0021] Thus and advantageously, the use of specific anti-NPRl antibodies according to the present invention makes it possible to detect, quantify and evaluate, in a simple, rapid and moderately costly manner, the activation of the defense mechanisms of a plant. Brief description of the drawings

[0022] Other features, details and advantages of the invention will become apparent from reading the detailed description below and from analyzing the accompanying drawings, in which: Fig. 1

[0023] [Fig. 1] shows the position of the two degenerate consensus peptides used to produce the antibodies according to the present invention and directed against the consensus NPR1 protein of different plant species. Antipeptide 0 (antipep0) was produced using the degenerate peptide 0 of consensus sequence SEQ ID NO 1 by injecting peptides of sequences SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 into rabbits. Antipeptide 1 (antipepl) was produced using peptide 1 degenerate of consensus sequence SEQ ID NO 6, by injection of peptides of sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 in rabbits. Fig. 2

[0024] [fig.2] represents recombinant proteins (prot T, P and I) according to their positions relative to the NPR1 protein of A. thaliana. Fig. 3

[0025] [fig.3] is a western blot performed using antipeptideO on plant extracts of A. thaliana whose defenses were activated using Bion® (1) or Salicylic Acid SA (2) or not activated using water (3). A 37kDa band is detected (1 and 2) only when the defenses are activated and this band is not detected when the defenses are not activated (3). Fig. 4

[0026] [fig.4] shows western blots performed by hybridization of the different proteins recombinant proteins with the anti-histidine tag antibody (1, 3, and 5) and with the anti-peptideO antibody (2, 4, and 6). A band corresponding to protein T is detected with both antibodies (1 and 2). Bands of the same size corresponding to protein P (3 and 4) and protein I (5 and 6) are also hybridized by these same antibodies to the corresponding protein extracts. Therefore, the same bands are detected with these two antibodies when different forms of recombinant proteins possessing a histidine tag are used. Fig. 5

[0027] [fig.5] shows western blots carried out by hybridization of extracts of A. thaliana, Plants were treated or untreated with various defense activators, including the commercial anti-NPR1 antibody (1, 3, and 5) and antipepO (2, 4, and 6). Defenses involving the JA / Et pathway were activated using methyl jasmonate (Me-JA), while those involving the SA pathway were activated using SA. Water-treated plants were used as a negative control for defense activation. The same band was observed regardless of the antibody used and whether the defenses were activated with Me-JA (3 and 4) or SA (5 and 6). This band is not found (or with very low intensity) when the plants have been treated with water (1 and 2). These differences in intensity are not due to the amount of charged proteins, as shown by the corresponding protein profiles (7 and 8) obtained by Coomassie blue staining. Fig. 6

[0028] [fig.6] represents two western blots carried out by hybridization of extracts of A. thaliana, whose defenses were activated using SA, with antipeptides 0 and 1 (antipepO and antipepl). The same band is found at 37kDa (1 and 2) regardless of the antipeptide used. Fig. 7

[0029] [fig.7] shows the western blots carried out by hybridization of the eluates Immunoprecipitation (IP) with antipeptide O. The antibodies used during IP (anti-pep O and commercial antibody) are released during elution and are therefore detected (1, 2, and 3). These antibodies enabled immunoprecipitation of the 37 kDa protein when extracts of A. thaliana whose defenses had been activated using SA were used (4 and 6). Anti-pep O is also capable of immunoprecipitating protP (5), whereas the commercial antibody does not, as shown by the absence of a band of the expected size (7). Fig. 8

[0030] [fig.8] shows the expression level of a defense marker gene (PR5) of Different samples measured by qPCR (1, 2, 3, and 4) and the intensity of the 37 kDa band detected by Western blot on these same samples (5, 6, 7, and 8) were analyzed. The amount of protein deposited does not explain the difference in the intensity of these bands, as shown by Coomassie blue staining of the membrane (9). The level of immune activation measured by qPCR is correlated with the intensity of the band observed by Western blot. Fig. 9

[0031] [fig.9] shows a western blot performed by hybridization of tomato extracts, of which the Defenses were stimulated or not by spraying with SA, Me-JA, or water, along with antipepO. A band was detected around 40 kDa with a higher intensity on plant extracts treated with SA (2) and Me-JA (3) than on plants treated with water (1). The amount of protein deposited did not explain the difference in the intensity of these bands, as shown by Coomassie blue staining of the membrane (4). Detailed description

[0032] The present invention therefore relates to an anti-NPR1 antibody characterized in that said antibody binds specifically to the monomeric form of the NPR1 protein.

[0033] NPR1 The NPR1 (Non-expressor of Pathogenesis Related protein 1) protein, also known as niml and sail, is a protein involved in various immune signaling pathways, in systemic acquired resistance (SAR), involving the phytohormone salicylic acid (SA), systemic induced resistance (ISR) involving the phytohormone pair jasmonic acid / ethylene (JA / Et) as well as local acquired resistance (LAR).

[0034] NPR1 exists in dynamic equilibrium between high molecular weight oligomers (linked via intermolecular disulfide bridges), which are inactive, and monomers, which are the active form of the protein. The protein's status is tightly controlled by redox changes in plant cells that are triggered by external stresses such as temperature, UV radiation, infection, pathogens, or treatment with substances such as salicylic acid (SA), methyl jasmonate (Me-Ja), or Bion®. NPR1 in its oligomeric, i.e., inactive form, is sequestered in the cytosol. NPR1 transitions from an oligomeric to a monomeric state by the reduction of intermolecular disulfide bridges. The NPR1 protein in its monomeric, i.e., active form, then enters the nucleus to control gene expression related to the SAR (Mou et al., Inducers of Plant Systemic Acquired Resistance Regulate NPR1 Function through Redox Changes, Cell, Volume 113, Issue 7, 27 June 2003, pages 935-944). .

[0035] Thus, the antibodies according to the present invention bind specifically to the monomeric form, i.e., to the active form of NPR1.

[0036] Specificity Anti-NPR1 antibodies recognize and bind specifically to the monomeric form of the NPR1 protein rather than the oligomeric form. Thus, for the purposes of the present invention, an "anti-NPR1 antibody that binds specifically to the monomeric form of the NPR1 protein" is understood to be an antibody that exhibits high affinity for its target molecule, i.e., for the monomeric form of the NPR1 protein.

[0037] The terms "binds specifically" or "recognizes specifically" are used here to indicate that the antibody has the ability to recognize and interact with the monomeric form of NPR1, while having relatively few detectable interactions with the oligomeric form of NPR1.The antibody binds specifically to the monomeric form of NPR1 if its affinity is significantly higher for the monomeric form of NPR1 than for the oligomeric form of NPR1; preferably, the anti-NPRL antibody according to the present invention does not bind to the oligomeric form of NPR1.

