INCREASED FUNGAL RESISTANCE IN PLANTS VIA MODULATION OF A WALL-ASSOCIATED KINASE OR BENZOXAZINOIDS

Abstract

The present invention relates to methods for producing plants with increased fungal resistance, preferably seedling resistance against Northern Corn Leaf Blight. Further provided are methods for introducing, modifying, or modulating at least one wall-associated kinase (WAK) in(to) a plant cell, tissue, organ, or whole plant and thereby causing a reduced synthesis of benzoxazinoid and in turn increased fungal resistance. There are further provided methods to identify and/or modify downstream effector molecules in a WAK signalling cascade. Finally, plant cells, tissues, organs or whole plants having increased fungal resistance and methods using substances to activate signalling pathways in a targeted way are provided. The present invention thus relates to WAKs as master regulators and crucial signaling mediators in plant defense against fungal disease and the regulation and cross-talk mechanisms in the WAK signaling cascade and further gives examples for establishing novel anti-fungal strategies relevant for a series of crop plants.

Claims

1.-15. (canceled)

16. A method for selecting a maize plant having Helminthosporium turcicum resistance, wherein the Helminthosporium turcicum resistance is regulated by at least one wall-associated kinase, the method comprising: (i) providing at least one maize plant cell, tissue, organ, or whole plant comprising at least one gene encoding a wall-associated kinase in the genome of said maize plant cell, tissue, organ, or whole plant; (ii) determining a reduced synthesis of at least one benzoxazinoid in response to a Helminthosporium turcicum pathogen infection in at least one maize plant cell, tissue, organ, or whole plant, wherein said plant cell, tissue, organ, or whole plant exhibit an amount of at least one benzoxazinoid which is reduced by at least 25% as compared to a corresponding control plant cell, control tissue, control organ, or control whole plant of the same genotype, but lacking the reduced expression level of the at least one molecule within the signaling pathway from the at least one wall-associated kinase to the synthesis of at least one benzoxazinoid or within the synthesis pathway of at least one benzoxazinoid, wherein the synthesis of the at least one benzoxazinoid is regulated by the at least one wall-associated kinase and reduced synthesis is caused by a reduced expression level of at least one molecule within a signalling pathway from the at least one wall-associated kinase to the synthesis of at least one benzoxazinoid or within a synthesis pathway of at least one benzoxazinoid in said maize plant cell, tissue, organ, or whole plant, the reduced expression level being relative to the expression level of the at least one molecule within the signalling pathway in the control plant cell, control tissue, control organ, or control whole plant, wherein the at least one wall-associated kinase: a) is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, b) is encoded by a nucleic acid molecule comprising the nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 1, c) is encoded by a nucleic acid molecule comprising the nucleotide sequence coding for the amino acid sequence of SEQ ID NO: 2, d) is encoded by a nucleic acid molecule comprising the nucleotide sequence coding for an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, e) comprising the amino acid sequence of SEQ ID NO: 2, or f) comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the benzoxazinoid whose synthesis is regulated by the at least one wall-associated kinase, is selected from at least one of 7,8-dimethoxy-2,4-dihydroxy-1,4(2H)-benzoxazin-3-one (DIM.sub.2BOA)-glucoside, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), DIMBOA-glucoside, 2-O-Glucosyl-7-methoxy-1,4(2H)-benzoxazin-3-one (HMBOA)-glucoside, and any combination thereof, and wherein the at least one molecule within the signalling pathway from the at least one wall-associated kinase to the synthesis of at least one benzoxazinoid or within the synthesis pathway of at least one benzoxazinoid is selected from the group consisting of: (i) the nucleic acid sequences of any one of SEQ ID NOs: 10, 12, and 16, or (ii) a nucleic acid sequence having identity of at least 98% to the nucleic acid sequence of any one of SEQ ID NOs: 10, 12, and 16, over the full length of the coding regions, or (iii) the amino acid sequences of SEQ ID NOs: 11, 13, and 17, or (iv) an amino acid sequence having identity of at least 98% to the amino acid sequence of anyone of SEQ ID NOs: 11, 13, and 17, over the full length of the sequence.

17. The method according to claim 16, wherein the fungal resistance against which resistance to a fungus is increased, or a disease caused by said fungus is selected from a fungus of the order of Pleosporales, comprising Helminthosporium turcicum causing northern corn leaf blight (NCLB), particularly affecting maize and wheat plants, or comprising Bipolaris maydis causing southern corn leaf blight, the order of Pucciniales causing rust disease, comprising Puccinia sorghi causing common rust, or Diploida macrospora causing Diploida leaf streak/blight, or Colletotrichum graminicola causing Anthracnose, or Fusarium spp. causing Fusarium stalk rot, or Gibberella spp. causing Giberella stalk rot, or Sphacelotheca reiliana causing maize head smut.

18. The method according to claim 16, wherein the at least one gene encoding at least one wall-associated kinase is stably integrated into the genome of the at least one plant cell, tissue, organ, or whole plant, or wherein the at least one gene encoding at least one wall-associated kinase is transiently introduced into a plant cell, tissue, organ, or whole plant, or wherein the at least one gene encoding at least one wall-associated kinase is stably integrated into the genome of the at least one plant cell, tissue, organ, or whole plant, wherein the introduction of the at least one gene encoding at least one wall-associated kinase comprises the introgression of the at least one gene during plant breeding.

19. The method according to claim 16, wherein the reduced synthesis of at least one benzoxazinoid is achieved by providing at least one wall-associated kinase, an allelic variant, a mutant or a functional fragment thereof, or a gene encoding the same, wherein the at least one wall-associated kinase comprises a sequence which can directly or indirectly influence the benzoxazinoid pathway and at least one further plant metabolic pathway, wherein the plant metabolic pathway is selected from the group consisting of the jasmonic acid pathway, the ethylene pathway, the lignin synthesis pathway, a defense pathway, a receptor-like kinase pathway, and a cell wall associated pathway.

20. The method according to claim 16, wherein modification of the at least one gene encoding at least one wall-associated kinase is performed by at least one of a site-specific nuclease (SSN) or a catalytically active fragment thereof, or a nucleic acid sequence encoding the same; oligonucleotide directed mutagenesis; chemical mutagenesis; or TILLING.

21. The method according to claim 16, wherein the at least one plant cell, tissue, organ, or whole plant provided in step (i) is selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum oficinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza saliva, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Cofea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanmfolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the aforementioned plants.

22. A plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, obtainable by a method according to claim 16.

23. A method for identifying at least one gene involved in increased pathogen resistance in a plant cell, tissue, organ, whole plant, or plant material the method comprising: (i) determining the genotype of at least one plant cell, tissue, organ, whole plant, or plant material with respect to the presence of at least one gene encoding a wall-associated kinase in the genome of said plant cell, tissue, organ, whole plant or plant material; (ii) optionally determining the benzoxazinoid signature of the at least one plant cell, tissue, organ, whole plant, or plant material of step (i); (iii) exposing the at least one plant cell, tissue, organ, whole plant, or plant material of step (i) or (ii) to a stimulus, optionally wherein the stimulus is correlated with the benzoxazinoid signature in the at least one plant cell, tissue, organ, whole plant, or plant material; (iv) performing an analysis of at least one analyte obtained from the at least one plant cell, tissue, organ, whole plant, or plant material of step (i) or (ii) after exposition to the stimulus; (v) determining at least one gene being regulated upon exposition to a stimulus according to step (iii) in at least one cell of the at least one plant cell, tissue, organ, whole plant, or plant material as derivable from the analysis of at least one analyte as defined in step (iv), (vi) subjecting the at least one gene as determined in step (v) to a functional characterization; and (vii) providing at least one gene involved in increased pathogen resistance in a plant cell, tissue, organ, whole plant, or plant material.

24. A plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, obtainable by introducing at least one gene as provided by the method according to claim 23 into at least one cell of at least one of a plant cell, tissue, organ, or whole plant.

25. A plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, wherein the introduction of at least one gene into at least one cell of at least one of a plant cell, tissue, organ, or whole plant according to the method according to claim 23 is a stable introduction.

26. A method of increasing pathogen resistance in a plant cell, tissue, organ, whole plant, or plant material, the method comprising: (i) providing at least one plant cell, tissue, organ, whole plant or plant material; (ii) (a) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance neutralizing an effect of at least one benzoxazinoid, and/or (ii) (b) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance activating the signalling pathway downstream of at least one wall-associated kinase; and/or (ii) (c) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance modulating or modifying the activity of at least one promoter or at least one regulatory sequence of at least one gene of the at least one plant cell, tissue, organ, whole plant or plant material of step (i), wherein said promoter or said regulatory sequence is involved in the regulation of transcription of at least on gene involved in the signalling pathway of, or downstream of at least one wall-associated kinase or involved in the synthesis pathway of at least one benzoxazinoid; (ii) (d) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance inhibiting the synthesis of at least one benzoxazinoid; and (iii) reducing an amount of at least one benzoxazinoid and thereby increasing pathogen resistance in at least one plant cell, tissue, organ, whole plant, or plant material.

27. The method of claim 26, wherein the method is effective to increase fungal resistance in at least one plant cell, tissue, organ, whole plant, or plant material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] FIG. 1 (FIGS. 1 A and B) shows (FIG. 1 A) the structure of benzoxazinone (BXD) in the aglucone form. Derivatives from the BXD basic structure of FIG. 1 A are: HBOA (R.sup.1═H, R.sup.2═H, R.sup.3═H), DHBOA (R.sup.1═H, R.sup.2═OH, R.sup.3═H), HMBOA (R.sup.1═H, R.sup.2═OMe, R.sup.3═H), HM.sub.2BOA (R.sup.1═H, R.sup.2═OMe, R.sup.3═OMe), DIBOA (R.sup.1═OH, R.sup.2═H, R.sup.3═H), TRIBOA (R.sup.1═OH, R.sup.2═OH, R.sup.3═H), DIMBOA (R.sup.1═OH, R.sup.2═OMe, R.sup.3═H), DIM.sub.2BOA (R.sup.1═OH, R.sup.2═OMe, R.sup.3═OMe), HDMBOA (R.sup.1═OMe, R.sup.2═OMe, R.sup.3═H), or HDM.sub.2BOA (R.sup.1═OMe, R.sup.2═OMe, R.sup.3═OMe). FIG. 1 B shows the basic structure of a benzoxazolinone, a natural degradation product of BXDs and also comprised by the term BXD as used herein. Specific benzoxazolinones are BOA (R.sup.2═H, R.sup.3═H), MBOA (R.sup.2═OMe, R.sup.3═H), or M.sub.2BOA (R.sup.2═OMe, R.sup.3═OMe).

[0078] FIG. 2 (FIGS. 2 A and B) shows the rate of successful penetration events for fungal penetrations as described in Example 9. (FIG. 2 A) Hyphae detected inside of host tissues. In the upper and lower panels the focus is on the epidermis and hyphae, respectively. The arrows in the lower image of Fig. A indicate the hyphae inside the host tissues. (FIG. 2 B) RLK1b, RLK1d and RLK1f are ZmWAK-RLK1 mutants with compromised NCLB resistance that were produced in RP3Htn1, while RLK1b-wt, RLK1d-wt and RLK1f-wt are the corresponding sister lines, respectively. Statistics was conducted using Student's t test, based on three independent experiments. The asterisks represent a significant difference of ** p<0.01 or * p<0.05. Error bars indicate f standard error.

[0079] FIG. 3 (FIG. 3 A to C) shows the disease phenotype of Htn1-NILs and the corresponding parents as detailed in Example 10 below. (FIG. 3 A) The disease symptom of the second leaves at 16 dpi (from left to right for the lines w22, w22Htn1, B37 and B37Htn1). (FIG. 3 B) Rate of infected of tested plants B37, w22, w22Htn1, and B37Htn1. (FIG. 3 C) Area under the disease progress curve (AUDPC) for the isogenic lines detailed below the x-axis. * AUDPC in this panel was calculated as described in Hurni et al. 2015, based on calculating sum of rate of infected plants (%).

[0080] FIG. 4 shows the result of a transcriptome analysis in Htn1-NILs, which revealed a set of DEGs. FIG. 4 shows the number of DEGs in the two Htn1 NILs compared to the corresponding susceptible lines as further described in Example 10 below.

[0081] FIG. 5 (FIG. 5 A to F) shows the content of BXDs in w22 and w22Htn1. (FIG. 5 A) shows the proposed bio-synthesis pathway of secondary metabolites BXDs. The genes encoding the proteins catalyzing each step of enzymatic reactions are presented besides and below the arrows. The contents of BXDs compounds DIMBOA-Glc (FIG. 5 B), DIMBOA (FIG. 5 C), HMBOA-Glc (FIG. 5 D), DIM.sub.2BOA-Glc (FIG. 5 E) and HDMBOA-Glc (FIG. 5 F) were determined at before inoculation, at 3 dpi and 10 dpi. The statistics were conducted using Tukey's HSD (P=0.05) in eight biological replicates. The ns stands for no significance. Error bars are f SE. See also Example 10 below.

[0082] FIG. 6 (FIG. 6 A to D) shows that the presence of ZmWAK-RLK1 results in decreased DIM.sub.2BOA-Glc content possibly by down-regulating Ig1 expression in the maize RP3 genetic background (see also Example 11). (A) Content of DIM.sub.2BOA-Glc (n=8). Expression analysis of Bx1 (B), Ig1 (C) and ZmWAK-RLK1 (D) in Htn1 mutants and sister lines before infection at 21 days after sowing (n=5). Statistics was conducted using Student's t test. The asterisks represent a significant difference of ** p<0.01 or * p<0.05. Error bars are f SE.

[0083] FIG. 7 (FIG. 7 A to E) shows the content of BXDs compounds DIMBOA-Glc (A), DIMBOA (B), HMBOA-Glc (C), and DIM.sub.2BOA-Glc (D), HDMBOA-Glc (E) in second leaves at 10 dpi. Further information is provided in Example 12 below. The statistics were conducted using Student's t test (P=0.05) in eight biological replicates. The asterisks represent a significant difference of ** p<0.01 or * p<0.05. The ns stands for no significance. Error bars are f SE.

[0084] FIG. 8 (FIG. 8 A to D) (see also Example 12) shows that compromising the biosynthesis of BXDs decreases the susceptibility of NCLB disease at the seedling stage. (FIGS. 8 A and B): Visual symptoms and quantified NCLB disease severity in bx mutants. (FIG. 8 C): The transcriptional level of ZmWAK-RLK1 at 10 dpi. (FIG. 8 D) shows the proposed model of ZmWAK-RLK1 underlying NCLB disease resistance. The resistance allele suppressed the biosynthesis of major BXDs compounds, which likely served as the susceptibility component for promoting NCLB disease. Statistic test was conducted using Student's t test. The asterisks represent a significant difference of ** p<0.01 or * p<0.05. The ns stands for no significance. Error bars are f SE.

[0085] FIG. 9 (FIG. 9 A to D) shows the content of BXDs compounds DIMBOA-Glc (A), DIMBOA (B), HMBOA-Glc (C), and HDMBOA-Glc (D) in second leaves of Htn1 NILs and mutants at 21 days after sowing. Further information is provided in Example 12 below. The statistics were conducted using Student's t test (P=0.05) in eight biological replicates. The asterisks represent a significant difference of ** p<0.01 or * p<0.05. The ns stands for no significance. Error bars are f SE.

[0086] FIG. 10 shows relative RLK1 expression in tissues. These samples were harvested from 21 days old seedlings without pathogen inoculation. Different lower case letters in the graphs indicate a difference which is statistically different. See also Example 13 below.

[0087] FIG. 11 (FIGS. 11 A and P) shows the transcription levels of genes in Htn1-NILs and the corresponding parental lines. The expression of genes (FIG. 11 A) ZmWAK-RLK1, (FIG. 11 B) Bx1, (FIG. 11 C) Ig1, (FIG. 11 D) Bx2, (FIG. 11 E) Bx3, (FIG. 11 F) Bx4, (FIG. 11 G) Bx5, (FIG. 11 H) Bx6, (FIG. 11 I) Bx7, (FIG. 11 J) Bx8, (FIG. 11 K) Bx9, (FIG. 11 L) Bx10/11, (FIG. 11 M) Bx12, (FIG. 11 N) Bx13, (FIG. 11 O) Glu1 and (FIG. 11 P) Glu2 were quantified. The different colors and patterns of the bars indicate timepoints before and after infection as shown in the legend for FIG. 11 A which also applies for FIG. 11 B to P. The statistics were conducted separately in w22 and B37 genetic background using Tukey's HSD (P=0.05) in four biological replicates. Error bars are f SE. Different lower case letters in the graphs indicate a difference which is statistically different. See also Example 10 below.

