The role of pectin degradation in pathogenesis of Botrytis cinerea

Botrytis cinerea is a fungal plant pathogen that causes soft rot in many plant species. During the infection process, from the moment a conidium lands on the plant surface until complete host colonization, the fungus secretes numerous enzymes and metabolites that may contribute to virulence. Among the extracellular enzymes that are produced are pectin-degrading enzymes (pectinases) that facilitate the penetration of the plant surface and growth into the middle lamella, and contribute to decomposition of plant tissue and its conversion into fungal biomass. The main objective of the work was to elucidate the process of cell wall-degradation by B. cinerea, by studying when the genes were expressed during infection and determining which of the genes played an important role in the infection (functional analysis). Therefore, a PCR-based targeted mutagenesis method was developed. Using this method, many B. cinerea mutants were created in which endopolygalacturonase (Bcpg) or pectin methylesterase (Bcpme) genes were lacking. We showed that Bcpg2 played an important role in the infection from the moment that the fungus is penetrating the plant tissue. Other genes tested played no important role during the infection. Furthermore, we showed that BcPG enzymes each have specific biochemical characteristics and can degrade pectin in distinct ways. We tested whether each of the enzymes was capable of causing damage to healthy plant tissue. Especially BcPG1 and BcPG2 caused major damage resulting in rapid tissue collapse. This visible damage was caused by the enzymatic activity of BcPG2, not due to the response to protein recognition. The natural variation among Arabidopsis thaliana (thale cress) in their responses to infiltration with purified BcPGs was genetically analyzed in segregating progenies from crosses between parents that strongly differed in sensitivity to BcPGs. A genetic region that controls the response to BcPG2, BcPG3, BcPG4 and BcPG6 was identified. The identified genetic region is a starting point to identify and isolate the gene involved in response to BcPGs and study its role in resistance to B. cinerea