Highly Activated RNA Silencing Via Strong Induction of Dicer By

Home , Hypovirus, Megabirnaviridae, Victorivirus

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 replication 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) infection 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 without 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 mutants of the bench-top and photographed. (B) Northern analyses of mRNAs of viruses C. parasitica EP155 (Δdcl2 and Δagl2), CHV1-wt, CHV1-Δp69, and host dcl2 and agl2 genes. ssRNA fractions were extracted from virus- and MyRV1 commonly showed more severe symptoms than those infected C. parasitica strains and subjected to the assays. Counterpart virus-free in EP155 (4, 24). In contrast, RnVV1-infected mutant hosts strains were analyzed in parallel. Upper panels show accumulation of viral exhibited similar colony morphology to their virus-free counterparts genomic and/or messenger RNAs (g/mRNAs or mRNAs) (CHV1, MyRV1,and (Fig. 1A). Note that RnVV1 initially induced symptoms in Δdcl2, RnVV1), and Lower panels show host gene expression levels (dcl2 and agl2). Significant transcriptional induction of the dcl2 gene is marked by a red ar- which, however, was mitigated during repeated subculture (32). rowhead. Loading control is the ethidium bromide (EtBr)-stained ribosomal In these antiviral RNA silencing-deficient hosts, viruses were RNAs (rRNAs) in this and all the following figures for Northern analyses. consistently accumulated at considerably or slightly higher levels relative to those in RNA silencing-competent EP155 (Fig. 1B, upper blots). EP155 induced dcl2 and agl2 expression upon in- Interference in RnVV1 Multiplication by MyRV1 and CHV1-Δp69 in fection by MyRV1 and CHV1-Δp69 significantly compared with C. parasitica. We have previously found virus strain-specific syn- RnVV1 infection (Fig. 1B, lower blots). CHV1-wt carrying the p29 ergistic and apparently antagonistic interactions: CHV1-wt en- RSS canceled such induction (26). The agl2 induction was never hances the multiplication of RnVV1, and CHV1-Δp69 represses observed in the Δdcl2 background, at least following infection by RnVV1 in EP155 (32). To further analyze this phenomenon, we CHV1-wt, CHV1-Δp69, and MyRV1, consistent with previous devised an assay as shown in Fig. 2, which was used throughout the observations that agl2 induction depends upon dcl2 (24) (Fig. 1B, study. Two fungal mycelia were inoculated on a single PDA me- lower blots). However, RnVV1-infected Δdcl2 showed a marked dium side-by-side and grown for 7–10 d (coculture) to allow viruses increase in agl2 mRNA level. Furthermore, although Sun et al. (24) to move between two fungal strains (horizontal transmission) reported the requirement of agl2 for the induction of dcl2 upon during hyphal fusion. In this system, either side (L or R) of the two CHV1-wt infection (thus vice versa), dcl2 induction apparently colonies harbored RnVV1, whereas the other side usually carried occurred in the Δagl2 host infected by MyRV1, and relatively virus(es) that might affect RnVV1 replication and transmission. weakly in hosts infected by CHV1-Δp69 and RnVV1. Therefore, Mycelial plugs were taken from designated positions (N, near; M, dcl2 transcription could be induced independently of AGL2. middle; F, far from the hyphal contact junction), placed on new CHV1-wt with the p29 RSS successfully interfered with dcl2 and PDA media, and further subcultured in potato dextrose broth agl2 induction in all host backgrounds tested (Fig. 1B). (PDB) media for single-stranded RNA (ssRNA) extraction and These combined results showed that dcl2 and agl2 induction subsequent Northern blot and RT-PCR analyses. are regulated by mycovirus infections; they are highly activated When Δdcl2/RnVV1 (L side) was fused with either the re- by MyRV1 and CHV1-Δp69 but apparently less so by CHV1-wt cipient EP155 fungus (R side) uninfected or infected by MyRV1 (through p29 RSS activities) and RnVV1 in EP155. It is striking or CHV1-Δp69, Northern blotting of RnVV1 showed different that the highest induction of dcl2 wasobservedinMyRV1-infected RnVV1 detection profiles (Fig. 3). RnVV1 was successfully EP155 and Δagl2 and in CHV1-Δp69–infected EP155 (Fig. 1B, transmitted to and accumulated in originally virus-free EP155 red arrowheads). (Fig. 3, set 1, RnVV1). However, RnVV1 was undetectable in

E4912 | www.pnas.org/cgi/doi/10.1073/pnas.1509151112 Chiba and Suzuki Downloaded by guest on September 29, 2021 PNAS PLUS

Fig. 2. Experimental procedure used in the virus transmission assay. Experimental flow is schematically represented. For virus transmissions, two mycelial plugs (original paired strains) selected from fungal strains such as those shown in Fig. 1A were inoculated on a large PDA plate, side by side, and grown on the bench-top for 7∼10 d. The left-side (L side) colony was mostly Δdcl2 carrying RnVV1 to be transmitted to the other side, and the right-side (R side) colony was WT, Δdcl2,orΔagl2 in which virus accumulation and host responses were analyzed. After coculture (horizontal virus transmission period), mycelial plugs were taken from the growing edge positioned in the F, M, or N position relative to the hyphal border between the L and R side colonies, and subcultured onto new small PDA plates. These subisolates were further cultured in PDB liquid media in flasks in the dark for 7 d, and ssRNA fractions were recovered for Northern blotting and RT-PCR analyses.