[0088] FIG. 12 (FIG. 12 A to C) shows ZmWAK-RLK1 localization to the plasma membrane. (FIG. 12 A-B) Fluorescent signals in onion epidermal cells after transient expression of ZmWAK-RKL1-eGFP (SEQ ID NO: 9) and the positive control PIP2A-mCherry that is known to localize to the plasma membrane (Kammerloher. Werner, et al. “Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system.” The Plant Journal 6.2 (1994): 187-199). Signals are shown before (FIG. 12 A) and after (FIG. 12 B) plasmolysis with 0.8 M mannitol. (FIG. 12 C) Fluorescent signals in N. benthamiana leaves two days after infiltration. Notably, the signals shown in white in the respective graphs correspond to the originally green (column 1 RLK_eGFP), red (column 2 PIP2A_mCherry), and yellow (merge, column 4) fluorescent signal. Column 3 (DIC) represents the differential interference contrast to visualize the cellular structures as control.

[0089] FIG. 13 is a table showing the logFC and annotation of 215 the differentially expressed genes (DEGs) detected in B37Htn1/B37 and w22Htn1/w22 in at least one of timepoints.

DETAILED DESCRIPTION

[0090] Based on the experiments and data underlying the present invention, it was found that the genes involved in the biosynthesis of benzoxazinoids (BXDs) and derivatives thereof, mainly hydrolysis based derivatives like hydroxamic acids, are not only involved in the defense mechanism against E. turcicum but rather can also effect and mediate plant-defense, i.e., resistance mechanisms against various other fungal pathogens in a series of crop plants. The present invention thus implements both the link between a wall associated kinases (WAK), downstream signaling molecules, such as Bx1, Bx2, Bx6, Bx14 and Ig1, or any other enzyme involved in the benzoxazinoid synthesis pathway, and the decrease of BXD secondary metabolites and further technically implements the finding that this decrease of BXD secondary metabolites is associated with increased fungal resistance.

[0091] The present invention thus provides in a first aspect a method for producing a plant having increased fungal resistance, wherein the fungal resistance is regulated by at least one wall-associated kinase, the method comprising: (i) (a) providing at least one plant cell, tissue, organ, or whole plant having a specific genotype with respect to the presence of at least one gene encoding a wall-associated kinase in the genome of said plant cell, tissue, organ, or whole plant; or (i) (b) introducing at least one gene encoding at least one wall-associated kinase into the genome of at least one cell of at least one of a plant cell, tissue, organ, or whole plant; and (ii) (a) modifying at least one gene encoding at least one wall-associated kinase in the at least one plant cell, tissue, organ, or whole plant; and/or (ii) (b) modulating the expression level of at least one wall-associated kinase and/or the transcription level, the expression level, or the function of at least one molecule within the signaling pathway from the at least one wall-associated kinase to the synthesis of at least one benzoxazinoid or within the synthesis pathway of at least one benzoxazinoid in the at least one plant cell, tissue, organ, or whole plant; and (iii) producing a population of plants from the at least one plant cell, tissue, organ, or whole plant; and (iv) selecting a plant having an increased fungal resistance, from the plant population, based on the determination of a reduced synthesis of a benzoxazinoid preferably in response to a fungal infection, wherein the selected plant have an increased fungal resistance based on the reduced synthesis of a benzoxazinoid, and/or wherein the synthesis of the benzoxazinoid is regulated by the at least one wall-associated kinase.

[0092] Wall associated kinases (WAKs) have recently been identified as major components of fungal and bacterial disease resistance in several cereal crop species. However, the molecular mechanisms of WAK-mediated resistance are presently largely unknown. According to this invention, the function of the maize gene ZmWAK-RLK1 (Htn1) that confers quantitative resistance to northern corn leaf blight (NCLB) caused by the hemibiotrophic fungal pathogen Exserohilum turcicum was investigated. ZmWAK-RLK1 (Htn1) was found to localize to the plasma membrane and its presence resulted in a modification of the infection process by specifically reducing pathogen penetration into host tissues. Furthermore, the ubiquitous expression of ZmWAK-RLK1 and the findings on the signaling pathway downstream of ZmWAK-RLK1 demonstrate the function of this and associated wall-associated kinases as master regulators and crucial signaling mediators in plant defense against fungal disease.

[0093] A transcriptome analysis of near-isogenic lines (NILs) differing for ZmWAK-RLK1 revealed that several genes involved in the biosynthesis of the secondary metabolites benzoxazinoids (BXDs) were differentially expressed in the presence of ZmWAK-RLK1. Particularly the content of BXD compounds DIMBOA-Glc, DIMBOA, HMBOA-Glc and DIM.sub.2BOA-Glc were significantly lower in the NILs with ZmWAK-RLK1. Furthermore, DIM.sub.2BOA-Glc, which is an inactive glucoside of BXDs, was significantly elevated in ZmWAK-RLK1 mutants with compromised NCLB resistance. In addition, maize mutants that were affected in BXDs biosynthesis showed reduced susceptibility to E. turcicum infection at the seedling stage. We thus conclude that BXD biosynthesis increases susceptibility to E. turcicum infection and that the ZmWAK-RLK1-mediated NCLB resistance results from a reduction of these compounds. These findings indicate a novel link between WAKs underlying quantitative disease resistance and the defense mechanism mediated by the secondary metabolites BXDs that have been known for their involvement in cereal insect resistance. The term “WAK” as used herein may comprise a plant receptor-like kinase associated with the signal transduction directly or indirectly effecting the biosynthesis of genes involved in the BXD synthesis, or interacting with signaling mechanism and/or protein-protein interactions being involved in the BXD synthesis.

[0094] The plant immune response “caused” by a WAK can thus be of direct or indirect nature. As it is known to the skilled person, receptor-like kinases usually comprise at least one extracellular signaling domain, e.g., for sensing PAMPs and/or DAMPs, a transmembrane domain, and an intracellular kinase domain. The kinase domain allows the WAK to transform the extracellular signal into an intracellular response transferred via a cascade of proteins involved in the downstream signal transduction. Usually, receptor kinases thus indirectly initiate the activation and transfer into the nucleus/organelle of a transcription factor which regulates the transcription of a target gene. Furthermore, plant WAKs can trigger defense responses such as reactive oxygen species (ROS) accumulation through the activation of a NADPH oxidase, nitric oxide production, callose deposition, besides a MAP kinase-mediated activation of defense gene expression. The terms “causing” or “caused” as used herein in the context of a WAK or another plant receptor kinase is thus to be construed broadly to comprise any direct or indirect effect the activity of the WAK can have on downstream signaling molecules, wherein the molecules can be selected from at least one amino acid sequence, preferably an enzyme in the signal transduction cascade downstream of the WAK or a peptide being able to stimulate or inhibit complex formation downstream of the WAK or signal transduction downstream of a WAK, a metabolite, such as any secondary metabolite produced by a plant, a ROS, or an indirect effect on the regulation of the transcription and/or translation of another downstream gene/protein. A physical interaction of the WAK in the form of a signaling complex may occur to cause an action. In another embodiment, the action caused by the WAK is mediated by a downstream molecule, e.g., a downstream kinase phosphorylating another molecule, in an indirect way. In the terminal part of the WAK signaling cascade, a transcription activator or repressor can be induced to regulate the transcription of a target gene of a WAK, preferably a target gene in the jasmonic acid and/or BXD biosynthesis pathway. Besides a protein-DNA interaction, WAK signaling can also imply protein-protein interactions influencing the BXD biosynthesis pathway.

[0095] In another aspect, the methods of the present invention further may comprise the step of introducing, modifying and/or modulating at least one further or other gene into at least one plant cell, tissue, organ, or whole plant to provide a synergistic effect in increasing fungal disease by decreasing the synthesis of at least one BXD compound associated with fungal resistance. In preferred embodiments, the at least one further or other gene is selected from a bx), bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene (SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24 or 26, respectively), or a homologous gene thereof, or the respective proteins encoded by said genes as set forth exemplary in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25 or 27, respectively, or homologs thereof. As disclosed herein, certain genes involved in the jasmonic acid pathway, the ethylene pathway, the lignin synthesis pathway, a plant defense pathway, a further receptor-like kinase pathway, or a cell wall pathway, and preferably certain genes involved in the jasmonic acid pathway, contribute to the signaling pathway of at least one functional WAK, wherein there may be a synergistic effect provided by the presence of a specific functional WAK and a specific non-functional or less functional gene of the jasmonic acid pathway, as the presence of both will contribute to an even significantly reduced amount of a BXD compound of interest and thus a more than additive increase in fungal resistance.

[0096] The present invention thus provides specific target genes which can be modulated in addition or alternatively to the at least one WAK of interest to provide a significantly improved fungal defense strategy for a plant of interest. These results are based on different functional studies including comparative transcriptome analysis in defined specific WAK genotypes, namely in two pairs of near isogenic lines, w22 and W22Htn1 as well as B37 and B37Htn1 (see Example 10 below), after fungal specific stimuli by analyzing the RNA sequencing datasets. Furthermore, additional RT-qPCR experiments and systematic RNA sequencing were conducted to decipher the plant immune network as triggered by a WAK, e.g., Htn1, (Examples 6 and 10 and Tables 1 and 2). These data demonstrated a cross-talk between the WAK and the benzoxazinoid synthesis and jasmonic acid pathway and thus provided new candidates to provide new elite plant lines comprising both a specific WAK as well as a specific genotype with respect to enzymes involved in the benzoxazinoid synthesis and jasmonic acid pathway.

[0097] In one embodiment according to the present invention, the method for producing a plant having increased fungal resistance may comprise the modification of at least one gene encoding at least one wall-associated kinase, and optionally at least one further or other gene, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, in the at least one plant cell, tissue, organ, or whole plant. The modification can be conducted by any means of plant breeding, including classical and modern methods of plant breeding, and/or techniques of molecular biology. Classical plant breeding methods may comprise the deliberate interbreeding (crossing) of closely or distantly related species to produce new crops with desirable properties. Plants are crossed to introduce traits/genes from a particular variety into a new genetic background to provide plants having modified and/or increased quality, yield, tolerance (against abiotic stress), resistance (against biotic stress), etc., characteristics. Breeding nowadays also includes methods like marker-assisted selection, reverse breeding and the targeted combination with molecular biology tools known and available to the skilled person.

[0098] In one embodiment according to the present invention, the modulation or modulating of at least one wall-associated kinase, and/or of at least one further or other gene, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, can thus comprise at least one of modulating the expression level of at least one wall-associated kinase, preferably increasing the expression at least one wall-associated kinase, and/or modulating the function or activity of and/or activity of at least one wall-associated kinase, for example, by providing at least one molecule interacting with the extracellular signalling domain of at least one WAK, e.g., an activator, or by providing at least one molecule interacting with the intracellular signalling domain of at least one WAK, such as a molecule inducing or inhibiting kinase activity. In one embodiment, the modulation or modulating of at least one wall-associated kinase, and/or of at least one further or other gene, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, can comprise the targeted introduction of at least one mutation into a WAK and/or a further or other gene of interest to modulate the activity of the WAK and the further or other protein encoded by the at least one further or other gene in a targeted way. Embodiments comprising the modulation of at least one wall-associated kinase thus aim at influencing the activity of the at least one WAK within without modifying the nucleic acid sequence and thus possibly the amino acid sequence of a WAK of interest. In certain embodiments, wherein at least one further or other gene, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, is modulated, the modulation may aim at reducing the activity of a at least one allele of a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene to decrease the amount of BXD compound synthesized. For example, BX1 and IGL enzymes are accountable for the bulk of BX biosynthesis. Therefore, inhibiting the presence of a functional BX enzyme, preferably a BX1, BX2 or BX6 enzyme, or an Ig1 enzyme can contribute to the provision of a reduced BXD synthesis and thus an increased fungal resistance in a plant of interest.

[0099] The modulation according to the present invention can comprise any direct or indirect interaction between two molecules, i.e., a receptor-ligand interaction, a transcription factor-transcription factor binding site interaction, an interaction of an enzyme, e.g., a kinase with its target site, an interaction of a peptide or nucleic acid modulator with a target site, an antibody-antigen interaction, an interaction with a DNA or histone binding protein and its cognate ligand (DNA or histone), a hybridization between two nucleic acid sequences/molecules and the like.

[0100] In one embodiment, the transcription level of at least one WAK within at least one cell of at least one of a plant cell, tissue, organ, or whole plant can be modified or modulated by specifically influencing a regulatory sequence of a WAK gene. In another embodiment, the modulation affects at least one gene, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene. This modulation or modification can comprise the introduction of at least one specific mutation, for example to activate a promoter of interest, or the modulation or modification can be in trans by providing a transcription factor modulating the transcription of at least one WAK gene, wherein the at least one WAK gene according to all embodiments of the present invention may comprise an endogenously occurring WAK gene, or a WAK gene introduced into at least one cell of at least one of a plant cell, tissue, organ, or whole plant.

[0101] According to the various aspects and embodiments of the present invention, a signalling pathway from the at least one wall-associated kinase to the synthesis of at least one benzoxazinoid in at least one plant cell, tissue, organ, or whole plant thus implies the whole chain of molecular actions downstream of a WAK as sensing molecule triggering a signalling cascade involving various different effectors until the synthesis of a BXD compound.

[0102] In one embodiment according to the various aspects of the present invention, the at least one wall-associated kinase is WAK-RLK1, preferably selected from Htn1, Ht2, or Ht3, or an allelic variant thereof, a mutant or a functional fragment thereof, or a gene encoding the same, preferably wherein the at least one wall-associated kinase a) is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 7, or a functional fragment thereof, b) is encoded by a nucleic acid molecule comprising the nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to nucleotide sequence of SEQ ID NO: 1 or 7, preferably over the entire length of the sequence, c) is encoded a nucleic acid molecule hybridizing with a complementary sequence to a) or b) under stringent conditions, d) is encoded by a nucleic acid molecule comprising the nucleotide sequence coding for an amino acid sequence of SEQ ID NO: 2 or 8, or a functional fragment thereof, e) is encoded by a nucleic acid molecule comprising the nucleotide sequence coding for an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid sequence of SEQ ID NO: 2 or 8, preferably over the entire length of the sequence, f) comprising the amino acid sequence so of SEQ ID NO: 2 or 8, or g) comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid sequence of SEQ ID NO: 2 or 8, preferably over the entire length of the sequence, provided that any sequence of a) to g), optionally after expression, still encodes at least one functional Htn1, Ht2, or Ht3, or an allelic variant, a mutant, or a functional fragment thereof. In a preferred embodiment, the at least one wall-associated kinase is selected from Htn1 (RLK1) or an allelic variant, a mutant or a functional fragment thereof, or a gene encoding the same. Variants may further comprise any functional splice variant of a WAK gene. As it is known to the skilled person, eukaryotic mRNA comprising introns is spliced during processing from a precursor mRNA into a mature mRNA giving rise to a protein after translation (protein biosynthesis).

[0103] “Functional” or “functional fragment” or “variant” as used in connection with a WAK or any other receptor-like kinase or any at least one further gene/protein, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, or the corresponding proteins Bx1, Bx2, Ig1, Bx6, Bx11, Bx14, Opr2, Lox3 or Aoc1, according to the present disclosure means a fragment of an amino acid or nucleic acid sequence with reference to the respective (longer) sequence occurring in the natural environment of a plant genome of interest, whereas the functional fragment still comprises—optionally after transcription, processing and translation—at least one function of the respective parent sequence. The functional fragment may be less sterically demanding and thus more convenient for certain approaches. Furthermore, the functional fragment may be fused to another domain to create a fusion molecule for functional assays, e.g., a fusion with a gene encoding a protein having fluorescence activity. In another embodiment, the functional fragment may be fused to a tag and the like. Therefore, a functional fragment may also comprise a sequence comprising codon optimizations on the nucleic acid level, or comprising certain mutations, said mutations not influencing the activity or function of a WAK, or another receptor-like kinase of interest.

[0104] Preferably, any function variant at least comprises a truncated form of the extracellular signalling domain of a WAK and an active intracellular kinase domain, wherein the intracellular kinase domain is able to initiate downstream signalling. Notably, the extracellular domain, the transmembrane domain and/or the intracellular kinase domain of a WAK according to the present invention can comprise at least one mutation. Said mutation may lead to an increased signalling activity to represent a functional variation or functional mutation in the sense of the present invention.

