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

Pauline Mochama,Shin-Yi Lee Marzano,Achal Neupane,Prajakta Jadhav

doi:10.3390/v10040214

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.

Highlights

RNA-directed gene silencing down-regulates gene expression at the transcriptional and post-transcriptional level
Nested PCR was used to rule out heterokaryotic mutation in which both the original dicer genes and disrupted genes occur in different nuclei within fungal hyphae (Table S1)
Our study demonstrated that a robust RNA silencing mechanism does exist in the plant pathogenic fungus, Sclerotinia sclerotiorum, and established the vital role played by dicer genes in this regulatory pathway

Summary

Introduction

RNA-directed gene silencing down-regulates gene expression at the transcriptional and post-transcriptional level. RNA (sRNA) duplexes of 18–30-nt in length. These sRNA duplexes are separated into two strands with one of the strands being loaded onto Argonaute proteins to target complementary nucleic acids in a sequence-specific manner. There are two main biological functions of RNA silencing: the first is endogenous gene regulation in development, stress response, and suppression of transposons and repetitive elements to maintain genome integrity. The second role is to confer defense against invasive nucleic acids including viruses [1,2,3]. Endogenous gene regulation through RNA silencing has been confirmed in plants and animals but is still debatable for fungi because RNA-silencing gene disruption mutants often do not Viruses 2018, 10, 214; doi:10.3390/v10040214 www.mdpi.com/journal/viruses

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