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WO2019134759A1 - Identification de réseaux extracellulaires de récepteurs kinases à répétition riches en leucine - Google Patents

Identification de réseaux extracellulaires de récepteurs kinases à répétition riches en leucine Download PDF

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WO2019134759A1
WO2019134759A1 PCT/EP2018/050360 EP2018050360W WO2019134759A1 WO 2019134759 A1 WO2019134759 A1 WO 2019134759A1 EP 2018050360 W EP2018050360 W EP 2018050360W WO 2019134759 A1 WO2019134759 A1 WO 2019134759A1
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lrr
rks
interactions
centrality
network
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Youssef BELKHADIR
Elwira SMAKOWSKA-LUZAN
Karin GRÜNWALD
David Stuart GUTTMAN
Darrell Desveaux
Glenn Adam MOTT
Mehdi LAYEGHIFARD
Cyril Boris ZIPFEL
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GREGOR MENDEL INSTITUTE OF MOLECULAR PLANT BIOLOGY GmbH
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GREGOR MENDEL INSTITUTE OF MOLECULAR PLANT BIOLOGY GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance

Definitions

  • This invention lies in the field of interaction platforms of extracellular domains of cell surface receptors and allows identification of an leucine-rich-repeat (LRR)-based Cell Surface Interaction network (CSI LRR ) to predict and validate the function of uncharacterized leucine-rich-repeat receptor kinases (LRR-RKs).
  • LRR leucine-rich-repeat
  • CSI LRR Cell Surface Interaction network
  • Extracellular domains (ECDs) of cell surface receptors serve as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability.
  • discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions 5 .
  • the largest of these families in Arabidopsis thaliana consists of 225 evolutionarily-related leucine-rich repeat receptor kinases (LRR-RKs) that function in microbe sensing, cell expansion, stomata development and stem cell maintenance.
  • LRR-RKs are key in recognizing and transmitting exogenous (non-self) as well as endogenous (self) molecules that act as vital signals in plants defenses and growth. LRR-RKs are involved in developmental processes, immune reactions upon
  • ligands pathogenic attacks, growth, cell death, abscission and ripening. They are involved in specific biological processes, and bind through the extracellular domain families of related specific signal-molecules, which are termed ligands.
  • LRR-RKs are composed of a multi-domain architecture in which each domain provides a specific function for the receptor.
  • LRR-RKs On the intracellular side LRR-RKs have a protein kinase domain that enzymatically catalyzes the transfer of a phosphate group to a substrate protein.
  • phosphate groups to intracellular substrates results in the initiation of a signaling cascade that triggers a cellular response, for example involving immunity related genes in order to fight off a
  • the kinase domain could be classified as the output domain of LRR-RKs.
  • the Kinase and the LRR domains are interconnected by a single
  • transmembrane pass the latter spanning the cellular membrane, which allow the receptor to be membrane bound.
  • the ECD of the receptor contains a leucine rich repeat (LRR) domain.
  • the domain is named according to a series of repeating sequence motifs comprising about 20 to 30 amino acids with a high content of leucine.
  • Stereotypical LRR-RKs include the steroid receptor BRASSINOSTEROID INSENSITIVE1 (BRI1 ) as well as the immune receptors FLAGELLIN SENSING 2 (FLS2), Elongation Factor Tu Receptor (EFR) and PEP RECEPTORS 1 /2
  • EPR1 /2) 1 ,11 The EFR and FLS2 receptors function as immune receptors recognizing the presence of their ligands as "non-self” and initiating a defense response in the plant cell.
  • Ligand-induced activation of BR11 , FLS2 or PEPR1 /2 signaling requires physical interaction with the LRR-RK co-receptor BRI1 -ASSOCIATED KINASE 1 (BAK1 ) 12 15 .
  • BAK1 LRR-RK co-receptor BRI1 -ASSOCIATED KINASE 1
  • interactions between ECDs can take signaling pathways to either low- or high functioning points 2 . Yet, the full range of these interactions remains unmapped.
  • Other defense response related receptors include XA21 from rice, CERK1 and AtPEPRI , the latter recognizing endogenous danger signals related to wound healing processes.
  • LRR-RK leucine-rich repeat receptor kinases
  • a screening assay for the identification of the level of connectivity of leucine-rich repeat receptor kinases (LRR- RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • the bidirectional interactions are high- confidence interactions (HCIs), characterized by absorbance values of the pairwise interactions obtained in a) with a geometric mean product of modified Z-scores of greater than 2.5.
  • HCIs high- confidence interactions
  • a screening assay for the identification of interactive leucine-rich repeat receptor kinases (LRR-RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • HCIs bidirectional interactions
  • a centrality score of the algorithm in the first quartile represents highly interactive LRR-RKs
  • a centrality score in the second quartile represents medium-high interactive LRR-RKs
  • a centrality score in the third quartile represents medium-low interactive LRR-RKs
  • a centrality score in the fourth quartile represents low interactive LRR-RKs.
  • said receptor kinase interaction assay is selected from the group consisting of extracellular interactome assay (ECIA), avidity-based extracellular interaction screening (AVEXIS), yeast two hybrid assay (Y2H), co-immunoprecipitation assays with and without crosslinking, bimolecular fluorescence complementation, label transfer assays, and phage display.
  • said receptor kinase interaction assay is selected from the group consisting of an extracellular interactome assay and avidity- based extracellular interaction screening. Even more preferably, said receptor kinase interaction assay is ECIA.
  • said community detection algorithm is selected from the group consisting of WalkTrap, Fastgreedy, Infomap, Leading Eigenvector, Label Propagation, Multilevel, Markov Clustering and Edge Betweenness.
  • said community detection algorithm is a WalkTrap algorithm.
  • said algorithm using centrality measures to identify said level of connectivity is selected from the group consisting of PageRank, Degree Centrality, Subgraph Centrality, Katz Centrality, Closeness Centrality, Betweenness Centrality, Eccentricity Centrality, Dynamical Importance and Authority Score.
  • said algorithm using centrality measures is a PageRank algorithm.
  • a screening assay for the identification of the level of connectivity of leucine-rich repeat receptor kinases (LRR-RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • ECIA extracellular interactome assay
  • HCIs high-confidence bidirectional interactions
  • CSI-LRR operates as a unified regulatory network in which the LRR-RKs most critical for its overall structure are required to prevent aberrant signaling of receptors that are several network-steps away.
  • plants have evolved LRR-RK networks to process extracellular signals into carefully balanced response.
  • a screening assay for the identification of interactive leucine-rich repeat receptor kinases (LRR-RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • ECIA extracellular interactome assay
  • HCIs high-confidence bidirectional interactions
  • a PageRank algorithm to identify said interactive LRR-RKs, wherein a PageRank score in the first quartile represents highly interactive LRR-RKs, a PageRank score in the second quartile represents medium-high interactive LRR- RKs, a PageRank score in the third quartile represents medium-low interactive LRR- RKs and a PageRank score in the fourth quartile represents low interactive LRR-RKs.
  • the first quartile comprises the top 25% PageRank scores in the set of PageRank scores identified by the method provided herein. Specifically, the highest PageRank score represents a 100% score, the lowest PageRank score represents a 0% score. Specifically, the second quartile comprises the 50 to 75% PageRank scores and the third quartile comprises the 25 to 50% scores. Specifically, the fourth quartile comprises PageRank scores from 0 to 25%.
  • the extracellular interactome assay is performed as previously described by Ozkan et al 3 . Specifically, said ECIA is performed with slight modifications. Specifically, said ECIA is applied to perform an all-by-all screen of the extracellular domains (ECDs) of cell surface receptors of a given plant species.
  • the ECIA screen may comprise all known ECDs of a given plant species. Specifically, at least 50, 60, 65, 70, 75, 80, 85, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 % of all known ECDS of a given plant species are subjected to the ECIA screen.
  • the ECIA screen results in a CSI-LRR map comprising a plethora of bidirectional interactions of LRR-RKs, of which the high-confidence interactions have to be identified.
  • bidirectional interactions are interactions that give a positive test result in both orientations.
  • LRR-RK-A is subjected to the ECIA to test whether it interacts with LRR-RK-B.
  • a positive bidirectional interaction is achieved if LRR-RK-A gives a positive interaction result with LRR-RK-B and LRR-RK- B gives a positive interaction result with LRR-RK-A.
  • the CSI-LRR does not include interaction pairs that tested positive in only one orientation as these are considered of the lowest confidence.
  • the modified Z-score used herein is (i) nonparametric and makes minimal distributional assumptions, (ii) minimizes measurement bias due to positional effects during the screening procedure and (iii) is resistant to statistical outliers.
  • the geometric mean modified Z-score of the interaction is calculated as measured in the bait-prey and prey-bait orientations. Specifically, any value, for which the geometric mean product of the Z-scores is greater than at least 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5 is considered significant for the purposes of detecting the interactions of the network construction.
  • the re-test screen is an ECIA.
  • the corresponding intracellular domains are tested for interactions using other methods known in the art such as yeast-two hybrid assay (Y2H) or co-immunoprecipitation assay.
