RNA Silencing Functions As an Antiviral Defense Mechanism in Fungi

Evidence that RNA silencing functions as an antiviral defense mechanism in fungi

Gert C. Segers*, Xuemin Zhang, Fuyou Deng, Qihong Sun, and Donald L. Nuss†

Center for Biosystems Research, University of Maryland Biotechnology Institute, Shady Grove Campus, Rockville, MD 20850

Edited by Reed B. Wickner, National Institutes of Health, Bethesda, MD, and approved June 21, 2007 (received for review March 19, 2007) The role of RNA silencing as an antiviral defense mechanism in involved in incorporating siRNA into the RNA-induced silenc- fungi was examined by testing the effect of dicer gene disruptions ing complex, a RecQ helicase, QDE-3, and two Dicer orthologs, on mycovirus infection of the chestnut blight fungus Cryphonectria DCL-1 and DCL-2 (15–20). However, efforts to demonstrate a parasitica. C. parasitica dicer-like genes dcl-1 and dcl-2 were cloned role for RNA silencing in antiviral defense in N. crassa have been and shown to share a high level of predicted amino acid sequence limited by the absence of a well developed mycovirus experi- identity with the corresponding dicer-like genes from Neurospora mental system. crassa [Ncdcl-1 (50.5%); Ncdcl-2 (38.0%)] and Magnaporthe oryzae The chestnut blight fungus Cryphonectria parasitica is phylo- [MDL-1 (45.6%); MDL-2 (38.0%)], respectively. Disruption of dcl-1 genetically related to N. crassa and genetically tractable because and dcl-2 resulted in no observable phenotypic changes relative to of the haploid nature of its genome and the availability of a wild-type C. parasitica. Infection of ⌬dcl-1 strains with hypovirus robust DNA transformation protocol (21, 22). Moreover, C. CHV1-EP713 or reovirus MyRV1-Cp9B21 resulted in phenotypic parasitica has been shown to support the replication of members changes that were indistinguishable from that exhibited by wild- of five RNA virus families: Hypoviridae, Reoviridae, Narnaviri- type strain C. parasitica EP155 infected with these same viruses. In dae, Partitiviridae, and Chrysoviridae (23). A reverse genetics stark contrast, the ⌬dcl-2 and ⌬dcl-1/⌬dcl-2 mutant strains were system has been developed for members of the family Hypoviri- highly susceptible to mycovirus infection, with CHV1-EP713- dae (reviewed in ref. 21), and the hypovirus-encoded papain-like infected mutant strains becoming severely debilitated. Increased protease p29 recently was reported to suppress RNA silencing in viral RNA levels were observed in the ⌬dcl-2 mutant strains for a both C. parasitica and a heterologous plant system (24). We now hypovirus CHV1-EP713 mutant lacking the suppressor of RNA report the use of the C. parasitica/mycovirus experimental silencing p29 and for wild-type reovirus MyRV1-Cp9B21. Comple- system to investigate the role of RNA silencing as an antiviral mentation of the ⌬dcl-2 strain with the wild-type dcl-2 gene defense pathway in fungi. resulted in reversion to the wild-type response to virus infection. These results provide direct evidence that a fungal dicer-like gene Results functions to regulate virus infection. Cloning of C. parasitica dicer-like Genes dcl-1 and dcl-2. RNA silencing is eliminated in N. crassa by disruption of both dicer genes Cryphonectria parasitica ͉ Dicer ͉ hypovirus ͉ mycoreovirus ͉ Ncdcl-1 and Ncdcl-2 (16) and in Magnaporthe oryzae by disrup- double-stranded RNA tion of one of two dicer genes, MDL-2 (25). To examine whether RNA silencing plays a role in antiviral response in C. parasitica, NA-mediated, sequence-specific suppression of gene expres- we cloned and disrupted the endogenous dicer-like gene homo- Rsion, termed RNA silencing, has been described as post- logues to determine the effect of pathway disruption on myco- transcriptional gene silencing in plants (1, 2), RNA interference virus infection. (RNAi) in animals (3), and quelling in fungi (4). A common Degenerate PCR primers, based on conserved regions in feature of RNA silencing in these different organisms is the fungal dicer-like proteins from N. crassa (16), M. oryzae (25), and processing of structured or dsRNA into small interfering RNAs Fusarium graminearum (FG09025.1 and FG04408.1), were used (siRNAs) of 21–24 nt by RNase III-like endonucleases termed to amplify