Highly activated RNA silencing via strong induction of PNAS PLUS dicer by one virus can interfere with the replication of an unrelated virus Sotaro Chiba1 and Nobuhiro Suzuki2 Institute of Plant Science and Resources, Okayama University, Kurashiki, Okayama 710-0046, Japan Edited by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved July 21, 2015 (received for review May 12, 2015) Viruses often coinfect single host organisms in nature. Depending and interferes with the replication of a closely related severe strain on the combination of viruses in such coinfections, the interplay of the same virus (14). This phenomenon is also referred to as between them may be synergistic, apparently neutral with no effect cross-protection, and some attenuating plant viruses have been on each other, or antagonistic. RNA silencing is responsible for many commercialized as biological control agents (15). Most, if not all, cases of interference or cross-protection between viruses, but such cross-protection phenomena in plants are shown to be due to RNA antagonistic interactions are usually restricted to closely related silencing(14,16).Therefore,RNA silencing-mediated cross- strains of the same viral species. In this study, we present an protection in plants is reminiscent of acquired immunity-mediated unprecedented example of RNA silencing-mediated one-way in- cross-protection between closely related viruses in vertebrates (17). terference between unrelated viruses in a filamentous model The Cryphonectria parasitica (the chestnut blight fungus) has fungus, Cryphonectria parasitica. The replication of Rosellinia necatrix been established as a model filamentous fungus for studying victorivirus 1 (RnVV1; Totiviridae) was strongly impaired by coinfec- virus/virus and virus/host interactions (18). This C. parasitica/virus tion with the prototypic member of the genus Mycoreovirus (MyRV1) system provides a unique platform for the exploration of RNA si- or a mutant of the prototype hypovirus (Cryphonectria hypovirus 1, lencing and virus replication (18–20). The fungus supports diverse CHV1) lacking the RNA silencing suppressor (CHV1-Δp69). This inter- fungal viruses within six different families: Hypoviridae, Narnavir- ference was associated with marked transcriptional induction of key idae, Reoviridae, Partitiviridae, Megabirnaviridae,andTotiviridae. genes in antiviral RNA silencing, dicer-like 2 (dcl2) and argonaute-like These viruses are from not only the homologous host fungus but 2(agl2), following MyRV1 or CHV1-Δp69 infection. Interestingly, the also heterologous fungi belonging to different orders (18, 21). The inhibition of RnVV1 replication was reproduced when the levels of fungus has two Dicer-like genes (dcl1 and dcl2), four Argonaute-like dcl2 and agl2 transcripts were elevated by transgenic expression of a genes (agl1 to agl4), and four RNA-dependent RNA polymerase hairpin construct of an endogenous C. parasitica gene. Disruption of (RDR) genes (rdr1 to rdr4) (22, 23). Among these RNA silencing- dcl2 completely abolished the interference, whereas that of agl2 did related genes, only dcl2 and agl2 play roles in antiviral defense at the not always lead to its abolishment, suggesting more crucial roles of cellular level (4, 24). No redundant roles have been observed in dcl2 in antiviral defense. Taken altogether, these results demon- other dcl or agl homologs, unlike those in plants (25). Interestingly, MICROBIOLOGY strated the susceptible nature of RnVV1 to the antiviral silencing in dcl2 and agl2 were considerably up-regulated (over 10-fold) by C. parasitica activated by distinct viruses or transgene-derived double- transgenic expression of exogenous dsRNA and infection by mu- stranded RNAs and provide insight into the potential for broad-spec- tants of the prototype hypovirus Cryphonectria hypovirus 1 (CHV1) trum virus control mediated by RNA silencing. (24, 26), a member of the expanded picornavirus superfamily (27). A multifunctional protein encoded by CHV1, p29, inhibits the RNA silencing | dicer | argonaute | virus | Cryphonectria parasitica Significance NA silencing is a homology-dependent RNA degradation Rmechanism that is conserved in eukaryotic organisms across RNA silencing-mediated virus interference or cross-protection kingdoms. Briefly, double-stranded RNA (dsRNA), generated generally occurs between closely related strains of a single from transgenes, endogenous genes, or molecular parasites such virus species. Here, we show strong virus interference between as viruses and transposable elements, is processed into small in- unrelated RNA viruses in the filamentous ascomycetous fun- – terfering RNAs (siRNA) of 21 26 nucleotides by Dicer or Dicer- gus, Cryphonectria parasitica. Lateral