MyRV1-infected or CHV1-Δp69–infected EP155 by Northern Moreover, dcl2 expression levels were comparable between sin- blotting and was only faintly detected or undetected by RT-PCR gle and double infections, indicating that the antiviral RNA si- (Fig. 3, sets 2 and 3, RnVV1). Note that all subcultures from the lencing was fully activated by these viruses and that each virus three positions—N, M, and F—in recipient EP155 showed a tolerates it. similar detection pattern. Importantly, MyRV1 and CHV1-Δp69 moved to Δdcl2/RnVV1 (L side), indicating the occurrence of proper hyphal fusion that would allow for RnVV1 transmission in the reverse direction (Fig. 3, sets 2 and 3, MyRV1 and CHV1- Δp69). Another important observation is that mRNA levels of

dcl2 were substantially increased in EP155 infected by MyRV1 MICROBIOLOGY and CHV1-Δp69 but not in virus-free or RnVV1-transmitted EP155 (Fig. 3, dcl2). These combined results suggest the in- volvement of activated host antiviral RNA silencing in the in- hibitory effects on RnVV1 multiplication and transmission.

Invasive MyRV1 and CHV1-Δp69 Eliminate RnVV1. To further confirm the above observations, we took an inverse approach: MyRV1 and CHV1-Δp69 were transmitted from Δdcl2 (L side) into EP155 infected by RnVV1 (R side). As shown in Fig. 4, RnVV1 was detectable by Northern blotting in EP155 (set 2, R side) and was transmitted to Δdcl2 (set 2, L side), when cocultured with virus- free Δdcl2. In contrast, when cocultured with Δdcl2 carrying MyRV1 (set 1, L side), RnVV1 in EP155 was no longer detectable by Northern blotting but faintly detectable or undetectable by RT- PCR (set 1, R side). In this combination, MyRV1 invaded EP155 preinfected by RnVV1 and strongly interfered with RnVV1 replication. Although we expected RnVV1 transmission to the L side (MyRV1-carrying Δdcl2) given the coinfection established in the previous experiment in the Δdcl2 background (Fig. 3, set 2, L Fig. 3. Inhibition of RnVV1 accumulation by MyRV1 and CHV1-Δp69 in the side), such transmission was not observed (Fig. 4, set 1, L side, Δ Δ WT strain (EP155) of C. parasitica. The RnVV1-carrying C. parasitica dcl2 RnVV1). Similar results were obtained using CHV1- p69 as an strain (L side) was cocultured with virus-free (set 1), MyRV1-infected (set 2), invader (Fig. S1). These suggest that the lateral transfer of RnVV1 or CHV1-Δp69–infected (set 3) EP155 (R side). Their subculture strains were from EP155 to Δdcl2 was blocked before the establishment of obtained from the N, M, and F positions of the R sides and from the M coinfection with CHV1-Δp69 or MyRV1 in the Δdcl2 background. position of the L side (Δdcl2/RnVV1). The original paired strains were also It seems that RNA silencing was activated upon virus infection in included in the assay. Northern blotting was performed in this and sub- heterokaryotic cells with the EP155 and Δdcl2 karyons during sequent figures to detect mRNAs of RnVV1, CHV1-Δp69, given segment(s) of anastomosis and led to RnVV1 clearance. It should be noted that MyRV1 (segment S1 in this figure), and the host dcl2 and/or agl2 gene (Northern blotting). Detection targets are specified on the right of each during coculture karyons move from one colony to another more panel. Supporting RT-PCR analysis to detect RnVV1 and host actin mRNAs is extensively than previously thought (32). Δ presented at the bottom (RT-PCR). Sizes of DNA fragments are shown on the We also examined interference between CHV1- p69 and left. Arrows above the lanes indicate the theoretical direction of virus MyRV1, but these viruses established coinfection in EP155 with movement, and bars refer to originally virus-free strains in this and sub- similar accumulation levels to those of single infections (Fig. S2). sequent figures. DI-RNA, defective-interfering RNA.

Chiba and Suzuki PNAS | Published online August 17, 2015 | E4913 Downloaded by guest on September 29, 2021 parts. In the Δdcl2 background, RnVV1 and each of the two other viruses were bidirectionally transmitted and established coinfections (Fig. 5A). RnVV1 accumulated at high levels regardless of single or double infections. The expression levels of agl2 varied, suggesting that AGL2 has no effect on RnVV1 accumulation levels in the absence of DCL2. The same experiment in the Δagl2 background showed interesting differences. Firstly, RnVV1 failed to establish coinfection with MyRV1 in Δagl2 (Fig. 5B,set2), unlike in Δdcl2, but RnVV1 was successfully transmitted to virus- free and CHV1-Δp69–infected Δagl2 (Fig. 5B,sets1and3).Sec- ondly, the dcl2 expression level in Δagl2/CHV1-Δp69 was considerably lower than that in Δagl2/MyRV1 (Fig. 5B and Fig. 1B, dcl2). Thus, maximal up-regulation of the dcl2 expression appears to be required for RnVV1 interference, and this can occur in the absence of agl2 (Fig. 5B, set 2). This observation was accordingly confirmed by a cotransmission analysis in which Δdcl2 coinfected by RnVV1 and MyRV1 was fused with virus-free EP155, Δdcl2,or Δagl2. Consequently, Δdcl2 allowed for cotransmission, but not EP155 or Δagl2 recipients, in which dcl2 expression was highly induced (Fig. S3).

