Mycoviruses as Triggers and Targets of RNA Silencing in White Mold Fungus Sclerotinia sclerotiorum

Abstract

This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in <i>Sclerotinia sclerotiorum</i> by infecting wild-type and RNA-silencing-deficient strains of the fungus with an RNA virus and a DNA virus. Key silencing-related genes were disrupted to dissect the RNA silencing pathway. Specifically, dicer genes (<i>dcl-1, dcl-2</i>, and both <i>dcl-1</i>/<i>dcl-2</i>) were displaced by selective marker(s). Disruption mutants were then compared for changes in phenotype, virulence, and susceptibility to virus infections. Wild-type and mutant strains were transfected with a single-stranded RNA virus, SsHV2-L, and copies of a single-stranded DNA mycovirus, SsHADV-1, as a synthetic virus constructed in this study. Disruption of <i>dcl-1</i> or <i>dcl-2</i> resulted in no changes in phenotype compared to wild-type <i>S. sclerotiorum</i>; however, the double dicer mutant strain exhibited significantly slower growth. Furthermore, the <i>&Delta;dcl-1/dcl-2</i> double mutant, which was slow growing without virus infection, exhibited much more severe debilitation following virus infections including phenotypic changes such as slower growth, reduced pigmentation, and delayed sclerotial formation. These phenotypic changes were absent in the single mutants, <i>&Delta;dcl-1</i> and <i>&Delta;dcl-2</i>. Complementation of a single dicer in the double disruption mutant reversed viral susceptibility to the wild-type state. Virus-derived small RNAs were accumulated from virus-infected wild-type strains with strand bias towards the negative sense. The findings of these studies indicate that <i>S. sclerotiorum</i> has robust RNA silencing mechanisms that process both DNA and RNA mycoviruses and that, when both dicers are silenced, invasive nucleic acids can greatly debilitate the virulence of this fungus.