  • the WalkTrap algorithm is used to identify subnetworks in the CSI-LRR network.
  • a subnetwork is a group of densely connected LRR-RKs with few connections to LRR-RKs outside of said group.
  • a subnetwork is characterized by a higher level of interconnectedness when compared to the rest of the CSI-LRR network, wherein the interconnectedness is characterized by the number of bidirectional interactions. Specifically, the level of interconnectedness is increased, when the number of interactions is increased by at least, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5-fold, or 2-fold.
  • a WalkTrap algorithm is a community detection operation, wherein groups of densely connected LRR-RKs with few connections to LRR-RKs outside of the group are detected. Specifically, said groups represent subnetworks.
  • the PageRank algorithm defines the number and quality of connections to an LRR-RK, hence establishing its importance and assigning a score value to it.
  • the PageRank score identified by the PageRank algorithm is used to determine the level of connectivity of an LRR-RK.
  • the method provided herein further comprises the step of identifying one or more articulation points (AP), which AP is an LRR-RK in a subnetwork, the removal of which disconnects the subnetwork from the network.
  • AP articulation points
  • an AP is an LRR-RK in a subnetwork, the removal of which disconnects at least two subnetworks from the network.
  • the LRR-RK the removal of which results in the disconnection of the most subnetworks, is considered the most important AP for network integrity.
  • a recombinant plant cell with improved stress resistance heterologously expressing an LRR-RK obtained by the screening assay provided herein.
  • said plant cell is selected from the group consisting of Oryza sp., Triticum sp., Glycine sp., Solanum sp., Saccharum sp., Zea mays sp., Coffea sp., Vicia sp., Phaseolus sp., Vigna sp., Arachis sp., Malus sp., Lactuca sp., Theobroma sp., Helianthus sp. and Daucus sp.
  • said plant cell is selected from the group consisting of oryza sativa, oryza glaberrima, triticum aestivum, glycine soja, glycine max, glycine canescens, glycine tomentella, solanum lycopersicum, solanum tuberosum, solanum melongena, saccharum barberi, saccharum officinarum, saccharum edule, saccharum ravennae, saccharum spontaneum, zea mays amylacea, zea mays everta, zea mays indentata, zea mays indurata, zea mays saccharata, zea mays rugosa, zea mays ceratina, zea mays tunicate, coffea canephora, coffea arabica, malus pumila, lactuca sativa, theobroma cacao, helianthus annuus, he
  • the recombinant plant cell has improved biomass, yield, growth rate, biotic or abiotic stress resistance.
  • the recombinant plant cell provided herein is of the Solanum
  • Lycopersicum origin and the heterologously expressed LRR-RK is selected from the group consisting of APEX, AT1 G14390, AT5G51560, PXC2, NIK3, AT2G02780, BIR4, SRF7, AT 1 G64210, RLK7/KUK2, AT3G47580, AT5G24100, BRL1 , AT2G28960, AT1 G49100, PRK4, AT5G49770 and NIK1 and orthologues thereof.
  • orthologues are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologues retain the same function in the course of evolution. Preferably, orthologues share at least 40, 50, 60, 65, 70, 75, 80, 85, 90 or 95 % sequence identity in two species. Preferably, orthologues share the same biological function, i.e. they are functional orthologues.
  • orthologues of APEX are selected from the group consisting of Solyc05g056370, Solyd 1 g008960, Solyc03g078520, KHN16920.1 glycine soja and KRH40996.1 glycine max.
  • orthologues of AT5G51560 are selected from the group consisting of Solyc01 g091230, Solyc08g079460, and Solyc03g083510.
  • heterologous gene expressing the LRR-RK is derived from Arabidopsis thaliana.
  • the heterologous gene expressing the LRR-RK is derived from one or more species selected from the group consisting of Arabidopsis arenicola, Arabidopsis arenosa, subspecies arenosa and borbasii, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis halleri, subspecies halleri, ovirensis and gemmifera, Arabidopsis lyrata, subspecies lyrata, petraea and kamchatica, Arabidopsis neglecta, Arabidopsis pedemontana, Arabidopsis suecica and Arabidopsis thaliana.
  • a recombinant Arabidopsis sp. comprising a modification in its apex gene (SEQ ID NO. 1 , AT5G63710) is provided.
  • the species comprising a modification in its apex gene SEQ ID NO.
  • Arabidopsis arenicola is selected from the group consisting of Arabidopsis arenicola, Arabidopsis arenosa, subspecies arenosa and borbasii, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis halleri, subspecies halleri, ovirensis and gemmifera, Arabidopsis lyrata, subspecies lyrata, petraea and kamchatica, Arabidopsis neglecta, Arabidopsis pedemontana, Arabidopsis suecica and Arabidopsis thaliana.
  • said modification is the deletion of the apex gene (SEQ ID NO. 1 ).
  • said modification comprises introducing one or more mutations into the apex gene (SEQ ID NO. 1 ).
  • said gene is modified by site-directed mutagenesis, thereby introducing one or more point mutations, nucleotide insertions, deletions or substitutions.
  • CRISPR/Cas9 multiplex automated genome engineering (MAGE) using l-red recombination, conjugative assembly genome engineering (CAGE), the Argonaute protein family (Ago) or a derivative thereof, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, tyrosine/serine site- specific recombinases (Tyr/Ser SSRs), hybridizing molecules, sulfurylases,
  • RNA polymerases recombinases, nucleases, DNA polymerases, RNA polymerases or TNases.
  • mutation of the apex gene is a loss-of-function mutation.
  • a recombinant Arabidopsis sp. characterized by overexpression of the APEX protein is provided.
  • overexpression indicates an increase in the level of active APEX protein present in the cell or plant in comparison with a level of APEX expression in a wild type plant. Such an increase is typically of about 2-fold, 3-fold, 4-fold, 5-fold, 10- fold, 100-fold, or more.
  • said overexpression is induced by deletion, insertion and/or substitution of one or more nucleotides of the APEX gene or its regulatory elements, site-specific mutagenesis, ethyl methanesulfonate (EMS) mutagenesis or targeting induced local lesions in genomes (TILLING); induction of the APEX gene promoter; insertion of heterologous promoter; or introduction into said plant of an expression cassette comprising a nucleic acid sequence coding for apex or its orthologue.
  • EMS ethyl methanesulfonate
  • a recombinant Solarium According to a further specific embodiment, a recombinant Solarium
  • Lycopersicum characterized by expression of the Arabidopsis thaliana APEX gene (SEQ ID NO. 1 ) is provided. Specifically, said recombinant organism is generated using molecular cloning. According to a further specific embodiment, a recombinant plant cell, comprising at least one modification in at least one of its articulation points identified by the screening assay provided herein, is provided.
  • said at least one modification improves the recombinant plant cell’s biomass, yield, growth rate, stress resistance or pathogen resistance.
  • a plant cell’s biomass or yield is improved if the generation of biomass or yield is increased by at least 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 %.
  • a plant cell’s growth rate increased by at least 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 % compared to non-modified plants of the same species is considered improved.
  • a plant cell’s stress resistance is considered improved if said plant’s resistance to drought or nutrient deprivation is improved.
  • pathogen resistance refers to viral, fungal and bacterial resistance.
  • the method for producing a plant having increased resistance to pathogens thus comprises the following steps:
  • nucleic acid construct comprising a nucleic acid sequence encoding an AP, or an orthologue thereof, under the control of a promoter, preferably constitutive promoter, enabling the expression of said nucleic acid sequence;
  • step (b) optionally, selection of plant cells of step (b) which express the AP, or an orthologue thereof;
  • Figure 1 CSI-LRR interaction map and functional validation a, Interaction heat map organized by phylogenetic subgroups pf LRR-RKs (roman numeral, XIV and XV are merged) 5 b, Hypocotyl length ratios of seedlings grown in the presence (T) or absence (NT) of 500 nM brassinolide (BL). c, flg22-induced SGI.
  • Wild-type (WT), mutant lines targeting the HCI (top) and LCI (bottom) partners for BRI1 and FLS2 are indicated on the bottom and ordered by decreasing interaction score from left to right; Dots represent individual observations from six independent experiments; Box plots display the 1 st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling. The symbols on top of the boxes indicate the results of a post hoc unpaired f-test corrected with the Holm method for multiple testing (ns (not significant), ** P ⁇ 0.01 ).
  • Figure 3 Table of LRR-RK subnetworks defined by the WalkTrap algorithm.
  • FIG. 4 APEX interacts with PEPR1 and PEPR2 to regulate danger peptide signaling a-b, Nicotiana benthamiana leaves expressing FLAG-tagged variants of PEPR1/2 either alone or together with a YFP-tagged APEX were treated with water (-) or Pep2 (+).
  • Western blot analyses of PEPR1 /2-APEX co-)immunoprecipitations (Co- IP/IP).
  • anti-FLAG and anti-YFP antibodies were used to analyse lysates. These experiments were repeated three times with similar results c, Pep2-induced oxidative bursts represented as total photon counts over 40 mins. Genetic backgrounds are indicated on the bottom. Dots represent individual observations from three
  • whiskers extend to include the max/min values. Statistical significance was determined by linear mixed effect.
