ABSTRACT
RNA silencing or RNA interference (RNAi) is an essential mechanism in animals, fungi, and plants that functions in gene regulation and defense against foreign nucleic acids. In fungi, RNA silencing has been shown to function primarily in defense against invasive nucleic acids. We previously determined that mycoviruses are triggers and targets of RNA silencing in Sclerotinia sclerotiorum. However, recent progresses in RNAi or dsRNA-based pest control requires more detailed characterization of the RNA silencing pathways in S. sclerotiorum to investigate the utility of dsRNA-based strategy for white mold control. This study elucidates the roles of argonaute enzymes, agl-2 and agl-4, in small RNA metabolism in S. sclerotiorum. Gene disruption mutants of agl-2 and agl-4 were compared for changes in phenotype, virulence, viral susceptibility, and small RNA profiles. The Δagl-2 mutant but not the Δagl-4 mutant had significantly slower growth and virulence prior to virus infection. Similarly, the Δagl-2 mutant but not the Δagl-4 mutant, showed greater debilitation under virus infection compared to uninfected strains. The responses were confirmed in complementation studies and revealed the antiviral role of agl-2. Gene disruption mutants of agl-2, agl-4, Dicer-like (dcl)-1, and dcl-2 did not change the stability of the most abundant endogenous small RNAs, which suggests the existence of alternative enzymes/pathways for small RNA biogenesis in S. sclerotiorum. Furthermore, in vitro synthesized dsRNA targeting agl-2 showed a significantly reduced average lesion diameter (P < 0.05) on canola leaves with agl-2 down-regulated compared to controls. This is the first report describing the effectiveness of RNA pesticides targeting S. sclerotiorum RNA silencing pathway for the control of the economically important pathogen.
ABSTRACT
This study aimed to demonstrate the existence of antiviral RNA silencing mechanisms in Sclerotinia sclerotiorum 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 (dcl-1, dcl-2, and both dcl-1/dcl-2) 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 dcl-1 or dcl-2 resulted in no changes in phenotype compared to wild-type S. sclerotiorum; however, the double dicer mutant strain exhibited significantly slower growth. Furthermore, the Δdcl-1/dcl-2 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, Δdcl-1 and Δdcl-2. 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 S. sclerotiorum 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.