[0038] Antibody The terms "antibody" and "immunoglobulin" are equivalent and can be used interchangeably. An antibody or immunoglobulin is a glycoprotein synthesized in response to an antigen, capable of recognizing and binding to the antigen responsible for its production.

[0039] Thus, the term “antibody” refers to immunoglobulins or immunologically active parts of immunoglobulin, that is, molecules that contain an antigen-binding site that binds immunospecifically to said antigen. As such, the term antibody encompasses not only antibody molecules Immunoglobulin molecules can be found in whole antibodies, as well as antibody fragments and variants (including derivatives). Classically, immunoglobulin molecules have a basic structure of four polypeptide chains linked by disulfide bonds. Each light chain consists of approximately 220 amino acids and has a molecular mass of about 25 kilodaltons (kDa). Each heavy chain consists of approximately 400 amino acids and has a molecular mass of 50 to 70 kDa. For each class or subclass of immunoglobulins, the heavy chains are structurally distinct. Both the heavy and light chains contain two different regions. The constant regions (CL and CH) have amino acid sequences that do not vary significantly between antibodies of the same class. The variable regions (VL and VH) of antibodies have different sequences.Folded together, the variable regions (VL and VH) form the antibody binding site (Microbiology, L. Prescott 2002).

[0040] According to one embodiment, the anti-NPR1 antibody is a polyclonal antibody.

[0041] Polyclonal antibodies have the advantage of being generated rapidly (for example, in a few weeks), more easily, and at a lower cost compared to monoclonal antibodies. Furthermore, since they recognize several epitopes, polyclonal antibodies generally have a broader spectrum of activity than monoclonal antibodies.

[0042] Polyclonal antibodies are preferably non-human polyclonal antibodies.

[0043] Polyclonal antibodies can be selected from the group consisting of rabbit polyclonal antibodies, mouse polyclonal antibodies, rat polyclonal antibodies, guinea pig polyclonal antibodies, chicken polyclonal antibodies, goat polyclonal antibodies, cow polyclonal antibodies, sheep polyclonal antibodies and their combinations.

[0044] Preferably, the polyclonal antibodies are rabbit polyclonal antibodies.

[0045] A person skilled in the art may use any technique to develop polyclonal or monoclonal antibodies specifically directed against the monomeric form of the NPR1 protein, so that said antibodies bind specifically to an NPR1 antigen.

[0046] Antigen The term "antigen" refers to any substance, such as proteins, nucleoproteins, polysaccharides, peptides, and certain glycolipids, that induces an immune response and reacts with the products of that response. Each antigen can have several antigenic determinants or epitopes. Epitopes are the regions of the antibody that bind to the antigen-binding site on an antigen. specific (according to Microbiology, L. Prescott 2002).

[0047] The inventors of the present invention have advantageously achieved a sequence alignment of the NPR1 protein from several plant species (beetroot, alfalfa, cotton, cocoa, tomato, potato, castor bean, chili pepper, poplar, tobacco, sweet potato, papaya, grapevine, brown mustard, rapeseed, apple tree, etc.) as well as that of Arabidopsis thaliana

[0048] By this multiple alignment, a consensus sequence, of SEQ ID NO: 1, was then identified.

[0049] In addition, the "hidden" part of the protein, corresponding to an inaccessible part in its inactive oligomeric form, was also identified (between cysteine ​​82 and cysteine ​​216 for the A. thaliana sequence) by bioinformatics, allowing the generation of a second consensus sequence of SEQ ID NO: 6.

[0050] Consensus degenerate peptides based on the alignment of different NPR1 proteins from the main plant species of agronomic interest were then synthesized. These peptides were then injected into rabbits to produce antibodies or antipeptides.

[0051] These peptides are said to be degenerate because two or three amino acids in their sequence are variable, and consensus peptides because the different sequences thus created correspond to the NPR1 sequences conserved in different plant species. These synthetic peptides have thus been used as antigens to immunize rabbits and to produce antibodies or antipeptides.

[0052] The term "antipeptides" may be used alternatively. It is used to specify that these are antibodies created using consensus degenerate peptides. These antipeptides are polyclonal, but the strategy implemented could just as easily be carried out using monoclonal antibodies.

[0053] Thus, two consensus peptide sequences targeting the active form of the NPR1 protein were proposed, respectively SEQ ID NO: 1 and SEQ ID NO: 6.

[0054] The consensus sequence of SEQ ID NO: 1 comprises two degenerate amino acids (XI and X2): His Val His Arg Cys XI Leu Ser Ala Arg Ser X2 Phe Phe, XI can be either a Valine (Val) or an Isoleucine (Ile), and X2 can be a Serine (Ser) or a Proline (Pro).

[0055] 4 peptides of SEQ ID NO: 2, 3, 4 and 5 were generated on the basis of the peptide SEQ ID NO consensus: 1 and as reproduced below:

[0056] [Tables 1] SEQ ID NO SEQ ID NO: 2 His Val His Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe SEQ ID NO: 3 His Val His Arg Cys Ile Leu Ser Ala Arg Ser Ser Phe Phe SEQ ID NO: 4 His Val His Arg Cys Val Leu Ser Ala Arg Ser Pro Phe Phe SEQ ID NO: 5 His Val His Arg Cys Ile Leu Ser Ala Arg Ser Pro Phe Phe

[0057] The consensus sequence of SEQ ID NO: 6 comprises three degenerate amino acids (X3, X4 and X5): His Ala Leu Gin Leu Leu Ser Asn Ser X3 can be either a Valine (Val) or an Isoleucine (Ile), X4 can be a Serine (Ser) or a Proline (Pro) and X5 can be a Serine (Ser) or a Proline (Pro).

[0058] 8 peptides of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 were generated on the basis of the SEQ consensus peptide ID NO: 6 and as reproduced below:

[0059] [Tables2] SEQ ID NO SEQ ID NO: 7 His Ala Leu Gin Leu Leu Ser Asn Ser Val Glu Ser Ser Ser SEQ ID NO: 8 His Ala Leu Gin Leu Leu Ser Asn Ser Val Glu Ser Pro Ser SEQ ID NO: 9 His Ala Leu Gin Leu Leu Ser Asn Ser Val Glu Ser Pro Pro SEQ ID NO: 10 His Ala Leu Gin Leu Leu Ser Asn Ser Val Glu Ser Ser Pro SEQ ID NO: 11 His Ala Leu Gin Leu Leu Ser Asn Ser Ile Glu Ser Ser Ser SEQ ID NO: 12 His Ala Leu Gin Leu Leu Ser Asn Ser Ile Glu Ser Pro Ser SEQ ID NO: 13 His Ala Leu Gin Leu Leu Ser Asn Ser Ile Glu Ser Pro Pro SEQ ID NO: 14 His Ala Leu Gin Leu Leu Ser Asn Ser Ile Glu Ser Ser Pro

[0060] Thus, the present invention also relates to an anti-NPR1 antibody characterized in that said antibody binds to at least one epitope of the monomeric form of NPR1, of sequence chosen from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.

