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Establishment a gene silencing system in Verticillium dahliae and identification of a novel gene required for microsclerotia formation and virulence

Dianguang Xiong: Beijing Forestry University

<div><strong>Establishment a gene silencing system in <em>Verticillium dahliae</em> and identification of a novel gene required for microsclerotia formation and virulence </strong></p> <p><strong> </strong>Dianguang Xiong, Yonglin Wang, Chengming Tian</p> <p> The Key Laboratory for Silviculture and Conservation of Ministry of Education, College of Forestry, Beijing Forestry University, Beijing, China</p> <p> * Corresponding author: Chengming Tian,<;/p> <p><strong>Abstract</strong></p> <p>The genome of <em>Verticillium dahliae</em> has been sequenced and annotated, but incorrect gene annotations and unidentified transcribed regions persist. In this study, we established a dsRNA-induced gene silencing system with a plasmid carrying two convergent opposing RNA polymerase II promoters in <em>V. dahliae</em>. Subsequently, using RNA-Seq combined with reverse-transcription PCR, we identified a novel transcribed gene, Nag1, located in a cluster of putative secondary metabolic genes whose roles remain unknown. Functional analysis of Nag1 by dsRNA-mediated gene silencing revealed that loss of Nag1 significantly decreased fungal growth and conidial production. In addition, Nag1-silenced mutants exhibited obvious defects in microsclerotia formation and fungal virulence. Consistent with phenotypic observation of the reduction in microsclerotia formation, melanin production and expression of genes involved in melanin biosynthesis were markedly reduced in Nag1-silenced mutants. Overall, our data suggest that Nag1 acts as an important regulator of fungal development, pathogenicity, microsclerotia formation, and secondary metabolism in <em>V. dahliae</em>.</p> <p><strong>Key words: </strong>Novel transcript region, RNAi-mediated silencing, microsclerotia formation, melanin biosynthesis.</div>