Nonviral dsRNA-Induced dcl2/agl2 Expression Reduced RnVV1 Accumulation. Transgene-generating dsRNAs (hairpin RNAs) have been reported to induce dcl2 and agl2 expression in this fungus (24). We examined whether the transgene-induced dcl2 and agl2 result in the reduction of RnVV1 transmission/accumulation as observed above. A well-characterized endogenous, mitogen-activated protein kinase (CpMK1) gene (35) was targeted Fig. 4. Clearance of preinfected RnVV1 from EP155 by invasive MyRV1. The with a hairpin construct in EP155, and we obtained CpMK1- MyRV1-infected (set 1) or virus-free (set 2) C. parasitica Δdcl2 strain (L side) knockdown lines (EP155/CpMK1-KD) in which CpMK1 expres- was cocultured with the RnVV1-infected EP155 strain (R side, EP155/RnVV1), sion levels were reduced compared with WT EP155 (Fig. S4 A and and subcultures were obtained from the N and F positions of the R sides. The B). The dcl2 mRNA levels in the EP155/CpMK1-KD were higher subcultures from the L side (Δdcl2/MyRV1 and Δdcl2, M position) and orig- than those in EP155, and thus, dcl2 induction was triggered by the inal cocultured strains were included as controls. Northern blot and RT-PCR hairpin RNAs (Fig. S4B, dcl2). Using one of these nonvirally dcl2- analyses were performed as described in Fig. 3. induced strains, we performed RnVV1 transmission assay from Δdcl2/RnVV1 (L side) into EP155/CHV1-wt, EP155/Twtp29 (the CHV1 p29-expressing strain), EP155, EP155/CpMK1-KD, and DCL2 but Not AGL2 Is Essential for the RnVV1 Interference. We were EP155/MyRV1 (R sides) to analyze the behavior of RnVV1. As interested in defining the functional roles of AGL2 and DCL2 in shown in Fig. 6, Northern analysis revealed various levels of Δ this RnVV1 interference. In cocultures, RnVV1-infected dcl2 RnVV1 accumulation and virus levels declined basically in inverse and Δagl2 (L side) were allowed to undergo hyphal fusion with proportion to host dcl2 mRNA accumulation. The RnVV1- virus-free, MyRV1-infected, or CHV1-Δp69–infected counter- mRNAs were most abundantly accumulated in the Δdcl2 background,

Fig. 5. Bidirectional transmission of RnVV1 and MyRV1 or CHV1-Δp69 in Δdcl2 and Δagl2 hosts. (A) Transmission in Δdcl2. The RnVV1-infected C. parasitica Δdcl2 (L sides, Δdcl2/RnVV1) was cocultured with virus-free (set 1), MyRV1-infected (set 2), or CHV1-Δp69–infected (set 3) Δdcl2 (R sides) and allowed to undergo horizontal transmission. The subcultures were obtained from the M positions of both the L and R sides and subjected to Northern analysis. The original cocultured strains were analyzed in parallel. (B) Transmission in Δagl2. The same experiment as in A (Δdcl2) was conducted with Δagl2.

E4914 | www.pnas.org/cgi/doi/10.1073/pnas.1509151112 Chiba and Suzuki Downloaded by guest on September 29, 2021 slightly less in EP155 under CHV1-wt infection, even less in the RnVV1 avoid their host RNA silencing systems. Segment S10 of PNAS PLUS p29-expressing host, and much less in EP155 overall. In EP155/ MyRV3, a member of the genus Mycoreovirus with a 12-segment CpMK1-KD and EP155/MyRV1, RnVV1 was no longer detect- genome, encodes an RSS (39). Although the suppressor activities able and these fungi consistently showed elevated dcl2 and agl2 of MyRV3 S10-coded VP10 against transgene silencing have been expression. Note that RnVV1 can replicate in a knockout mutant validated, those against virus infection remain enigmatic. In- ofCpMK1(CpMK1-KO)(35)toacomparableextentasinEP155 terestingly, MyRV1 (11-segment genome) has no counterpart to (Fig. S4C), thus eliminating the possibility that the RnVV1 inter- MyRV3 VP10. In addition, no synergistic interactions were found ference in CpMK1-KD was mediated by reduced expression levels between MyRV1 and CHV1-Δp69 (Fig. S2), unlike between of CpMK1. CHV1-wt and RnVV1 (Fig. 6). These results suggest that MyRV1 Overall, we conclude that the strong interference with RnVV1 lacks an RSS. RnVV1 appears to be targeted by a steady-state level by CHV1-Δp69, MyRV1, and transgenically expressed dsRNA of RNA silencing (Figs. 1 and 3), but there is no evidence that specifically occurs in conjunction with massive dcl2 induction. RnVV1 encodes an RSS. In support of this, no RSS activities were detected in another totivirus, Magnaporthe oryzae virus 2 (MoV2) Discussion (40). The same authors identified MoV2 siRNAs derived from Various interactions have been reported in the C. parasitica/virus both the virus negative and positive strands. This suggests that system: one-way synergism between CHV1 [nonsegmented (+) genomic dsRNA serves as a template for DCL in the host RNA genome] and MyRV1 (11-segmented dsRNA genome) (36), fungus, Magnaporthe oryzae, but it then remains unclear how and the generation of MyRV1 genome rearrangements by CHV1 virus dsRNA, believed to be confined within particles, is ex- coinfection; transgenic expression of its RSS, p29; or deletion of posed to DCL. Highly structured regions of viral mRNA appear host dcl2 or agl2 (37, 38). This study focused on different virus/ to serve as DCL templates (41). Whether the same is true for virus interplays involving RnVV1 (undivided dsRNA genome) and totivirus mRNA also remains elusive. showed that (i) MyRV1 and an RSS-lacking CHV1 mutant, Considering the aforementioned points, we propose a model CHV1-Δp69, interfere with the replication and lateral transmission to account for the RnVV1 interference observed here in view of of unrelated RnVV1 in WT C. parasitica;(ii) this interference is RNA silencing levels. In this model (Fig. 7), RNA silencing also mediated by transgene-derived dsRNA; (iii) disruption of dcl2 levels are determined based primarily on expression levels of completely abolished the interference, whereas disruption of agl2 AGL2 and DCL2, which are influenced by infection with CHV1- did not always do so; and (iv) the interference coincided with high Δp69 and MyRV1. There are several key unanswered questions. levels of accumulation of the dcl2 and agl2 transcripts. Taken to- Firstly, how do DCL2 and AGL2 exert differential effects on gether, these results indicate that RnVV1 interference is mediated RnVV1 interference? Different levels of contribution of key by RNA silencing activated by the interfering viruses or transgenic RNA silencing players to antiviral defense were noted earlier in hairpin RNA, in which the primary player is DCL2. Such virus Drosophila melanogaster in which AGO2 and Dicer-2 (DCR2) interference appears to operate at the intracellular replication play major roles in antiviral defense. AGO2 appears to make a level, as supported by the observation that invasive MyRV1 and greater contribution to defense against cricket paralysis virus CHV1-Δp69 inhibit replication of the preexisting RnVV1 in the (CrPV) than DCR2, when examined according to survival rate WT EP155 host background (Fig. 4 and Fig. S1). The apparent (42), whereas mutation of DCR2 has a slightly greater positive

