Analysis of VELVET mutants reveals important roles of host tissue acidification and protein secretion for pathogenesis of Botrytis cinerea

Matthias Hahn 1 Nathalie Müller 1 Michaela Leroch 1 Julia Schumacher 2 David Scheuring 1
1Biology, University of Kaiserslautern, Kaiserslautern, Germany
2Institute of Plant Biology and Biotechnology, Westfälische Wilhelms-Universität Münster, Münster, Germany

The necrotroph Botrytis cinerea kills host tissue by secretion of lytic enzymes and necrosis-inducing metabolites and proteins. Loss of BcVEL1, BcVEL2 or BcLAE1 from the VELVET regulatory complex results in disturbed light-dependent development and impaired virulence. The mutants were unable to acidify the host tissue, and showed strongly reduced release of citrate, the major acid secreted by the wild type. In contrast to previous reports, no significant secretion of oxalic acid was observed in the first days of lesion formation, and an oxalic acid deficient BcoahA mutant showed no major reduction in virulence. Keeping infection sites of VELVET mutants artificially low at pH 3 resulted in increased lesion formation, whereas alkalinizing wild type infection sites to pH 6-7 strongly suppressed lesion expansion, which confirmed that acidification is required for optimal infection.

VELVET mutants showed coordinate changes in transcriptome profiles in planta compared to the wild type. A common set of genes encoding secreted proteins were underexpressed in all VELVET mutants during infection, and many of these genes were induced in planta in the wild type strain. Quantitative analysis of in planta secretomes using 15N metabolic labeling revealed a high correlation of changes in mRNA and protein levels in the mutants, indicating that B. cinerea regulates infection-related protein secretion mainly by transcript abundance. The VELVET mutants showed drastically reduced secretion of proteases, and significantly impaired degradation of proteins released from killed host cells. Furthermore, the phytotoxic activity of the mutant secretomes was strongly reduced, due to lower expression of phytotoxic proteins and increased pH.

Taken together, VELVET controls major aspects of pathogenic development of B. cinerea by mediating acidification of the host tissue and activating secretion of virulence-related proteins. Current studies are focusing at functional and mutational dissection of B. cinerea secretome proteins and their effects on host killing and defence.