[0105] In certain aspects according to the present invention, at least one further or other gene, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, is introduced, and/or modified and/or modulated according to the methods of the present invention, variants or mutants representing “loss-of-function”, or having reduced activity might be specifically preferred for the purpose of the present invention in case that the at least one variant or mutant results in a decreased BXD synthesis. Particularly, it was found according to the present invention that there is a cross-talk between the WAK signaling pathway so and the BXD synthesis pathway, mainly the BXD synthesis pathway as mediated by Bx1, Bx2, Bx6, Bx11 and BX14 and/or Ig1, wherein the targeted insertion, modulation or modification of at least one WAK, or the gene encoding the same, and a further effector, or the gene encoding the same, for example a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, contribute to a enhanced fungal resistance, in particular NCLB resistance, in a plant as the regulation of both pathways leads to a reduced BXD signature. Wherein the WAK pathway plays the role of the general “pacemaker” in this regulatory network which senses and forwards signals due to its recognition and kinase function, there is also a feedback regulation between the further effectors involved in the jasmonic acid and BXD synthesis pathway. The master regulator function of WAK is demonstrated by the fact that the combined expression of Bx1 and Ig1 was consistently lower in genotypes with ZmWAK-RLK1 (FIGS. 11 B and C and Example 10) demonstrating that ZmWAK-RLK1 and other WAKs have the capacity to induce a concerted action also regulating the benzoxazinoid pathway and the jasmonic acid pathway.

[0106] In one embodiment according to the various aspects of the present invention, the method for producing a plant having increased fungal resistance thus comprises the introduction and/or modification and/or modulation of at least one WAK, or a gene encoding the same, wherein the WAK at least comprises a functional intracellular kinase domain, for example a sequence selected from SEQ ID NOs: 1, 2, 7, or 8, or an allelic variant or mutant thereof, and wherein the method further comprises the introduction and/or modification and/or modulation of at least one BX or Ig1 protein, or a gene encoding the same, wherein the corresponding bx gene, or the Ig1 gene comprises at least one mutation, or wherein the bx gene, or the Ig1 gene is of a specific genotype is knocked-out, so that the WAK activity and the decreased or deleted BX protein or Ig1 protein activity results in a decreased BXD biosynthesis.

[0107] Receptor-like kinases can be further divided into RD and non-RD kinases, depending on the presence or absence of an arginine residue at the catalytic site of the kinase domain. ZmWAK-RLK2 contains an RD kinase and ZmWAK-RLK1 has a non-RD kinase domain (cf. for instance positions 505 and 506 of SEQ ID NO: 2, amino acids F (phenylalanine) and D (aspartic acid), respectively). Most receptor-like kinases involved in plant immunity identified so far belong to the non-RD kinases, whereas RD kinases are thought to play a role in other processes such as development. Variants of SEQ ID NO: 2 have been constructed (cf. SEQ ID NOs: 3 to 6 and Hurni et al., 2015). It was found that mutations at positions M455, G497 and G548 (with reference to SEQ ID NO: 2) may result in a higher susceptibility to NCLB. All said positions reside in the serine threonine kinase domain of ZmWAK-RLK1. A functional variant according to the present invention will thus avoid any mutation or combination of mutations in the kinase domain of a WAK which results in decreased fungal resistance. Exemplary mutants of SEQ ID NO: 2 are presented with SEQ ID NOs: 3 and 4 (RLK1b, M4551) and SEQ ID NOs: 5 and 6 (RLK1d, G497E). A further mutant analyzed herein, RLK1f, comprises a mutation G548R in comparison to the wild-type sequence according to SEQ ID NO: 2. All mutants were tested in comparison to the respective sister lines as described herein. Based on these structural data the importance of a functional intracellular kinase domain of a WAK could be deduced. Therefore, a functional variant or a functional mutant of a WAK may comprise at least one mutation in comparison to the cognate wild-type sequence which at least one mutation does not disturb the downstream signaling of the WAK in that sense that a functional mutant or variant will decrease the level of a specific BXD compound to in turn increase fungal resistance of a plant, plant cell, tissue, or organ comprising such a functional variant of a WAK, or the sequence encoding the same.

[0108] According to certain embodiments of the present invention, more than one gene encoding a WAK, or a functional fragment thereof, or the sequence encoding the same, can be introduced into, or modulated or modified in at least one plant cell, tissue, organ, or whole plant. The introgression of several WAKs can have a synergistic effect in providing enhanced fungal resistance, particularly in case an elite line can be established based on the staggering of more than one WAK into the genome of a plant of interest according to the disclosure of the present invention. As described herein, WAKs represent the key signalling molecules initiating an immune cascade downstream of and mediated by the intracellular kinase domain of the WAKs. Therefore, more than one WAK may thus have a dosage effect positively downregulating BXD synthesis and thus increasing fungal resistance in a plant, in particular a crop plant, of interest. Furthermore, at least one further gene or protein, preferably being selected from any one of SEQ ID NOs: 10 to 27 or homologous genes or homologs thereof, can be additionally or alternatively modified as detailed above to provide a plant cell, tissue, organ or whole plant as material for producing a plant with improved fungal resistance properties, preferably resistance against NCLB. Further target sequences to be modified having an implication in the cross-talk between WAK signalling and BXD biosynthesis are disclosed in Tables 1 and 3 herein.

[0109] In a further embodiment according to the present invention, there is provided a method, wherein the reduced synthesis of at least one benzoxazinoid is achieved by providing at least one wall-associated kinase, an allelic variant, a mutant or a functional fragment thereof, or a gene encoding the same, wherein the at least one wall-associated kinase comprises a sequence which can directly or indirectly influence the benzoxazinoid pathway and at least one further plant metabolic pathway, preferably a disease resistance associated pathway, wherein the plant metabolic pathway is selected from the group consisting of the jasmonic acid pathway, the ethylene pathway, the lignin synthesis pathway, a defense pathway, a receptor-like kinase pathway, a cell wall associated pathway, preferably, wherein the at least one further plant metabolic pathway is the jasmonic acid pathway and wherein the reduced synthesis of at least one benzoxazinoid is achieved by an decreased or down-regulated Ig1 and/or Bx1 expression as induced by at least one WAK of interest.

[0110] Several differentially expressed genes (DEGs) identified by the inventors of the present invention belonged to several different immune networks and to different disease resistance associated pathways including benzoxazinoids (BXDs) biosynthesis, (phytohormone) jasmonic acids (JAs), ethylene, lignin, defense and receptor-like kinases as well as cell wall were found in Hin1 NILs (Example 10, FIG. 13). Surprisingly, six genes of the BXDs biosynthesis pathway showed differential expression in at least one timepoint, including Bx1 (SEQ ID NOs: 10 and 11), Bx2 (SEQ ID NOs: 12 and 13), Ig-like (SEQ ID NOs: 14 and 15), Bx6 (SEQ ID NOs: 16 and 17), Bx11 (SEQ ID NOs: 18 and 19) and Bx14 (SEQ ID NOs: 20 and 21) (see also FIG. 13, column 2 of table for further reference to publicly available data base entries for gene IDs and names) demonstrating a cross-regulatory network between the WAK and further pathways in fungal defense. This was particularly surprising because BXDs as secondary metabolites have so far not been associated with defense against fungi mediated by WAK kinases. More surprisingly, the transcriptome data and functional assays also, for the first time, revealed DEGs that are part of immune networks including the phytohormone jasmonic acids that plays a central role in regulating resistance against hemibiotrophic and necrotrophic diseases. JAs treatment can induce the accumulation of BXD compounds (Oikawa, Akira, Atsushi Ishihara, and Hajime Iwamura. “Induction of HDMBOA-Glc accumulation and DIMBOA-Glc 4-O-methyltransferase by jasmonic acid in poaceous plants.” Phytochemistry 61.3 (2002): 331-337; Oikawa, Akira. et al. “Accumulation of HDMBOA-Glc is induced by biotic stresses prior to the release of MBOA in maize leaves.” Phytochemistry 65.22 (2004): 2995-3001). The present invention thus provides evidence that there is an additional link between the WAK kinase signaling pathway and the jasmonic acid pathway which paves the way for a variety of new disease and particularly fungal resistance strategies as disclosed herein. The enzymatic properties of IGL are similar to BX1, but the transcriptional regulation of their corresponding genes is different. Like other Bx genes, Bx1 is constitutively expressed during the early developmental stages of the plant, which correlates with endogenous BX levels. Plants carrying the mutant alleles of the Bx1 gene produce only a fraction of the BXs that are found in Bx1 wild-type plants. Therefore, a WAK according to the present invention may act as a master regulator bridging anti-fungal signalling with the effectors of the jasmonic acid pathway and other pathways, preferably an effector selected from the group consisting of SEQ ID NOs: 10 to 27 or homologous genes or homologs thereof.

[0111] In one embodiment, the introduction at least one additional gene encoding at least one wall-associated kinase into at least one cell of at least one of a plant cell, tissue, organ, or whole plant may comprise the introduction of a nucleic acid sequence, comprising DNA and/or RNA in a single stranded and/or double stranded form, or an amino acid sequence, by means of breeding techniques, or by means of molecular biology to transfer a functional WAK of interest, or an additional functional WAK of interest, or the sequence encoding the same, into at least one cell of interest. Said at least one additional gene can be also any gene, wherein the resulting protein/enzyme is involved in the BXD biosynthesis pathway or in a jasmonic acid pathway, such as Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Ig1, Glu1, Glu2, OPR2, LOX3 or AOC1 (see FIG. 13 and SEQ ID NOs: 10 to 27), or any variant thereof, or a combination of the aforementioned genes/proteins. Preferably, the at least one additional gene comprises at least one mutation which changes the function of the naturally so occurring respective additional gene, wherein the mutation, in the coding or within a regulatory region, causes decreased synthesis of the respective BXD compound, or wherein the mutation, in a regulatory region, such as a promoter region, or in a coding region, causes a reduced signal transduction from a WAK kinase located upstream in the signalling cascade so the said mutation results in a decreased synthesis of a BXD compound. In another embodiment, the gene encoding at least one of Bx1, Bx2, 3s Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Ig1, Glu1, Glu2, OPR2, LOX3 or AOC1 may be deleted or partially deleted within the genome of a plant cell of interest, or the gene may be modified in a targeted way.

[0112] Further enzymes involved in the regulation of the BXD synthesis which can be modulated, introduced or modified according to the methods of the present invention to achieve an increased fungal resistance in a plant cell, plant or plant material pathway are selected from the group of jasmonic synthesis pathway enzymes, including 12-oxo-phytodienoic acid reductase 2 (OPR2), Lipoxygenase 3 (LOX3) or Allene oxide cyclase 1 (AOC1), ethylene pathway enzymes, such as S-adenosylmethionine synthase, lignin pathway enzymes, such as, for example, Caffeoyl-CoA O-methyltransferase 1 (OMT1) or OMT2, enzymes and proteins involved in plant defense mechanisms, such as, for example SAF1—Safener induced 1; Glutathione S-transferase, and any combination thereof.

[0113] Presently, WAKs are the only known proteins that can physically link the cell wall to the plasma membrane (Brutus, Alexandre, et al. “A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides.” Proceedings of the National Academy of Sciences 107.20 (2010): 9452-9457). Therefore, further structurally and functionally related cell wall spanning or associated kinases are suitable as WAKs according to the present invention, e.g., maize qHSR1 (Zuo, Weiliang, et al. “A maize wall-associated kinase confers quantitative resistance to head smut.” Nature genetics 47.2 (2015): 151-157), or rice OsWAK/Xa4 gene conferring quantitative rice blight resistance by strengthening the cell wall (Hu et al. 2017).

[0114] In another embodiment according to the present invention, the step of introducing at least one gene into at least one cell of at least one of a plant cell, tissue, organ, or whole plant may comprise the introduction of a gene, wherein the amino acid sequence or enzyme encoded by said gene is involved in the catalytic pathway downstream of a WAK kinase, wherein the additional gene is introduced alone, or together with at least one gene encoding a WAK kinase or a variant thereof.

[0115] “Benzoxazinoids” or “BXDs” are a class of indole-derived plant chemical defenses comprising compounds with a 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton and their derivatives. BXDs have been described as phytochemicals in monocots, including grasses, including important cereal crops such as maize, wheat and rye, as well as a certain dicot species. The term “BXDs” as used herein refers to both benzoxazinones (glucosides and corresponding aglucones containing a 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton) and their downstream derivative products during metabolic pathways, benzoxazolinones, as well as any intermediates. The term BXD may thus also comprise a derivative being the result of the activity of hydrolyzing glucosidases found in plastids, cytoplasm, and cell walls, or derivatives and intermediated being the result of degradation to benzoxazolinones via oxo-cyclo/ring-chain tautomerism. Further comprised are downstream metabolites directly being derivable from any benzoxazolinone. The term “BXDs” shall further comprise any open form, nitrenium form or complex, e.g., a metal complex from a BXD. BXD basic structures are represented in FIG. 1.

[0116] By the term “reduced/decreased synthesis of a benzoxazinoid” or “reduced synthesis of at least one benzoxazinoid” or “reducing the amount of at least one benzoxazinoid” or “reduction on BXDs content” or “reduced amount of a BXD compound” or the like, is meant that the plant cell, tissue, organ, or whole plant according to the present invention exhibit an amount of a benzoxazinoid, at least one benzoxazinoid or the benzoxazinoid of interest which is reduced by at least 10%, 15%, 20% or 25%, preferably by at least 30%, 35%, 40% or 45%, more preferably by at least 50%, 60% or 70% as compared to a corresponding control plant cell, control tissue, control organ, or control whole plant of the same genotype, but lacking the modification of the at least one gene encoding at least one wall-associated kinase or the modulation of the expression level of at least one wall-associated kinase and/or the transcription level, the expression level, or the function of at least one molecule within the signaling pathway from the at least one wall-associated kinase to the synthesis of at least one benzoxazinoid or within the synthesis pathway of at least one benzoxazinoid. In one embodiment according to the various aspects of the present invention, the benzoxazinoid whose synthesis is regulated by the at least one wall-associated kinase and optionally regulated by the at least one further enzyme of the jasmonic acid and/or benzoxazionoid pathway is selected from at least one of DIM.sub.2BOA, DIMBOA, HMBOA, HM.sub.2BOA, HDMBOA, HDM.sub.2BOA, HBOA, DHBOA, DIBOA or TRIBOA, the aforementioned benzoxazinoid being in the glucoside or aglucone form, or a benzoxazolinone, or any combination of the aforementioned benzoxazinoids, preferably wherein the benzoxazinoid whose synthesis is regulated by the at least one wall-associated kinase is selected from at least one of DIM.sub.2BOA, DIMBOA, HMBOA or HDMBOA, the aforementioned benzoxazinoid being in the glucoside or aglucone form, or any combination of the aforementioned benzoxazinoids.

[0117] In one embodiment according to the various aspects of the present invention, a reduced content of BXDs can be achieved by introducing at least one gene encoding at least one wall-associated kinase into at least one cell of at least one of a plant cell, tissue, organ, or whole plant, wherein the at least one wall-associated kinase causes a reduced synthesis of at least one BXD. More than one WAK encoding gene and different allelic variants of a WAK gene may be introduced into a cell of interest in addition to a WAK gene potentially already being present in the genome of a plant cell of interest. The presence of several WAKs or receptor-like kinases involved in the BXD synthesis may thus be favourable in order to increase the copy number and thus the dosage effect of a gene of interest.

[0118] In one embodiment, quantitative NCLB disease resistance is based on a decrease of the biosynthesis of at least one secondary metabolite BXDs, preferably DIM.sub.2BOA-Glc, DIMBOA, HMBOA, DIMBOA-Glc or HMBOA-Glc, and the methods according to the various aspects of the present invention comprise the addition of a scavenger molecules interacting with and this neutralizing the activity of at least one secondary metabolite BXD to reduce the amount of the of at least one secondary metabolite BXD susceptibility component to decrease fungal infection at least one plant cell, tissue, organ, or whole plant.

[0119] According to the present invention, there are thus provided methods for producing a plant having increased fungal resistance, wherein the fungal resistance is regulated by at least one wall-associated kinase. “Regulated” in this context thus implies a direct or indirect regulation mediated by at least one wall-associated kinase. This regulation may imply a signalling cascade initiated by the at least one wall-associated kinase and proceeding through further molecules involved in the signalling cascade. The regulation can be on a protein, RNA or nucleic acid level. Furthermore, the regulation may imply a cross-talk or feedback regulation, for example implying a Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Ig1, Glu1, Glu2, OPR2, LOX3 or AOC1 enzyme, or the gene encoding the same, or the transcriptional regulation of such a further gene encoding Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Ig1, Glu1, Glu2, OPR2, LOX3 or AOC1, or a modulation or modification of a gene encoding Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Ig1, Glu1, Glu2, OPR2, LOX3 or AOC1.

