The regulation of D-galacturonic acid utilization in Botrytis cinerea

The plant cell wall is the first barrier to pathogen invasion. The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an important cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. D-galacturonic acid is the most abundant component of pectin and effective utilization of D-galacturonic acid is important for virulence of B. cinerea. The D-galacturonic acid catabolic pathway comprises three enzymatic steps, involving D-galacturonate reductase (encoded by Bcgar1 and Bcgar2), L-galactonate dehydratase (encoded by Bclgd1), and 2-keto-3-deoxy-L-galactonate aldolase (encoded by Bclga1). Therefore, an effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete pectin utilization by B. cinerea. In this study, RNA sequencing was performed to compare genome wide transcriptional profiles in B. cinerea grown in media containing glucose and pectate as sole carbon sources. We identified 31 genes that are significantly upregulated in pectate containing culture, including Bcgar2, Bclga1, and a putative monosaccharide transporter. In addition, conserved cis-regulatory elements were predicted in the promoters of genes involved in pectate decomposition and D-galacturonic acid utilization. Functional analysis was carried out of the bidirectional promoter of the Bcgar2-Bclga1 gene cluster to study which of the cis-regulatory elements is required for induction by D-galacturonic acid. Furthermore, potential regulatory protein(s) were isolated by DNA-protein pull down assays using one important cis-regulatory element.