inhibition of RnVV1 transmission (Figs. 3 and 5B and Fig. S3)is impact on flock house virus accumulation (10). In C. parasitica, MICROBIOLOGY considered to be a manifestation of blocked RnVV1 replication. RNA silencing-mediated RnVV1 interference did not always The mechanisms for RNA silencing suppression by CHV1 are require AGL2 (Fig. 5 and Fig. S3), a phenomenon more prom- well-studied, but there is little understanding of how MyRV1 and inent than the precedents in insect hosts. As long as dcl2 expression was sufficiently elevated, RnVV1 multiplication was impaired even in the absence of AGL2. AGL2 appears to in- directly contribute to RnVV1 interference by enhancing dcl2 transcription levels in certain conditions rather than by directly degrading RnVV1 mRNA. CHV1-Δp69 requires AGL2 for full- scale dcl2 elevation, but not MyRV1 (Fig. 5B, compare sets 2 and 3). The requirement for AGL2 for induction of dcl2 mRNA by CHV1-wt was reported by Sun et al. (24). An investigation of the mechanism underlying the augmentation of agl2 and dcl2 transcription in C. parasitica is a future challenge. Secondly, is the virus interference specific for certain virus/ virus combinations? This study showed RnVV1 to be severely affected by RNA silencing, which was activated by the other two viruses. It is obvious that MyRV1 and CHV1-Δp69 can survive and establish coinfection at highly activated RNA silencing levels (Fig. S2). However, RnVV1 is highly susceptible to RNA silencing such that its accumulation was often below the detection limit for RT-PCR (Figs. 3 and 4). This may be associated with the apparent lack of an RnVV1 RSS, as discussed earlier. In this regard, how CHV1-Δp69 and MyRV1, apparently lacking RSSs, Fig. 6. Hierarchic reduction of the RnVV1 accumulations in inverse pro- tolerate the activated antiviral state remains elusive. The high sus- portion to dcl2 expression levels. Northern analysis of mRNAs for RnVV1 and ceptibility of RnVV1 to RNA silencing may also be related to the the host dcl2 and agl2 in EP155 variants. RnVV1 was horizontally transmitted fact that RnVV1 is from a heterologous fungus, R. necatrix (43), Δ Δ from dcl2 ( dcl2/RnVV1, donor) into the EP155 variants (RnVV1 recipients): and has not adapted to the new host, C. parasitica.Toexaminethis CHV1-wt–infected strain (EP155/CHV1-wt), p29-expressing strain (EP155/ Twtp29), virus-free strain (EP155), CpMK1-KD strain (EP155/CpMK1-KD), and possibility, it would be interesting to compare the genome se- MyRV1-infected strain (EP155/MyRV1). Two independent transmission assays quences and tolerance to RNA silencing of RnVV1 maintained for (two lanes per recipient) were conducted for each EP155 variant, and sub- a long term by repeated subculturing in the natural and experi- cultures from the M positions were analyzed for RnVV1 accumulation mental hosts. Alternatively, RnVV1 may by nature be highly sus- (RnVV1) and dcl2 and agl2 expression levels (dcl2 and agl2). ceptible to RNA silencing. Support for this comes from the

