BcSpl1, a cerato-platanin family protein, contributes to Botrytis cinerea virulence and elicits the hypersensitive response in the host

Proteins belonging to the cerato-platanin family are small proteins with phytotoxic activity. A member of this family, BcSpl1, is one of the most abundant proteins in the Botrytis cinerea secretome. Expression analysis of the bcspl1 gene revealed that the transcript is present in every condition studied, showing the highest level in planta at the late stages of infection. Expression of a second cerato-platanin gene found in the B. cinerea genome, bcspl2, was not detected in any condition. Two bcspl1 knock-out mutants were generated and both showed reduced virulence in a variety of hosts. bcspl1 was expressed in Pichia pastoris and the recombinant protein was able to cause a fast and strong necrosis when infiltrated in tomato, tobacco and Arabidopsis leaves, in a dose-dependent manner. The BcSpl1-treated plant tissues showed symptoms of the hypersensitive response such as induction of reactive oxygen species, electrolyte leakage, cytoplasm shrinkage, and cell autofluorescence, as well as the induction of defense genes considered to be markers of the hypersensitive response. The Arabidopsis bak1 mutation partially prevented the induction of necrosis in this plant by BcSpl1. Two different BcSpl1-derived 40-amino acids peptides were also active in inducing necrosis.