  • a-b Box plots display the 1 st and 3rd quartiles, split by the median (line); whiskers extend to include the max/min values. Statistical significance was determined by linear mixed effect modelling.
  • CBB Colloidal brilliant blue
  • Figure 7 SEQ ID NOs of APEX and FIR. The nucleotide sequences and the amino acid sequences of APEX and FIR are shown.
  • Figure 8 SEQ ID NOs of APEX, AT1 G14390, AT5G51560, PXC2, NIK3, AT2G02780, BIR4, SRF7, AT1 G64210, RLK7/KUK2, AT3G47580, AT5G24100, BRL1 , AT2G28960, AT1 G49100, PRK4, AT5G49770 and NIK1.
  • the nucleotide sequences and the amino acid sequences are shown.
  • LRR-RKs extracellular leucine-rich repeat receptor kinases
  • LRR-RKs are typically characterized by a series of extracellular LRRs (eLRRs), a single transmembrane pass and, in most cases, a functional intracellular kinase domain.
  • the kinase domains of LRR-RKs can be further divided into RD and non-RD families, based on the presence or absence of an arginine located prior to a catalytic aspartate residue.
  • Non-RD kinases lack the strong autophosphorylation activities of RD kinases and display lower enzymatic activities.
  • the eLRRs control the assembly of signaling competent LRR RK complexes by conferring ligand specificity and recruiting appropriate signaling partners.
  • LRR-RKs Analysis of LRR numbers demonstrates a bimodal distribution that separates the LRR- RKs into two groups with a median number of LRRs of 6 and 22, respectively.
  • the two types of LRR-RKs are termed short and long LRR-RKs, respectively.
  • node as used herein, is meant to refer to a LRR-RK within the CSI- LRR network.
  • CSI-LRR Leucine-rich repeat cell surface interaction
  • CSI LRR CSI LRR
  • a CSI-LRR network refers to a network of interactions between LRR-RKs in plant species.
  • Extracellular domains (ECDs) of LRR-RKs serve as interaction platforms, and as regulatory modules of receptor activation. Identification of such a network of interactions previously proved difficult due to the low biochemical tractability the interactions between ECDs and due to the massive expansion of receptor families in plants. Only with the highly sensitive high-throughput interaction assay provided herein was it possible to identify reactive LRR-RKs and uncover their complex interactions within the CSI-LRR network.
  • CSI-LRR operates as a unified regulatory network in which the LRR-RKs most critical for its overall structure are required to prevent aberrant signaling of receptors that are several network-steps away. It could therefore, surprisingly, be shown that plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses and that these networks including their subnetworks can be uncovered by the highly sensitive high-throughput interaction assay provided herein.
  • the term“level of connectivity” refers to the number and quality of bidirectional interactions of a given LRR-RK.
  • the level of connectivity can be defined as the number of pair-wise interactions an LRR-RK established with other LRR-RKs.
  • the quality of bidirectional interactions is characterized by the identification of a bidirectional interaction in an extracellular interactome assay and subsequent confirmation as a high confidence interaction.
  • the quality of a bidirectional interaction of LRR-RKs is higher, the higher the scores in the ECIA and the greater the modified Z-score.
  • the higher the PageRank score the higher the level of connectivity.
  • the higher the level of connectivity the higher the possibility for a certain LRR-RK to be an articulation point, i.e. being essential for the connection of a subnetwork to the network.
  • the term“receptor kinase interaction assay” refers to an assay, which identifies extracellular and/or intracellular pairwise interaction between receptors. Specifically, said assay identifies interactions between receptor kinases in plants, specifically between plant LRR-RKs. Specifically, the receptor kinase interaction assay is selected from the group of techniques for detecting protein-protein interactions. This group includes but is not limited to methods such as extracellular interactome assay (ECIA), avidity-based extracellular interaction screen (AVEXIS), Yeast Two Hybrid assays (Y2H), Co-immunoprecipitation assays with and without crosslinking, Bimolecular fluorescence complementation, label transfer assays, and phage displays.
  • ECIA extracellular interactome assay
  • AVEXIS avidity-based extracellular interaction screen
  • Y2H Yeast Two Hybrid assays
  • Co-immunoprecipitation assays with and without crosslinking Bimolecular flu
  • said receptor kinase interaction assay is an extracellular interactome assay.
  • a centrality score assigns relative scores to all nodes in a network based on the concept that connections to high-scoring nodes contribute more to the score of the node in question than equal connections to low-scoring nodes.
  • extracellular interactome assay or“ECIA” refer to a high-throughput assay comprised of the following components.
  • Bait and prey proteins which are LRR- RKs of the plant species to be investigated. Bait proteins are fused with human dimeric Fc, which allows for easy capture, and immobilized on plates preferably coated with Protein A.
  • Prey proteins are oligomerized into pentamers by fusing the ECDs to a pentameric helical region of rat COMP (cartilage oligomeric matrix protein). This avidity enhancement is a powerful means of capturing low affinity interactions and also recovering interacting pairs when the prey is only expressed at low protein
  • Prey proteins are further fused with human placental alkaline phosphatase (AP) for facile enzymatic detection using colorigenic substrate.
  • AP placental alkaline phosphatase
  • the sensitivity in detection is amplified by the pentamerization of the AP tag.
  • the term“avidity-based extracellular interaction screen” or“AVEXIS” refers to an assay that enables the detection of very weak protein interactions with a low false positive rate.
  • the assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format. It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions.
  • the recombinant protein libraries are produced using a convenient and high-level mammalian expression system, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added.
  • Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (b-lactamase) prey.
  • the bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA.
  • the pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected.
  • COMP cartilage oligomeric matrix protein
  • extracellular pairwise interactions of LRR-RKs synonymously used with“pairwise LRR-RK interactions” or“bidirectional interactions of LRR-RKs” or simply“pairwise interactions” or“bidirectional interactions”, refers to the positive bidirectional interactions identified by any of the receptor kinase interaction assays described herein, specifically by the ECIA.
  • ECIA plates are incubated with AP substrate and AP activity is assessed by measuring the absorbance at 650 nm.
  • Each LRR-RK is subjected to the ECIA once as bait and once as prey protein for each interaction tested. Only those interactions where the LRR-RKs gave positive results as bait and prey are considered positive.
  • the term“high-confidence interaction” or“HCI” refers to bidirectional
  • HCI interactions are also subjected to a re-test screen and/or a yeast-two hybrid screen (Y2H).
  • the re-test screen is a repeat of the ECIA, wherein potential HCI interactions are compared to a positive reference set and, optionally, a negative reference set.
  • potential HCIs are validated on the intracellular level.
  • Intracellular domains corresponding to the extracellular domains of potential HCIs are tested for bidirectional interactions.
  • Z-score is a measure of how many standard deviations below or above the population mean a rawscore is.
  • the rawscores referred to herein are the absorbance values obtained by the ECIA. The complete set of absorbance values for each pairwise interaction was combined into a data matrix. To make measurements comparable across plates and eliminate any bias in the data arising from the
  • the MAD is the median of the absolute values of the residuals (deviations) from the data’s median.
  • the MAD is used for the calculation of modified Z-scores for each individual interaction measured.
  • the modified Z-score used here is (i) nonparametric and makes minimal distributional assumptions, (ii) minimizes measurement bias due to positional effects and (iii) is resistant to statistical outliers.
  • the modified Z-score usually excludes control measurements altogether under the assumption that most interactions in a screen such as CSI LRR would be unproductive and thus serve as controls.
  • each 96-well plate contains two mock prey negative control wells and one well with the positive control interaction pair BAK1 -BIR4 31 .
  • the geometric mean modified Z-score of the interaction is calculated. Any value for which the geometric mean product of the Z-scores is greater than or equal to at least 1 .0, 1.5, 2.0, 2.5, 3.0 or 3.5 is considered significant for the purposes of network construction.
  • the term“community detection algorithm” refers to network analysis algorithms, such as WalkTrap, Fastgreedy, Infomap, Leading Eigenvector, Label Propagation, Multilevel, Markov Clustering and Edge Betweenness algorithm, which can be used for community, i.e. subnetwork, identification.
  • the WalkTrap algorithm is applied for subnetwork detection.
  • the term“WalkTrap algorithm” refers to a community detection operation, wherein groups of densely connected LRR-RKs with few connections to LRR-RKs outside of the group are detected. Specifically, said groups represent subnetworks.
  • “Walktrap” refers to a method that uses random walks based on the assumption that a random walk will likely get trapped in a highly interconnected community of LRR-RKs.
  • subnetwork refers to a region within the CSI-LRR network which is characterized by a higher level of interconnectedness when compared to the rest of the CSI-LRR network, wherein the level of interconnectedness is characterized by the number of interactions.
  • the subnetworks are clusters of interacting proteins in the network.
  • a subnetwork is a collection of interactions within the entire network, which are connected to the rest of the network by a lower number of interactions compared to the number of interactions within the subnetwork. Determination of such subnetworks is essential for the identification of Articulation Points since an Articulation Point is defined as an LRR-RK the removal of which disconnects the subnetwork from the network.
