THE CRISPR/CAS9 EDITING OF A WRKY GENE AND THE OVEREXPRESSION OF A LIPOXYGENASE GENE FOR IMPROVING PATHOGEN RESISTANCE IN MAIZE

Fusarium verticillioides (Fv) is a major cereal pathogen causing stalk rot\r\nand ear rot in maize, negatively affecting crop productivity, and\r\ncompromising food safety by producing the secondary metabolites fumonisins.\r\nSeveral studies were conducted to identify maize genes associated with host\r\nplant resistance to Fv infection and fumonisin accumulation. The maize WRKY\r\ntranscription factors and the lipoxygenases (ZmLOXs) are well recognized as\r\nimportant players in plant defense against pathogens, and it is known that\r\nthe host-pathogen lipid cross-talk influences the pathogenesis. In this\r\nregard, previous RNA-seq experiments reported the enhanced expression of\r\nZmLOX genes in maize resistant genotypes and GWAS resulted in one SNP\r\nsignificantly associated with ZmWRKY125.\r\nThe Clustered Regularly Interspaced Short Palindromic Repeat/associated\r\nCas9 (CRISPR/Cas9) editing of ZmWRKY125 and the transgenic overexpression\r\nof ZmLOX4 genes were carried out to investigate the possible implication of\r\nthese two genes in the resistance mechanisms against Fv. Before cloning\r\nexperiments, protein domain conservation and different splicing products\r\nhave been analyzed comparing homologues and orthologues for both genes.\r\nAs regards ZmWRKY125, the CRISPR cloning was based on a double cloning\r\nusing two different guides (sgRNA) for one gene target. Agrobacterium\r\ntumefaciens mediated transformation was used for introducing the construct\r\nunder the maize promoter ZmpUBI in the binary vector p1609 in maize A188\r\nline. Mutants from three different transformation events were obtained. For\r\neach event, T2 plants will be genotyped to find homozygous for the mutation\r\nthat in turn will be phenotyped for Fv resistance and fumonisin content.\r\nAs regards ZmLOX4, the gene was cloned under an overexpressed promoter\r\ninvolved in kernel development in the vector L1781, and the same\r\ntransformation conditions adopted for the CRISPR/Cas9 editing of ZmWRKY125\r\nwere used. Mutants from two different transformation events were obtained.\r\nFor each event, T2 plants were genotyped in order to find homozygous for\r\nthe mutation. Homozygous plants will be further evaluated for Fv\r\nresistance, fumonisin accumulation, oxylipin content as well as for the\r\nexpression analysis of the main genes involved in the jasmonic acid pathway.