Exploiting Wild Genetic Resources: Characterization of PR Genes from Sinapis alba for Resistance to Alternaria Blight

Alternaria blight, caused by the necrotrophic fungi Alternaria brassicicola and A. brassicae, is one of the most devastating and widespread diseases of oilseed Brassicas worldwide. Conventional breeding efforts to develop resistant cultivars have been unsuccessful due to the lack of suitable resistance sources among cultivated species. Sinapis alba, a crop wild relative (CWR) of oilseed Brassica spp., has been reported to exhibit considerable resistance to Alternaria blight. In the present study, we attempted to clone and characterize two important pathogenesis-related (PR) genes from S. alba, endochitinase and glucan endo-1,3-beta-glucosidase, and designed constructs for their functional characterization through overexpression. The genes were selected from a transcriptomic dataset of differentially expressed genes (DEGs), generated in a previous study, based on their expression patterns in S. alba and B. rapa following inoculation with A. brassicicola. The differential expression patterns of the PR genes were validated through qPCR. Furthermore, gene ontology analysis and protein–protein co-expression network studies provided insights into the functional roles of these genes in defense against the necrotroph. The complete coding sequences (CDSs) of the genes were then isolated from S. alba via PCR amplification, cloned into the pGEM-T Easy cloning vector, and subsequently into the pCAMBIA1301 binary vector. The constructs were validated by transient expression assays through agroinfiltration in Nicotiana benthamiana and B. rapa, followed by qPCR analysis. The constructs prepared for the overexpression of endochitinase and glucan endo-1,3-beta-glucosidase will further be used for stable transformation in B. rapa for functional validation through appropriate bioassays.