  • a subnetwork comprises a higher level of interconnectedness when the number of interactions is increased by at least, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
  • centrality measure is a measure of the influence of a node in a network. It assigns relative scores to all nodes in the network based on the concept that connections to high-scoring nodes contribute more to the score of the node in question than equal connections to low-scoring nodes.
  • said algorithm using centrality measures is selected from the group consisting of PageRank, Degree Centrality, Subgraph Centrality, Katz Centrality, Closeness Centrality, Betweenness Centrality, Eccentricity Centrality, Dynamical Importance and Authority Score algorithm.
  • PageRank algorithm refers to an algorithm that operates by counting the number and quality of links to a node hence establishing its importance and assigning a‘weight’ value to it. In simpler terms, PageRank measure node connectivity via the number of connections to other nodes.
  • PageRank algorithm is an example of link-analysis algorithms which are iterative and interactive data-analysis techniques and operate with the underlying assumption that nodes with higher scores are likely to be more connected to other nodes when compared to nodes with lower scores.
  • the PageRank algorithm outputs a probability distribution used to represent the likelihood that a node will be linked to a particular node.
  • PageRank can be calculated for collections of values of any size. It is assumed in several research papers that the distribution is evenly divided among all values in the collection at the beginning of the computational process.
  • the PageRank computations require several passes, called “iterations”, through the collection to adjust approximate PageRank score values, also called PageRank scores, to more closely reflect the theoretical true value.
  • a probability is expressed as a numeric score value between 0 and 1.
  • a 0.5 probability is commonly expressed as a "50% chance" of something happening.
  • a PageRank of 0.5 means there is a 50% chance that a node will be linked to the node with the 0.5 PageRank.
  • the term“quartiles” refers to the four equal groups of a data set, each comprising a quarter of the data.
  • the highest PageRank score represents a 100% score, whereas the lowest PageRank score represents a 0% score.
  • the first quartile comprises the top 25% PageRank scores in the set of PageRank scores identified by the method provided herein.
  • the second quartile comprises the 50 to 75% PageRank scores and the third quartile comprises the 25 to 50% scores.
  • the fourth quartile comprises PageRank scores from 0 to 25%.
  • a PageRank score in the first quartile represents highly interactive LRR-RKs
  • a PageRank score in the second quartile represents medium-high interactive LRR-RKs
  • a PageRank score in the third quartile represents medium-low interactive LRR-RKs
  • a PageRank score in the fourth quartile represents low interactive LRR-RKs.
  • AP refers to an LRR-RK the removal of which disconnects a subnetwork from the network. Therefore, APs are nodes whose removal results in the formation of at least two disconnected subnetworks. The AP most important for network integrity is the one whose removal results in the loss of the most nodes from the core structure of the CSI-LRR. The chances for highly interactive LRR- RKs to be an Articulation Point are higher compared to medium-high, medium-low and low interactive LRR-RKs, but not all highly interactive LRR-RKs are APs. Surprisingly, APs are also identified among medium-high and medium-low interactive LRR-RKs. While no APs were identified among low interactive LRR-RKs, this does not
  • LRR-RKs could never serve as AP.
  • APs are highly likely to play an integral role in plant development and survival. APs most important for network integrity are even more likely to do so.
  • recombinant plant cells refers to any plant cell comprising foreign DNA, which was introduced into the plant cell by molecular cloning methods known in the art.
  • said foreign DNA is an Articulation Point identified by the method provided herein.
  • heterologous expression refers to the expression of a gene or part of a gene in a host organism, which does not naturally have this gene or gene fragment. Insertion of the gene in the heterologous host is performed by recombinant DNA technology.
  • the methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, microorganism-mediated gene transfer such as Agrobacterium mediated transformation, microprojectile-mediated transformation, electroporation, micro-injection, direct gene transfer, sonication, liposome or spheroplast fusions, protoplast transformation, macroinjection, DNA uptake by germinating pollen and DNA uptake in embryos by swelling, and biolistic bombardment.
  • the DNA introduced is integrated into the genome of the plant cell, it is generally considered stable and is also retained in the progeny of the originally transformed cell. It can contain a selection marker which mediates, for example, resistance to a biocide such as phosphinothricin or an antibiotic such as kanamycin, G 418, bleomycin or hygromycin, to the transformed plant cells or which permits selection via the presence or absence of certain sugars or amino acids.
  • the marker chosen should therefore allow the selection of transformed cells over cells which lack the DNA introduced.
  • these cells can be used to regenerate transgenic plants in any manner available to the art. Seeds may be obtained from the plant cells. Two or more generations can be grown in order to ensure that the phenotype characteristic is stably retained and inherited. Also, seeds can be harvested in order to ensure that the phenotype in question or other characteristics have been retained.
  • the term“recombinant plant cell” refers to plant cells comprising genetic modifications.
  • Such modifications comprise deletions of a gene and introducing one or more mutations into a gene.
  • said gene is an Articulation Point identified by the method provided herein.
  • genes are modified by site-directed or random mutagenesis, thereby introducing one or more point mutations which are any of nucleotide insertions, deletions or substitutions.
  • Site-directed mutagenesis is typically performed by enzymatic modification, employing any one or more of methyltransferases, kinases, CRISPR/Cas9, multiplex automated genome engineering (MAGE) using l-red recombination, conjugative assembly genome engineering (CAGE), the Argonaute protein family (Ago) or a derivative thereof, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, tyrosine/serine site- specific recombinases (Tyr/Ser SSRs), hybridizing molecules, sulfurylases,
  • RNA polymerases recombinases, nucleases, DNA polymerases, RNA polymerases or TNases.
  • overexpressed or overexpression indicates an increase in the level of mRNA that should translate in an increase in active protein levels present in the cell or plant in comparison with the level of mRNA/protein expression in the wild type plant. Such an increase is typically of about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10- fold, 100-fold, or more.
  • overexpression also designates a modified expression profile of a protein or its orthologues, such as a constitutive expression.
  • Overexpression of an Articulation Point or orthologues thereof may be obtained by techniques known per se in the art such as, without limitation, by genetic means, enzymatic techniques, chemical methods, or combinations thereof. Overexpression may be conducted at the level of DNA, mRNA or protein, and induce the expression (e.g., transcription or translation) or the activity of the Articulation Point. A preferred overexpression method affects expression and leads to the increased production of a functional LRR-RK in the cells. It should be noted that the induction may be transient or permanent. Overexpression of an AP or its orthologues may be induced by any mutation in the corresponding gene or its orthologues, for example point mutation, deletion, insertion and/or substitution of one or more nucleotides in a DNA sequence.
  • EMS ethyl methanesulfonate
  • TILLING targeting induced local lesions in genomes
  • a particular approach is gene overexpression by insertion of a DNA sequence through transposon mutagenesis using mobile genetic elements called transposons, which may be of natural or artificial origin.
  • DNA mutations may also be introduced within the gene or its orthologues, or within a sequence of the promoter of the AP gene or its orthologues. Mutations in the coding sequence may result in gain of function by increasing biological activity and efficacy of the AP protein. Alternatively, introducing mutations into the promoter sequence may result in a gain or loss of function by either induction or reduction of the promoter activity by controlling and enhancing or reducing the transcription of the AP gene.
  • Overexpression of an AP may also be induced by introduction into a plant an expression cassette comprising a nucleic acid sequence coding for an AP or its orthologue under control of a promoter enabling the expression of said nucleic acid sequence.
  • AP overexpression may also be performed transiently, e.g., by applying (e.g., spraying) an exogenous agent to the plant, for example molecules that induce AP expression or activity.
  • Preferred overexpression is a constitutive expression under control of a constitutive promoter. Such constitutive expression, leads to a drastic increase in the expression of an active AP protein in the plant, while the plant is still viable.
  • the promoter is a heterologous promoter functional in plant cells.
  • Useful promoters include, but are not limited to, constitutive, inducible, temporally regulated, developmental ⁇ regulated, spatially-regulated, chemically regulated, stress- responsive, tissue-specific, viral and synthetic promoters.
  • An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus.
  • An isolated promoter sequence that is a strong promoter for heterologous nucleic acid is advantageous because it provides for a sufficient level of gene expression to allow for easy detection and selection of transformed cells and provides for a high level of gene expression when desired.
  • promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. In some cases, expression in multiple tissues is desirable. While in others, tissue-specific, e.g., leaf-specific, seed-specific, petal- specific, anther-specific, or pith- specific, expression is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally
  • dicotyledonous promoters are selected for expression in dicotyledons.
  • monocotyledonous promoters for expression in monocotyledons. There is, however, no restriction to the origin or source of a selected promoter. It is sufficient that the promoters are operational in driving the expression of a desired nucleotide sequence in the particular cell. Further examples of such promoters include, but are not limited to, constitutive promoters, plant tissue-specific promoters, plant development-stage- specific promoters, inducible promoters, viral promoters as well as synthetic or other natural promoters. Specifically, the promoter is a constitutive or an inducible promoter.
