ABSTRACTRNA silencing acts as a defense mechanism against virus infection in a wide variety of organisms. Here, we investigated inductions of RNA silencing against encapsidated double-stranded RNA (dsRNA) fungal viruses (mycoviruses), including a partitivirus (RnPV1), a quadrivirus (RnQV1), a victorivirus (RnVV1), a mycoreovirus (RnMyRV3), and a megabirnavirus (RnMBV1) in the phytopathogenic fungusRosellinia necatrix. Expression profiling of RNA silencing-related genes revealed that a dicer-like gene, an Argonaute-like gene, and two RNA-dependent RNA polymerase genes were upregulated by RnMyRV3 or RnMBV1 infection but not by other virus infections or by constitutive expression of dsRNA inR. necatrix. Massive analysis of viral small RNAs (vsRNAs) from the five mycoviruses showed that 19- to 22-nucleotide (nt) vsRNAs were predominant; however, their ability to form duplexes with 3′ overhangs and the 5′ nucleotide preferences of vsRNAs differed among the five mycoviruses. The abundances of 19- to 22-nt vsRNAs from RnPV1, RnQV1, RnVV1, RnMyRV3, and RnMBV1 were 6.8%, 1.2%, 0.3%, 13.0%, and 24.9%, respectively. Importantly, the vsRNA abundances and accumulation levels of viral RNA were not always correlated, and the origins of the vsRNAs were distinguishable among the five mycoviruses. These data corroborated diverse interactions between encapsidated dsRNA mycoviruses and RNA silencing. Moreover, a green fluorescent protein (GFP)-based sensor assay inR. necatrixrevealed that RnMBV1 infection induced silencing of the target sensor gene (GFP gene and the partial RnMBV1 sequence), suggesting that vsRNAs from RnMBV1 activated the RNA-induced silencing complex. Overall, this study provides insights into RNA silencing against encapsidated dsRNA mycoviruses.IMPORTANCEEncapsidated dsRNA fungal viruses (mycoviruses) are believed to replicate inside their virions; therefore, there is a question of whether they induce RNA silencing. Here, we investigated inductions of RNA silencing against encapsidated dsRNA mycoviruses (a partitivirus, a quadrivirus, a victorivirus, a mycoreovirus, and a megabirnavirus) inRosellinia necatrix. We revealed upregulation of RNA silencing-related genes inR. necatrixinfected with a mycoreovirus or a megabirnavirus but not with other viruses, which was consistent with the relatively high abundances of vsRNAs from the two mycoviruses. We also showed common and different molecular features and origins of the vsRNAs from the five mycoviruses. Furthermore, we demonstrated the activation of RNA-induced silencing complex by mycoviruses inR. necatrix. Taken together, our data provide insights into an RNA silencing pathway against encapsidated dsRNA mycoviruses which is differentially induced among encapsidated dsRNA mycoviruses; that is, diverse replication strategies exist among encapsidated dsRNA mycoviruses.
Diverse Partitiviruses From the Phytopathogenic Fungus, Rosellinia necatrix