[0120] In one embodiment, the pathogen according to the present disclosure is a fungal pathogen infesting a plant. The disease caused by a fungal pathogen and the respective fungus may be selected from Plume blotch Septoria (Stagonospora) nodorum, Leaf blotch (Septoria tritici), Ear fusarioses (Fusarium spp.), Late blight (Phytophthora infestans), Anthrocnose leaf blight or Anthracnose stalk rot (Colletotrichum graminicola (teleomorph: Glomerella graminicola Politis) Glomerella tucumanensis), Curvularia leaf spot (Curvularia clavata, C. eragrostidis, =C. maculans (teleomorph: Cochliobolus eragrostidis), Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolus intermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus), Curvularia pallescens (teleomorph: Cochlobolus pallescens), Curvularia senegalensis, C. tuberrulata (teleomorph: Cochliobolus tuberculatus), Didymella leaf spot (Didymella exitalis), Diplodia leaf spot or streak (Stenocarpella macrospora=Diplodialeaf macrospora), Brown stripe downy mildew (Sclerophthora rayssiae var. zeae), Crazy top downy mildew (Sclerophthora macrospora=Sclerospora macrospora), Green ear downy mildew (Sclerospora graminicola), Leaf spots (various minor leaf spots) (Alternaria alternata, Ascochyta maydis, A. tritici, A. zeicola, Bipolaris victoriae=Helminthosporium victoriae (teleomorph: Cochliobolus victoriae), C. sativus (anamorph: Bipolaris sorokiniana=H. sorokinianum=H. sativum), Epicoccum nigrum, Exserohilum prolatum=Drechslera prolata (teleomorph: Setosphaeria prolata) Graphium penicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.), Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola, S. zeina, Northern corn leaf blight (Setosphaeria turcica (anamorph: Exserohilum turcicum=Helminthosporium turcicum), Northern corn leaf spot (Cochliobolus carbonum (anamorph: Bipolaris zeicola=Helminthosporium carbonum)), Phaeosphaeria leaf spot (Phaeosphaeria maydis=Sphaerulina maydis), Rostratum leaf spot (Setosphaeria rostrata, (anamorph: Helminthosporium rostratum)), Java downy mildew (Peronosclerospora maydis=Sclerospora maydis), Philippine downy mildew (Peronosclerospora philippinensis=Sclerospora philippinensis), Sorghum downy mildew (Peronosclerospora sorghi=Sclerospora sorghi), Spontaneum downy mildew (Peronoscierospora spontanea=Scierospora spontanea), Sugarcane downy mildew (Peronoscierospora sacchari=Sclerospora saccharn), Sclerotium ear rot (southern blight) (Sclerotium rolfsii Sacc. (teleomorph: Athelia rolfsii)), Seed rot-seedling blight (Bipolaris sorokiniana, B. zeicola=Helminthosporium carbonum, Diplodia maydis, Exserohilum pedicillatum, Exserohilum turcicum=Helminthosporium turcicum, Fusarium avenaceum, F. culmorum, F. monikforme, Gibberella zeae (anamorph: F. graminearum), Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.), Selenophoma leaf spot (Selenophoma sp.), Yellow leaf blight (Ascochyta ischaemi, Phyllosticta maydis (teleomorph: Mycosphaerella zeae-maydis), Zonate leaf spot (Gloeocercospora sorght).

[0121] Further plant pathogenic fungi include Plasmodiophoromycota, such as Plasmodiophora brassicae (clubroot of crucifers), Spongospora subterranea, Polymyxa graminis, Oomycota, such as Bremia lactucae (downy mildew of lettuce), Peronospora (downy mildew) in snapdragon (P. antirrhini), onion (P. destructor), spinach (P. effusa), soybean (P. manchurica), tobacco (“blue mold”; P. tabacina) alfalfa and clover (P. trifolium), Pseudoperonospora humuli (downy mildew of hops), Plasmopara (downy mildew in grapevines) (P. viticola) and sunflower (P. halstedii), Sclerophthora macrospora (downy mildew in cereals and grasses), Pythium (for example damping-off of Beta beet caused by P. debaryanum), Phytophthora infestans (late blight in potato and in tomato and the like), Albugo spec., Ascomycota, such as Microdochium nivale (snow mold of rye and wheat), Fusarium, Fusarium graminearum, Fusarium culmorum (partial ear sterility mainly in wheat), Fusarium oxysporum (Fusarium wilt of tomato), Blumeria graminis (powdery mildew of barley (sp. hordei) and wheat (f.sp. tritici)), Erysiphe pisi (powdery mildew of pea), Nectria galligena (Nectria canker of fruit trees), Uncinula necator (powdery mildew of grapevine), Pseudopeziza tracheiphila (red fire disease of grapevine), Claviceps purpurea (for example, rye and grasses), Gaeumannomyces graminis (take-all on wheat, rye and other grasses), Magnaporthe grisea, Pyrenophora graminea (leaf stripe of barley), Pyrenophora teres (net blotch of barley), Pyrenophora tritici-repentis (leaf blight of wheat), Venturia inaequaiis (apple scab), Sclerotinia scierotium (stalk break, stem rot), Pseudopeziza medicaginis (leaf spot of alfalfa, white and red clover), Basidiomycetes, such as Typhula incarnata (typhula blight on barley, rye, wheat), Ustilago maydis (blister smut on maize), Ustilago nuda (loose smut on barley), Ustilago tritici (loose smut on wheat, spelt), Ustilago avenae (loose smut on oats), Rhizoctonia solani (rhizoctonia root rot of potato), Sphacelotheca spp. (head smut of sorghum), Melampsora lini (rust of flax), Puccinia graminis (stem rust of wheat, barley, rye, oats), Puccinia recondita (leaf rust on wheat), Puccinia dispersa (brown rust on rye), Puccinia hordei (leaf rust of barley), Puccinia coronata (crown rust of oats), Puccinia striiformis (yellow rust of wheat, barley, rye and a large number of grasses), Uromyces appendiculatus (brown rust of bean), Scierotium rolfsii (root and stem rots of many plants), Deuteromycetes (Fungi imperfecti), such as Septoria (Stagonospora) nodorum (glume blotch) of wheat (Septoria tritici), Pseudocercosporella herpotrichoides (eyespot of wheat, barley, rye), Rynchosporium secalis (leaf spot on rye and barley), Alternaria solani (early blight of potato, tomato), Phoma betae (blackleg on Beta beet), Cercospora beticola (leaf spot on Beta beet), Alternaria brassicae (black spot on oilseed rape, cabbage and other crucifers), Verticillium dahliae (verticillium wilt), Colletotrichum, such as Colletotrichum lindemuthianum (bean anthracnose), Phoma lingam (blackleg of cabbage and oilseed rape), Botrytis cinerea (grey mold of grapevine, strawberry, tomato, hops and the like).

[0122] Preferred fungal diseases to be prevented and the corresponding causative pathogens which can be combated based on the disclosure of the present invention in a crop plant of interest are selected from a fungus from the order of Pleosporales, comprising E. turcicum/H. turcicum causing northern corn leaf blight (NCLB), particularly affecting maize and wheat plants, or comprising Bipolaris maydis causing southern corn leaf blight, the order of Pucciniales causing rust disease, comprising Puccinia sorghi causing common rust, or Diploida macrospora causing Diploida leaf streak/blight, or Colletotrichum graminicola causing Anthracnose, or Fusarium spp., preferably Fusarium verticilioides causing Fusarium stalk rot, or Gibberella spp., e.g., Gibberella zeae causing Giberella stalk rot, or Sphacelotheca reiliana causing maize head smut are thus plant diseases caused by pathogenic fungi which can be prevented in the plants and by the methods of the present invention.

[0123] In one embodiment according to the various aspects of the present invention the at least one gene encoding at least one wall-associated kinase may be stably integrated into the genome of the at least one plant cell, tissue, organ, or whole plant, or the at least one gene encoding at least one wall-associated kinase may transiently introduced into a plant cell, tissue, organ, or whole plant.

[0124] In another embodiment according to the various aspects of the present invention at least one further gene encoding at least one enzyme within the signalling cascade downstream of a wall-associated kinase may be stably integrated into the genome of the at least one plant cell, tissue, organ, or whole plant, or the at least one further gene encoding at least one enzyme within the signalling cascade downstream of a wall-associated kinase may transiently introduced into a plant cell, tissue, organ, or whole plant.

[0125] Methods for introducing a gene of interest into a plant cell of interest by means of molecular biology or conventional and modem breeding and associated tools and methodologies are disclosed herein and are known to the skilled person.

[0126] In one embodiment, the transient introduction may comprise the direct introduction of an amino acid effector instead of the introduction of a gene of interest.

[0127] In one embodiment according to the various aspects of the present invention the at least one gene encoding at least one wall-associated kinase may be stably integrated into the genome of the at least one plant cell, tissue, organ, or whole plant, wherein the introduction of the at least one gene encoding at least one wall-associated kinase comprises the introgression of the at least one gene during plant breeding.

[0128] Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

[0129] Since expression of the genes or nucleic acids of the invention may lead to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product. Thus, the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a transgenic or mutant plant of the invention and another plant. The desired effects, e.g., expression of the at least one WAK gene or a mutant allele of the invention to produce a plant having a modified BXD synthesis profile, or a modulated Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Ig1, Glu1, Glu2, OPR2, LOX3 or AOC1 profile, can be enhanced when both parental plants express the genes or mutant alleles of the invention, or if both allels are modified or even deleted, depending on the target to be modified in accordance with the disclosure of the present invention. The desired effects can be passed to future plant generations by standard propagation means. “Introgressing”, as also detailed above, thus means the integration of a gene or allele in a plant's genome by natural means, i.e. by crossing a plant comprising the gene or allele of interest described herein with a plant not comprising said gene or allele. The offspring can be selected for those comprising the gene or allele of interest.

[0130] Furthermore, the methods of the present invention can result in the creation or provision of a plant material, comprising grains or seeds, relating to any means known in the art to produce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods, such as, for example, air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, or twin-scaling, sexual reproduction, comprising crossing with another plant, and asexual reproduction, such as e.g. apomixis, somatic hybridization and the like.

[0131] In one embodiment according to the various aspects of the present invention, the modification of the at least one gene encoding at least one wall-associated kinase within step (ii) (a) or (ii) (b) of the method for producing a plant having increased fungal resistance, or a modification of a gene encoding Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Ig1, Glu1, Glu2, OPR2, LOX3 or AOC1, may be performed by at least one of a site-specific nuclease (SSN) or a catalytically active fragment thereof, or a nucleic acid sequence encoding the same, oligonucleotide directed (ODM) mutagenesis (ODM), chemical mutagenesis, or TILLING.

[0132] TILLING, initially a functional genomics tool in model plants, has been extended to many plant species and become of paramount importance to reverse genetics in crops species. A major recent change to TILLING has been the application of next-generation sequencing (NGS) to the process, which permits multiplexing of gene targets and genomes. NGS will ultimately lead to TILLING becoming an in silico procedure. Because it is readily applicable to most plants, it remains a dominant non-transgenic method for obtaining mutations in known genes and thus represents a readily available method for non-transgenic approaches according to the methods of the present invention. As it is known to the skilled person, TILLING usually comprises the chemical mutagenesis, e.g., using ethyl methanesulfonate (EMS), or UV light induced modification of a genome of interest, together with a sensitive DNA screening-technique that identifies single base mutations in a target gene, wherein the target gene may encode a protein being selected from the group of a receptor-like kinase, such as a WAK, an enzyme involved in benzoxazinoid synthesis or metabolism, defense, the lignin pathway, the jasmonic acid synthesis pathway, or a transcription factor involved in one of the aforementioned metabolic and/or signalling pathways.

[0133] SSNs and ODM mutagenesis both are suitable techniques for precision genome engineering in plant cells. As it is known to the skilled person, ODM offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. ODM is a precision genome editing technology, which uses oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of plant systems.

[0134] In one embodiment according to the various aspects and embodiments of the present invention, the at least one site-specific nuclease (SSN), or the nucleic acid sequence encoding the same, may be selected from at least one of a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, a base editor complex, a restriction endonuclease, including FokI or a variant thereof, or two site-specific nicking endonucleases, or a variant or a catalytically active fragment thereof. Said targeted genome engineering SSNs can be suitable for both, the introduction of a gene of interest not yet present in a specific genotype, as well as the targeted mutagenesis of a gene of a given specific genotype to modulate (up- or downregulate) the activity of an enzyme encoded by a gene of interest to be modified in a highly precise way.

[0135] SSNs meanwhile emerged as indispensable prerequisite for site-directed genome engineering. SSNs are (programmable) nucleases, which can be used to break a nucleic acid of interest at a defined position to induce either a double-strand break (DSB) or one or more single-strand breaks. Alternatively, said nucleases can be chimeric or mutated variants, no longer comprising a nuclease function, but rather operating as recognition molecules in combination with another enzyme. Those nucleases or variants thereof are thus key to any gene editing or genome engineering approach. In recent years, many suitable nucleases, especially tailored endonucleases have been developed comprising meganucleases, a base editor complex, zinc finger nucleases, TALE nucleases, and CRISPR nucleases, comprising, for example, Cas, Cpf1, CasX or CasY nucleases as part of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. The use of those SSNs and the necessary accessory molecules, for example crRNAs, tracrRNAs, or gRNAs, and delivery systems are thus envisaged for performing the methods according to the present invention.

[0136] A “base editor” as used herein refers to a protein or a fragment thereof having the same catalytical activity as the protein it is derived from, which protein or fragment thereof, alone or when provided as molecular complex, referred to as base editing or base editor complex herein, has the capacity to mediate a targeted base modification, i.e., the conversion of a nucleotide base of interest resulting in a point mutation of interest which in turn can result in a targeted mutation, if the base conversion does not cause a silent mutation, but rather a conversion of an amino acid encoded by the codon comprising the position to be converted with the base editor. Preferably, the base editor is temporarily or permanently linked to at least one site-specific effector, or optionally to a component of at least one site-specific effector complex. The linkage can be covalent and/or non-covalent. Multiple publications have shown targeted base conversion, primarily cytidine (C) to thymine (T), using a CRISPR/Cas9 nickase or non-functional nuclease linked to a cytidine deaminase domain, Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC1), e.g., APOBEC derived from rat. The deamination of cytosine (C) is catalyzed by cytidine deaminases and results in uracil (U), which has the base-pairing properties of thymine (T). Most known cytidine deaminases operate on RNA, and the few examples that are known to accept DNA require single-stranded (ss) DNA.

[0137] A “CRISPR nuclease” according to the present invention can be a CRISPR-based nuclease, or the nucleic acid sequence encoding the same, which is selected from the group consisting of (a) Cas9, including SpCas9, SaCas9, SaKKH-Cas9, VQR-Cas9, StlCas9, or (b) Cpf1, including AsCpf1, LbCpf1, FnCpf1, (c) CasX, or (d) CasY, or any variant or derivative of the aforementioned CRISPR-based nucleases, or a CRISPR-based nuclease comprising a mutation in comparison to the respective wild-type sequence so that the resulting CRISPR-based nuclease is converted to a single-strand specific DNA nickase, or to a DNA binding effector lacking all DNA cleavage ability. A “CRISPR(-based) nuclease”, as used herein, is thus any nuclease which has been identified in a naturally occurring CRISPR system, which has subsequently been isolated from its natural context, and which preferably has been modified or combined into a recombinant construct of interest to be suitable as tool for targeted genome engineering. Any CRISPR-based nuclease can be used and optionally reprogrammed or additionally mutated to be suitable for the various embodiments according to the present invention as long as the original wild-type CRISPR-based nuclease provides for DNA recognition, i.e., binding properties. Said DNA recognition can be PAM dependent. CRISPR nucleases having optimized and engineered PAM recognition patterns can be used and created for a specific application. The expansion of the PAM recognition code can be suitable to target the site-specific effector complexes to a target site of interest, independent of the original PAM specificity of the wild-type CRISPR-based nuclease. Cpf1 variants can comprise at least one of a S542R, K548V, N552R, or K607R mutation, preferably mutation S542R/K607R or S542R/K548V/N552R in AsCpf1 from Acidaminococcus (cf. SEQ ID NO: 24). Furthermore, modified Cas variant, e.g., Cas9 variants, can be used according to the methods of the present invention as part of a base editing complex, e.g. BE3, VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, SaKKH-BE3 (see Kim et al., Nat. Biotech., 2017, doi:10.1038/nbt.3803). Therefore, according to the present invention, artificially modified CRISPR nucleases are envisaged, which might indeed not be any “nucleases” in the sense of double-strand cleaving enzymes, but which are nickases or nuclease-dead variants, which still have inherent DNA recognition and thus binding ability. Other suitable Cpf1-based effectors for use in the methods of the present invention are derived from Lachnospiraceae bacterium (LbCpf1, e.g., NCBI Reference Sequence: WP_051666128.1), or from Francisella tularensis (FnCpf1, e.g., UniProtKB/Swiss-Prot: A0Q7Q2.1). Variants of Cpf1 are known (cf. Gao et al., BioRxiv, dx.doi.org/10.1101/091611). Variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in vivo are thus envisaged as site-specific effectors according to the present invention. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 and thus provide a useful addition to the CRISPR/Cas genome engineering toolbox.

