Maize resistance to Fusarium verticillioides by CRISPR/CAS9 gene editing approach of a WRKY gene and overexpression of a lipoxygenase gene

The maize WRKY transcription factors and the lipoxygenases (ZmLOXs) are well recognized as\r\nimportant players in plant defense against pathogens. In this regard, previous RNA-seq\r\nexperiments reported the enhanced expression of ZmLOX genes in maize resistant genotypes\r\nand GWAS resulted in one SNP significantly associated with ZmWRKY125.\r\nThe Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9)\r\nediting of ZmWRKY125 and the transgenic overexpression of ZmLOX4 genes were carried out\r\nto investigate the possible implication of these two genes in the resistance mechanisms against F.\r\nverticillioides.\r\nAs regards ZmWRKY125, the CRISPR cloning was based on a double cloning using two sgRNA\r\nfor one gene target. Agrobacterium tumefaciens mediated transformation was used for\r\nintroducing in maize A188 line the construct under the maize promoter ZmpUBI in the binary\r\nvector p1609. Mutants from three different transformation events were obtained. For each event,\r\nT2 plants will be genotyped to find homozygous for the mutation that in turn will be phenotyped\r\nfor F. verticillioides resistance and fumonisin content.\r\nAs regards ZmLOX4, the gene was cloned under a promoter involved in kernel development in\r\nthe vector L1781, and the same transformation conditions adopted for the CRISPR/Cas9 editing\r\nof ZmWRKY125 were used. Mutants from two different transformation events were obtained. For\r\neach event, T2 plants were genotyped in order to find homozygous for the mutation.\r\nHomozygous plants will be further evaluated for F. verticillioides resistance, fumonisin accumulation,\r\noxylipin content as well as for the expression analysis of the main genes involved in the jasmonic\r\nacid pathway.