fragments from C. parasitica genomic DNA, resulting Dicers. These siRNAs then are incorporated into an RNA- in the identification and characterization of two dicer-like genes. induced silencing complex that guides sequence-specific degra- Sequence alignment analysis revealed high levels of deduced dation or translational repression of homologous RNA in the amino acid sequence identity of the two C. parasitica dicer-like cytoplasm or DNA or histone methylation of target sequences in genes with Ncdcl-1/MDL-1 (50.5%/45.6%) and Ncdcl-2/MDL-2 the nucleus (reviewed in refs. 5 and 6). (38.0%/39.3%) of N. crassa and M. oryzae, respectively, resulting In plants and animals, the RNA silencing pathway also in designations of dcl-1 and dcl-2 for the two C. parasitica genes. produces microRNAs (miRNAs) from genome-encoded RNA The dcl-1 ORF encodes a protein of 1,548 aa, and the size of hairpins that are involved in developmental regulation (reviewed the predicted protein encoded by dcl-2 is 1,541 aa. Both DCL-1 in refs. 7 and 8). miRNAs have not been identified in fungal and DCL-2 proteins contain domains characteristic of the Dicer genomes (9, 10). Thus, RNA silencing in fungi generally is thought to serve primarily as a defense mechanism against Author contributions: D.L.N. designed research; G.C.S., X.Z., F.D., and Q.S. performed invasive nucleic acids and viruses (10). RNA silencing plays a key research; G.C.S., X.Z., F.D., Q.S., and D.L.N. analyzed data; and G.C.S. and D.L.N. wrote the antiviral defense role in plants (reviewed in refs. 11 and 12) and paper. recently has been demonstrated to influence virus replication in The authors declare no conﬂict of interest. animal cells (reviewed in ref. 13). Although silencing of trans- This article is a PNAS Direct Submission. posons has been reported in fungi (14), there currently are no Abbreviation: PDA, potato dextrose agar. reports of RNA silencing functioning as a fungal antiviral Data deposition: The sequences reported in this paper have been deposited in the GenBank defense mechanism. database (accession nos. DQ186989 and DQ186990). Mechanisms underlying RNA silencing in fungi have been *Present address: Monsanto Company, Chesterﬁeld, MO 63017. elucidated primarily through studies with the model fungus †To whom correspondence should be addressed at: Center for Biosystems Research, Uni- Neurospora crassa. Cellular components of RNA silencing in this versity of Maryland Biotechnology Institute, Shady Grove Campus, 9600 Gudelsky Drive, fungus include the RNA-dependent RNA polymerases QDE-1 Rockville, MD 20850. E-mail: [email protected]. and Sad-1, the Argonaute-2 orthologs QDE-2 and Sms-2 that are © 2007 by The National Academy of Sciences of the USA

12902–12906 ͉ PNAS ͉ July 31, 2007 ͉ vol. 104 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702500104 Downloaded by guest on September 24, 2021 A iW epytdl lcd 1- l suco X oh I Xho I ohX I pb006 rP o eb 0 9. bK detpursiD lcd 1- sucol IohX hph IohX IohX 1 9.3 K b IohX IohX but IohX IohX IohX 2 Fig. 1. Conserved polypeptide domains for C. parasitica dicer-like proteins 1.2 bK 3.1 K b DCL-1 and DCL-2. The location of each domain along the predicted amino acid d lc sequence is indicated above the corresponding box representing the domain. liW epytd 2- l co su 006 bp The percentage amino acid identity relative to N. crassa DCL-1 and M. oryzae Nsi I N Ied N Iis IefM rP o eb MDL-2 is shown to the right of each domain for C. parasitica DCL-1 and DCL-2, respectively (16, 25). DEAD, DEAD box helicase; HelC, helicase C-terminal .4 0 bK domain; DUF238, Domain of Unknown Function 283; RNIIIa and RNIIIb, RNase fM e I N Iis sN Ii III a and b domains; dsrm, dsRNA-binding domain. hph .2 0 bK rsiD u detp lcd -2 ucol s protein family (Fig. 1). These include a DEAD box helicase domain near the N terminus followed by a helicase C-terminal B domain and a Domain of Unknown Function 283 (DUF), which C is found in most Dicer proteins. Two RNase III domains are present in the C-terminal region of both the predicted DCL-1 and DCL-2 sequences. C. parasitica DCL-2 also contains a predicted dsRNA-binding domain at the C terminus that also is found in C-terminal regions of NcDCL-2, MDL-1, and MDL-2 but not in C. parasitica DCL-1 or NcDCL-1 (16, 25).