[0138] Due to the fact that receptor-like kinases and BX enzymes (cf. SEQ ID NOs: 10 to 13 and 16 to 21), Ig1 (SEQ ID NOs: 14 and 15), OPR2 (SEQ ID NOs:22 and 23), LOX3 (SEQ ID NOs:24 and 25), and AOC1 (SEQ ID NOs:26 and 27) are ubiquitously found in a variety of plants, particularly monocotyledonous plants (monocots) and dicotyledonous plants (dicots) of agronomic interest, the methods according to the present invention can be used for the targeted optimization of several important monoct and dicot crop plants.

[0139] In one embodiment according to the various aspects of the present invention, the at least one plant cell, tissue, organ, or whole plant provided in step (i) (a) may be selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum oficinarium, Zea spp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp., including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Cofea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine nexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa, Raphanus sativus, Brassica juncacea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanmfolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the aforementioned plants, preferably wherein the plant cell, tissue, organ, or whole plant in step (i) is selected from Zea mays or Triticum spp., or any variety or subspecies belonging to one of the aforementioned plants.

[0140] In one aspect of the present invention, there is thus disclosed a plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, obtainable by any one of the methods according to the various aspects disclosed herein. The plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof obtained according to the present invention will have at least one optimized agronomic trait, wherein this trait is disease resistance or tolerance, preferably fungus resistance or tolerance, more preferably resistance or tolerance against NCLB caused by E. turcicum or a related fungal diseases caused by any one of the related fungal pathogens disclosed herein. Based on the disclosure provided herein demonstrating the functional mechanism of a WAK induced quantitative NCLB resistance, said resistance being associated with a reduction of the BXD biosynthesis, which in turn inhibits the hemibiotrophic fungus E. turcicum and related fungi, the teachings provided herein can be used to provide a plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof having a favourable BXD content, preferably a reduced BXD content, so that the plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof has an increased resistance against fungal infection, i.e., fungal infestation and persistence.

[0141] In yet a further embodiment according to the present invention, more than one agronomic property of a plant cell or plant of interest can be modified in addition to the introduction, modulation and/or modification of a WAK or WAK gene of interest. Said agronomic properties are selected from seed emergence, vegetative vitality, stress tolerance, disease resistance or tolerance against a further fungus, or against another pathogen, comprising a virus, bacterium, a nematode, an insect etc., herbicide resistance, branching tendency, flowering time, seed clusters, seed density, stability and storability, threshing capability (uniform ripening), lodging resistance, increased yield (seed size, yield etc.), or a modified composition of a molecule of agronomic importance (e.g. starch, carbohydrate, protein etc.) of interest, and the like.

[0142] In another aspect according to the present invention there is provided method for identifying at least one gene involved in increased pathogen resistance, preferably increased fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material the method comprising: (i) determining the genotype of at least one plant cell, tissue, organ, whole plant, or plant material with respect to the presence of at least one gene encoding a wall-associated kinase in the genome of said plant cell, tissue, organ, whole plant or plant material; (ii) optionally: determining the benzoxazinoid signature of the at least one plant cell, tissue, organ, whole plant, or plant material of step (i); (iii) exposing the at least one plant cell, tissue, organ, whole plant, or plant material of step (i) or (ii) to a stimulus, optionally wherein the stimulus is correlated with the benzoxazinoid signature in the at least one plant cell, tissue, organ, whole plant, or plant material, preferably wherein the stimulus is associated with a fungal pathogen infection; (iv) performing an analysis of at least one analyte obtained from the at least one plant cell, tissue, organ, whole plant, or plant material of step (i) or (ii) after exposition to the stimulus; (v) determining at least one gene being regulated upon exposition to a stimulus according to step (iii) in at least one cell of the at least one plant cell, tissue, organ, whole plant, or plant material as derivable from the analysis of at least one analyte as defined in step (iv), (vi) subjecting the at least one gene as determined in step (v) to a functional characterization; and (vii) providing at least one gene involved in increased pathogen resistance, preferably increased fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material.

[0143] In one embodiment, the determination of the genotype of at least one plant cell, tissue, organ, whole plant, or plant material with respect to the presence of at least one gene encoding a wall-associated kinase may be performed by determining in the genome of a plant cell, tissue, organ, whole plant, or plant material of interest the presence and/or transcript level of a WAK gene of interest, preferably a WAK gene comprising a nucleotide sequence according to SEQ ID NO: 1 or 7, or comprising a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to one of the nucleotide sequence according to SEQ ID NO: 1 or 7, preferably over the entire length of the sequence, or comprising a nucleotide sequence hybridizing with a nucleotide sequence complementary to the nucleotide sequence according to SEQ ID NO: 1 or 7 preferably under stringent conditions, or comprising a nucleotide sequence encoding for an amino acid sequence of SEQ ID NO: 2 or 8 or for an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to one of SEQ ID NO: 2 or 8.

[0144] In another embodiment, determining the benzoxazinoid signature comprises a step of determination of so the presence and/or the transcript level of at least one gene from the BXD biosynthesis pathway and/or the jasmonic acid pathway, The gene may be selected from any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24 or 26, or a variant, homologous gene, allel or mutant or a fragment thereof. Bioinformatic tools for the determination and/or alignment of sequences of interest are disclosed herein, or are readily available to the skilled person.

[0145] In one embodiment, the determination of the genotype of at least one plant cell, tissue, organ, whole plant, or plant material with respect to the presence of at least one gene encoding a wall-associated kinase may also comprise the sequencing of a gene having a certain sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to one of the gene sequences disclosed herein, which gene has not yet been annotated in a publicly available genome database to determine the precise sequence of said gene by means of molecular biology, e.g., PCR techniques.

[0146] In one embodiment, the method for identifying at least one gene involved in increased pathogen resistance, preferably increased fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material may comprise the determination of the benzoxazinoid (BXD) signature of the at least one plant cell, tissue, organ, whole plant, or plant material of step (i). The benzoxazinoid signature means the qualitative and/or quantitative determination of at least one BXD secondary metabolite of interest as disclosed herein. This determination can provide a reference value for any subsequent analysis. The benzoxazinoid signature determination, which can be performed at different timepoints and with or without the addition of a stimulus, thus can provide information on the background level of a specific BXD present before and after addition of a stimulus. Furthermore, the BXD signature may provide data on the total amount of mixed BXD compounds synthesized in a plant, plant cell, tissue, organ or whole plant under suitable and defined conditions. The BXD signature may thus serve as reference value to have a benchmark for any subsequent modifications and/or modulations performed in accordance with the methods of the present invention. Due to the fact that BXD synthesis depends on the action of different enzymes in the terminal branch of the synthesis pathway, more than one different BXD compound may be analyzed to provide a BXD signature of a plant cell, tissue, organ, or whole plant of interest representing a full picture of the different BXD compounds synthesized by the plant under defined conditions (timepoint, stimulus, stimulus amount and environmental factors, for example, biotic or abiotic stress).

[0147] In one embodiment, the method for identifying at least one gene involved in increased pathogen resistance, preferably increased fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material may comprise the exposition of the at least one plant cell, tissue, organ, whole plant, or plant material of step (i) or (ii) to a stimulus, optionally wherein the stimulus is correlated with the so benzoxazinoid signature in the at least one plant cell, tissue, organ, whole plant, or plant material, preferably wherein the stimulus is associated with a fungal pathogen infection.

[0148] A “stimulus” in this context refers to any naturally occurring, endogenous or exogenous, or non-naturally occurring substance chemical substance stimulating a plant cell, tissue, organ, whole plant. Preferably, the stimulus is a stimulus derived from or associated with a pathogen, preferably a fungal pathogen. The stimulus may be a known PAMP or DAMP triggering an immune response mediated by a receptor-like kinase in a plant cell, tissue, organ, whole plant. The correlation may be of direct or indirect nature. The “stimulus” may also be an endogenous substance, e.g., a BXD or jasmonic acid, or a synthetic variant thereof, as BXD compounds and jasmonic acids may induce feedback regulation mechanisms in a plant cell. The “stimulus” may the pathogen by itself causing the desired response in a plant.

[0149] The stimulus may thus be any environmental stimulus which will cause a response in a plant, wherein the response is effected by a signal cascade, or reaction within a plant cell, tissue, organ, whole plant, e.g., resulting in a different transcriptome profile in comparison to the transcriptome profile of a non-stimulated plant. Preferably, the stimulus is correlated with a benzoxazinoid signature in at least one plant cell, tissue, organ, whole plant, or plant material. In one embodiment, where the correlation between a stimulus and the BXD signature is not known, a correlation between a stimulus of interest and the BXD signature can be easily determined by measuring the up- or down-regulation of genes within the BXD signalling pathway upon addition of a stimulus of interest to determine a direct or indirect correlation.

[0150] In a preferred embodiment, the stimulus is associated with a fungal pathogen, but is not restricted thereto. As it is known in the field of plant pathophysiology, plants evolved sophisticated strategies to respond to a stimulus as provided by a variety of different plant pathogens to initiate defense responses. Certain response may be highly specific for a pathogen, or one specific molecule associated or produced by said pathogen, whereas other defense strategies are part of a global regulatory network as associated by a stimulus of interest. According to the methods of the present invention it is thus possible to analyze the effect of a stimulus of interest a pathway of interest to identify any implication in the BXD or jasmonic acid biosynthesis pathway having a favourable effect on plant fungal response as disclosed herein in a highly targeted way to identify new target genes contributing to a favourable fungal defense response in a plant cell, tissue, organ, or whole plant of interest.

[0151] In one embodiment, the method for identifying at least one gene involved in increased pathogen resistance, preferably increased fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material according to the present invention can comprise an additional step of electronically transmitting and/or electronically storing data on a computer readable medium.

[0152] An “analyte” obtained from the at least one plant cell, tissue, organ, or whole plant may comprises a nucleic acid, including DNA and RNA, an amino acid sequence, or a plant metabolite.

[0153] In one embodiment, a transcriptome analysis, i.e., an analysis of the sum total of all the messenger RNA molecules expressed from the genes of an organism, using RNA obtained from the at least one plant cell, tissue, organ, or whole plant of step (ii) after exposition to the stimulus is performed to obtain data on any changes in the transcription profile of certain genes in a plant cell, tissue, organ, whole plant treated with a stimulus of interest in comparison to plant cell, tissue, organ, or whole plant not treated with the respective stimulus. A variety of different tools to perform a transcriptome analysis of genome-wide differentially expressed RNA and to analyze altered gene expression/transcription is available to the skilled person. In one embodiment, the determination of at least one gene being regulated upon exposition to a stimulus according to step (iii) of the above method for identifying at least one gene involved in increased pathogen resistance in at least one cell of the at least one plant cell, tissue, organ, whole plant thus comprises the determination of the transcription level of a gene. Preferably, differentially regulated, or highly regulated genes, e.g., genes being significantly up- or down-regulated in comparison to a non-treated plant or plant cell, may be further analyzed.

[0154] In another embodiment, a proteome analysis, i.e., an analysis of the entire complement of proteins that is or can be expressed by a plant cell, tissue, or organism, using amino acids obtained from the at least one plant cell, tissue, organ, or whole plant of step (ii) after exposition to the stimulus is performed to obtain data on any changes in the transcription profile in a plant cell, tissue, organ, whole plant treated with a stimulus of interest in comparison to plant cell, tissue, organ, or whole plant not treated with the respective stimulus. Several methods for quantitative and qualitative proteome analysis, of the whole proteome or parts thereof, are available to the skilled person.

[0155] In yet another embodiment, an analysis of a metabolite, e.g., a substance produced by the at least one plant cell, tissue, organ or whole plant and representing an intermediate or product of its metabolism, is performed to identify the effect of a stimulus has on the overall constitution and production level with respect to said metabolite of interest.

[0156] In one embodiment of the method for identifying at least one gene involved in increased pathogen resistance, preferably increased fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material, a gene of interest determined, said gene being regulated upon exposition to a stimulus, preferably a stimulus influencing a BXD signature, may be subjected to a functional characterization.

[0157] The functional characterization may comprise an in silico analysis, an in vitro analysis, an in vivo analysis, or a combination of the aforementioned analyses. The in silico analysis may comprise the determination of any known function of said gene in different plant, or information on available allelic variants of said gene in different plants or different germplasm. Furthermore, the in silico analysis may comprise the determination of the locus of a gene such determined in the genome of a plant of interest, or the determination of regulatory sequences associated with the gene of interest. An in vitro analysis so or manipulation may comprise the cloning, sequencing and characterization of the gene of interest and/or the creation of an expression construct, or vector, or a fusion construct, or the creation of mutants of a gene of interest. An in vitro analysis or manipulation may further comprise the introduction of a gene of interest, comprised by a suitable construct, into a target plant, tissue, organ or whole plant of interest by a suitable delivery vector. The in vivo analysis may comprise the analysis of different plants or plant cells, tissues or organs from different species, cultivars or varieties comprising or not comprising the gene of interest in their genome to provide a functional characterization of the phenotype the gene of interest may participate in, optionally by subjecting the different plants or plant cells, tissues or organs from different species, cultivars or varieties to different stimuli und controlled conditions to be able to compare the respective results.

[0158] In one embodiment, at least one gene involved in increased pathogen resistance as identified according to the methods of the present invention can be further subjected to directed mutagenesis studies and subsequent functional analyses to identify mutations positively or negatively effecting a phenotype of interest, wherein the phenotype is a change in the BXD signature, or a change of fungal resistance in comparison to the respective wild-type. Methods to introduce (multiple) site-directed mutations into a given gene of interest are available to the skilled person.

[0159] In one aspect of the present invention there is provided a plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, obtainable by introducing at least one gene as provided by the method for identifying at least one gene involved in increased pathogen resistance, preferably increased fungal resistance into the genome of at least one cell of at least one of a plant cell, tissue, organ, or whole plant.

[0160] In one embodiment, the plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, comprises at least one wall-associated kinase (WAK) selected from Htn1, Ht2, or Ht3, or an allelic variant, a mutant or a functional fragment thereof, or a gene encoding the same, preferably wherein the at least one wall-associated kinase a) is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 7, or a functional fragment thereof, b) is encoded by a nucleic acid molecule comprising the nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to nucleotide sequence of SEQ ID NO: 1 or 7, preferably over the entire length of the sequence, c) is encoded a nucleic acid molecule hybridizing with a complementary sequence to a) or b) under stringent conditions, d) is encoded by a nucleic acid molecule comprising the nucleotide sequence coding for an amino acid sequence of SEQ ID NO: 2 or 8, or a functional fragment thereof, e) is encoded by a nucleic acid molecule comprising the nucleotide sequence coding for an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid so sequence of SEQ ID NO: 2 or 8, preferably over the entire length of the sequence, f) comprising the amino acid sequence of SEQ ID NO: 2 or 8, or g) comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to amino acid sequence of SEQ ID NO: 2 or 8, preferably over the entire length of the sequence, provided that the sequence, optionally after expression, still encodes at least one functional Htn1, Ht2, or Ht3, or an allelic variant, a mutant, or a functional fragment thereof. In a preferred embodiment, the plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, comprises at least one wall-associated kinase or an allelic variant, a mutant or a functional fragment thereof, or a gene encoding the same, which has been introduced or introgressed, or which at least one wall-associated kinase or an allelic variant, a mutant or a functional fragment thereof, or a gene encoding the same, comprises at least one mutation enhancing the kinase activity of the at least one WAK.

