Functional analysis of Botrytis cinerea nep-like proteins

Necrosis and ethylene-inducing proteins (NEP) have been described in bacteria, oomycetes and fungi, and have been proposed to act as phytotoxin in dicotyledons. However, they have not been shown to exhibit any phytotoxic activity in monocotyledons. The mechanism of action has thus far not been elucidated. This ever-expanding family of related proteins is characterised by a highly conserved sequence motif (GHRHDWE) of unknown function. The Botrytis cinerea genome contains two genes, encoding NEP-like proteins, namely BcNEP1 and BcNEP2. Knockout mutants in the Bcnep1 or Bcnep2 gene do not show a reduced virulence on tomato or Nicotiana benthamiana. To study the in planta expression pattern of Bcnep genes, constructs of Green Fluorescent Protein (GFP), under control of the native promoters of either Bcnep1 or Bcnep2, were transformed to B. cinerea, and GFP expression was followed in time, after infection on flower petals, using confocal laser scanning and wide-field fluorescence microscopy. Both BcNEP proteins have been produced by heterologous production in Pichia pastoris. Infiltration of the purified proteins into Nicotiana benthamiana leads to a rapid induction of ethylene in a dose-dependent manner. BcNEP1 is capable of inducing ethylene at much lower concentrations than BcNEP2. Both proteins contain a number of cysteine residues, which are predicted to form disulfide bridges. They also possess several potential post-translational modification motifs for phosphorylation, N-glycosylation, O-glycosylation. In order to study which amino acid residues are important for the phytotoxic activity of the B. cinerea NEP proteins, site directed mutagenesis was performed. Preliminary results suggest that mutation of certain cysteine residues or post-translational modification motifs does not lead to total loss of function, whereas mutation of the conserved heptapeptide motif completely abolishes the phytotoxic activity. A comprehensive update of the results will be presented