Disruption of C. parasitica dicer-like Gene dcl-2 but Not dcl-1 Results in Severe Symptoms After Mycovirus Infection. Strains containing null-mutations of dcl-1, dcl-2, or both genes (⌬dcl-1/⌬dcl-2) were constructed by homologous recombination. Southern blotting analysis confirmed that the endogenous gene copies were re- Fig. 2. Disruption of C. parasitica dicer-like genes dcl-1 and dcl-2.(A) Genomic placed by the disruption construct (Fig. 2), whereas real-time organization and disruption constructs for C. parasitica dicer-like genes. Disrup- RT-PCR with dcl-1- and dcl-2-specific probes showed the ab- tion of dcl-1 was performed with the PCR-based strategy of Davidson et al. (44) sence of the corresponding gene transcripts in the respective as described by Deng et al. (45). The PCR fragment used for disruption transfor- mutant strains (data not shown). Phenotypic analysis of the mation extended 1,020 bp upstream and 1,422 bp downstream of the dcl-1 single and double disruption mutant strains revealed no obvious coding region and contained the hygromycin resistance cassette substituted for phenotypic consequences of dicer-like gene disruption. As a region of the coding region extending from nucleotide 2018 to nucleotide 2024 (construct 1). Double mutants (⌬dcl-1/⌬dcl-2) were constructed by disrupting shown in Fig. 3 Top, the dicer disruption mutants exhibited a dcl-1 in a ⌬dcl-2 background by using a PCR disruption fragment that contained colony morphology indistinguishable from the wild-type strain the benomyl resistance cassette (construct 2). The gene-replacement plasmid EP155. The mutants were found to be male and female fertile construct for C. parasitica dcl-2 was made by using genomic clone dcl-2, which and able to produce viable ascospores (data not shown). Addi- contains the complete dcl-2 ORF with 5Ј and 3Ј flanking regions of Ϸ5 and 2 kb, tionally, no differences in virulence on dormant chestnut stems respectively. The 2,690-bp MfeI–NdeI fragment that contains 910 bp of upstream or in the production of asexual spores were found between the sequence and 1,780 bp of dcl-2 coding sequence was replaced with a cassette dicer mutant and wild-type strains (data not shown). conferring hygromycin resistance (46). The resulting dcl-2 gene disruption construct was digested with NotI to release the insert before transformation of To test whether the C. parasitica dicer-like proteins are wild-type strain EP155. At least two independent deletion mutants were gener- involved in an antiviral response, the dicer deletion strains were ated and characterized for each dicer-like gene and the double dicer knockout. infected independently with hypovirus CHV1-EP713 and reo- (B) Southern blotting analysis of C. parasitica dicer-like gene disruption mutants virus MyRV1-Cp9B21 either by transfection of spheroplasts with with a probe specific for dcl-1. Genomic DNA, prepared from strain EP155 and viral transcripts (CHV1-EP713) or anastomosis with a virus- mutants ⌬dcl-1, ⌬dcl-2, and ⌬dcl-1/⌬dcl-2, was digested with XhoI and hybridized infected strain (CHV1-EP713 and MyRV1-Cp9B21). As shown with the dcl-1-specific probe shown in A. Note that the 0.9-kb XhoI fragment in in Fig. 3 Middle, CHV1-EP713 infection of ⌬dcl-1 resulted in a the lanes containing DNA from the wild-type strain EP155 (lane 1) and the ⌬dcl-2 ⌬ phenotype indistinguishable from that of CHV1-EP713-infected mutant (lane 3) was replaced by a 3.9-kb band in the dcl-1 mutant (lane 2) and two bands of 2.1 and 1.3 kb predicted for disruption of dcl-1 in the double dicer wild-type strain EP155. In stark contrast, CHV1-EP713-infected ⌬ ⌬ ⌬ mutant (lane 4) with the benomyl-cassette-containing disruption construct. (C) dcl-2 and dcl-1/ dcl-2 were severely debilitated. Complemen- Southern blotting analysis of disruption mutants with a probe specific for dcl-2. tation of the ⌬dcl-2 strain with the wild-type dcl-2 gene, strain Genomic DNA for strains indicated in B was digested with NsiI and hybridized ⌬dcl-2C, resulted in reversion to the wild-type response to with the dcl-2-specific probe shown in A. Note that the 4-kb NsiI band present in CHV1-EP713 infection (Fig. 3 Middle). A similar set of results the lanes containing DNA from strain EP155 (lane 1) and the ⌬dcl-1 mutant (lane was obtained for reovirus MyRV1-Cp9B21-infected mutant 2) was replaced by a 2-kb band in the lanes containing DNA from the ⌬dcl-2 (lane strains (Fig. 3 Bottom). MyRV1-Cp9B21-infected strains EP155 3) and double dicer (lane 4) mutants because of the presence of a NsiI site in the hph cassette of the integrated disruption construct. and ⌬dcl-1 were indistinguishable, whereas the ⌬dcl-2- and ⌬dcl-1/⌬dcl-2-infected strains exhibited a reduced growth and altered colony morphology but were not debilitated to the extent Unexpectedly, CHV1-EP713 dsRNA (agarose gel analysis)

observed for the corresponding CHV1-EP713-infected mutant and total viral RNA [RT-PCR analysis (26)] levels were not MICROBIOLOGY strains (Fig. 3 Middle and Bottom). found to increase significantly in accumulation in CHV1-EP713-

Segers et al. PNAS ͉ July 31, 2007 ͉ vol. 104 ͉ no. 31 ͉ 12903 Downloaded by guest on September 24, 2021 Fig. 3. Effect of mycovirus infection on C. parasitica dicer gene deletion mutants. (Top) Colony morphology for uninfected wild-type strain EP155, dicer gene disruption mutants ⌬dcl-1, ⌬dcl-2, and double mutant ⌬dcl-1/ Fig. 4. Quantitation of mycovirus RNA in dicer mutant C. parasitica strains. ⌬dcl-2 are shown. (Middle) Corresponding strains and a complemented ⌬dcl-2 (A) Agarose gel analysis of relative dsRNA accumulation for hypovirus CHV1- strain, ⌬dcl-2C, infected with hypovirus CHV1-EP713 (marked at left as CHV1 EP713 and mutant strain ⌬p29, which lacks suppressor of RNA silencing p29, infected) are shown. The ⌬dcl-2 deletion mutant was complemented with a in wild-type strain EP155 and the ⌬dcl-2 deletion mutant strain. Lane M, 1-kb genomic DNA clone of the dcl-2 coding region cloned into plasmid pCPXNBn1 DNA ladder size markers; lane 1, CHV1-EP713-infected control strain EP155; to generate the complementation plasmid pCDCL2, which contains the beno- lane 2, ⌬p29-infected strain EP155; lane 3, ⌬p29-infected ⌬dcl-2 mutant strain. myl resistance cassette and the C. parasitica glyceraldehyde-3-phosphate The migration positions of hypovirus dsRNAs are shown at the right. The dehydrogenase promoter (24) to drive expression of the inserted dcl-2 coding slowest migrating band represents full-length viral dsRNA, whereas the faster region. Cultures were grown for 7 days on PDA. (Bottom) Corresponding migrating bands represent internally deleted viral dsRNAs commonly gener- strains infected with reovirus MyRV1–9B21 (marked at left as MyRV1 infected) ated by hypoviruses (47). The migration positions of rRNAs are indicated by are shown. Cultures were grown for 9 days on PDA. asterisks. Results of semiquantitative RT-PCR analysis of total viral RNA in the corresponding CHV1-EP713 and ⌬p29-infected strains relative to 18S rRNA sequences are shown in the chart below the agarose gel. The values are infected ⌬dcl-2 strains relative to the levels found in the infected normalized to the viral RNA accumulation in ⌬p29-infected strain EP155 (set control strain EP155 (data not shown). Because deletion of the to a value of 1), with the standard deviation based on three independent region encoding the p29 suppressor of RNA silencing in the measurements of two independent RNA preparations indicated by the error ⌬ bars. (B) Agarose gel analysis of viral dsRNA accumulation in reovirus MyRV1- context of the CHV1-EP713 infectious cDNA clone, virus p29, Cp9B21-infected strain EP155 (lane 1), infected mutant ⌬dcl-1 (lane 2), in- previously was shown to result in a 70–80% reduction in viral fected mutant ⌬dcl-1 (lane 3), and infected double mutant ⌬dcl-1/⌬dcl-2 (lane RNA accumulation (26), we asked whether ⌬p29 RNA levels 4). Lane M contains 1-kb DNA ladder size markers. The migration position of increased in ⌬dcl-2 mutant strains. Infection of the ⌬dcl-2 the three largest MyRV1-Cp9B21 dsRNA segments are shown at the right. mutant strain with the ⌬p29 virus resulted in a similar level of Results of semiquantitative RT-PCR analysis of MyRV1-Cp9B21 RNA in the debilitation as observed for the CHV1-EP713 parent virus (data corresponding strains are shown in the chart below the agarose gel. In this not shown), but as shown in Fig. 4, the level of ⌬p29 viral total case, the relative amount of total viral RNA was estimated by measuring the and dsRNAs (Fig. 4A) increased to a level approaching that amount of segment S3-speciﬁc RNA relative to 18S rRNA (42). Values were normalized to the amount of S3-speciﬁc RNA in strain EP155 (set to a value of observed for wild-type CHV1-EP713 in wild-type strain EP155, Ϸ 1), with the standard deviation based on three independent measurements of i.e., an 4-fold increase. Also consistent with disruption of two independent RNA preparations indicated by the error bars. cellular RNA silencing and increased virus-mediated symptom expression, reovirus MyRV1-Cp9B21 RNA levels were observed to increase on the order of 3- to 4-fold in the ⌬dcl-2 mutant genes in antiviral defense. The phenotypic consequences of strains relative to that observed in the EP155 parent stain but not CHV1-EP713 infection were indistinguishable for wild-type in the ⌬dcl-1 mutant strain (Fig. 4B). strain EP155 and the ⌬dcl-1 deletion mutant, whereas the double dicer mutant showed the same level of severe symptoms as the Discussion ⌬dcl-2 single mutant (Fig. 3). Our previous report that hypovirus CHV1-EP713-encoded pro- None of the C. parasitica single or double dicer mutants tein p29 can suppress RNA silencing (24) provided circumstan- exhibited any detectable phenotypic change from wild type in the tial evidence that RNA silencing serves as an antiviral defense absence of virus infection. This differs from the reports that mechanism in fungi. The demonstration here that the C. para- the M. oryza MDL-1 mutant formed abnormal conidia and the sitica dcl-2, but not dcl-1, disruption mutants are highly debili- MDL-2 mutant showed reduced colony growth (25). Deletion of tated upon hypovirus infection provides more direct evidence for the dicer-like gene dcp-1 in Mucor circinelloides also was reported this conclusion and identifies DCL-2 as a primary component of to result in reduced growth rate and altered hyphal morphology the antiviral pathway. These results extend the role of fungal (27). The phenotypic characteristics of the N. crassa dicer dicer-like genes to include protection against virus infection. The mutants were not described. However, N. crassa dcl-1 has been enhanced symptoms and increased virus RNA accumulation reported to participate in the N. crassa meiotic silencing pathway observed in reovirus MyRV1-Cp9B21-infected ⌬dcl-2 mutant (MSUD for meiotic