[0061] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least two epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.

[0062] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least three epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.

[0063] According to one embodiment, the anti-NPRl antibody binds to a consensus sequence epitope SEQ ID NO:1 where XI is a valine or an isoleucine and X2 is a serine or a proline.

[0064] According to one embodiment, the anti-NPR1 antibody binds to the SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 sequences of the monomeric form of the NPR1 protein.

[0065] The present invention also relates to an anti-NPR1 antibody characterized in that said antibody binds to at least one epitope of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0066] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least two epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0067] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least three epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0068] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least four epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0069] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least five epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0070] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least six epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0071] According to one embodiment, the anti-NPR1 antibody is characterized in that said antibody binds to at least seven epitopes of the monomeric form of NPR1 of sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0072] According to one embodiment, the anti-NPRl antibody binds to a consensus sequence epitope SEQ ID NO:6 where X3 is a valine or an isoleucine, X4 is a serine or a proline and X5 is a serine or a proline.

[0073] According to one embodiment, the anti-NPR1 antibody binds to the sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 of the monomeric form of the NPR1 protein.

[0074] Polyclonal antibodies are preferably obtained or can be obtained from at least one biological sample from an animal immunized with at least one antigen comprising preferably or consisting of at least one sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and / or of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0075] In one embodiment, polyclonal antibodies can be obtained by immunizing a non-human animal with an antigenic composition comprising a mixture of antigens of sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and / or a mixture of antigens of sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0076] The term “antipeptide 0” or “antipep 0” means a non-human polyclonal antibody obtained by immunizing a non-human animal with an antigen composition comprising a mixture of peptides of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

[0077] The term “antipeptide 1” or “antipep 1” means a non-human polyclonal antibody obtained by immunizing a non-human animal with an antigen composition comprising a mixture of peptides from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0078] Another object of the invention is a method for producing polyclonal antibodies as defined above, in which said method comprises the steps of: - providing a biological sample from an animal immunized with at least one antigen comprising preferably or consisting of at least one sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and / or of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; - collect or purify the polyclonal antibodies of said biological sample.

[0079] Method for detecting the activation of a plant's defenses The present invention also relates to a method for detecting the activation of a plant's defenses comprising detecting the monomeric form of the NPR1 protein in a sample of said plant, using an anti-NPR1 antibody according to the present invention.

[0080] The monomeric form of the NPR1 protein can also be quantified in a sample of said plant, using an anti-NPR1 antibody according to the present invention.

[0081] Advantageously, the use of specific anti-NPR1 antibodies according to the present invention makes it possible to target the active form of the NPR1 protein of different plant species in order to detect the defenses put in place by the plants.

[0082] By "activation of plant defense mechanisms" is meant the development of a set of biological modifications that confer on the plant i) immediate resistance, in particular LAR (local acquired resistance), ii) induced systemic resistance (ISR) and / or SAR (systemic acquired resistance), and / or iii) pre-sensitization of the potentiation type, thereby enabling it to respond more effectively to subsequent biotic or abiotic stress. Activation of plant defense mechanisms also refers to the activated or non-activated state of the various molecular pathways involved in natural defense mechanisms, particularly the pathway involving salicylic acid, but also those involving jasmonic acid. Activation of plant defense mechanisms also refers to stress resistance, that is, the ability of a plant to cope with biotic and / or abiotic stresses.

[0083] Biotic stress is caused by a living organism. This includes any harmful organism that impairs the health of a plant. These are all living organisms capable of attacking a cultivated plant. They include pests (insects, nematodes, spiders), disease-causing agents (fungi, oomycetes, bacteria, viruses, viroids), and all weeds. Animals such as rodents or ruminants may also be included.

[0084] Abiotic stress is caused by environmental conditions (excluding living organisms) such as drought, cold, ultraviolet rays... (Methodological guide for evaluating the effectiveness of Plant Defense Stimulators (PDS).

[0085] By "plant" we mean for example a plant at any stage of development whatsoever, in particular embryo or any other seedling stage or of the adult plant.

[0086] By way of illustration, the plants are chosen from among fruit trees, crops garden crops, vines, cereals and oilseeds, protein crops, oilseed and protein crops, tobacco, plants belonging to the Brassicaceae family and ornamental plants.

[0087] Preferably, the plants will be chosen from Arabidopsis thaliana, beetroot, alfalfa, cotton, cocoa, tomato, potato, castor bean, chili pepper, poplar, tobacco, sweet potato, papaya, vine, brown mustard, rapeseed, apple tree, wheat, strawberry, cabbage, lettuce.

[0088] Even more preferably, the plant will be the vine.

[0089] The term "sample" means any sample that allows for the detection and quantification of the monomeric form of NPR1. The sample may be obtained from the whole plant (e.g., a seed or a seedling), from a part of the plant, or from a plurality of plants or parts of plants, such as a batch of plants or leaves. Those skilled in the art know how to obtain such samples for the detection and quantification of a protein.

[0090] By part of the plant, we mean for example pollen, ovules, embryos, aerial parts (stems and leaves), leaves, anthers, stems, petioles, roots, fruits, seeds, flowers, buds, protoplasts, calluses, cells and cellular tissues.

[0091] Preferably, the aerial parts will be used.

[0092] A person skilled in the art is aware of the methods for detecting and / or quantifying the monomeric form of the NPR1 protein using a given antibody from a given sample. Thus, any means for detecting and / or quantifying the monomeric form of NPR1 in a sample may be used.

[0093] Typically, the monomeric form of the NPR1 protein can be detected and / or quantified in the method according to the present invention by Western blot, an ELISA (Enzyme Link ImmunoSorbent Assay) test or an LFD (Lateral Flow Device or Lateral Flow Immunchromatographic Assay) type strip test.

[0094] According to one embodiment, the detection of the monomeric form of NPR1 by the specific antibodies according to the present invention makes it possible to identify a plant exhibiting an induction of the activation of its defense mechanisms.