transmission and repli- like proteins (DCLs). siRNAs are recruited into an RNA-induced cation of a totivirus with an undivided dsRNA genome was silencing complex (RISC), whose major constituent is Argonaute severely inhibited by a silencing suppressor deletion mutant (AGO) or Argonaute-like protein (AGL), an effector molecule of the prototype hypovirus with a positive-strand RNA genome – (1 3). RISC cleaves target RNAs using siRNAs as guides. RNA or the prototype mycoreovirus with an 11-segmented dsRNA silencing is part of the host defense mechanism operating pri- genome, and even by transgenic expression of hairpin RNA of marily against viruses (2, 4, 5). As a counterdefense tool, viruses an endogenous fungal gene. This interference required high- encode RNA silencing suppressors (RSSs) that inhibit different level expression of the key RNA silencing gene, dicer-like 2 steps of the silencing pathway (6, 7). Therefore, any defects in (dcl2), but not necessarily argonaute-like 2 (agl2). This study RNA silencing components typically make hosts supersusceptible provides insight into broad-spectrum virus control. or nonhosts susceptible to infection (8–11). There are three types of interplay between coinfecting viruses in Author contributions: S.C. and N.S. designed research; S.C. performed research; S.C. and asinglehost—synergistic, neutral (with no effects on each other), N.S. analyzed data; and S.C. and N.S. wrote the paper. and antagonistic interactions—many of which involve RNA si- The authors declare no conflict of interest. lencing. In synergistic infections, the RSS(s) of one virus plays an This article is a PNAS Direct Submission. important role in facilitating the multiplication of unrelated coin- 1Present address: Asian Satellite Campuses Institute, Graduate School of Bioagricultural fecting viruses. This is exemplified by the observation that potyvi- Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. ruses (HC-Pro RSS) increased potexvirus multiplication and 2To whom correspondence should be addressed. Email: [email protected]. pathogenicity in plants (12, 13). In antagonistic interactions, a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. preinfecting virus, usually a very mild strain, incites RNA silencing 1073/pnas.1509151112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1509151112 PNAS | Published online August 17, 2015 | E4911–E4918 Downloaded by guest on September 29, 2021 up-regulation of these host genes as an RSS (24). This protein shows phylogenetic affinity to and plays a role similar to potyvirus HC-Pro as a protease, a symptom determinant, and an RSS (28–31). It remains unknown whether the up-regulation of agl2 and dcl2 is transgene sequence-specific or virus-specific and to what degree these transcriptional inductions contribute to antiviral defense. During the course of studying antiviral RNA silencing in C. parasitica, we have found distinct induction patterns of the key RNA silencing genes upon infection by different viruses. That is, some viruses markedly induce the expression of dcl2 and agl2, whereas others do not. In this study, we provide solid evidence for RNA silencing-mediated, sequence-independent virus interference among unrelated RNA viruses. Lateral transmission and the rep- lication of a totivirus, Rosellinia necatrix victorivirus 1 (RnVV1) (32), which has an undivided dsRNA genome, was abolished by an RSS-deletion mutant of CHV1 or a reovirus with an 11-segmented dsRNA genome, and even by transgenic expression of hairpin RNA of an endogenous fungal gene. This interference coincided with high expression of two key genes for RNA silencing, dcl2 and agl2. A difference was also noted in the level of contribution to the interference between the two host genes. Results Behavior of CHV1, MyRV1, and RnVV1 in Wild-Type and RNA Silencing- Deficient C. parasitica. Wild-type (WT) CHV1 (CHV1-wt) in- fection of C. parasitica caused reduced pigmentation and reduced asexual sporulation in the WT standard strain EP155, whereas MyRV1 and a CHV1 mutant lacking most of 5′-proximal ORF A (CHV1-Δp69) induced a similar subset of symptoms, reducing the growth of aerial hyphae and increasing brown pigmentation with- out affecting sporulation considerably on potato dextrose agar (PDA)media(33,34)(Fig.1A). Infection of C. parasitica by Δ RnVV1, originally isolated from another phytopathogenic asco- Fig. 1. Infections of C. parasitica WT (EP155) and mutant strains ( dcl2 or Δagl2) by RnVV1, MyRV1, CHV1-wt, and CHV1-Δp69. (A) Phenotypes of virus- mycete (Rosellinia necatrix), did not induce macroscopic alterations free and -infected C. parasitica strains. Colonies were grown on PDA for 8 d on (32) (Fig. 1A). In antiviral RNA silencing-deficient