[0161] In another preferred embodiment, the plant cell, tissue, organ, whole plant or plant material, or a derivative or a progeny thereof, comprises at least one further introduced or introgressed enzyme, or the gene encoding the same, wherein the at least one further gene or enzyme is selected from a bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene (SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24 or 26, respectively), or a homologous gene thereof, or the respective proteins encoded by said genes (SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25 or 27, respectively), or a homolog thereof or an allelic variant or mutant thereof, preferably a mutant resulting in decreased transcription and/or translation of the bx1, bx2, ig1, bx6, bx11, bx14, opr2, lox3 or aoc1 gene or protein, respectively. The at least one mutation may thus reside in a regulatory region of such a gene leading to a reduced transcription, or the mutation may result in at least one point mutation affecting the catalytic activity of the translated protein so that said protein or enzyme has a decreased capability to synthesize a BXD compound.

[0162] In one embodiment, the introduction of at least one gene into plant cell, tissue, organ, whole plant or plant material, obtainable by introducing at least one gene as provided by the method for identifying at least one gene involved in increased pathogen resistance, preferably increased fungal resistance, is a stable introduction, preferably a stable introduction mediated by plant breeding, or a stable introduction mediated by means of molecular biology, comprising Agrobacterium-mediated transformation, genome editing, or a combination thereof.

[0163] In one embodiment, the introduction may be effected by introgression of the at least one gene identified, and/or the introduction may be effected may any means of molecular biology. In one embodiment the introduction of a gene or allele determined can take place by recombination between two donor genomes, e.g., in a fused protoplast, wherein at least the donor protoplast carries the gene allele of interest in its genome. In any case, any progeny or derivatives comprising the gene allele of interest can then be subjected to repeated back-crossing steps with a plant line carrying a genetic background of interest to select for the gene allele of interest in the resulting derivatives or progeny. The result may be the fixation of the gene allele of interest such introgressed in a selected genetic background. The whole process of introgression can, for example, take place by a mixture of breeding strategies and techniques of molecular biology to achieve at a genotype/phenotype of interest for a given germplasm, plant, plant cell or plant material.

[0164] In one embodiment, there is thus provided an improved donor source of germplasm having, e.g. by introgression, enhanced resistance to a fungus of interest, preferably wherein the fungus resistance against which resistance is increased, or the disease caused by said fungus is selected from a fungus of the order of Pleosporales, comprising E. turcicum/H. turcicum causing northern corn leaf blight (NCLB), particularly affecting maize and wheat plants, southern corn leaf blight (Bipolaris maydis), the order of Pucciniales causing rust disease, comprising common rust (Puccinia sorghi), or Diploida leaf streak/blight (Diploida macrospora/Stenocarpella macrospora), or Colletotrichum graminicola, or Fusarium spp., preferably Fusarium verticilloides causing Fusarium stalk rot, or Gibberella spp., e.g., Gibberella zeae causing Giberella stalk rot, rust, stalk rot, maize head smut (Sphacelotheca reiliana), and Diploida leaf streak/blight. This germplasm can then serve as basis for further breeding steps.

[0165] In another embodiment, the introduction of at least one gene as identified and provided by the method for identifying at least one gene involved in increased pathogen resistance into at least one plant cell, tissue, organ, whole plant may be effected by at least one means of molecular biology, comprising the use of a delivery vehicle or vector. Optionally, the method can further comprise the modification or modulation of a gene of interest using at least one of a site-specific nuclease (SSN) or a catalytically active fragment thereof, or a nucleic acid sequence encoding the same, oligonucleotide directed mutagenesis, chemical mutagenesis, or TILLING, wherein the at least one site-specific nuclease (SSN), or the nucleic acid sequence encoding the same, is selected from at least one of a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, a base editor complex, a restriction endonuclease, including FokI or a variant thereof, or two site-specific nicking endonucleases, or a variant or a catalytically active fragment thereof.

[0166] In yet a further aspect according to the present invention, there is provided a method of increasing pathogen resistance, preferably fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material, the method comprising: (i) providing at least one plant cell, tissue, organ, whole plant or plant material; (ii) (a) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance neutralizing the effect of at least one benzoxazinoid, and/or (ii) (b) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance activating the signalling pathway downstream of at least one wall-associated kinase; and/or (ii) (c) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance modulating or modifying the activity of at least promoter or at least one regulatory sequence of at least one gene of the at least one plant cell, tissue, organ, whole plant or plant material of step (i), wherein said at least promoter or at least one regulatory sequence is involved in the regulation of transcription of at least on gene involved in the signalling pathway of, or downstream of at least one wall-associated kinase or involved in the synthesis pathway of at least one benzoxazinoid; (ii) (d) treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance inhibiting the synthesis of at least one benzoxazinoid; (iii) reducing the amount of at least one benzoxazinoid and thereby increasing pathogen resistance, preferably fungal resistance, in at least one plant cell, tissue, organ, whole plant, or plant material.

[0167] A “a substance neutralizing the effect of at least one benzoxazinoid” as used herein is to be construed broadly and comprises any naturally occurring or synthetic molecule, which can interact with a BXD compound to decrease the natural effect of the BXD compound said BXD compound would exert (endogenously and/or exogenously) on a plant or the plant environment. Preferably, the substance neutralizing the effect of at least one benzoxazinoid can be added to a plant cell, tissue, organ, or whole plant, optionally coated or together with a suitable delivery vehicle, so that the substance can be transferred into a plant cell of interest. Alternatively, the substance can be added to a plant cell, tissue, organ or whole plant to neutralize the effect of a volatile compound released by a plant cell, tissue, organ or whole plant. Preferably, the substance neutralizing the effect of at least one benzoxazinoid is not toxic to the plant cell, tissue, organ, or whole plant, or to the environment. As it is known that jasmonic acid (JA) treatment can induce the accumulation of BXD compounds (Oikawa et al, 2002 and 2004), a substance according to the present invention may also be a substance scavenging or reducing the amount of jasmonic acid to decrease the accumulation of a BXD compound, which in turn leads to the increased fungal resistance of a plant cell, tissue, organ or whole plant of interest. The substance may also interfere with the transcription of at least one Bx, Ig1 or a further gene involved in the BXD or jasmonic acid biosynthesis pathway.

[0168] In a further embodiment, alone or in combination with the use of a neutralizing substance, at least one plant cell, tissue, organ, whole plant or plant material can be treated with a substance activating the signalling pathway downstream of at least one wall-associated kinase. As used in the context of molecular biology, the terms “upstream” and “downstream” can refer to the temporal and mechanistic order of cellular and molecular events. For example, in signal transduction cascade, the second messenger or an intracellular kinase acts downstream to—that is to say, temporally after—activation of cell membrane receptors, for example a WAK. The other way around, activation of cell membrane receptors occurs upstream of—that is to say, prior to—the production of second messengers or the activation or inhibition of further enzymes acting later and intracellularly in the signalling cascade. Such an activating substance can be selected from a substance acting from the exterior of a plant or plant cell, for example, a substance being a PAMP or DAMP for a receptor-like kinase, e.g., a WAK, so that a stronger signal is received and the receptor mediated response is enhanced. Furthermore, the substance may activate the kinase function of a WAK, or any kinase downstream of the WAK. Finally, the substance may act at the interface between the WAK and a further BXD or jasmonic acid biosynthesis pathway.

[0169] For the wheat WAK gene TaWAK/Snn1 it was shown that it is hijacked by the necrotrophic effector SnTox1 that triggers programmed cell death allowing a pathogen to feed and grown on the dead tissue (Shi et al. 2016). Furthermore, these data show that elicitors recognized by WAKs can both be cell wall derived degraded polysaccharides (e.g. OGs) or pathogenic short peptides (SnTox1) (Brutus et al. 2010; Shi et al. 2016). Thus, there is increasing evidence for a complex nature and functional divergence of WAKs in perception of types of ligands and in their role of interacting with biotic diseases in a direct as well as an indirect way. Preferably, an activating substance according to the present invention is a substance directly activating a WAK of interest which in turn, directly or indirectly, leads to a decreased synthesis of at least one BXD compound, which in turn increases the fungal resistance of a plant cell, tissue, organ or whole plant.

[0170] The inventors of the present invention demonstrated that the WAK ZmWAK-RLK1 functions upstream of the BXDs biosynthesis pathway and decreases the content of secondary metabolites BXDs compounds, e.g., DIM.sub.2BOA-Glc. As the BXD class of secondary metabolites has been found in many of cereal species such maize, wheat and rice, which are the most important food crops worldwide, the methods according to the various aspects of the present invention can thus be used to effect the WAK signaling cascade intrinsically linked to the BXD synthesis, which in turn was found to be key to provide new strategies in fungal defense in plants, preferably for reducing susceptibility to northern corn leaf blight already at the seeding stage. For example, the storage glucoside DIM.sub.2BOA-Glc was found to be constantly lower in susceptible ZmWAK-RLK1 mutants, which suggested DIM.sub.2BOA-Glc severed as a candidate susceptibility compound for promoting E. turcicum infection. Knock-out of this compound has been shown to slightly increase the performance of corn leaf aphids Rhopalosiphum maidis (Handrick, Vinzenz, et al. “Biosynthesis of 8-O-methylated benzoxazinoid defense compounds in maize.” The Plant Cell 28.7 (2016): 1682-1700), a completely different class of plant pathogens, not infecting, yet feeding on a plant, whereas the functional mechanism of DIM.sub.2BOA-Glc in interaction with phloem-feeding insects as presently known is possibly different and antagonistic. The methods and findings according to the present invention and mainly the new insights in gap bridge of WAKs and the secondary defense metabolite BXDs can also be used to provide new defense mechanisms against aphids and other phloem feeding insects to a plant, preferably a crop plant.

[0171] In one preferred embodiment, the methods comprise the modulation or modification of at least one so further gene from a BXD and/or jasmonic acid biosynthesis pathway as disclosed herein to further decrease the content of secondary metabolites BXDs compounds, e.g., DIM.sub.2BOA-Glc and thus to enhance fungal resistance in a plant.

[0172] In yet a further embodiment of the method of increasing pathogen resistance, preferably fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material according to the present invention, the method comprises treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance modulating the activity of at least promoter or at least one regulatory sequence of at least one gene of the at least one plant cell, tissue, organ, whole plant or plant material of step (i), wherein said at least promoter or at least one regulatory sequence is involved in the regulation of transcription of at least on gene involved in the signalling pathway of, or downstream of at least one wall-associated kinase, or involved in the synthesis pathway of at least one benzoxazinoid.

[0173] By modulating or modifying the activity of a promoter or regulatory sequence, the transcription level of a gene of interest and in turn the expression level of a protein of interest can be influenced in a targeted way on a molecular level, or by introducing a transcription factor, preferably a synthetic transcription factor like TAL effector activator/repressor or CRISPR-dCas9 activator/repressor, for a given promoter/gene into a cell. In embodiments, where a promoter is modified in a targeted way, the modification is performed by at least one of a site-specific nuclease (SSN) or a catalytically active fragment thereof, or a nucleic acid sequence encoding the same, oligonucleotide directed mutagenesis, chemical mutagenesis, or TILLING.

[0174] In one embodiment, the at least one site-specific nuclease (SSN), or the nucleic acid sequence encoding the same, is selected from at least one of a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, a meganuclease, a base editor complex, a restriction endonuclease, including FokI or a variant thereof, a recombinase, or two site-specific nicking endonucleases, or a variant or a catalytically active fragment thereof. Preferably, a targeted point mutation is introduced modifying the promoter region, wherein the modification can make use of a transient introduction of the site-specific nuclease tools to obtain a non-transgenic plant cell, tissue, organ or whole plant.

[0175] In yet another or further embodiment of the method of increasing pathogen resistance, preferably fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material according to the present invention, the method comprises treating the at least one plant cell, tissue, organ, whole plant or plant material according to step (i) with a substance inhibiting the synthesis of at least one benzoxazinoid. A substance inhibiting the synthesis of at least one benzoxazinoid can be a double stranded RNA (dsRNA) which is suitable to reduce the expression level of at least on gene involved in the signalling pathway of, or downstream of at least one wall-associated kinase, or involved in the synthesis pathway of at least one benzoxazinoid, wherein by said reduction of the expression level the synthesis or the amount of at least one benzoxazinoid and thereby increasing pathogen resistance, preferably fungal resistance, in at least one plant cell, tissue, organ, whole plant, or plant material. This down regulating of gene expression is well-known to a person skilled in art as RNAi approach or miRNA interference approach (Fire, A, Xu, S, Montgomery, M, Kostas, S, Driver, S, Mello, C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391 (6669): 806-811). Preferably the substance inhibiting the synthesis of at least one benzoxazinoid is at least one siRNA or an siRNA library directed to at least on gene involved in the signalling pathway of, or downstream of at least one wall-associated kinase, or involved in the synthesis pathway of at least one benzoxazinoid. The siRNA or siRNA library can be part of one or more expression cassettes. The siRNA may comprise a first strand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′ end, wherein the first strand is complementary to at least 15 nucleotides of the at least on gene involved in the signalling pathway of, or downstream of at least one wall-associated kinase, or involved in the synthesis pathway of at least one benzoxazinoid, and an second strand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′ end, and wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a small interfering RNA (siRNA) duplex under physiological conditions, and wherein the siRNA silences the at least on gene involved in the signalling pathway of, or downstream of at least one wall-associated kinase, or involved in the synthesis pathway of at least one benzoxazinoid.

[0176] The various embodiments of the aspect of the present invention being directed to a method of increasing pathogen resistance, preferably fungal resistance, in a plant cell, tissue, organ, whole plant, or plant material, alone or in combination, may result in the targeted reduction of the amount of at least one benzoxazinoid and thereby may lead to an increased pathogen resistance, preferably fungal resistance, in at least one plant cell, tissue, organ, whole plant, or plant material. In yet a further aspect according to the present invention there is thus provided use of a substance a for increasing pathogen resistance, preferably fungal resistance, in at least one plant cell, tissue, organ, whole plant, or plant material. The substance may act as a plant protective agent and may be applied to a plant exogenously, or the substance may be a scavenger of any plant molecule or material, or the substance may act as a modulator of WAK, of the downstream signalling cascade, or of a cellular pathway disclosed herein being related to the WAK signalling pathway, preferably a BXD and/or jasmonic acid biosynthesis pathway, or the substance may act on the transcription of any gene involved in the WAK or an associated pathway as disclosed herein, wherein the substance can thus directly or indirectly influence, preferably decrease, the amount of a BXD compound produced and stored in a plant cell. Thereby the use of the substance according to the present invention will lead to a decreased BXD level and thus an increased fungal resistance in a plant cell, tissue, organ, or whole plant of interest.

[0177] Delivery Methods:

[0178] A variety of suitable delivery techniques suitable according to the methods of the present invention for introducing genetic material into a plant cell are known to the skilled person., e.g. by choosing direct delivery techniques ranging from polyethylene glycol (PEG) treatment of protoplasts (Potrykus, Ingo, et al. “Direct gene transfer to cells of a graminaceous monocot.” Molecular and General Genetics MGG 199.2 (1985): 183-188), procedures like electroporation (D'Halluin, Kathleen, et al. “Transgenic maize plants by tissue electroporation.” The plant cell 4.12 (1992): 1495-1505), microinjection (Neuhaus, G., et al. “Transgenic rapeseed plants obtained by the microinjection of DNA into microspore-derived embryoids.” Theoretical and Applied Genetics 75.1 (1987): 30-36), silicon carbide fiber whisker technology (Kaeppler, H. F., et al. “Silicon carbide fiber-mediated stable transformation of plant cells.” Tag Theoretical and Applied Genetics 84.5 (1992): 560-566), viral vector mediated approaches (Gelvin, Nature Biotechnology 23, “Viral-mediated plant transformation gets a boost”, 684-685 (2005)) and particle bombardment (see e.g. Sood et al., 2011, Biologia Plantarum, 55, 1-15).