[0095] According to one embodiment, the monomeric form of NPR1 will be quantified. The quantification of the monomeric form of NPR1 can be expressed in an arbitrary unit to reflect the amount of the monomeric form of the NPR1 protein in the sample.

[0096] According to one embodiment, the value may consist of the concentration of the monomeric form of the NPR1 protein, measured by any method of protein quantification known to those skilled in the art, such as by Western blot.

[0097] According to one embodiment, the method for detecting / quantifying activation The study of a plant's defenses may include an additional step of comparison with a reference sample, in order to determine or quantify the activation state of the plant's defenses.

[0098] A "reference sample" is defined as a sample obtained from a plant or plant parts whose state of defense activation is known. Thus, reference samples may be taken from plants or plant parts that are untreated or unstressed, or that have been previously subjected to a biotic or abiotic stress.

[0099] The term "active substance capable of activating a plant's defenses," "Plant Defense Stimulator," "PDS," "natural defense stimulators," or "NDS" refers to any non-pathogenic substance or living microorganism which, when applied to a plant, is capable of promoting a significantly higher state of resistance to biotic or even abiotic stresses compared to an untreated plant. A PDS does not generally act directly on pests and diseases; it is perceived by the plant as an alert message. The plant will react by preparing or activating various defense mechanisms, thereby contributing to its increased resistance to pest and disease attacks.Thus, a product effective on a plant-pathogen pair, which does not have a significant direct effect on the pathogen at the effective dose on the plant, and which is capable of inducing known defense markers (PR proteins, lipoxygenase, phenylalanine ammonia-lyase, etc.) under these conditions, can be considered as an SDP.

[0100] SDPs are described, for example, in application WO2011 / 161388 and can be classified into two main families: (i) so-called "direct stimulator" compounds, which, once applied to the plant, lead to a complete activation of defense reactions, whether or not pathogens are present, and ii) so-called “potentiating” compounds, which, after application to the plant, trigger only the aforementioned “potentiation” phenomenon (defense reactions only being activated following an attack by a pathogen or stress). Most of these SDP products are still known as "elicitors" or "resistance inductors".

[0101] Typically, approved SDPs in France are as described below:

[0102] [Tables 3] Active ingredients Commercial products Supplier Targets COS-OGA Pytosave Garden Messager Jouffray Driaud Syngeta - downy mildew, powdery mildew: vines - powdery mildew: vegetable crops Laminarin lodus 2 specialty crops lodus 2 cereals Vacciplant Jardin Goëmar - powdery mildew: vines, wheat, barley, strawberries - pieti-loft, septoria leaf blotch: wheat - helminthosporium leaf blotch: barley - downy mildew: lettuce - fire blight, scab: fruit crops and gardens Potassium phosphites Pertinan / Etonan LBG-O1F34 De Sangosse downy mildew: vines and vegetable crops Sodium phosphonates Redeli Syngenta Downy mildew: vines Bacillus subtilis strain QST 713 Serenade Max Texio Bayer SBM development - grey rot: vines - ornamental vegetable crops, aromatic, food, medicinal and condiment plants Bacillus amyloliquefaciens strain MBI 600 Integral Pro BASF Seed treatment: fungi other than Pythiaaceae: oilseed crucifers Cerevisiane Actileaf Romeao Agrauxine BASF Downy mildew, powdery mildew,Grey rot: vines, vegetable crops. Fenugreek extract Stifenia SOFT - powdery mildew: vines - aerial parts treatment: melon. Acybenzolar-S-methyl Bion WG Syngenta - aerial parts treatment: banana, Ornamental crops - powdery mildew: wheat - bacterial blight: tomato

[0103] By way of illustration, to obtain a reference sample representative of a biotic stress, Bion® can be sprayed on plants or parts of plants, to obtain a reference sample representative of a bacterial stress.

[0104] By way of illustration, to obtain a reference sample representative of the activation of the SA pathway, salicylic acid (SA) can be applied to the plant or part of the plant.

[0105] By way of illustration, to obtain a reference sample representative of the activation of the jasmonic acid (JA) pathway, Methyl-Jasmonate can be applied to the plant or part of the plant.

[0106] By way of illustration, to obtain a reference sample representative of an abiotic stress, the plant or part of a plant can be exposed to UV radiation, heat, cold, ...

[0107] To obtain an untreated reference sample, water can be sprayed on the plants or parts of plants.

[0108] To obtain an unstressed reference sample, the plant or part of a plant can be isolated in a preserved and controlled environment.

[0109] By way of illustration, to obtain a reference sample representative of a treatment with an SDP, an SDP can be applied to the plant or part of the plant.

[0110] Typically, the detection of the monomeric form of the NPR1 protein will be significant of the activation of a plant's defenses.

[0111] Typically, a significant increase in the concentration of the monomeric form of the NPR1 protein, compared to a reference sample, allows the identification of a plant exhibiting induction of the activation of its defense mechanisms.

[0112] Thus, and in general, the method according to the present invention makes it possible to detect and / or measure the activation of plant defense mechanisms by detecting and / or quantifying the monomeric form of the NPR1 protein. Typically, the method will make it possible to detect / measure the activation of plant defense mechanisms in the field before treatment to facilitate decision support and the positioning of plant protection products (PPPs) according to the reactivity of the targeted plants.

[0113] The method according to the invention also makes it advantageous to demonstrate the activation of defenses following the application of a product as requested in the context of the method of the Commission for Biological Testing (CEB) on the "general principles of experimentation of plant defense stimulators" (method general MG14), necessary for the approval process and regulating the placing of the product on the market.

[0114] SDP screening method The present invention also relates to a method for screening active substances capable of activating a plant's defenses, comprising: a) the treatment of a sample of said plant with at least one substance; b) the detection in said sample of the monomeric form of the NPR1 protein according to the detection method according to the present invention.

[0115] The present invention also relates to a method for screening active substances capable of activating the defenses of a plant comprising a) the treatment of a sample of said plant with at least one substance; b) the quantification in said sample of the monomeric form of the NPR1 protein according to the detection method according to the present invention.

[0116] Advantageously, the present screening method allows for the screening of potentially SDP molecules in the laboratory to enable the emergence of new active substances with SDP activity. The detection and / or quantification of the monomeric form of NPR1 by the antibodies according to the present invention will make it possible to determine the efficacy of a molecule as an SDP.

[0117] Thus, it is possible to make a discrimination between i) SDP products which effectively activate plant defenses, and ii) products which have no effect.