  • Constitutive promoters may include, for example CaMV 35S promoter (Odell et al. (1985) Nature 313, 9810-812), rice actin promoter (McElroy et al. (1990) Plant Cell 2: 163-171 ) and ubiquitin promoter (Christian et al. (1989) Plant Mol. Biol. 18 (675- 689); pEMU (Last et al. (1991 ) Theor Appl. Genet. 81 : 581 -588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. Application Seriel No.
  • a nucleic acid molecule may be introduced into a plant cell by any means, including transfection, transformation, transduction, electroporation, particle
  • the introduced nucleic acid molecule may be maintained in the plant cell stably.
  • the introduced nucleic acid molecule may be transiently expressed or transiently active. Selection of a plant which overexpresses the AP can be made by techniques known in the art, such as, but not limited toPCR, hybridization, use of a selectable marker gene, protein dosing, western blot, etc.).
  • APEX function is herein understood the ability of APEX to function as an Articulation Point.
  • Articulation Points are LRR-RKs central to the integrity of a subnetwork of a plant’s CSI-LRR. Since a plant’s CSI-LRR is integral for the plant’s response to environmental changes or threats, such as droughts and exposure to pathogens, modification of APs in the CSI-LRR modify the plant’s response to such environmental changes or threats. Therefore, modifications of APs can improve a plant’s stress resistance.
  • modifications in the AP can improve the recombinant plant’s or plant cell’s biomass, yield, growth rate, stress resistance or pathogen resistance.
  • a plant’s or plant cell’s biomass or yield is considered improved if the generation of biomass or yield is increased by at least 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 %.
  • a plant or plant cell’s growth rate increased by at least 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 % compared to non- modified plants of the same species is considered improved.
  • a plant cell’s stress resistance is considered improved if said plant’s resistance to heat, pest infestation, drought or nutrient deprivation is improved.
  • pathogen resistance refers to fungal, viral, and bacterial resistance.
  • biomass refers to material derived from recently living organisms. This includes plants, animals and their by-products. For example, manure, garden waste and crop residues are all sources of biomass. It is a renewable energy source based on the carbon cycle.
  • APEX is the term used to refer to a small LRR-RK with the locus name
  • AT5G63710 in Arabidopsis thaliana It was identified as highly important Articulation Point for network integrity of the CSI-LRR of Arabidopsis thaliana by the screening assay provided herein.
  • Deletion of APEX affects the functions of BRI1 and FLS2, two receptors that reside multiple network steps away from APEX. Elimination of APEX has destabilizing effects in otherwise well-balanced LRR-RK signaling pathways.
  • Nucleotide sequence of apex is provided in SEQ ID NO 1
  • amino acid sequence of APEX is provided as SEQ ID NO 3.
  • AT5G51560 PXC2 (At5g01890), NIK3 (At1 g60800), AT2G02780, BIR4 (At1 g69990), SRF7 (At3G14350), AT1 G64210, RLK7/KUK2 (At1 g09970), AT3G47580, AT5G24100, BRL1 (At1 g55610), AT2G28960, AT1 G49100, PRK4 (At3g20190), AT5G49770 and NIK1 (At5g16000).
  • orthologous genes are homologous genes that diverged after evolution gives rise to different species, an event known as speciation.
  • orthologues maintain a similar function to that of the ancestral gene that they evolved from.
  • the ancestral gene and its function is maintained through a speciation event, though variations may arise within the gene after the point in which the species diverged.
  • Alkaopsis spJ refers to the genus Arabidopsis, which can be grouped into four common species ⁇ A. thaliana, A. halleri, A. lyrata and A. arenosa ), three species with limited geographic distribution (A. croatica, A. cebennensis and A. pedemontana) and two allotetraploid species ⁇ A. suecica and A. kamchatica).
  • Arabidopsis thaliana is the species which is most commonly used in plant biology.
  • Lycopersicon there are currently 13 species recognized in the Solanum section Lycopersicon. Three of these species— S. cheesmaniae, galapagense, and pimpinellifolium— are fully cross compatible with domestic tomato. Four more species— S. chmielewskii, S. habrochaites, S. neorickii, and S. pennelli— can be readily crossed with domestic tomato, with some limitations.
  • the Lycopersicon section has not been fully sampled within wild species in the South American range, so new species may be added in the future.
  • the present invention also refers to following items:
  • a screening assay for the identification of the level of connectivity of leucine-rich repeat receptor kinases (LRR-RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • a screening assay for the identification of interactive leucine-rich repeat receptor kinases (LRR-RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • a centrality score of the algorithm in the first quartile represents highly interactive LRR-RKs
  • a centrality score in the second quartile represents medium-high interactive LRR-RKs
  • a centrality score in the third quartile represents medium-low interactive LRR-RKs
  • a centrality score in the fourth quartile represents low interactive LRR-RKs.
  • receptor kinase interaction assay is selected from the group consisting of extracellular interactome assay (ECIA), avidity-based extracellular interaction screening (AVEXIS), yeast two hybrid assay (Y2H), co-immunoprecipitation assays with and without crosslinking, bimolecular fluorescence complementation, label transfer assays, and phage display.
  • ECIA extracellular interactome assay
  • AVEXIS avidity-based extracellular interaction screening
  • Y2H yeast two hybrid assay
  • co-immunoprecipitation assays with and without crosslinking bimolecular fluorescence complementation
  • label transfer assays and phage display.
  • a screening assay for the identification of the level of connectivity of leucine-rich repeat receptor kinases (LRR-RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • HCIs high-confidence bi-directional interactions
  • a screening assay for the identification of interactive leucine-rich repeat receptor kinases (LRR-RK) within a plant Leucine-Rich Repeat Cell Surface Interaction (CSI-LRR) network comprising the sequential steps of
  • HCIs high-confidence bi-directional interactions
  • a PageRank algorithm to identify said interactive LRR-RKs, wherein a PageRank score in the first quartile represents highly interactive LRR-RKs, a PageRank score in the second quartile represents medium-high interactive LRR- RKs, a PageRank score in the third quartile represents medium-low interactive LRR- RKs and a PageRank score in the fourth quartile represents low interactive LRR-RKs.
  • the recombinant plant cell of item 9 wherein the recombinant plant cell is of Solanum Lycopersicum origin and the heterologously expressed LRR-RK is selected from the group consisting of APEX, AT1 G14390, AT5G51560, PXC2, NIK3, AT2G02780, BIR4, SRF7, AT1 G64210, RLK7/KUK2, AT3G47580, AT5G24100, BRL1 , AT2G28960, AT1 G49100, PRK4, AT5G49770 and NIK1 and orthologues thereof.
  • a recombinant Arabidopsis sp. comprising a modification in its apex gene (SEQ ID NO. 1 ). 13. The recombinant Arabidopsis sp. of item 12, wherein said modification is a partial or full deletion of the apex gene (SEQ ID NO. 1 ).
  • a recombinant Arabidopsis sp. characterized by overexpression of the apex gene (SEQ ID NO. 1 ).
  • a recombinant Solarium Lycopersicum characterized by expression of the Arabidopsis thaliana apex gene (SEQ ID NO. 1 ).
  • a recombinant plant cell comprising at least one modification in at least one of its articulation points identified by the screening assay of item 8.
  • Example 1 Identification of a CSI LRR and characterization of level connectivity of identified LRR-RKs
  • the ECDs of 200 LRR-RKs from Arabidopsis were cloned into bait and prey expression vectors for recombinant protein production in Drosophila Schneider S2 cells.
  • Table 1 shows LRR-RKs defined as articulation points in CSI.
  • Table 2 provides the list of pairwise ECD interactions that passed statistical cut offs in the primary screen (high-confidence interactions (HCI) which comprises the data of the CSI LRR map.
  • HCI high-confidence interactions
  • a random reference set was chosen instead, composed of 248 protein pairs that showed no interaction in the primary screen and that have no evidence of interaction in the literature, which was termed the Low Confidence Interaction (LC ⁇ ) negative control set. While it must be noted that it is nearly impossible to construct a set of negative interacting pairs with absolute confidence, this approach allows us to benchmark the validation assay 4,59 . To benchmark the performance of the validation assay used for the retest, all PRS and RRS protein pairs were tested. In the retest, all PRS interactions were successfully confirmed, whereas 12.5% of the LC ⁇ interactions showed evidence of interaction, which is the assay background. Thus, the retest shows a sensitivity of 100% and a specificity of 87.5% (1 -assay background).
  • the confirmation rate of the CSI LRR dataset (92%) is comparable to that of the PRS, and markedly different than that of the LCi, once again demonstrating the high quality of the data.
  • the correlation between the results for the LCi, and HCi sets in both the primary and retest screens were examined too.
  • the Spearman correlation coefficients between the CSI LRR and the retest is 0.769. This high correlation between independent assays performed with independently produced protein samples shows clearly the robustness of the data. Having benchmarked the performance of the validation assay, and demonstrated the reproducibility of the technique, we then assessed the quality of the data contained in CSI LRR .
