VdNEP, an Elicitor from Verticillium dahliae , Induces Cotton Plant Wilting

ABSTRACT Verticillium wilt is a vascular disease of cotton. The causal fungus, Verticillium dahliae , secretes elicitors in culture. We have generated ∼1,000 5′-terminal expressed sequence tags (ESTs) from a cultured mycelium of V. dahliae . A number of ESTs were found to encode proteins harboring putative signal peptides for secretion, and their cDNAs were isolated. Heterologous expression led to the identification of a protein with elicitor activities. This protein, named V. dahliae necrosis- and ethylene-inducing protein (VdNEP), is composed of 233 amino acids and has high sequence identities with fungal necrosis- and ethylene-inducing proteins. Infiltration of the bacterially expressed His-VdNEP into Nicotiana benthamiana leaves resulted in necrotic lesion formation. In Arabidopsis thaliana , the fusion protein also triggered production of reactive oxygen species and induced the expression of PR genes. When added into suspension cultured cells of cotton ( Gossypium arboreum ), the fusion protein elicited the biosynthesis of gossypol and related sesquiterpene phytoalexins at low concentrations, and it induced cell death at higher concentrations. On cotton cotyledons and leaves, His-VdNEP induced dehydration and wilting, similar to symptoms caused by a crude preparation of V. dahliae elicitors. Northern blotting showed a low level of VdNEP expression in the mycelium during culture. These data suggest that VdNEP is a wilt-inducing factor and that it participates in cotton- V. dahliae interactions.