[0118] The term "substance" means any non-pathogenic substance or living microorganism whose properties for promoting a significantly higher state of resistance compared to an untreated plant to biotic or even abiotic stresses are not known. The method according to the present invention thus makes it possible to screen any active compound or active composition, whether biological or chemical.

[0119] By way of illustration, the detection of the monomeric form of the NPR1 protein will be significant of the effectiveness of the substance tested, and will allow the said substance to be classified as SDP.

[0120] According to one embodiment, the screening method may include an additional step of comparison with a reference sample, of the monomeric form of the NPR1 protein according to the detection method according to the present invention.

[0121] Typically, the amount of the monomeric form of the NPR1 protein in the plant sample can be compared to the amount of the monomeric form of the NPR1 protein in the reference sample. Generally, a significant increase in the amount of the monomeric form of the NPR1 protein in the plant sample compared to the reference sample is synonymous with the efficacy of the substance as an SDP and may allow the said substance to be classified as an SDP, while the absence of significant variation may be synonymous with the absence of efficacy as an SDP.

[0122] Method for evaluating the sensitivity of a plant to a SDP The invention also relates to a method for evaluating the sensitivity of a plant to a molecule capable of stimulating the plant's defenses, comprising: a) treating a sample of said plant with a molecule capable of stimulating plant defenses; b) the detection in said sample of the monomeric form of the NPR1 protein according to the present invention.

[0123] The invention also relates to a method for evaluating the sensitivity of a plant to a molecule capable of stimulating the defenses of a plant comprising: a) treating a sample of said plant with a molecule capable of stimulating plant defenses; b) the quantification in said sample of the monomeric form of the NPR1 protein according to the present invention.

[0124] Typically, the detection of the monomeric form of the NPR1 protein will allow us to conclude on the sensitivity of the plant to the molecule capable of stimulating the defenses of a plant.

[0125] According to one embodiment, the method for evaluating the sensitivity of a plant to a molecule capable of stimulating the defenses of a plant may include an additional step of comparison with a reference sample, in order to determine or evaluate the activation state of said plant from the concentration of the monomeric form of NPR1 obtained in the quantification step.

[0126] Typically, the amount of the monomeric form of the NPR1 protein in the plant sample can be compared to the amount of the monomeric form of the NPR1 protein in the reference sample. Generally, a significant increase in the amount of the monomeric form of the NPR1 protein in the plant sample compared to the reference sample indicates sensitivity of the plant to the molecule capable of stimulating plant defenses.

[0127] The present evaluation method thus makes it advantageous to select varieties highly receptive to SDPs, within the framework of varietal selection or to optimize plant variety / SDP pairs.

[0128] Method for selecting a plant exhibiting improved resistance The present invention also relates to a method for selecting a plant exhibiting a state of activated defenses likely to confer improved resistance to at least one biotic and / or abiotic stress, including: (i) the application of said stress(s) to a sample of said plant, ii) the detection in said sample of the monomeric form of the NPR1 protein according to the detection method of the present invention.

[0129] The present invention also relates to a method for selecting a plant exhibiting a state of defense activation capable of conferring improved resistance to at least one biotic and / or abiotic stress comprising: i) the application of said stress(s) to a sample of said plant, ii) the quantification in said sample of the monomeric form of the NPR1 protein according to the detection method of the present invention.

[0130] Typically, the detection of the monomeric form of the NPR1 protein in the plant sample will be synonymous with improved resistance to at least one biotic and / or abiotic stress and will allow the plant to be selected for its improved defense activation properties.

[0131] The selection method may further include a comparison step with a reference sample to determine or evaluate the state of stimulation of the plant's natural defenses in order to select said plant if the plant has a state of defense activation likely to confer improved resistance to at least one biotic or abiotic stress.

[0132] Typically, a significantly increased concentration of the monomeric form of the NPR1 protein, compared with a reference sample not subjected to stress and / or SDP, allows for the identification of plants or plant parts exhibiting a state of defense activation and improved resistance.

[0133] Kit for implementing the methods according to the invention The present invention also relates to a kit for implementing the detection, screening and activation methods according to the present invention comprising an anti-NPRl antibody according to the invention. Examples

[0134] Materials and Methods

[0135] In the following examples, the materials and methods detailed below have been used.

[0136] Immunization The production of the consensus degenerate peptides and antibodies according to the present invention was carried out by the company GENEPEP. To this end, they first synthesized the two peptides (pepO) and (pepl) as shown in [Fig. 1] based on the two consensus sequences SEQ ID NO: 1 and SEQ ID NO: 6. The peptides of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, were used as immunogenic agents in order to to induce an immune response against these peptides in rabbits. To this end, peptides with the sequences defined above were injected into two rabbits each, five times during the rabbits' lives. All of their blood was then collected to extract the serum containing antibodies recognizing the injected peptides.

[0137] Purification and concentration of anti-NPR1 antibodies The rabbit serum obtained is used to purify the antipeptides (antipeptide 0 or antipepO and antipeptide 1 or antipepl) specific to the active form of NPR1, according to the following method: 50 ml of solution of a mixture of peptides of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 at 0.5 mg / ml is loaded onto a membrane to purify antipeptide O and a mixture of peptides of sequence SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, at 0.5 mg / ml is loaded onto another membrane to purify antipeptide 1. For each mixture of peptides, the membrane is a 1.5 x 5 cm Polyvinylidene difluoride (PVFD) membrane previously activated for 10 min in absolute ethanol and then dried. This membrane is then transferred to a hemolysis tube and saturated with 1 ml of 0.1% phosphate-tween buffer (PBST) + 5% milk (Régilait® skimmed milk powder) by shaking for 1 hour at 4°C. One milliliter of the corresponding serum is added before overnight incubation with shaking at 4°C. The membrane is then washed 4 times with 2 ml of PBST by shaking for 15 minutes at 4°C.The antipeptides are then eluted by acid shock (1 ml of 50 mM glycine, 500 mM NaCl, 0.1% Tween 20, 1% BSA, pH 3) for 1 min with stirring at 4°C. The 1 ml eluate is collected and neutralized with 100 ml of Tris Base IM. This elution is repeated 5 times, and eluates 2 and 3 (the most concentrated in antipeptide) are collected for further concentration. This operation is performed on 10 membranes in parallel, and each membrane is used twice. 10 mL of antipeptide solution is thus obtained for the two eluates of interest. Two 4 mL samples of each solution are concentrated by ultrafiltration using the Amicon lOkDa ultrafiltration system (10,000 rpm, 10 min, 4°C). Between 500 and 600 µL of concentrated antipeptide solution are thus produced for each eluate.

