EXPLORING THE ROLE OF A WRKY GENE BY CRISPR/CAS EDITING TO ENHANCE PATHOGEN RESISTANCE IN MAIZE

Maize is severely affected by Fusarium verticillioides (Fv), a major cereal pathogen that causes stalk rot and ear rot. By producing secondary metabolites known as fumonisins, Fv also compromises food safety and reduces crop productivity. Various studies have sought to identify maize genes linked to resistance in host plants against Fv infection and fumonisin accumulation. Maize WRKY transcription factors are known to be crucial in plant defense against pathogens. In this context, a previous GWAS analysis pinpointed a SNP significantly associated with ZmWRKY125. To explore the potential role of ZmWRKY125 in the resistance mechanisms against Fv, CRISPR/Cas9 gene editing was performed. Prior to cloning experiments, analyses of protein domain conservation and different splicing products were conducted by comparing homologous and orthologous genes. The CRISPR cloning process utilized a double cloning strategy with two distinct guides (sgRNA) targeting one gene. Agrobacterium tumefaciensmediated transformation was employed to introduce the construct, under the maize promoter ZmpUBI, into maize A188 line using the binary vector p1609. Mutants from two separate transformation events were successfully generated. For each event, T2 plants were genotyped by sequencing to find homozygous for the mutation. The events were selected based on sequencing results. In one event, the editing resulted in 1 bp deletion from one guide, and no mutation from the other guide. In the second event, a deletion of 48 bp and 16 bp was observed for the two guides, respectively. The two events were chosen to further perform disease assay, RNA-Seq, hormonal and fumonisin accumulation analysis. Hormonal analyses were carried out on seeds of the two WRKY-edited lines and A188 at 3 and 7 days post inoculation (dpi) with Fv and mock-inoculated. Interestingly, a significant higher accumulation of abscisic acid, jasmonic acid and cis-12- oxo-10-phytoenoic acid was measured in the two WRKY-edited lines after Fv inoculation. RNA-Seq analysis was conducted on seeds of the same genotypes collected at 3 dpi. More than 967 million of bp paired-end reads were generated for inoculated and mock-inoculated plants. Pairwise differential expression analysis was performed between the transcriptomes of the three genotypes and inoculated vs. mock-inoculated transcriptomes revealing a total number of 11,297 differentially expressed genes (DEGs). Results regarding DEG functions and their connections with hormonal pathways are currently being analyzed to better clarify resistance mechanisms towards Fv.