Functional analysis of a melanin biosynthetic gene using RNAi-mediated gene silencing in Rosellinia necatrix

• RNAi is a useful tool for functional analysis of target genes in R. necatrix.
• RNAi knockdown identified the melanin biosynthesis gene, RnPKS1, in R. necatrix.
• Melanin is not essential for R. necatrix pathogenesis.
• Melanin is required for R. necatrix pseudosclerotia and conidiomata development.
• Melanin increases soil survival in R. necatrix.

Rosellinia necatrix causes white root rot in a wide range of fruit trees and persists for extended periods as pseudosclerotia on root debris. However, the pathogenesis of this disease has yet to be clarified. The functions of endogeneous target genes have not been determined because of the inefficiency in genetic transformation. In this study, the function of a melanin biosynthetic gene was determined to examine its role in morphology and virulence. A polyketide synthase gene (termed as RnPKS1) in the R. necatrix genome is homologous to the 1,8-dihydroxynaphthalene (DHN) melanin biosynthetic gene of Colletotrichum lagenarium. Melanin-deficient strains of R. necatrix were obtained by RNA interference-mediated knockdown of RnPKS1. The virulence of these strains was not significantly reduced compared with the parental melanin-producing strain. However, knockdown strains failed to develop pseudosclerotia and were degraded sooner in soil than the parental strain. Microscopic observations of albino conidiomata produced by knockdown strains revealed that melanization is involved in synnema integrity. These results suggest that melanin is not necessary for R. necatrix pathogenesis but is involved in survival through morphogenesis. This is the first report on the functional analysis of an endogenous target gene in R. necatrix.