[0138] Extraction of plant proteins a) Extraction of proteins from A, thaliana A. thaliana plants are grown in soil for 3 weeks in a long-day growth chamber (18 hours of light, 60% humidity, 21°C during the day and 19°C at night) and then sprayed with water (negative control), Bion® 0.015%, or ImM salicylic acid (SA) (control for SA pathway activation), or with 0.1M methyl jasmonate (Me-JA) in 0.1% dimethyl sulfoxide (DMSO) (control for Jasmonic acid (JA) pathway activation). The aerial parts of the plants are The plants were then harvested 48 hours after spraying and ground in liquid nitrogen. The total protein content of these plants was extracted using 1 µl of extraction buffer (Tris-HCl pH 7.5 50 mM, NaCl 150 mM, MgCl 10 mM, EDTA 5 mM, glycerol 10%, lo-doacetamide 4%, protease inhibitor IX (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, ROCHE)) per mg of frozen powder. The samples were thawed in the buffer by vortexing regularly and then centrifuged (10 min, 14,000 g, 4°C). The supernatant was centrifuged again under the same conditions. The final supernatants were collected and constituted the plant protein extracts used for the various experiments. b) Extraction of proteins from other plants Tomato plants were grown in a greenhouse for four weeks before being sprayed with SA, Me-JA, or water to activate or suppress their defenses. The aerial parts of these plants were harvested 48 hours later, then ground into powder in liquid nitrogen and used to extract proteins. The total protein content of these plants was then extracted using 2 µL of extraction buffer (Tris-HCl pH 7.5 50 mM, NaCl 150 mM, EDTA 0.5 mM, Triton X100 0.1%, nP40 0.2% antiprotease IX (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, Roche)) per mg of powder. The samples were resuspended in the buffer by vortexing regularly and then centrifuged (10 min, 14,000 g, 4°C). The supernatant was centrifuged again under the same conditions. The final supernatants were collected and constituted the plant protein extracts used for the various experiments.

[0139] Western blots (WB) SDS-PAGE gels containing 10% acrylamides are used for protein extract migration. 30 µL of plant extract or 25 µL of immunoprecipitation eluate are incubated in 15 µL of Laemmli 2X for 10 min at 65°C with shaking and then dispensed by well. The gels are then run through RB IX buffer at a constant voltage (100 mV). The proteins, having undergone electrophoresis, are then transferred onto PVFD membranes pre-activated for 10 min in absolute ethanol, using the Bio-Rad Trans-Blot® Turbo™ Transfer System. The membranes are then rinsed three times in Phosphate Saline + 1% Tween (PBST) buffer and then saturated with milk protein in PBST + 5% milk (1 h with shaking at 40 rpm, room temperature). They are then incubated overnight in PBST+ 5% milk overnight at 4% in the presence of the primary antibody (1 / 5000°).The membranes are then washed in PBST (4 x 10 min + 1 x 1h, 40 rpm, room temperature) and then incubated with the secondary antibody (sigma anti-rabbit HRP, 1 / 10,000, 1h, 40 rpm, room temperature). They are then washed again in PBST (4 x 10 min + 1 x 1h, 40 rpm, room temperature) before being dried for 2 min on absorbent paper. Proteins hybridized by the primary antibody are then detected by chemiluminescence using the LAS 4000 (Fujifilm) and the Amersham ECL™ Prime substrate (luminol + peroxide) from GE Healthcare. The membranes are then stained with Coomassie blue for 1 hour at room temperature with stirring at 35 rpm. The protein profiles are then revealed by decolorizing the membranes with a 10% acetic acid and 10% ethanol decolorizing solution overnight at room temperature with stirring at 35 rpm.

[0140] Immunoprecipitation (IP) Protein A coupled magnetic beads (Dynabeads™ Protein A, Ther-moFisher No. 10001D) are used to perform immunoprecipitation (IP) of A. thaliana proteins or recombinant proteins using the antipeptide or a commercial antibody targeting NPR1 only in this plant species (Agrisera No. AS12 1854). Beads are prepared for each IP (Intraproximal Intracytoplasmic Sclerosis). Four 100 ml aliquots of bead solution are placed in 1.5 ml Eppendorf tubes and then placed on a magnetic rack for 2 minutes. The supernatant is discarded, and the beads are washed twice with 500 ml of PBST (Plant-Based Stem Cell Therapy). The tubes are placed back on the magnetic rack for 2 minutes, the supernatant is removed, and then 90 ml of PBST is added. Six microliters of commercial antibody (6 µg) and 0.5 ml of antipeptide (25 µg) are added to each tube (to the 100 ml PBST level), and the entire incubation is performed for 1 hour and 30 minutes at room temperature with shaking on a rotary mixer. The supernatant is removed, and the antibody-bound beads are washed three times with 500 ml of PBST with shaking (5 minutes) on a rotary mixer. The entire mixture is then resuspended in 100 ml of PBST. The antibodies are then covalently linked to the A proteins of the beads by washing them twice for 10 minutes with agitation using 1 ml of crosslink buffer (200 mM triethanolamine pH 8.2).The tubes are placed back on the magnetic rack (2 min) and then the supernatant is removed. One milliliter of cross-link buffer + DMP (200 mM triethanolamine pH 8.2 + 20 mM dimethyl pimelimidate dihydrochloride) is added to each tube, and the tubes are incubated for 30 min with shaking at room temperature. The covalent binding reaction is stopped by removing the supernatant (on the magnetic rack), adding 1 ml of 50 mM Tris-HCl pH 7.5 (15 min incubation at room temperature with shaking on a wheel), and then washing three times for 5 min with 500 µl of PBST. The beads covalently bound to the antibodies are then resuspended in 100 µl of PBST and stored at 4°C for PIs. The tubes are placed on the magnetic rack to remove the supernatant. 800 µl of total A. thaliana protein treated with SA or 150 µl of recombinant protein (Ipg for each) are loaded into the four tubes containing magnetic beads bonded to either the commercial antibody or the antipeptide. The four tubes are incubated overnight at 4°C on a wheel to hybridize the protein(s) recognized by the antibodies to the antibodies themselves. The beads coupled to the antibodies, which are themselves hybridized to the proteins, are washed five times. The proteins recognized by the antibodies are then eluted using 50 µL of Laemmli + dithiothreitol (DTT) 100 µM, first for 15 min at room temperature (eluate 1), then a second time, again using Laemmli + DTT 100 µM, but for 1 h at 95°C (eluate 2). These different eluates are then used in Western blotting and SDS-PAGE.