  • the Y2H screen data of the ICDs was used to attempt to provide further evidence of interaction between LRR-RKs.
  • the challenge of this approach is that no single technique can detect all interactions, leading to imperfect agreement between assays 16 .
  • even lower confirmation rates of the CSILRR dataset were expected, as the interaction pattern of the ECDs may not perfectly match the pattern of interaction between the ICDs in the absence of technical limitations.
  • the measured background was 10% (5 out of 50), although 2 of these interactions detected by Y2H scored positive as unidirectional hits.
  • the 27 T-DNA insertion mutants were at3g46350, at3g14840, at2g01210, At2g27060, at2g01210, peprl, fls2c efr-1, mik1, fei2, nik3, bam3, sr ⁇ 9, rlk, rpk1, rul1, sark, serk4, smk1, sobir1-13 , tmkH, pskrl, bak1-5, srf4, bri1- 301, pepr2, erl2 and hsl2 (Table 3).
  • FLS2-INTERACTING RECEPTOR FLS2-INTERACTING RECEPTOR (FIR, AT2G27060 SEQ ID NO 2), a previously uncharacterized LRR-RK, also interacted with the FLS2 co-receptor BAK1 in CSI LRR , suggesting that FIR may influence the FLS2-BAK1 signaling complex in vivo.
  • FLS2-BAK1 complex formation was reduced upon flg22 treatment in the fir mutant (FIG. 1 D), and this correlated with a reduction in flg22-induced ROS burst and FLG22-INDUCED RECEPTOR KINASE1 (FRK1) gene expression (FIG. 1 E).
  • flg22-induced root growth inhibition was also measured as well as resistance against the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), and it was found that both were significantly reduced in fir mutants.
  • FIR regulates FLS2 signaling and facilitates flg22-induced BAK1 -FLS2 complex formation.
  • Figure 1 shows the functional validation of the CSI-LRR interaction map.
  • FIG. 1 a shows Flypocotyl length ratios of seedlings grown in the presence (T) or absence (NT) of 500 nM brassinolide (BL).
  • Figure 1 b shows flg22-induced SGI.
  • Figures 1 b and c show Wild-type (WT), mutant lines targeting the HCI (top) and LCI (bottom) partners for BRI1 and FLS2 are indicated on the bottom and ordered by decreasing interaction score from left to right; Dots represent individual observations from six independent experiments; Box plots display the 1 st and 3rd quartiles, split by the median; whiskers extend to include the max/min values. Statistical significance was determined using linear mixed effect modelling.
  • Figure 1 c shows Western blot analyses of FLS2-BAK1 co-immunoprecipitations (Co-IP/IP) in seedlings treated with either water (-) or flg22 (+) for 10 min. anti-BAK1 or anti-FLS2 antibodies were used to analyse lysates from the genotypes indicated on the top. This experiment was repeated three times with similar results.
  • LRR- RKs have large (>12 LRR repeats) or small ECDs ( ⁇ 12 LRR repeats), and the sizes are typically associated with roles in ligand perception or regulation, respectively 1 ,21 .
  • the experimental pattern of interactions between these groups was compared to the expected distribution of interactions assuming random binding (FIG. 2).
  • LRR-RKs subnetworks were identified 22 (FIG. 2B), of which at least one is biologically relevant.
  • Network analysis helps de-convolute protein-protein interaction datasets by revealing their underlying modular structure.
  • communities also known as modules or clusters, are elementary units of biological networks that can provide information about local interaction patterns and their contribution to the overall structure and connectivity of a network.
  • the WalkTrap a graph-theoretical algorithm that uses a hierarchical clustering approach 22 , was used to identify functionally relevant subnetworks of LRR-RKs.
  • the WalkTrap identified one small subnetwork composed of 14 members (subnetwork 1 ) and three medium sized subnetworks comprising an average number of ⁇ 30 LRR-RKs
  • Figure 2a shows expected and observed percentages of interactions organized by interaction types and ECD sizes. The expected % were calculated assuming random interaction between observed proteins.
  • Figure 2b shows WalkTrap
  • FIG. 3 shows LRR-RK subnetworks defined by the WalkTrap algorithm.
  • the PageRank algorithm was then used to compare the contributions of small and large ECDs to CSI LRR connectivity 23 (Table 4). Nodes corresponding to small ECDs have significantly higher PageRank values and are thus more essential to the overall connectivity of the network (Fig. 2C). Notably, BAK1 (a small LRR-RK) was measured by PageRank as the most interconnected and important node in CSI LRR .
  • a PageRank score in the first quartile represents highly interactive LRR-RKs
  • a PageRank score in the second quartile represents medium-high interactive LRR-RKs
  • a PageRank score in the third quartile represents medium-low interactive LRR-RKs
  • a PageRank score in the fourth quartile represents low interactive LRR-RKs.
  • Articulation points are nodes whose removal from a network results in the formation of at least two disconnected subnetworks 24 . Removal of AT5G63710
  • APEX another small LRR-RK
  • CSI LRR core structure of CSI LRR
  • apex bak1 -5 double mutant plants were constructed 25 . While apex and bak1-5 single mutant plants were morphologically wild-type, apex bak1-5 double mutants were developmental ⁇ impaired. Thus, network properties defined in silico are relevant in living plants.
  • APEX interacted with PEPR1/2.
  • APEX associates with PEPR1/2 in the context of the full-length receptors.
  • co-immunoprecipitation assays were performed.
  • PEPR1/2 both associated with APEX in plant cells in the presence or absence of Pep2, a peptide ligand for PEPR1/2 26 (FIG. 4A, B).
  • a peptide ligand for PEPR1/2 26 FIG. 4A, B.
  • Figure 4 shows that APEX interacts with PEPR1 and PEPR2 to regulate danger peptide signaling.
  • Figures 4a and b show Nicotiana benthamiana leaves expressing FLAG-tagged variants of PEPR1/2 either alone or together with a YFP-tagged APEX were treated with water (-) or Pep2 (+).
  • Anti-FLAG and anti-YFP antibodies were used to analyse lysates. These experiments were repeated three times with similar results.
  • Figure 4c shows Pep2-induced oxidative bursts represented as total photon counts over 40 mins. Genetic backgrounds are indicated on the bottom. Dots represent individual observations from three independent experiments.
  • BRI1 and FLS2 functions were both altered in apex mutants, as indicated either by the low levels of hypocotyl elongation in response to BRs or by the enhanced flg22-induced ROS burst (FIG. 5A, B ). Notably, these aberrant ligand-induced signaling responses were both BAK1 -dependent (FIG. 5A, B ). Finally, apex mutants showed a striking increase in flg22-induced FLS2-BAK1 complex formation, mitogen-activated protein kinase (MAPK) activation and FRK1 expression (FIG. 5C-E). Thus, elimination of APEX has destabilizing effects in otherwise well- balanced LRR-RK signaling pathways.
  • MAPK mitogen-activated protein kinase
  • Figure 5 shows that CSILRR functions as a unified regulatory network.
  • Figures 5 a and b shows Box plots displaying the 1 st and 3rd quartiles, split by the median (line); whiskers extend to include the max/min values. Statistical significance was determined by linear mixed effect modelling.
  • Figure 5c shows Western blot analyses of FLS2-BAK1 co-immunoprecipitations (Co- I P/I P) in seedlings treated with either water (-) or flg22 (+). anti-BAK1 or anti-FLS2 antibodies were used to analyse lysates from the genotypes indicated on the top. This experiment was repeated three times with similar results.
  • Figure 5d shows flg22- induced activation of MAPKs in the genotypes indicated on top. The phosphorylated MPK3/6 proteins were detected with an antipERK antibody. This experiment was repeated four times with similar results.
  • CBB Colloidal brilliant blue
  • Figure 6 shows the modulation of BR signaling by AT5G51560 as measured by hypocotyl length fold changes. Representative seedlings grown for 7 days in the absence (NT) or presence of 500 nM brassinolide (BL), the most potent
  • LRR-RKs operate in a unified regulatory network governed by the following key guiding tenets: (i) ligand- induced signaling is modulated locally by the presence and/or activities of other LRR- RKs, (ii) small LRR-RKs, in addition to their function as coreceptors, act as regulatory scaffolds and organize their larger counterparts into a signaling network, and (iii) coupling of LRR-RK signaling to the overall stability of the network ensures appropriate response modulation by network-feedback mechanisms, an overlooked determinant of response specificity.
  • each ECD was the identification of the N- and C-terminal cysteine capping consensus motifs (CxxxxC and variations thereof) that borders most of the Arabidopsis ECDs. This was achieved by visual inspection of the primary amino acid sequences. These cysteine-caps are thought to play a role by capping the exposed edges of the hydrophobic core formed by the repetition of the LRRs and produce disulphide bonds that preserve the tertiary protein structure. In our hands, they were important for enhancing ECD solubility and preventing aggregation and proteolysis in vitro. For expression in Drosophila melanogaster Schneider 2 (S2) cells, each ECD was inserted into the pECIA-2 and the pECIA-14 expression vectors (a gift from Christopher K. Garcia) 3 . pECIA2/14 are derivatives of the pMT/BiP/V5 (Invitrogen,
  • V4130-20 which uses a copper-inducible Drosophila metallothionein promoter and have the signal sequence of the Drosophila BiP protein.
