Increased fungal resistance in crop plants

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.

1 .- 15 . (canceled) 
     
     
         16 . 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 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 a function 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 the at least one plant cell, tissue, organ, or whole plant;   (iii) producing a population of plants from the at least one plant cell, tissue, organ, or whole plant; and   (iv) selecting a plant having increased fungal resistance from the population of plants, optionally based on a determination of a reduced synthesis of at least one benzoxazinoid in response to a fungal pathogen infection, wherein the synthesis of the at least one benzoxazinoid is regulated by the at least one wall-associated kinase.   
     
     
         17 . The method according to  claim 16 , wherein the at least one wall-associated kinase is a WAK-RLK1 gene. 
     
     
         18 . The method of  claim 17 , 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,   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,   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,   provided that any sequence of a) to g), optionally after expression, still encodes at least one functional Htn1, Ht2, Ht3, or an allelic variant, a mutant, or a functional fragment thereof.   
     
     
         19 . The method according to  claim 16 , wherein the benzoxazinoid whose synthesis is regulated by the at least one wall-associated kinase, is selected from at least one of DIMBOA, DIMBOA, HMBOA, HM2BOA, HDMBOA, HDM2BOA, 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. 
     
     
         20 . 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. 
     
     
         21 . 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. 
     
     
         22 . The method according to  claim 16 , 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 the genes bx1 (SEQ ID NO: 10), bx2 (SEQ ID NO: 12), igl (SEQ ID NO: 14), bx6 (SEQ ID NO: 16), bxl1 (SEQ ID NO: 18), bxl4 (SEQ ID NO: 20), opr2 (SEQ ID NO: 22), lox3 (SEQ ID NO: 24) or aoc1 (SEQ ID NO: 26), or a homologous genes thereof, or the proteins BX1 (SEQ ID NO: 11), BX2 (SEQ ID NO: 13), IGL (SEQ ID NO: 15), BX6 (SEQ ID NO: 17), BX11 (SEQ ID NO: 19), BX14 (SEQ ID NO: 21), OPR2 (SEQ ID NO: 23), LOX3 (SEQ ID NO: 25) or AOC1 gene (SEQ ID NO: 27), or a homolog thereof. 
     
     
         23 . 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. 
     
     
         24 . The method according to  claim 16 , wherein the modification of the at least one gene encoding at least one wall-associated kinase within step (ii) (a) or (ii) (b) 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. 
     
     
         25 . 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 officinarium, 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, Coffea 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 cajanifolius, 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. 
     
     
         26 . 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 . 
     
     
         27 . 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.   
     
     
         28 . 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 27  into at least one cell of at least one of a plant cell, tissue, organ, or whole plant. 
     
     
         29 . 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 27  is a stable introduction. 
     
     
         30 . 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.   
     
     
         31 . The method of  claim 30 , wherein the method is effective to increase fungal resistance in at least one plant cell, tissue, organ, whole plant, or plant material.