[0141] Recombinant protein production Cloning The coding region of the A. thaliana NPR1 protein, as well as sub-regions thereof, were cloned into the bacterium E. coli using the Gateway® cloning technique. Three different constructs were thus created. The first produced the entire NPR1 protein (prot T), a protein of 560 amino acids. The second produced a partial protein (prot P), corresponding to the 260 amino acids in the N-terminus of NPR1. The third produced only the inaccessible portion of NPR1 in its inactive form, namely the 140 amino acids located between cysteine ​​82 and cysteine ​​216 of NPR1 (called prot I for protein of interest), as shown in [Fig. 2].

[0142] PCRs were performed using A. thaliana cDNA as a template with standard primers known to those skilled in the art. These primers amplify the DNA sequences encoding the three aforementioned proteins by integrating a TEV sequence at the 5' end, enabling protein cleavage by the TEV protease. The attB1 and attB2 sequences were also added to the primers for integration at the 5' and 3' ends, respectively, of the PCR products. These products were then assembled into the pDONR207 vector by BP reaction for cloning using the Gateway® technique. The resulting clones were validated by sequencing (GATC biotech) and subsequently used to purify the donor plasmids (pDONR207 + constructs) for further cloning.The donor plasmids were then used for the recombination reaction (LR step of the Gateway® protocol) with the expression plasmids (PetGlO-A), allowing the addition of a histidine tag-encoding sequence to the 5' end of the various sequences of interest. These sequences were then inducibly translated into protein (by adding d-Isopropyl[3-Dl-thiogalactopyranoside (IPTG)]. The resulting expression plasmids were then cloned into the bacterium E. coli dH5a for multiplication and purification, and subsequently cloned into E. coli Rosetta to produce the proteins with their histidine tag. A sequence representing the entire A. thaliana NPR1 protein (Pro T) including at the N-terminus a histidine tag followed by the amino acids corresponding to the attB 1 sequence necessary for cloning with the Gateway® method, as well as the TEV protease cleavage sequence for obtaining the entire or total NPR1 protein (Pro T T).

[0143] A sequence representing the partial NPR1 protein sequence of A. thaliana (Pro P) including in Nterminal a histidine tag followed by the amino acids corresponding to the attBl sequence required for cloning with the Gateway® method as well as the cleavage sequence of the TEV protease for obtaining the partial NPR1 protein (prot P).

[0144] A sequence representing the sequence of the inaccessible part in the inactive form of the partial A. thaliana NPR1 protein (Pro I) including in N-terminal a histidine tag followed by the amino acids corresponding to the attBl sequence required for cloning with the Gateway® method as well as the cleavage sequence of the TEV protease for obtaining the NPR1 protein of interest (prot I). b) Protein production The different bacteria possessing the constructs of interest were cultured in 21 ml of lysogenic broth + ampicillin 100 pg / ml + chloramphenicol 34 pg / ml until they reached an optical density of 0.6 at 600 nm, at 37°C. Protein production was then triggered by the addition of IPTG (0.5 mM for T and P and 1 mM for I). Bacteria were harvested 3 hours later for T and P and 4 hours later for I. The cultures were separated into six 40 ml portions and centrifuged (12,000 g for 15 min at 4°C). The pellets were frozen in liquid nitrogen for subsequent steps. The pellets are resuspended in a denaturing lysis buffer because the recombinant proteins are in inclusion bodies (NaH2PO4 100mM, Tris 10mM pH 8, NaCl 250mM, urea 8M, DTT ImM, imidazole 250mM, protease inhibitor IX (cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail, ROCHE)) and then lysed by sonication (12 pulses of 20 s at 50% amplitude interspersed with 30 s pauses, on 2°C ice). The extracts are then centrifuged (20,000 g, 15 min, 4°C) to remove cell debris. The supernatants are collected and used for the various experiments as recombinant protein.

[0145] Analysis of gene expression by q-RT-PCR A. thaliana plants are grown in soil for 3 weeks in a long-day growth chamber and then sprayed with water (negative control) and Bion® 0.015% (control for activation of the SA pathway). The aerial parts of the plants are then harvested 48 hours after spraying and ground in liquid nitrogen. The RNAs are then extracted with trizol and quantified to determine the RNA Ipg for the DNase and RT steps. The DNA present in the samples is degraded using DNase (RNA Ipg, Ipl of 10X buffer with MgCl2, Ipl of DNase, completed to 1Opl with DEPC water). The mixture is incubated for 45 min at 37°C in a water bath, then Ipl of 50mM EDTA is added before a 10 min incubation at 65°C, again in a water bath, to inactivate the DNase. These DNase-treated RNAs are then used for the reverse transcription (RT) step. Five microliters of RNA are incubated at 65°C for 5 min in a water bath with 1 µl of primers and 6.5 µl of diethyl pyrocarbonate (DEPC) treated water. Then, 4 µl of 5X RT buffer, 0.5 µl of Ribolock, 2 µl of 10 mM dNTPs, and 1 µl of M-MuLV reverse transcriptase are added per tube before incubation for 1 h at 37°C in a water bath. The reverse transcriptase is then inactivated for 10 min at 70°C. The resulting cDNAs are then diluted 10-fold (by adding 180 µl of water) for use in the quantitative PCR step. The qPCR reaction is carried out in lOpl, with 5pl of SybrGreen mix, 0.3pl of 0.3pM primer mix, 2.4pl of water and 2.5pl of cDNA. qPCRs were performed using a Bio-Rad CFX-96 Real Time PCR System thermocycler. The raw data were processed using Bio-Rad CFX Manager software. The normalized transcript quantity was then calculated by dividing the data obtained for the defense genes of interest by the data obtained for the constitutive gene used (here, the clathrin-encoding gene), taking into account the primer efficiency obtained using the dilution range and their quantity expressed in arbitrary units.

[0146] Example 1: Detection of the active form of NPR1 by anti-NPR1 antibodies according to the present invention

[0147] The objective of this example is to show that the antibodies according to the present invention can detect an active form of NPR1. Western blots were therefore performed on extracts of A. thaliana treated with Bion®, SA, or water. In the western blots performed with these plant extracts, a band is detected when the plants are treated with defense activators and is not detected when the plants are treated with water. This band is detected at a size of 37 kDa, as shown in [Fig. 3].

[0148] To demonstrate that the detected band corresponds to the NPR1 protein, sequencing was performed on bands cut from an acrylamide gel in parallel with a Western blot, using peptide sequencing performed by the PAPPSO platform (UMR MICALIS, PAPPSO, building 526, Domaine de Vilvert 78352, Jouy en Josas cedex). Between 100 and 160 proteins were then identified in the band detected by Western blot, including the NPR1 protein. NPR1 is therefore indeed present in the band detected by Western blot (at a size of 37 kDa).