[0179] Despite transformation methods based on biological approaches, like Agrobacterium transformation or viral vector mediated plant transformation, and methods based on physical delivery methods, like particle bombardment or microinjection, have evolved as prominent techniques for introducing genetic material into a plant cell or tissue of interest. Helenius et al. (“Gene delivery into intact plants using the Helios™ Gene Gun”, Plant Molecular Biology Reporter, 2000, 18 (3):287-288) discloses a particle bombardment as physical method for introducing material into a plant cell. Currently, there thus exists a variety of plant transformation methods to introduce genetic material in the form of a genetic construct into a plant cell of interest, comprising biological and physical means known to the skilled person on the field of plant biotechnology and which can be applied to introduce at least one gene encoding at least one wall-associated kinase into at least one cell of at least one of a plant cell, tissue, organ, or whole plant. Notably, said delivery methods for transformation and transfection can be applied to introduce the tools of the present invention simultaneously. A common biological means is transformation with Agrobacterium spp. which has been used for decades for a variety of different plant materials. Viral vector mediated plant transformation represents a further strategy for introducing genetic material into a cell of interest. Physical means finding application in plant biology are particle bombardment, also named biolistic transfection or microparticle-mediated gene transfer, which refers to a physical delivery method for transferring a coated microparticle or nanoparticle comprising a nucleic acid or a genetic construct of interest into a target cell or tissue. Physical introduction means are suitable to introduce nucleic acids, i.e., RNA and/or DNA, and proteins. Likewise, specific transformation or transfection methods exist for specifically introducing a nucleic acid or an amino acid construct of interest into a plant cell, including electroporation, microinjection, nanoparticles, and cell-penetrating peptides (CPPs). Furthermore, chemical-based transfection methods exist to introduce genetic constructs and/or nucleic acids and/or proteins, comprising inter alia transfection with calcium phosphate, transfection using liposomes, e.g., cationic liposomes, or transfection with cationic polymers, including DEAD-dextran or polyethylenimine, or combinations thereof. Said delivery methods and delivery vehicles or cargos thus inherently differ from delivery tools as used for other eukaryotic cells, including animal and mammalian cells and every delivery method has to be specifically fine-tuned and optimized so that a construct of interest for introducing and/or modifying at least one gene encoding at least one wall-associated kinase in the at least one plant cell, tissue, organ, or whole plant; and/or can be introduced into a specific compartment of a target cell of interest in a fully functional and active way. The above delivery techniques, alone or in combination, can be used for in vivo (in planta) or in vitro approaches.

[0180] In one embodiment, a regulatory sequence according to the present invention may be a promoter sequence, wherein the editing or mutation or modulation of the promoter comprises replacing the promoter, or promoter fragment with a different promoter (also referred to as replacement promoter) or promoter fragment (also referred to as replacement promoter fragment), wherein the promoter replacement results in any one of the following or any one combination of the following: an increased promoter activity, an increased promoter tissue specificity, a decreased promoter activity, a decreased promoter tissue specificity, a new promoter activity, an inducible promoter activity, an extended window of gene expression, a modification of the timing or developmental progress of gene expression in the same cell layer or other cell layer, for example, extending the timing of gene expression in the tapetum of anthers, a mutation of DNA binding elements and/or a deletion or addition of DNA binding elements. The promoter (or promoter fragment) to be modified can be a promoter (or promoter fragment) that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited. The replacement promoter or fragment thereof can be a promoter or fragment thereof that is endogenous, artificial, pre-existing, or transgenic to the cell that is being edited.

[0181] The present invention will now be illustrated by the following Examples, which are not construed to limit the scope of the present invention.

EXAMPLES

Example 1: Plant Material and Growth Conditions

[0182] Seventeen maize inbred lines were used, including: (1) historical cultivars B37 and w22, and the NILs B37Htn1 and w22Htn1 that contain the NCLB resistance gene Htn1; (2) Breeding line RP3 and its NIL line RP3Htn1 carrying Htn1 from KWS (see US 2016/0201080 A1); (3) three pairs of mutants RLK1b (S, compromising Htn1 resistance), RLK1d and RLK1f, and corresponding sister lines RLK1b-wt (R, carrying functional Htn1), RLK1d-wt, and RLK1f-wt, which were produced by EMS-mutagenesis in RP3Htn1 (Hurni et al. 2015); (4) three maize mutants (bx1, bx2 and bx6) and parental line w22, which were provided by Prof. Georg Jander (Cornell University, Ithaca, US). Two or three maize seeds were sown in each Jiffy pot (o 8 cm), and fifteen pots were placed in one tray. Seedling plants were grown in a greenhouse condition of 16 h at 20° C. in the day, 8 h at 18° C. in the night and approximately 60% relative humidity.

Example 2: NCLB Infection Tests in the Greenhouse

[0183] Testing for NCLB resistance using E. turcicum isolate Passau-1 was performed as previously described with minor modification (Hurni et al. 2015). After the second leaves had fully emerged, the newly emerged leaves were cut and removed until the end of each test experiment. Single spore inoculation and culture on PDA medium plate, harvest and quantification of progeny spores were described (Hurni et al. 2015). Instead of infection by dropping 80 μl spore suspension into the leaf sheath of the second leaf twice, here maize seedlings were infected once by spray (sprayer: ø 28 mm, Semadeni, Ostermundigen, Switzerland). Each 4 trays (ca. 60-80 seedlings) were sprayed with 4 ml of spore suspension (4.5×10.sup.4 spores/ml). A very high humidity mic-condition was produced by placing plastic hoods on top of each tray after infection. Each plant was scored for disease symptom between 11 and 25 days and the severity was evaluated by calculating the area under the disease progress curve (AUDPC) or by quantifying the diseased leaf area of the inoculated second leaves (PrimDLA). About 15 seedling plants were scored in each genotype in each experiment.

Example 3: Vector Construction and Subcellular Localization

[0184] The coding sequence of ZmWAK-RLK1 was amplified using a cDNA clone as template, which was initially amplified in NCLB resistance genotype RP1Htn1 (Hurni et al. 2015). The PCR fragment was introduced into the Gateway donor vector pDONR207 using the Gateway® BP Clonase® II Enzyme mix (Thermo Fisher Scientific, Wilmington, USA). The generated entry vector carrying the target ZmWAK-RLK1 sequence was inserted by recombination with the destination vector pUBC-GFP-DEST, to produce an in-frame ZmWAK-RLK1+c'-eGFP fusion protein construct driven by Arabidopsis ubiqutin-10 (UBQ10) gene promotor (Grefen, Christopher, et al. “A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies.” The Plant Journal 64.2 (2010): 355-365). The UBQ10::ZmWAK-RLK1-c'-eGFP construct (SEQ ID NO:9) together with a reference plasmid PIP2A-mCherry (Cutler et al., Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc. Natl Acad. Sci. USA 97, 3718-3723, (2000) contains 35S::PIP2A_c'_RFP construct, which is localized to the plasma membrane) were mixed with nanograde gold particles and co-bombarded into onion epidermal cells, which were subsequently incubated at 20° C. in the dark for 2-3 days until being ready for observation using a confocal microscope. Plasmolysis was induced by adding a 0.8 M mannitol solution. Furthermore, both plasmids were transformed into Agrobacterium GV3101 and co-infiltrated into 4-week-old N. bentaniana leaves, which were ready for observation 2 days post infiltration. The primers used for vector construct are provided in the below Table 1.

TABLE-US-00001 TABLE 1 Forward (F) and reverse (R) primers used for vector constructs PCR efficiency (E) R.sup.2 of Order Target genes Primers (5′ to 3′) Calibration curve slope Description 1 Actin F—SEQ ID NO:28 E = 109.8%, R.sup.2 = 0.995, Reference gene R—SEQ ID NO:29 Slope = −3.107 2 FPGS F—SEQ ID NO:30 E = 104.9%, R.sup.2 = 0.990, Reference gene R—SEQ ID NO:31 Slope = −3.209 3 ZmWAK-RLKI F—SEQ ID NO:32 E = 104.0%, R.sup.2 = 0.996, WAK-RLK1 = R—SEQ ID NO:33 Slope = −3.229 ZmWAK-RLKI herein. 4 BX1 (BX: benzoxazinless) F—SEQ ID NO:34 E = 100.1%, R.sup.2 = 0.994, Benzoxazinoid R—SEQ ID NO:35 Slope = −3.319 pathway 5 BX2 F—SEQ ID NO:36 E = 126.1%, R.sup.2 = 0.989, Benzoxazinoid R—SEQ ID NO:37 Slope = −2.821 pathway 6 BX3 F—SEQ ID NO:38 E = 107.4%, R.sup.2 = 0.994, Benzoxazinoid R—SEQ ID NO:39 Slope = −3.156 pathway 7 BX4 F—SEQ ID NO:40 E = 100.9%, R.sup.2 = 0.994, Benzoxazinoid R—SEQ ID NO:41 Slope = −3.300 pathway 8 BX5 F—SEQ ID NO:42 E = 109.7%, R.sup.2 = 0.992, Benzoxazinoid R—SEQ ID NO:43 Slope = −3.109 pathway 9 BX6 F—SEQ ID NO:44 E = 107.6%, R.sup.2 = 0.961, Benzoxazinoid R—SEQ ID NO:45 Slope = −3.153 pathway 10 BX7 F—SEQ ID NO:46 E = 106.9%, R.sup.2 = 0.989, Benzoxazinoid R—SEQ ID NO:47 Slope = −3.167 pathway 11 BX8 F—SEQ ID NO:48 E = 114.0%, R.sup.2 = 0.987, Benzoxazinoid R—SEQ ID NO:49 Slope = −3.027 pathway 12 BX9 F—SEQ ID NO:50 E = 117.3%, R.sup.2 = 0.999, Benzoxazinoid R—SEQ ID NO:51 Slope = −2.967 pathway 13 BX10 + BX11 F—SEQ ID NO:52 E = 109.5%, R.sup.2 = 0.999, Benzoxazinoid R—SEQ ID NO:53 Slope = −3.114 pathway 14 BX12 F—SEQ ID NO:54 E = 95.4%, R.sup.2 = 0.996, Benzoxazinoid R—SEQ ID NO:55 Slope = −3.437 pathway 15 BX13 F—SEQ ID NO:56 E = 99.3%, R.sup.2 = 0.961, Benzoxazinoid R—SEQ ID NO:57 Slope = −3.340 pathway 16 IGL (Indole Glycerol F—SEQ ID NO:58 E = 111.8%,R.sup.2 = 0.996, Benzoxazinoid Phosphate Lyase) R—SEQ ID NO:59 Slope = −3.069 pathway 17 GLU1 (GLU: beta F—SEQ ID NO:60 E = 110.6%, R.sup.2 = 0.998, Benzoxazinoid glucosidase) R—SEQ ID NO:61 Slope = −3.092 pathway 18 GLU2 F—SEQ ID NO:62 E = 110.4%, R.sup.2 = 0.985, Benzoxazinoid R—SEQ ID NO:63 Slope = −3.095 pathway 19 ZmWAK-RLK1 F—SEQ ID NO:64 plasmid construction R—SEQ ID NO:65

Example 4: Mycelium Development

[0185] The second leaves of 21-day seedling plants were harvested and cut into 2×2 cm.sup.2 leaf segments, which were placed and incubated on the phytoagar plates. A spore suspension (4.5×10.sup.4 spores/ml) was painted using swabs on the leaf surface. The petri dishes carrying samples were sealed using PARAFILM and incubated 24 hours at room temperature until harvest. Trypan blue straining was conducted as previously described (Chung. Chia-Lin. et al. “Resistance loci affecting distinct stages of fungal pathogenesis: use of introgression lines for QTL mapping and characterization in the maize-Setosphaeria turcica pathosystem.” BMC plant biology 10.1 (2010): 103). The infected segments at 1 dpi were incubated overnight in an acetic acid:ethanol (1:3, v/v) solution, and then in a mixed solution of acetic acid:ethanol:glycerol (1:5:1, v/v/v) for 4 hours. The samples were stained overnight in 0.01% (w/v) trypan blue lactophenol solution, and then washed once using ddH.sub.2O and stored in 60% glycerol ready for use. Specimens were placed on slides and examined under the ZEISS Axio Imager 2 microscope system (CARL ZEISS, Jena, Germany). The numbers of germinated spores, germ tubes, appressoria and successful penetrations (hyphae inside of cell or between cell walls) were counted. Three independent experiments were performed.

Example 5: RNA Extraction, RNA Sequencing and Data Analysis

[0186] The second leaves of seedling plants were harvested with four biological replicates at 0, 9-hpi, 3-dpi and 10-dpi, which corresponded to before inoculation, the germination/penetration, biotrophic growth and necrotrophic growth, respectively (Jennings, P. R., and A. J. Ullstrup. “A HISTOLOGICAL STUDY OF 3 HELMINTHOSPORIUM LEAF BLIGHTS OF CORN.” Phytopathology 47.12 (1957): 707-714; Hilu, H. M., and A. L. Hooker. “Host-pathogen relationship of Helminthosporium turcicum in resistant and susceptible corn seedlings.” (1964): 570-5). Forty-eight samples (4 genotypes, 4 time points, 3 biological replicates) were subjected for total RNA extraction using SV Total RNA Isolation Kits (Promega, Dübendorf, Switzerland). 1 μl of total RNA was checked by Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA) to estimate the RNA concentration. Meanwhile, 15 plants in each genotype were evaluated for the AUDPC value to control if the infection worked.

[0187] The quantity and quality in RNA sequencing were determined using Qubit® 1.0 Fluorometer (Thermo Fisher Scientific, Wilmington, USA) and Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., Hayward, USA) was used for library preparation. 1 μg of total RNA per sample was ribosome depleted and then subjected for synthesizing double-strand cDNA. Each cDNA sample was fragmented, end-repaired, polyadenylated and then ligated with TruSeq adaptor that contains the index for multiplexing. The cDNA fragments containing TruSeq adapters at the both ends were enriched with PCR reaction. The enriched libraries were quantified and qualified, and then normalized to 10 nM. The TruSeq SR Cluster Kit v4 cBot (Illumina, Inc., Hayward, USA) was used for cluster generation using 8 pM of pooled normalized libraries. Sequencing was performed on the Illumina HiSeq2500 at single end 125 bp using the TruSeq SBS Kit v4 (Illumina, Inc., Hayward, USA).

[0188] The maize reference genome Zea_mays.AGPv3.27 and the corresponding annotation were downloaded (maizegdb.org/). The RNA sequencing reads were mapped on the reference genome with STAR (Dobin, Alexander, et al. “STAR: ultrafast universal RNA-seq aligner.” Bioinformatics 29.1 (2013): 15-21) allowing one mismatch per 100 bp and no multimapper with the following command: STAR—outFilterMultiMapNmax 1—outFilterMismatchNoverLmax 0.01—alignIntronMax 10000. Read counts were determined from the mapping files with featureCounts 1.4.6 (Liao, Yang, Gordon K. Smyth. and Wei Shi. “featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.” Bioinformatics 30.7 (2013): 923-930). Statistical analyses were done with the R package edgeR and genes were tested for differential expression with pairwise comparisons and tagwise estimation of dispersion. A gene was considered to be expressed when at least 10 reads were mapped on it and a gene was considered to be differentially expressed with log.sub.2FC≤|2| and FDR <0.01. First, pairwise comparisons were performed between Htn1 and no Htn1 plants for each genotype and each time points separately. The results were then compared between time points and then between the two genotypes. The Gene Ontology analysis for differentially expressed genes (DEGs) was conducted by using online software agriGO (Du, Zhou, et al. “agriGO: a GO analysis toolkit for the agricultural community.” Nucleic acids research 38.suppl_2 (2010): W64-W70). The significant terms were colored if adjusted p≤0.05.

Example 6: RT-qPCR Assay

[0189] 1 μg total RNA was subjected for first strand cDNA synthesis using the iScript Advanced cDNA kit (172-5038, Rio-Rad). 1:20 diluted cDNA was applied for quantifying expression using a Real-Time System C1000™ Thermal cycler (% or 384 wells, Bio-Rad). The expression of targets was normalized by the reference genes FPGS and Actin as described (Hurni et al. 2015). The primers for expression analysis are shown in Table 1 above.

Example 7: Benzoxazinoids (BXDs) Extraction and Measurement

[0190] 60-100 mg leaves (without veins) of the seedling plants were harvested and freezing immediately in liquid nitrogen, grinded and added the extraction buffer (1 mg sample+10 μl extraction buffer). The samples were mixed thoroughly and centrifuged at 13,000 rpm under 4° C. The supernatant was transferred into new tube and centrifuged once more under same condition, to remove the possible leaf particles. The supernatant was collected being ready for BXDs measurement.

