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 important players in plant defense against pathogens. In this regard, previous RNA-seq experiments reported the enhanced expression of ZmLOX genes in maize resistant genotypes and GWAS resulted in one SNP significantly associated with ZmWRKY125. The Clustered Regularly Interspaced Short Palindromic Repeat/associated Cas9 (CRISPR/Cas9) editing of ZmWRKY125 and the transgenic overexpression of ZmLOX4 genes were carried out to investigate the possible implication of these two genes in the resistance mechanisms against F. verticillioides. As regards ZmWRKY125, the CRISPR cloning was based on a double cloning using two sgRNA for one gene target. Agrobacterium tumefaciens mediated transformation was used for introducing in maize A188 line the construct under the maize promoter ZmpUBI in the binary vector p1609. Mutants from three different transformation events were obtained. For each event, T2 plants will be genotyped to find homozygous for the mutation that in turn will be phenotyped for F. verticillioides resistance and fumonisin content. As regards ZmLOX4, the gene was cloned under a promoter involved in kernel development in the vector L1781, and the same transformation conditions adopted for the CRISPR/Cas9 editing of ZmWRKY125 were used. Mutants from two different transformation events were obtained. For each event, T2 plants were genotyped in order to find homozygous for the mutation. Homozygous plants will be further evaluated for F. verticillioides resistance, fumonisin accumulation, oxylipin content as well as for the expression analysis of the main genes involved in the jasmonic acid pathway.