An auxiliary activity family 9 protein, VdAA91, is required for the penetration structure formation in Verticillium dahliae

Abstract

Verticillium dahliae is a soil-borne fungal pathogen that can cause severe vascular wilt in many plant species; however, the role of some proteins secreted during infection needs to be explored. The fungal genome contains multiple auxiliary activity family 9 (AA9) genes that encode a conserved lytic polysaccharide monooxygenase domain, and some AA9 genes are required for the virulence of plant pathogenic fungi. To investigate the potential roles of AA9 genes, genome-wide identification and characterization were performed. A total of 20 AA9 genes were identified in the V. dahliae genome. According to a gene knockout experiment, VdAA91 is essential for the pathogenicity of V. dahliae. Furthermore, sequence analysis revealed that VdAA91 contains three conserved amino acid residues (His17, His114, and Tyr193) in the AA9 protein family. Subcellular localization and western blotting analyses revealed that VdAA91 is a secretory protein that is localized in the apoplast space of host cells. VdAA91 mutants showed defects in virulence and failures in root colonization based on observation of green fluorescent protein-labeled V. dahliae. We also observed penetration structure and its derived septin neck formation under microscopy. The VdAA91 mutants were incapable of producing penetration pegs and a VdSep5-GFP ring signal. Taken together, our results showed deletion of VdAA91 impairs the formation of the penetration structure and ultimately compromises V. dahliae colonization in the vascular bundles in the xylem of cotton.