Chiba and Suzuki PNAS | Published online August 17, 2015 | E4915 Downloaded by guest on September 29, 2021 viruses for degradation. Thus, the protective and challenge viruses must be closely related at the nucleotide sequence level, and the spectrum of this interference is rather narrow. However, a wide spectrum of RNA silencing-mediated antiviral defenses in plants was recently hypothesized by Ding and colleagues (53). These authors showed that virus infection activates the production of various siRNAs targeting over 1,000 host genes that include defense-related genes. As mentioned earlier, the virus interference in C. parasitica differs in several ways. In C. parasitica, as long as the dcl2 transcription level is sufficiently elevated by infection by heterologous viruses or transgenic expression of dsRNA (Fig. 7), no sequence homology is required between interfering elements and susceptible viruses. Viral inhibition most probably relies on the activities of DCL2 to cleave viral dsRNA or structured ssRNA sequence independently and does not always require AGL2 (Figs. 5B and 7). However, such RNA silencing-based effects can be impaired by viral counterdefense via RSSs such as CHV1 p29, which is able to sup- Fig. 7. A model for the RnVV1 interference mediated by host RNA silenc- press dcl2 and agl2 expression (Figs. 6 and 7). There are many plant ing. Activities of host (C. parasitica) RNA silencing are influenced by viruses. genes whose transcription is responsive to and up-regulated by virus CHV1-Δp69, MyRV1, and transgenic dsRNA activate antiviral RNA silencing infection. The use of their promoters to generate transgenic plants by transcriptional induction (up-regulation) of the dcl2 and agl2 genes. The overexpressing major antiviral RNA silencing genes may also result total mass of DCL2 and AGL2 is correspondingly increased or decreased in tolerance against a wide range of virus infections. along with transcription levels, and thus silencing activity should be significantly different. DCL2 and AGL2 may be involved in the positive feedback Materials and Methods regulation for activation of RNA silencing. RnVV1 infection does not induce such events as in the case of the virus-free state (basal RNA silencing). RnVV1 Fungal and Viral Materials. C. parasitica standard strain EP155 WT and its RNA Δ Δ is highly susceptible to the C. parasitica RNA silencing pathway. RnVV1 silencing-deficient derivatives dcl2 and agl2 were used. The RnVV1 Δ Δ dsRNAs and possibly structured mRNAs are effectively digested by DCL2, and transfectant strain of C. parasitica dcl2 ( dcl2/RnVV1) was previously Δ this results in strong interference. This interference is possibly independent named A12 (54). The EP155 strains infected with CHV1-wt, CHV1- p69, or of the ssRNA cleavage activity by AGL2. The whole picture illustrates that MyRV1 were regenerated from freeze stocks (EP155/CHV1-wt, EP155/CHV- RnVV1 replication is influenced by host RNA silencing activity, which is Δp69, and EP155/MyRV1) (33, 34). These viruses were horizontally trans- modulated by other viruses and dsRNAs. ferred into either EP155, Δdcl2,orΔagl2 from respective donor strains at least three times sequentially to eliminate possible heterokaryon formation (32). The host and viral strains used in this study are summarized in Table S1. observation by Drinnenberg et al. (44) that Saccharomyces cer- The fungi were cultured at 22–27 °C on PDA plates (6 cm in diameter) on the bench-top for maintenance and phenotypic observations and in PDB liquid evisiae virus L-A (SsV-L-A) is highly susceptible to RNA silencing media (20 mL) in the dark for RNA preparation. restored in S. cerevisiae. SsV-L-A is one of the best studied fungal viruses, which belongs to the same family, Totiviridae, as RnVV1 Plasmid Construction and Artificial RNA Silencing Induction. The CpMK1 gene (45, 46). It should also be determined whether other viruses of (accession no. AY166687, coding region 492–1773 consisting of four exons and C. parasitica origin are subject to RNA silencing-mediated in- three introns) was knocked down by transformation of EP155 using a hairpin terference in the same way as RnVV1. construct, pCPXHY2-CpMK1-IR, which was constructed based on a conven- Similarly, whether this interference is specific for the fungal tional expression vector pCPXHY2 (55). This construct expresses an inverted- host C. parasitica is another interesting question to address. The repeat messenger of the CpMK1 gene (targeting the 5′ region of the ORF) responsiveness of dcl2 and agl2 homologs upon virus infection carrying sense (1–1748/Nco I) and directly connected antisense (492–1200) se- has been poorly explored in other fungus/virus systems. In quences (Fig. S4A). Transformation of C. parasitica protoplasts and subsequent Neurospora crassa, these genes are induced by transgenically selection with hygromycin B were performed as described previously (56). All transgenic strains were subjected to single conidial spore isolation, and rep- expressed hairpin RNAs. However, no virus has been isolated as resentative homokaryon strains were selected for use (Fig. S4B). an infectious entity in this fungus (18). Plants generally carry larger numbers of DCL and AGO genes than fungi, and some of Horizontal Virus Transmission. Horizontal viral transmission via hyphal anas- them are functionally redundant. Plants have RDR genes to tomosis was allowed by coculture of viral-donor and -recipient fungal strains amplify sequence-specific silencing signals by converting aber- (Fig. 2). Mycelial plugs from paired colonies were inoculated on a new PDA rant RNAs into dsRNA as substrates for DCLs. These genes plate (9 cm in diameter) side-by-side and cultured for 7–10 d. Mycelial plugs might be regulated transcriptionally and posttranscriptionally. from the growing edge of the donor and recipient sides were transferred to However, only some genes have been reported to be up-regu- new PDA plates and grown for 3–5 d. These were further subjected to co- lated, generally by less than 10-fold, by infections by specific culture when required. viruses (47, 48). This smaller magnitude of up-regulation of RNA Extraction. Mycelia-containing PDB media (20 mL) were cultured in the genes in plants may be compensated by the RDR6/DCL4- dark at 24 °C for 7 d, and fungal flora were obtained by filtration with mediated amplification of viral siRNA that is functionally sub- Miracloth (Calbiochem). These were homogenized in liquid nitrogen, and stitutable for RNA silencing activation in C. parasitica. Insects the resultant powders were mixed with 2.5 mL of the RNA extraction buffer operate antiviral RNA silencing with a similar number of genes [200 mM NaCl, 100 mM Tris∙HCl (pH 8.0), 4 mM EDTA-2Na, 2% SDS (wt/vol)] (DCR2, AGO2, R2D2) to those of C. parasitica (49). Insect genes and an equal volume of phenol/chloroform in a mortar. Aqueous layers were responsible for antiviral RNA silencing are generally up-regu- clarified with phenol/chloroform and subsequently with chloroform once lated by some viruses or hairpin RNAs (50–52). Importantly, each, followed by 2-propanol (equal volume) or lithium chloride (final 2 M) there are, however, no examples of RNA silencing-mediated precipitation to obtain total RNA or ssRNA fractions, respectively. The resulting pellets were washed in 70% (vol/vol) ethanol and dissolved in 50 μL interference among unrelated viruses in plants or insects. of sterilized water, and their concentrations were calculated by NanoDrop In plants, the RNA silencing-mediated cross-protection requires 2000 (Thermo Fisher Scientific). The ssRNA samples were subjected to sequence homology between a preexisting, protective mild strain Northern hybridization and RT-PCR analyses, whereas the total RNA samples and a challenging severe strain (14, 16). siRNAs derived from an were simply analyzed by agarose gel electrophoresis with or without interfering virus and integrated into RISC-AGO target challenging nuclease treatments.

