Cloning and expression of necrosis and ethylene-inducing proteins (NEPs) from Neofusicoccum parvum

Neofusicoccum parvum, one of the causal agents of Botryosphaeria dieback of grapevine, is known as an aggressive fungus infecting a wide range of hosts. N. parvum virulence has been related to the ability of this fungus to colonize woody tissue combined with the production of several phytotoxins and also the expression of extracellular proteins with phytotoxic properties. However, a complete understanding of the pathogenicity mechanism is still lacking. Analy sis of the N. parvum genome led to the identification of several putative genes encoding necrosis and ethylene-inducing proteins (NEPs). These have a con- served GHRHDWE motif and have been found in a diversity of microorganisms with the ability to induce necrosis and trigger defense responses in their hosts. Consequently, analysis of these genes and their proteins can help in understanding their role in N. parvum pathogenicity. In order to attain our goal, 4 of the 6 NEP genes were successfully cloned from N. parvum as a His tag fusion protein and expressed in E. coli BL21 (DE3). Subsequently, the recombinant proteins were purified by affinity chromatography. In the future, pure recombinant proteins will be characterized according to their phytotoxic effect on grapevine thus providing additional insight into the pathogenicity mechanism of N. parvum.