Insights on the molecular mechanisms regulating the anti-botrytis activity of VvAMP-13, a defensin from grapevine

Defensins, encoded by DEFL genes, are antimicrobial peptides playing a crucial role in the plant response to biotic stress. The family of grapevine (Vitis vinifera) DEFL genes has been recently characterized and, based on cysteine pattern, the encoded peptides have been divided in four groups. In particular, the group 2 contains highly conserved sequences differing from conventional plant defensins in the number of Cys residues predicted to form disulphide bridges (10 instead of 8). Among these, DEFL-13 gene, previously shown to encode for a defensin peptide with anti-Botrytis activity was selected to elucidate the mode of actionof the whole group. Here, DEFL-13, named Vv-AMP13 (Vitis vinifera AntiMicrobial 13) is shown to display strong and specific anitmicrobial activity against Botrytis cinerea, inhibiting both conidia germination and mycelial growth. Treatment of Botrytis cinerea hyphae with FITC labelled-VvAMP13 indicated that the peptide is internalized by fungal cells, and SYTOX green assay demonstrated that VvAMP13 provokes membrane permeabilization of the hyphae. Finally, the genetic bases of Botrytis cinerea susceptibility was addressed by screening by means of recombinant VvAMP 13 a Botrytis cinerea mutant library in which single genes were knocked out. Susceptibility to VvAMP 13 action was drastically reduced in Botrytis cinerea mutants depleted in genes encoding for proteins with central roles in cell signalling and cell cycle regulation, such as CDC42 and Rho proteins. These data suggest that G-proteins play important roles in VvAMP13 sensitivity.