E4916 | www.pnas.org/cgi/doi/10.1073/pnas.1509151112 Chiba and Suzuki Downloaded by guest on September 29, 2021 Northern Blotting. Northern blot assays were performed as described by Chiba RT-PCR. The first cDNA strands were synthesized by M-MLV reverse tran- PNAS PLUS et al. (54). We heat-denatured 1–3 μg (for viruses) and 8–12 μg (for host gene scriptase (Takara) with virus-specific primers and oligo-(dT) from 1 μgof and transgene messages) of ssRNA samples (68 °C for 10 min) and electro- ssRNA as templates. PCR fragments of target sequences were amplified by – phoresed them in agarose gels containing formaldehyde in Mops buffer QuickTaq (Takara) polymerase in optimal conditions in 25 30 cycles and system. RNAs were capillary transferred onto nylon membranes (Hybond N+, were analyzed by agarose gel electrophoresis. Gels were stained by ethidium bromide (EtBr) and photographed under UV illumination in a Dolphin- Amersham) and fixed by UV Crosslinker CL-1000 (UVP). Specific DIG-labeled Chemi system (Wealtec). All oligonucleotide primers used in this study are cDNA probes were prepared by PCR (PCR DIG Labeling mix, Roche) and hy- available upon request. bridized with target RNAs in DIG easy Hyb buffer (Roche) at 42 °C overnight in a hybridization oven HB-500 Minidizer (UVP). The membranes were sequentially ACKNOWLEDGMENTS. We are grateful to Drs. Donald L. Nuss, Bradley I. washed and treated with anti-DIG antibody (Roche) at a 1/10,000 concentra- Hillman, Satoko Kanematsu, and Dae-Hyuk Kim for the generous gift of tion. Alkaline phosphatase-based chemiluminescent detection was then con- the fungal strains (EP155, Δdcl2, Δagl2, Cp9B21, W1029, and CpMK1-KO). ducted using CDP-Star (Roche) in a dark room with film development or We thank Drs. Ida Bagus Andika, Liying Sun, and Hideki Kondo for plasmid pCPXHY2-CpMK1-IR and their helpful comments on the manuscript. This through digital imaging in the ImageQuant LAS 4000 system (GE Healthcare study was supported in part by Yomogi Inc. and a Grant-in-Aid for Scientific Life Sciences), or by (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3′- Research from the Japanese Ministry of Education, Culture, Sports, Science, indolylphosphate p-toluidine)-based coloring. and Technology (KAKENHI 25252011 to N.S.).

