Overexpression of an NLP protein family member increases virulence of Verticillium dahliae

Verticillium dahliae is a xylem-invading fungal pathogen that causes vascular wilt in a wide range of angiosperms. The pathogen uses a variety of virulence factors to invade and colonize its hosts. Here, we report that VdNEP, an NLP (Necrosis and ethylene inducing peptide 1-Like Protein), functions as one such factor in multiple hosts. Eggplant leaves treated with VdNEP developed necrotic symptoms. Overexpression of VdNEP by incorporating extra copies of the VdNEP gene increased virulence to cotton, eggplant and tomato plants, suggesting its role as a virulence factor in diverse plants. Increased expression of VdNEP among the transformants did not correlate with the number of VdNEP inserts, suggesting that its expression was affected by the genomic context of the insertion sites. Interestingly, a transformant derived from a defoliating strain with high VdNEP transcript levels caused disease symptoms in tomato plants, whereas the corresponding wild-type strain did not cause visible symptoms. The amount of V. dahliae DNA in plants infected with this VdNEP-overexpressing transformant was 22 times higher than that in plants infected with the wild-type isolate, further supporting the critical role of VdNEP in infection. A VdNEP-EGFP fusion was constructed to follow its localization in fungal cells and during infection.