[0191] Benzoxazinoid contents were analyzed by an Acquity UPLC equipment (Waters) coupled to a UV detector and coupled to a mass spectrometer (Waters) (Meihls, Lisa N., et al. “Natural variation in maize aphid resistance is associated with 2, 4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one glucoside methyltransferase activity.” The Plant Cell Online 25.6 (2013): 2341-2355). An Acquity BEH C18 column (Waters) was used. The temperatures of the autosampler and column were 15° C. and 40° C., respectively. The mobile phase consisted of 99% water, 1% acetonitrile, and 0.1% Formic acid (A) and acetonitrile and 0.1% Formic acid (B). Flow rate was set to 0.4 ml min.sup.−1 with 3% A and 97% B followed by column reconditioning. The injection volume was 5 μl. The extracted trace at 275 nm was used for benzoxazinoids quantification. The following extracted ion chromatograms were used for quantification with a mass window of 0.01 D: mass-to-charge ratio (m/z) for DIMBOA (retention time [RT] 5.62 min) and DIMBOA-Glc (RT 5.64 min), m/z for HDMBOA-Glc (RT 8.19 min), m/z for HMBOA-Glc (RT 5.34 min) and DIM.sub.2BOA-Glc (RT 5.825 min). Benzoxazinoids absolute concentrations were determined by external calibration curves obtained from purified DIMBOA-Glc, DIMBOA and HDMBOA-Glc standards.

Example 8: ZmWAK-RLK1 Encodes a Plasma Membrane Localized Protein

[0192] To determine the subcellular localization of the ZmWAK-RLK1 protein, a fusion construct consisting of a full-length coding sequence fused to the sequence of an enhanced green fluorescence protein (eGFP) at the C terminus was generated (cf. SEQ ID NO:9 for the nucleic acid plasmid construct). The ZmWAK-RLK1 fusion protein localized to the plasma membrane before and after plasmolysis when transiently expressed in onion epidermal cells. Furthermore, infiltration into leaves of Nicotiana benthamiana confirmed the localization of ZmWAK-RLK1 to the plasma membrane two days after infiltration (cf. FIG. 12). These data, particularly confocal analysis of onion epidermal cells after transient expression of ZmWAK-RKL1-eGFP and the positive control PIP2A-mCherry that is known to localize to the plasma membrane demonstrate that ZmWAK-RLK1 is a plasma membrane protein.

Example 9: ZmWAK-RLK1 Reduced Fungal Penetrations

[0193] Spores of the hemibiotrophic fungus E. turcicum penetrate the maize epidermis mostly between 6-18 hours after inoculation (hpi) (Jennings and Ullstrup, 1957). To investigate if ZmWAK-RLK1 changes the outcome of fungal penetration attempts, we investigated the infection process at one day post inoculation (dpi) using trypan blue staining (data not shown). The number of successful penetration events were evaluated in three EMS-induced ZmWAK-RLK1 loss-of-function mutant lines (RLK1b, RLK1d and RLK1f) and their corresponding sister lines that were generated in the near isogenic line (NIL) RP3Htn1 (Hurni et al. 2015). No significant difference in the establishment of germ tubes and appressoria was observed in genotypes with/without ZmWAK-RLK1 (data not shown). In contrast, the number of successful penetration events was significantly lower if ZmWAK-RLK1 was functional compared to loss-of-function mutants as demonstrated in FIGS. 2 A and B. This indicates that ZmWAK-RLK1 leads to a reduction of pathogen penetrations into host tissues.

Example 10: Transcriptome and Metabolism Analysis Identified Alterations to the BXDs Biosynthesis Pathway in the Presence of ZmWAK-RLK1

[0194] To decipher the immune network specifically influenced by ZmWAK-RLK1, we performed a transcriptome analysis by RNA sequencing in two pairs of near isogenic lines, w22 and W22Htn1 as well as B37 and B37Htn1. NCLB development was significantly reduced in the presence of ZmWAK-RLK1 in both NILs (FIG. 3 A-C). Leaf samples were collected at 0 and 9 hpi (penetration stage) as well as 3 (biotrophic growth) and 10 dpi (necrotrophic growth) (Jennings and Ullstrup, 1957). Forty-eight samples were sequenced and 1.159 billion reads were obtained (Table 2). More than 820 million reads were uniquely mapped with an average of 17.08 million reads per sample (70.7%) (Table 2). No obvious difference of percentage of mapped reads in genotypes with or without (was observed. A total of 15,345 genes were expressed and they were used for further analysis. By conducting a multidimensional scaling (MDS) analysis using expression normalized by edgeR, the biological replicates for the same genotype-timepoints combination were mostly grouped together, suggesting the repeatability of replicates (data not shown). More differentially expressed gene (DEGs) were detected in B37Htn1/B37 compared to w22Htn1/w22 (FIG. 4). These genes differently expressed in both NILs in at least one of timepoints. To identify DEGs associated with ZmWAK-RLK1 and to rule out genetic background effects, only genes that were differentially expressed in both NIL pairs were considered.

TABLE-US-00002 TABLE 2 Statistics of RNA-seq reads sequenced and mapped Percentage Percentage Uniquely of uniquely of multi- Percentage Sample mapped mapped mapped of unmapped number Samples Raw reads reads.sup.a reads (%) reads (%) reads (%) 1 0-W22-1 19,894,158 12,757,882 64.13 12.66 20.34 2 0-w22-2 25,718,662 18,612,570 72.37 6.63 20.18 3 0-w22-3 24,937,383 17,948,211 71.97 6.41 20.85 4 0-w22Htn1-l 27,452,042 20,264,307 73.82 5.96 19.70 5 0-w22Htn1-2 19,815,200 14,439,891 72.87 6.07 20.44 6 0-w22Htn1-3 24,173,527 16,215,534 67.08 9.55 21.40 7 0-B37-1 22,180,888 14,689,311 66.23 5.27 28.03 8 0-B37-2 24,474,895 18,273,286 74.66 5.96 18.75 9 0-B37-3 27,774,311 20,221,020 72.80 6.73 19.74 10 0-B37Htn1-l 26,728,646 19,277,404 72.12 6.16 21.04 11 0-B37Htn1-2 24,336,731 17,640,859 72.49 6.06 20.82 12 0-B37Htn1-3 22,625,055 16,499,617 72.93 6.15 20.30 13 9h-w22-l 25,343,283 17,460,901 68.90 7.81 22.49 14 9h-w22-2 26,384,078 18,489,206 70.08 7.16 22.03 15 9h-w22-3 21,846,924 13,680,343 62.62 5.97 30.81 16 9h-w22Htn1-l 34,074,874 23,121,230 67.85 6.27 25.40 17 9h-w22Htn1-2 24,276,403 17,299,847 71.26 7.02 21.07 18 9h-w22Htn1-3 30,200,422 20,381,456 67.49 7.23 24.62 19 9h-B37-l 17,962,777 12,878,288 71.69 7.38 20.10 20 9h-B37-2 23,815,810 16,892,476 70.93 7.12 21.22 21 9h-B37-3 24,188,988 17,604,433 72.78 7.15 19.38 22 9h-B37Htn1-l 25,195,971 17,304,100 68.68 7.80 22.65 23 9h-B37Htn1-2 23,902,398 16,173,755 67.67 7.27 24.40 24 9h-B37Htn1-3 24,731,481 17,279,056 69.87 7.13 22.39 25 3d-w22-l 22,399,000 15,772,632 70.42 6.44 22.49 26 3d-w22-2 26,175,191 18,816,409 71.89 6.43 21.06 27 3d-w22-3 23,253,678 16,758,123 72.07 6.45 20.82 28 3d-w22Htn1-l 23,878,639 17,185,076 71.97 5.92 21.58 29 3d-w22Htn1-2 20,568,384 14,338,420 69.71 7.09 22.35 30 3d-w22Htn1-3 34,008,596 24,127,141 70.94 5.81 22.76 31 3d-B37-l 23,690,913 14,726,042 62.16 4.70 32.71 32 3d-B37-2 27,646,350 19,934,380 72.10 5.89 21.42 33 3d-B37-3 21,114,803 16,208,484 76.76 5.69 16.98 34 3d-B37Htn1-l 21,758,134 15,409,264 70.82 5.68 23.03 35 3d-B37Htn1-2 27,236,811 19,866,766 72.94 5.84 20.68 36 3d-B37Htn1-3 22,700,637 16,553,064 72.92 5.60 21.00 37 10d-w22-l 23,051,842 16,566,330 71.87 6.16 21.24 38 10d-w22-2 28,611,557 20,463,896 71.52 6.01 21.77 39 10d-w22-3 12,829,435 7,480,564 58.31 4.82 36.38 40 10d-w22Htn1-l 24,435,356 17,199,294 70.39 5.45 23.76 41 10d-w22Htn1-2 28,996,022 21,442,119 73.95 5.78 19.69 42 10d-w22Htn1-3 18,649,090 13,429,238 72.01 5.78 21.71 43 10d-B37-l 13,275,463 9,658,508 72.75 5.46 21.16 44 10d-B37-2 15,217,491 11,288,257 74.18 5.38 19.89 45 10d-B37-3 23,349,541 17,096,788 73.22 5.36 20.83 46 10d-B37Htn1-l 20,104,137 14,270,397 70.98 4.94 23.79 47 10d-B37Htn1-2 38,203,107 27,216,898 71.24 5.40 22.82 48 10d-B37Htn1-3 26,094,601 18,820,165 72.12 5.61 21.68 Total 1,159,283,685 820,033,238 70.74 NA.sup.b NA.sup.b Mean 24,151,743 17,084,026 70.74 NA.sup.b NA.sup.b .sup.aParameters for mapping: less than 1% mismatch, 1 locus mapped, intron size is less than 10 kb; .sup.bNA = not analyzed.

[0195] Two-hundred and fifteen common DEGs were identified across all time points (FIG. 13). 132 and 83 genes were induced and repressed in the ZmWAK-RLK1-containing NIL compared to the respective susceptible line (heat map and Venn diagram not shown). 108 DEGs were only found at timepoint 0 before inoculation, while 107 of DEGs were found only after infection. Twenty-nine DEGs were differently expressed at all time points including timepoint. An overrepresentation analysis using agriGO was performed to identify enriched Gene Ontology (GO) terms associated with ZmWAK-RLK1. The functional groups were enriched for terms of defense response (e.g. GO:0009814) and metabolic/biosynthetic process (e.g. GO:0006725) (data not shown).

[0196] To further analyze if the presence of ZmWAK-RLK1 is associated with BXDs biosynthesis (FIG. 5 A), the content of major BXDs in second leaves of w22Htn1 and w22 before and after infection was quantified (FIG. 5 B to F). The content of four BXDs DIMBOA-Glc, DIMBOA, HMBOA-Glc and DIM.sub.2BOA-Glc was significantly lower in w22Htn1 compared to w22 at all timepoints (data not shown), which indicated a constitutive reduction on BXDs content when ZmWAK-RLK1 is present. Furthermore, via RT-qPCR the transcriptional levels of ZmWAK-RLK1 and specifically the genes in the BXDs biosynthesis pathway before and after pathogen inoculation were determined. The expression of genes (A) ZmWAK-RLK1, (B) Bx1, (C) Ig1, (D) Bx2, (E) Bx3, (F) Bx4, (G) Bx5, (H) Bx6, (I) Bx7, (J) Bx8, (K) Bx9, (L) Bx10/11, (M) Bx12, (N) Bx13, (O) Glu1 and (P) Glu2 at different timepoints before and after infection was measured and statistics were conducted separately in w22 and B37 genetic background using Tukey's HSD (P=0.05) in four biological replicates.

[0197] The ZmWAK-RLK1 expression in NILs showed no significant difference (FIG. 11 A). Overall, the transcriptional levels of BXD biosynthesis genes were genotype-specific (FIG. 11 A to P)). For instance, BX1 and its protein homolog IGL can convert indole-3-glycerol phosphate into indole, which is the first step of BXD metabolism (Frey et al. 2000). Their coding genes Bx1 and Ig1 showed opposite contributions of gene expression in B37 and w22, but the combined expression of Bx and Ig1 was consistently lower in genotypes with ZmWAK-RLK1 (FIGS. 11 B and C).

Example 11: Mutations in ZmWAK-RLK1 is Associated with the Reduction of Secondary Metabolite DIM.SUB.2.BOA-Glc

[0198] To further analyze the role of different BXD biosynthesis genes as well as the metabolites of this pathway in NCLB resistance, the ZmWAK-RLK1 mutants (RLK1b, d and f, SEQ ID NOs: 3 to 6 and G548R mutant of SEQ ID NO:2) and their sister lines were used (Hurni et al. 2015). The transcript levels of several BXD genes were quantified and the content of major BXD compounds in mutants which lost the resistance caused by ZmWAK-RLK1 (FIG. 6 A to D). In contrast to the NILs, there was no difference in the DIMBOA-Glc, DIMBOA and HMBOA, HDMBOA-Glc concentration. However, the content of DIM.sub.2BOA-Glc was significantly lower when ZmWAK-RLK1 was intact (FIG. 6 A, and further data not shown). The reduced DIM.sub.2BOA-Glc correlated with a reduction in Ig1 transcript levels, while no obvious difference in the transcriptional levels of ZmWAK-RLK1 and Bx1 was detected in ZmWAK-RLK1 (FIG. 6 B to D) Bx6, Bx7 and Bx13 are key genes of the BXDs pathway to produce DIM.sub.2BOA-Glc, and these genes were slightly but not significantly upregulated in mutants (data not shown). This phenomenon can be explained by a feedback regulation. Therefore, ZmWAK-RLK1 clearly seems to be associated with the reduction of secondary metabolite DIM.sub.2BOA-Glc, possibly by reducing the expression levels of Bx and/or Ig1.

Example 12: Mutations in BXDs Biosynthesis Genes Decreased NCLB Susceptibility

[0199] To further analyze the role of BXDs in NCLB resistance/susceptibility, mutants in the three BXD biosynthesis genes Bx1, Bx2 and Bx6 were tested upon inoculation with E. turcicum. These mutants showed strong reduction in several BXDs compounds, including DIM.sub.2BOA-Glc (FIG. 7). Interestingly, all three mutants showed a strong reduction of NCLB susceptibility at the seedling stage (FIGS. 8 A and B). This confirmed a negative correlation of BXDs content and NCLB disease resistance. Furthermore, the ZmWAK-RLK1 expression at 10 dpi was checked (FIG. 8 C). No significant difference was detected in the bx mutants if compared to the wild-type. Considering the reduction of Bx and Ig1 co-expression in ZmWAK-RLK1 genotypes (FIG. 6 A, FIGS. 5 B and C, FIG. 9 B), the data suggest that the BXDs synthesis pathway likely acts downstream of ZmWAK-RLK1.

[0200] Therefore, ZmWAK-RLK1 underlying quantitative NCLB disease resistance is based on a decrease of the biosynthesis of secondary metabolite BXDs, and DIM.sub.2BOA-Glc served as a candidate susceptibility component for promoting fungal infection (FIG. 8 D).

Example 13: Tissue-Specific Expression of WAK-RLK1

[0201] Further to the determination of the subcellular localization of a ZmWAK-RLK1 protein (see Example 8 above), the tissue specific expression of RLK1 in different genotypes, and different time points was determined. The genes FPGS and ACTIN served as reference to normalize the expression. The results shown in FIG. 10 demonstrate that RLK1 is a ubiquitously and steadily expressed gene which further underpins the function of said kinase as master regulator in relevant plant metabolic pathways.

Example 14: TILLING

[0202] To develop and screen a mutant population of plant material, TILLING was performed. A TILLING mutant population can be created, e.g., starting from KWS line RP3Htn1 according to Kato (2000, The maize handbook, pp. 212-219)). Pollen is harvested from field-grown RP3Htn1 plants and treated with 0.1% EMS solution for 45 min. Silks of individual plants are then pollinated and emerging ears bagged. From 436 pollinated M0 plants seeds were harvested, in one experiment. An additional propagation and selfing led to 10,084 individual M1 plants. Leaf material from these M1 plants was collected for DNA isolation. DNA of dried leaf samples (10 leaf discs bunches/sample) was isolated from 10,000 M1 individuals with the CTAB extraction method (Traitgenetics, Gatersleben, Germany). DNA is then aliquoted to 100 μl with 20 ng/μl. Primer development for mutant screening is performed. The amplification assay consisted of 20 ng/μl DNA, 5× GoTaq-Buffer, 25 μM dNTPs, 10 μM forward Primer, 10 μM reverse Primer, 5 Units/μl GoTaq. After denaturation for 300 s at 94° C. the amplification cycles were performed with 35 cycles of 60 s at 94° C., 60 s at 60° C. and 60 s at 72° C. followed by a final elongation time for 600 s at 72° C. Next, Sanger-sequencing of PCR products is performed according to established protocols. Sequences are then assembled, for example with the help of the software Lasergene Seqman NGen (DNASTAR) and heterozygote SNPs called with the software default settings. Positive mutant plants were sequenced again with the Sanger-method in order to confirm the polymorphism.