  • the ECDs were cloned by Sequence and Ligation Independent Cloning (SLIC) between the existing BiP signal sequence and the C-terminal epitope tags specific to each vector. Sanger sequencing confirmed the presence of each insert. Primers were designed to have a sequence partially homologous to the desired boundaries of the ECDs followed by extensions for RecA-mediated SLIC strategy attached. Amplification was done using Phusion Flash Mastermix (Thermo Scientific) according to the manufacturer’s instructions for 2-step Polymerase Chain Reaction. 176 ECDs out of 200 were cloned from plasmid templates available from the Arabidopsis Biological Resource Center (ABRC) 28 .
  • SLIC Sequence and Ligation Independent Cloning
  • ECDs Twenty-four ECDs were cloned from Arabidopsis seedlings and mature leaves using RTPCR, followed by amplification as described above and by RecA-mediated SLIC cloning. Secreted expression of LRR-RK extracellular domains.
  • Protease inhibitors Sigma and 0.02% NaN3 were added to the medium (ESF 921 , Expression Systems) containing the recombinant ECDs and then stored at 4°C prior to use.
  • the cell supernatant was assessed for recombinant protein expression by western blotting using anti-V5 antibodies (Invitrogen) for the baits or by alkaline phosphatase activity quantification for the preys.
  • Protein-A coated plates were washed in a PBS solution containing 0.1 % Tween-20 prior to use.
  • the bait-coated plates were blocked with the equilibration buffer containing 1 % BSA for 3 hours at 4°C and subsequently washed.
  • the prey proteins fused to the alkaline phosphatase (AP) were then added to the wells and incubated for 2 hours at 4°C and then washed away prior to adding the AP substrate (KPL 50-88-02).
  • AP substrate KPL 50-88-02
  • plates were incubated for 2 hours at room temperature and AP activity monitored by measuring the absorbance at 650 nm using a Synergy H4 Multi-Mode plate reader (BioTek). Images of the 96-well plates were acquired for visual inspection. The complete set of raw absorbance values was combined into a binary dataset using an in-house designed script (Platero v0.1.4), and then subjected to post experimental statistical analysis to remove both false positive and false negative interactions
  • CSI LRR Data Analysis The complete set of absorbance values for each pairwise interaction was combined into a data matrix. To make measurements comparable across plates and eliminate any bias in the data arising from the differential background binding capacities of the baits and preys, a 2-way median polish was used 29,30 . The residuals were then used to calculate the median and median absolute deviation (MAD). The MAD is the median of the absolute values of the residuals (deviations) from the data’s median. The MAD was used for the calculation of modified Z-scores for each individual interaction measured. The modified Z-score used here is (i) nonparametric and makes minimal distributional assumptions, (ii) minimizes measurement bias due to positional effects and (iii) is resistant to statistical outliers.
  • the modified Z-score usually excludes control measurements altogether under the assumption that most interactions in a screen such as CSI LRR would be unproductive and thus serve as controls.
  • each 96-well plate contained two mock prey negative control wells and one well with the positive control interaction pair BAK1 -BIR4 31 .
  • CSI LRR retest screens All of the HCI in CSI LRR and a randomly selected subset of LCF SI were independently retested. Each ECD was newly expressed and all retested interactions were assayed in both bait-prey orientations. For each interaction tested, three prey-only negative control wells were included, to control for non-specific binding. Thus, a total of six negative controls were tested for each bidirectional interaction. One well containing the positive control interaction pair BAK1 -BIR4 was included on each plate. The 2-way median polish and modified Z-scoring system used in the initial screen depends upon large numbers of non-interactions to perform reliably.
  • the absorbance values were paired with the corresponding value from the CSI LRR and subjected to an interquartile range (IQR) normalization step to ensure the two data-sets could be accurately compared.
  • IQR interquartile range
  • the network was constructed using the igraph package in the R programming environment. To identify clusters of interacting proteins in the network, the WalkTrap algorithm was used, which is based on a concept that, if one performs random walks on a network, then the walks are more likely to stay within the densely connected parts of the network, thus
  • PageRank To measure the importance of each node within the network, the PageRank algorithm that operates by counting the number and quality of links to a node hence establishing its importance and assigning a‘weight’ value to it 23 , was applied. In simpler terms, PageRank measure node connectivity via the number of connections to other nodes.
  • PageRank algorithm is an example of link-analysis algorithms which are iterative and interactive data-analysis techniques and operate with the underlying assumption that nodes with higher scores are likely to be more connected to other nodes when compared to nodes with lower scores 23 .
  • articulation points or cut vertices in the network were identified.
  • An articulation point is any node in a unidirectional network the removal of which disconnects the network.
  • yeast two-hybrid assays with LRR-RK intracellular domains The yeast two- hybrid experiment was conducted according to Mukhtar et al 17 with some
  • LRR-RK ICDs cloned in both bait and prey plasmids was used 17 .
  • the ICDs of the LRR-RKs were fused to the GAL4 activation domain using a pDEST-AD-CHY2 vector with a tryptophan selection marker to form the prey constructs and to the GAL4 DNA binding domain using a pDEST-DB vector with a leucine selection marker to form the bait constructs.
  • Target prey and bait constructs were transformed into S. cerevisiae strains Y8800 (MATa) and Y8930 (MATa), respectively. Transformations were confirmed by selecting the haploid yeast strains on their corresponding selective media (SD-T and SD-L).
  • Haploid bait and prey strains were mated in liquid YEPD (yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L, adenine 100 mg/L) media overnight at 30°C.
  • the resulting diploid yeasts were selected in liquid SD-LT media for 48 hours at 30°C. Reconstitution of the GAL4 transcription factor through the interaction of the bait and prey led to the activation of a HIS3 reporter gene and subsequently biosynthesis of histidine. Since the pDEST-AD vector contains the CHY2 (a cycloheximide sensitive gene), any growth on the yeast media containing cycloheximide constitutes a false positive interaction.
  • Equal amounts of diploid yeasts were transferred to solid SD-LTH (positive selection plates) and SD- LH+ cycloheximide (20 mg/L) media ( de novo auto activation plates). Interactions were scored positive if there was growth on positive selection plates, but no growth on de novo auto activation plates. The retest on the random LCI Y2H pairs was performed in similar conditions.
  • HCI BRI1 top genes 1 st Rank: STRUBBELIG-RECEPTOR FAMILY 9 (SRF9) 32 , 2nd Rank: ERECT A-UKE 2 (ERL2) 33 , 3rd Rank: FIR/AT2G27060 (this study), 4th Rank: BAK1 (bakt -4 allele) 25 34 35 , 6th Rank: BARELY ANY MERISTEM 3 (BAM3) 38 , 7th Rank: SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 4 (SERK4) 35 37 , 8th Rank: RECEPTOR-LIKE PROTEIN KINASE 1 (RPK1) 38 , 9th Rank: HAESA-LIKE 2 (HSL2) 39 - 41 .
  • STRUBBELIG-RECEPTOR FAMILY 4 (SRF4) 32 , 200th Rank: TRANSMEMBRANE KINASE LIKE 1 (TMKL1) 48 . The following genes were not tested: 192th Rank:
  • RECEPTOR-LIKE KINASE 902 (RLK902) 49 and 199th Rank: TRANSMEMBRANE KINASE 1 (TMK1) 50 .
  • TMK1 TRANSMEMBRANE KINASE 1
  • ERECTA and 6th Rank ERECTA-LIKE 2 (ERL2) since the er mutant shows altered flg22-induced marker gene expression 33
  • T-DNA lines were not tested: 5th Rank: RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) 42 , 1 1 th Rank: RECEPTOR-LIKE KINASE 1 (RKLlf and 12th Rank: BAK1 INTERACTING RECEPTOR 4 (B!R4 because they were genotyped as wild-type despite their annotation as homozygous for the presence of a T-DNA insert.
  • PHYTOSULFOKINE PEPTIDE RECEPTOR 1 (PSKR1) 54 58 , 191 st Rank: PEPP2 57 , 192nd Rank: AT3G46350, 194th Rank: AT3G14840, 195th Rank: AT2G01210, 196th Rank: PEPR1 57 , 198 th Rank: EE/2 31 , 200th Rank: NSP-INTERACTING KINASE 3 (NIK3) 58 .
  • POX flg22 responses assays were carried out as in Mott et a!., 20 . Briefly, leaf discs were taken from 4-week-old A. thaliana plants. The discs were washed for 1 hour in 1 ml_ of 1 X MS solution with agitation. After washing, discs were transferred to individual wells of a clear 96-well assay plate avoiding the use of the edge wells to minimize evaporation effects. Each well received 50 1 pi of 1 X MS buffer alone, or supplemented with 1 mM of flg22 peptide. Plates were sealed with parafilm and incubated for 20 hours with agitation.