1. Aliyari R, Ding SW (2009) RNA-based viral immunity initiated by the Dicer family of a group of positive-strand RNA plant viruses. Proc Natl Acad Sci USA 88(23): host immune receptors. Immunol Rev 227(1):176–188. 10647–10651. 2. Bologna NG, Voinnet O (2014) The diversity, biogenesis, and activities of endogenous 29. Segers GC, van Wezel R, Zhang X, Hong Y, Nuss DL (2006) Hypovirus papain-like silencing small RNAs in Arabidopsis. Annu Rev Plant Biol 65:473–503. protease p29 suppresses RNA silencing in the natural fungal host and in a heterolo- 3. Baulcombe D (2004) RNA silencing in plants. Nature 431(7006):356–363. gous plant system. Eukaryot Cell 5(6):896–904. 4. Segers GC, Zhang X, Deng F, Sun Q, Nuss DL (2007) Evidence that RNA silencing 30. Suzuki N, Maruyama K, Moriyama M, Nuss DL (2003) Hypovirus papain-like protease functions as an antiviral defense mechanism in fungi. Proc Natl Acad Sci USA 104(31): p29 functions in trans to enhance viral double-stranded RNA accumulation and ver- 12902–12906. tical transmission. J Virol 77(21):11697–11707. 5. Csorba T, Pantaleo V, Burgyán J (2009) RNA silencing: An antiviral mechanism. Adv 31. Suzuki N, Chen B, Nuss DL (1999) Mapping of a hypovirus p29 protease symptom Virus Res 75:35–71. determinant domain with sequence similarity to potyvirus HC-Pro protease. J Virol 6. Wu Q, Wang X, Ding SW (2010) Viral suppressors of RNA-based viral immunity: Host 73(11):9478–9484. targets. Cell Host Microbe 8(1):12–15. 32. Chiba S, Lin YH, Kondo H, Kanematsu S, Suzuki N (2013) A novel victorivirus from a 7. Csorba T, Kontra L, Burgyan J (2015) Viral silencing suppressors: Tools forged to fine- phytopathogenic fungus, Rosellinia necatrix, is infectious as particles and targeted by tune host-pathogen coexistence. Virology 479-480:85–103. RNA silencing. J Virol 87(12):6727–6738. 8. Andika IB, et al. (2015) Differential contributions of plant Dicer-like proteins to 33. Suzuki N, Nuss DL (2002) Contribution of protein p40 to hypovirus-mediated modu- antiviral defences against potato virus X in leaves and roots. Plant J 81(5): lation of fungal host phenotype and viral RNA accumulation. JVirol76(15): – 781–793. 7747 7759. 9. Jaubert M, Bhattacharjee S, Mello AF, Perry KL, Moffett P (2011) ARGONAUTE2 me- 34. Hillman BI, Supyani S, Kondo H, Suzuki N (2004) A reovirus of the fungus Crypho- diates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis. Plant nectria parasitica that is infectious as particles and related to the coltivirus genus of – Physiol 156(3):1556–1564. animal pathogens. J Virol 78(2):892 898. 10. Aliyari R, et al. (2008) Mechanism of induction and suppression of antiviral im- 35. Park SM, et al. (2004) Characterization of HOG1 homologue, CpMK1, from munity directed by virus-derived small RNAs in Drosophila. Cell Host Microbe 4(4): Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress. Mol Microbiol 51(5): 387–397. – 11. Mourrain P, et al. (2000) Arabidopsis SGS2 and SGS3 genes are required for post- 1267 1277. MICROBIOLOGY 36. Sun L, Nuss DL, Suzuki N (2006) Synergism between a mycoreovirus and a hypovirus transcriptional gene silencing and natural virus resistance. Cell 101(5):533–542. mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713. 12. Anandalakshmi R, et al. (1998) A viral suppressor of gene silencing in plants. Proc Natl J Gen Virol 87(Pt 12):3703–3714. Acad Sci USA 95(22):13079–13084. 37. Sun L, Suzuki N (2008) Intragenic rearrangements of a mycoreovirus induced by the 13. Kasschau KD, Carrington JC (1998) A counterdefensive strategy of plant viruses: multifunctional protein p29 encoded by the prototypic hypovirus CHV1-EP713. RNA Suppression of posttranscriptional gene silencing. Cell 95(4):461–470. 14(12):2557–2571. 14. Ziebell H, Carr JP (2010) Cross-protection: A century of mystery. Adv Virus Res 76: 38. Eusebio-Cope A, Suzuki N (2015) Mycoreovirus genome rearrangements associated 211–264. with RNA silencing deficiency. Nucleic Acids Res 43(7):3802–3813. 15. Kosaka Y, et al. (2006) Effectiveness of an attenuated Zucchini yellow mosaic virus 39. Yaegashi H, Yoshikawa N, Ito T, Kanematsu S (2013) A mycoreovirus suppresses isolate for cross-protecting cucumber. Plant Dis 90(1):67–72. RNA silencing in the white root rot fungus, Rosellinia necatrix. Virology 444(1-2): 16. Ratcliff FG, MacFarlane SA, Baulcombe DC (1999) Gene silencing without DNA. rna- 409–416. mediated cross-protection between viruses. Plant Cell 11(7):1207–1216. 40. Himeno M, et al. (2010) Significantly low level of small RNA accumulation derived 17. Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence from an encapsidated mycovirus with dsRNA genome. Virology 396(1):69–75. against viruses. Nature 411(6839):834–842. 41. Molnár A, et al. (2005) Plant virus-derived small interfering RNAs originate pre- 18. Eusebio-Cope A, et al. (2015) The chestnut blight fungus for studies on virus/host and dominantly from highly structured single-stranded viral RNAs. JVirol79(12): virus/virus interactions: From a natural to a model host. Virology 477:164–175. 7812–7818. 19. Hillman BI, Suzuki N (2004) Viruses of the chestnut blight fungus, Cryphonectria 42. van Rij RP, et al. (2006) The RNA silencing endonuclease Argonaute 2 mediates parasitica. Adv Virus Res 63:423–472. specific antiviral immunity in Drosophila melanogaster. Genes Dev 20(21): 20. Nuss DL (2005) Hypovirulence: Mycoviruses at the fungal-plant interface. Nat Rev 2985–2995. – Microbiol 3(8):632 642. 43. Yaegashi H, et al. (2013) Appearance of mycovirus-like double-stranded RNAs in the 21. Kondo H, Kanematsu S, Suzuki N (2013) Viruses of the white root rot fungus, Rose- white root rot fungus, Rosellinia necatrix, in an apple orchard. FEMS Microbiol Ecol – llinia necatrix. Adv Virus Res 86:177 214. 83(1):49–62. 22. Nuss DL (2011) Mycoviruses, RNA silencing, and viral RNA recombination. Adv Virus 44. Drinnenberg IA, Fink GR, Bartel DP (2011) Compatibility with killer explains the rise of – Res 80:25 48. RNAi-deficient fungi. Science 333(6049):1592. 23. Zhang DX, Spiering MJ, Nuss DL (2014) Characterizing the roles of Cryphonectria 45. Wickner RB, Fujimura T, Esteban R (2013) Viruses and prions of Saccharomyces cer- parasitica RNA-dependent RNA polymerase-like genes in antiviral defense, viral re- evisiae. Adv Virus Res 86:1–36. combination and transposon transcript accumulation. PLoS One 9(9):e108653. 46. Wickner RB, Ghabrial SA, Nibert ML, Patterson JL, Wang CC (2011) Family Totiviridae. 24. Sun Q, Choi GH, Nuss DL (2009) A single Argonaute gene is required for induction of Virus Taxonomy: Ninth Report of the International Committee for the Taxonomy of RNA silencing antiviral defense and promotes viral RNA recombination. Proc Natl Viruses, eds King AMQ, Adams MJ, Carstens EB, Lefkowits EJ (Elsevier, Academic Press, Acad Sci USA 106(42):17927–17932. New York), pp 639–650. 25. Deleris A, et al. (2006) Hierarchical action and inhibition of plant Dicer-like proteins in 47. Du P, et al. (2011) Viral infection induces expression of novel phased microRNAs from antiviral defense. Science 313(5783):68–71. conserved cellular microRNA precursors. PLoS Pathog 7(8):e1002176. 26. Zhang X, Nuss DL (2008) A host dicer is required for defective viral RNA production 48. Shivaprasad PV, et al. (2008) The CaMV transactivator/viroplasmin interferes with and recombinant virus vector RNA instability for a positive sense RNA virus. Proc Natl RDR6-dependent trans-acting and secondary siRNA pathways in Arabidopsis. Nucleic Acad Sci USA 105(43):16749–16754. Acids Res 36(18):5896–5909. 27. Koonin EV, Wolf YI, Nagasaki K, Dolja VV (2008) The Big Bang of picorna-like virus 49. Bronkhorst AW, van Rij RP (2014) The long and short of antiviral defense: Small RNA- evolution antedates the radiation of eukaryotic supergroups. Nat Rev Microbiol 6(12): based immunity in insects. Curr Opin Virol 7:19–28. 925–939. 50. Garbutt JS, Reynolds SE (2012) Induction of RNA interference genes by double- 28. Koonin EV, Choi GH, Nuss DL, Shapira R, Carrington JC (1991) Evidence for common stranded RNA; implications for susceptibility to RNA interference. Insect Biochem Mol ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and Biol 42(9):621–628.