[0149] Furthermore, Western blots were performed with the same plant extracts using in parallel antipeptide 0 (antipep 0) and a commercial antibody targeting NPR1 only in A. thaliana (Agrisera No. AS12 1854). A band of the same size as the band observed in Western blots using the antibodies developed in this invention is detected when the commercial antibody is used, as shown in [Fig. 5].

[0150] To demonstrate that the developed antipeptide is capable of hybridizing to the NPR1 protein, the different forms of recombinant NPR1 produced in E. coli (including the targeted active portion of the protein) are used to perform western blots with the antipeptide (antipep 0) or with a specific antibody for the Histidine (His) tag used in these same recombinant proteins (His-tag). All three of these protein forms are detected with each of the antibodies, and at their expected size (see [Fig. 4]).

[0151] In addition, the two antipeptides (antipep 0 and antipep 1) allow the detection of a band of the same size in western blot on protein extracts of A. thaliana elicited by an SDP, the SA, as demonstrated in [fig.6].

[0152] Thus, and advantageously, the antibodies according to the present invention allow for the specific detection of the active form of the NPR1 protein, unlike the commercial anti-NPR1 antibody, which cannot discriminate between the active and inactive forms of NPR1. The anti-NPR1 antibody therefore cannot determine whether plant defense mechanisms are activated, since it indiscriminately detects both the active and inactive forms of NPR1.

[0153] Furthermore, immunization with different synthetic peptides, based on two different degenerate consensus peptides corresponding to interspecies consensus sequences of NPR1, yields different antipeptides detecting the same band. Highly advantageously, these antibodies allow targeting the active form of NPR1 in different plant species, unlike the commercial anti-NPR1 antibody, which only targets the NPR1 protein of A. thaliana.

[0154] Example 2: Presence of the active form of NPR1 during the activation of different defense pathways

[0155] To demonstrate that this active form of NPR1 is present in both activated hormonal defense pathways (the pathway involving SA or the JA / Et pair), Western blots were performed using A. thaliana plants treated with SA, Me-JA, or water. The band observed at 37 kDa in Western blots using the produced antibodies (antipepO) is detected whether defenses are activated with SA or Me-JA, as shown in [Fig. 5]. These results are consistent with the scientific literature highlighting the involvement of the NPR1 protein in both systemic plant defense pathways: systemic acquired resistance (SAR) and systemic induced resistance (ISR) (Pieterse et al. 1998).

[0156] The same type of result is obtained when tomato extracts are used instead of A. thaliana extracts, as shown in [fig.9].

[0157] Example 3: Sensitivity of the method and advantage compared to the commercial antibody.

[0158] Antipeptide 0 and the commercial antibody were used to perform a Immunoprecipitation (IP) (Dynabeads™ Protein A, ThermoFisher No. 10001D) on A. thaliana protein extract treated with SA. The 37 kDa band is observed on the electrophoresis of the IP eluate obtained with plant extracts whose defenses have been activated [fig.7].

[0159] These antibodies were also used to immunoprecipitate the recombinant P protein of known size. This protein is detected by Westem-blot on the IP eluate obtained with antipeptideO, but not with that obtained using the commercial antibody, as shown in [Fig. 7].

[0160] Thus, and advantageously, the antibodies according to the present invention have improved sensitivity compared to a commercial anti-NPR1 antibody and allow detection of NPR1 when the commercial antibody does not detect it.

[0161] Example 4: Quantification of plant defense activation by the antibody according to the present invention

[0162] To demonstrate that antipeptides can allow for relative quantification of defense activation, extracts of A. thaliana, known to exhibit different levels of defense activation (observed by qPCR on reporter genes of different defense pathways), were used by western blot (WB). The intensity of the detected band correlates with the level of defense activation observed by qPCR, as shown in [Fig. 8].

[0163] Thus and advantageously, the method according to the present invention makes it possible not only to detect the activation of plant defenses but also to quantify the level of activation of plant defenses.

[0164] Example 5: Detection of defense activation in different plant species by antibodies according to the present invention

[0165] To show that the strategy of using degenerate peptides can make it possible to obtain antibodies capable of detecting the activation of defenses in different plant species, protein extracts from tomato plants treated with SA, Me-JA or water were analyzed by western blot.

[0166] A detected band is found to be much more intense when the protein extract used comes from tomato plants treated with SA or Me-JA compared to the protein extract from the control group of plants treated with water, as shown in [fig-9].

[0167] Advantageously, the use of specific anti-NPR1 antibodies according to the present invention makes it possible to target the active form of the NPR1 protein of different plant species and also to detect plant defenses put in place via the salicylic acid pathway but also those involving the JA / Et pathway.

Claims

Demands

1. Anti-NPR1 antibody specifically binding to the monomeric form of the NPR1 protein, and characterized in that said antibody binds to an epitope of consensus sequence SEQ ID NO:1, where XI is a valine or an isoleucine and X2 is a serine or a proline.

2. Anti-NPR1 antibody specifically binding to the monomeric form of the NPR1 protein, and characterized in that said antibody binds to the consensus sequence epitope SEQ ID NO:6, where X3 is a valine or an isoleucine, X4 is a serine or a proline and X5 is a serine or a proline.

3. Anti-NPRl antibody according to claim 1 or 2 characterized in that said antibody is a polyclonal antibody.

4. Method for detecting the activation of plant defenses comprising detecting the monomeric form of the NPR1 protein in a sample of said plant using an antibody according to any one of claims 1 to 3.

5. Detection method according to claim 4, characterized in that the monomeric form of the NPR1 protein is detected by Western blot, by an ELISA test or by an LFD type strip test.

6. Detection method according to any one of claims 4 or 5, characterized in that the sample is a sample of the aerial parts of said plant.

7. A screening method for active substances capable of activating the defenses of a plant comprising: a) treating a sample of said plant with at least one substance; b) detecting in said sample the monomeric form of the NPR1 protein according to the detection method according to any one of claims 4 to 6.

8. Method for evaluating the sensitivity of a plant to a molecule capable of stimulating plant defenses, comprising: a) treating a sample of said plant with a molecule capable of stimulating plant defenses; b) detecting in said sample the monomeric form of the NPR1 protein according to the detection method according to any one of claims 4 to 6.

9. Method for selecting a plant exhibiting an activation state of defenses likely to confer improved resistance to at least one biotic and / or abiotic stress, including: i) the application of said stress or stresses to a sample of said plant, ii) the detection in said sample of the monomeric form of the NPR1 protein according to the detection method according to any one of claims 4 to 6.

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