  • the leaf discs were removed and each well received 50 1 pi of a 1 mg/ml_ solution of 5-aminosalicylic acid (A79809, Sigma-Aldrich) pH 6.0 with 0.01 % hydrogen peroxide.
  • the reaction proceeded for 1 -3 min and was stopped by the addition of 20 1 pi 2N NaOH prior to reading the OD600 on a POLARstar OPTIMA microplate reader (BMG Labtech).
  • Genscript NJ, USA.
  • Agrobacterium strains carrying the pB35GWF binary plant expression vector for the expression of the full-length coding regions of PEPR1 (S1 G73080BFF) 28 and PEPR2 (S1 G17750BFF) 28 fused to a C-terminal Flag epitope tag were constructed and used for immunoprecipitation and western blot assays.
  • pDONR-Zeo vector (Life Technologies) containing the cDNA of APEX (N5G63710ZEF) was used for gateway recombination in the binary plant expression vector pEarleyGate101 vector to generate the Cterminal YFP-HA tag fusion vector expressing APEX-YFP-HA under the control of the CaMV35S promoter.
  • the respective sets of agrobacterium strains were mixed 1 :1 and syringe infiltrated into 3-week-old N. benthamiana leaves of plants grown in short-day conditions (12h light/12 h dark).
  • Samples for protein extraction were harvested 3 days after infiltration before flash freezing in liquid nitrogen.
  • beads were washed four times with washing buffer containing 50 mM T ris-HCI pH7.5, 150 mM NaCI, 1 % PMSF, and 0.1 % IPEGAL.
  • washing buffer containing 50 mM T ris-HCI pH7.5, 150 mM NaCI, 1 % PMSF, and 0.1 % IPEGAL.
  • One hundred microliters of 5x SDS Laemmli buffer was added to the beads, and the beads were heated at 95°C for 10 min and subjected for further SDS PAGE and immunoblotting analysis.
  • Plant Cultivation, transgenic plants and mutants The wild-type (WT) used in all experiments was A. thaliana accession Columbia (Col-0). If not specified otherwise the apex-1 allele was used in this work. Plants were grown on soil or vertically on Petri dishes containing 0.5X Murashige and Skoog medium in long-day light conditions (1 6 h light/8 h dark). For Pto DC3000 pathogen assay and callose deposition upon flg22 treatment, plants were grown in short-day conditions (12h light/12 h dark). The mutant plant genotypes used in this work are listed in Table 3.
  • the 35S.APEX-YFP-HA construct was transformed separately into WT plants and more than 20 independent T1 lines were isolated and between three and eight representative mono-insertion lines were selected in the T2 generation.
  • DNA genotyping, epifluorescence microscopy and protein extraction were performed on segregating T2 to obtain homozygous T3 generation lines with maximal expression levels.
  • the double mutant apex-1 bak1 -5 was generated by crosses and genotyped for homozygosity using allele-specific primers for apex-1 and dCAPS marker for bak1-5 as described in 25 . Genotyping was repeated for two consecutive generations and confirmed by Sanger sequencing.
  • Protein Extraction and immunoprecipitation in Arabidopsis Fifteen to twenty seedlings were grown in each well of a 6-well plate for 2 weeks. Subsequently, seedlings were transferred to water and incubated overnight. The next day, flg22 was added at a final concentration of 100 nM and incubated for 10 min. Seedlings were then frozen in liquid nitrogen and subjected to protein isolation.
  • proteins were isolated in 50 mM Tris-HCI pH 7.5, 150 mM NaCI, 10% glycerol, 5 mM dithiothreitol, 1 % protease inhibitor cocktail (Sigma Aldrich), 2 mM Na 2 Mo0 4 , 2.5 mM NaF, 1 .5 mM activated Na 3 V0 4 , 1 mM phenylmethanesulfonyl fluoride, and 1 % IGEPAL.
  • anti-rabbit Trueblot agarose beads eBioscience
  • anti-FLS2 antibodies coupled with anti-FLS2 antibodies and incubated with the crude extract for 2-3 h at 4°C.
  • beads were washed 3 times with wash buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 1 mM phenylmethanesulfonyl fluoride, and 0.5 % IGEPAL) before adding Laemmli sample buffer and incubating for 10 min at 95°C. Analysis was carried out by SDS-PAGE and western blots using anti-FLS2 and anti- BAK1 antibodies 25 .
  • wash buffer 50 mM Tris-HCI pH 7.5, 150 mM NaCI, 1 mM phenylmethanesulfonyl fluoride, and 0.5 % IGEPAL
  • RNA isolation, cDNA synthesis and real-time PCR analysis Total RNA was isolated from 1 -week-old seedlings grown on 1/2 MS plates using either the
  • QRLSTGSRINSAKDDAAGLQIA QRLSTGSRINSAKDDAAGLQIA
  • pep2 SEQ ID NO: 6 DNKAKSKKRDKEKPSS GRPGQTNSVPNAAIQVYKED peptides were synthesized at >95% purity by the in- house protein chemistry facility and dissolved to a 10 mM stock in pure water.
  • Leaf disks (0 6mm) were cut out from 4- to 5-week-old plants. Single disks were placed adaxial side up into 96-well microtiter plates in which every well contained 200 pL of sterile MonoQ water. Floated disks were then vacuum infiltrated for 10 min.
  • the plates were incubated on a rocking table at 45 rpm in continuous light, at 21 °C for 5 hours.
  • the mix for elicitation was freshly prepared in falcon tubes wrapped with aluminum foil on ice.
  • 99,1 pL of sterile MonoQ water was mixed together with 4 pL of 500x HRP, 4 pL of 500x L-012 and appropriate peptide at a final concentration of 1 pM.
  • the water was carefully removed and replaced immediately with 100 pL of elicitation solution using a multichannel pipette. Relative luminescence measurements were started immediately after adding the elicitation mix using a BiTec Synergy 4 microplate reader.
  • Horseradish Peroxidase was purchased from Sigma-Aldrich and prepared at a 10 mg/mL (500X) concentration in sterile MonoQ water.
  • L-012 was purchased from Wako Chemicals GmbH. Preparation of a 500x L-012 stock solution containing 17mg/ml_ L-012 in sterile MonoQ water and was subsequently protected from light. Solutions were stored at - 20°C.
  • the models were constructed using the total RLU measured for the first 39 time points to ensure comparability across experiments. Root inhibition ratios were calculated on 7-day-old seedlings grown on plates left untreated or treated with 1 mM flg22.
  • Seedling Growth Inhibition Assay Seedlings of the noted A. thaliana lines were grown for 5 days on MS-Agar plates with 1 % sucrose prior to transfer of up to 10 seedlings to each well of a 6-well plate containing 1 ml of 0.5x MS medium with 1 % sucrose. The seedlings were treated with water (NT) or 100 nM flg22 (T) peptide and grown further for 7 days. The seedlings were removed, briefly dried, and weighed (fresh weight). The percentage of seedling growth inhibition was calculated by dividing the weight of individual treated seedlings by the mean weight of the NT seedlings of the same genotype.
  • Pathogens assays Assays with Pseudomonas syringae pv. tomato DC3000 ( Pto DC3000) have been previously described 19 . Bacterial growth in plant leaves was assessed by inoculating 4-weekold plants with a bacterial inoculum of 105 cfu/ml. Growth inhibition of Pto DC3000 by 1 mM flg22 was conducted as described 19 . Leaves were either left untreated or infiltrated with an elicitor solution containing 1 pM flg22.
  • Leaf discs were pooled and three samples were taken per data point (12 leaf discs in total). Leaf discs were bored from the infiltrated area and ground to homogeneity in 10 mM MgCI2. The titer was determined by plating and serial dilution.
  • each node was classified based on its ECD. Assuming equal frequency of a given node binding to any other node, the frequency for each class of binding event was calculated and divided into self-interactions between small ECDs (small-small homotypic), self interactions between large ECDs (large-large homotypic), interactions between two different small ECDs (small heterotypic), interactions between two different large ECDs (large heterotypic), and interactions between one small and one large ECD (small- large heterotypic).
  • Genomic screens identify a new phytobacterial microbe- associated molecular pattern and the cognate Arabidopsis receptor-like kinase that mediates its immune elicitation. Genome biology 17, 98, doi :10.1 186/s13059-016- 0955-7 (2016).
  • Nimchuk, Z. L. CLAVATA1 controls distinct signaling outputs that buffer shoot stem cell proliferation through a two-step transcriptional compensation loop.
  • Kang, Y. H. & Hardtke, C. S. Arabidopsis MAKR5 is a positive effector of BAM3-dependent CLE45 signaling. 17, 1145-1 154, doi :10.15252/embr.201642450 (2016).
  • PSY1 R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner.
  • RECEPTOR-LIKE KINASE1 protein complex includes BRASSINOSTEROID- INSENSITIVE1.

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Abstract

La présente invention concerne le domaine des plateformes d'interaction des domaines extracellulaires des récepteurs de la surface cellulaire et permet l'identification d'un réseau d'interaction en surface cellulaire basé sur LRR (CSILRR) pour prédire et valider la fonction des LRR-RK non caractérisées.
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