Chiba and Suzuki PNAS | Published online August 17, 2015 | E4917 Downloaded by guest on September 29, 2021 51. Liu J, Kolliopoulou A, Smagghe G, Swevers L (2014) Modulation of the transcriptional 55. Craven MG, Pawlyk DM, Choi GH, Nuss DL (1993) Papain-like protease p29 as a response of innate immune and RNAi genes upon exposure to dsRNA and LPS in silk- symptom determinant encoded by a hypovirulence-associated virus of the chestnut moth-derived Bm5 cells overexpressing BmToll9-1 receptor. JInsectPhysiol66:10–19. blight fungus. J Virol 67(11):6513–6521. 52. Xu Y, Zhou W, Zhou Y, Wu J, Zhou X (2012) Transcriptome and comparative gene 56. Choi GH, Nuss DL (1992) Hypovirulence of chestnut blight fungus conferred by an expression analysis of Sogatella furcifera (Horváth) in response to southern rice black- infectious viral cDNA. Science 257(5071):800–803. streaked dwarf virus. PLoS One 7(4):e36238. 57. Shapira R, Choi GH, Nuss DL (1991) Virus-like genetic organization and expression 53. Cao M, et al. (2014) Virus infection triggers widespread silencing of host genes by a strategy for a double-stranded RNA genetic element associated with biological con- distinct class of endogenous siRNAs in Arabidopsis. Proc Natl Acad Sci USA 111(40): trol of chestnut blight. EMBO J 10(4):731–739. 14613–14618. 58. Suzuki N, Supyani S, Maruyama K, Hillman BI (2004) Complete genome sequence of 54. Chiba S, Lin YH, Kondo H, Kanematsu S, Suzuki N (2013) Effects of defective in- Mycoreovirus-1/Cp9B21, a member of a novel genus within the family Reoviridae, terfering RNA on symptom induction by, and replication of, a novel partitivirus from isolated from the chestnut blight fungus Cryphonectria parasitica. J Gen Virol a phytopathogenic fungus, Rosellinia necatrix. J Virol 87(4):2330–2341. 85(Pt 11):3437–3448.

E4918 | www.pnas.org/cgi/doi/10.1073/pnas.1509151112 Chiba and Suzuki Downloaded by guest on September 29, 2021