silencing by unpaired DNA) (28). strains also indicates that the C. parasitica RNA silencing Disruption of the antiviral RNA silencing pathway in plant or pathway serves as a general antiviral defense response and is not invertebrate systems generally results in an increase in virus titer specific for hypovirus infections. (29, 30). The observed increase in reovirus MyRV1-Cp9B21 The C. parasitica DCL-2 homologue in M. oryzae, MDL-2, was RNA levels in the ⌬dcl-2 mutant strains (Fig. 4B) was consistent reported to be responsible for hairpin RNA silencing (25), with these reports. Thus, it was surprising, given the severe whereas both Ncdcl-1 and Ncdcl-2 of N. crassa have been debilitation observed for the ⌬dcl-2 mutant strain after CHV1- reported to be redundantly involved in RNA silencing (16). EP713 infection, not to see a significant increase in hypovirus There was no evidence of redundancy for the C. parasitica dicer RNA accumulation in ⌬dcl-2 cultures grown on potato dextrose

12904 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702500104 Segers et al. Downloaded by guest on September 24, 2021 agar (PDA) medium. However, a similar result has been re- chestnut twigs embedded in 2% water agar, as described by ported for flock house virus (FHV). A very modest increase in Anagnostakis (40). wild-type FHV RNA was observed in infected dicer-2 mutant Drosophila melanogaster (31, 32), even though the infected flies Nucleic Acid and Protein Preparation and Analysis. Fungal genomic showed enhanced mortality in response to FHV infection (31). DNA was extracted as described by Choi et al. (41). RNA was However, a mutant FHV lacking the suppressor of RNA silenc- prepared, and quantitative analysis of RNA accumulation was ing B2 did show a significant increase in RNA accumulation in performed via reverse transcription and quantitative PCR using the dicer-2 mutant animals (31, 32). This last result is the TaqMan reagents (Applied Biosystems, Foster City, CA) and equivalent of the rescue of ⌬p29 RNA accumulation levels in the a GeneAmp 5700 PCR apparatus (Applied Biosystems) as C. parasitica ⌬dcl-2 mutant shown in Fig. 4A and indicates that described (26). Hypovirus CHV1-EP713 RNA was quantified as p29 directly or indirectly counteracts DCL-2 function. described by Suzuki and Nuss (26), whereas the relative level of Mycovirus infections are widespread in the Kingdom Fungi MyRV1-Cp9B21 RNA accumulation was determined by quan- (33). The results presented here with the C. parasitica/mycovirus tifying the amount of segment S3-specific RNA (42). system demonstrate that RNA silencing provides one mechanism for antiviral defense in fungi. However, a number of Cloning of C. parasitica dcl-1 and dcl-2 Genes. Degenerate PCR filamentous fungi (e.g., Candida albicans and Ustilago maydis) primers were designed according to conserved regions in dicer- appear to lack all or most of the components required for RNA like proteins DCL-1 and DCL-2 from N. crassa (16), MDL-1 and silencing (10), suggesting the possibility of additional, as-yet- MDL-2 from M. oryzae (24), and FG09025.1 and FG04408.1 undetected, mechanisms used by filamentous fungi to modulate from F. graminearum with the program CODEHOP (43): C. viral infections. In this regard, the products of the SKI genes have parasitica dcl-1 primers (Dcl1-EF1, 5Ј-AAG TCC ATC GCC been identified as acting as an antiviral system in the yeast GAC GTN TGY GAR GC-3Ј and Dcl1-FR1, 5Ј-GGT TGG Saccharomyces cerevisiae by blocking the translation of viral AGA CCA TGG CCA TYT TRT GYT C-3Ј) and dcl-2 primers non-poly(A) mRNAs (34). An understanding of how mycovi- (Dcl2-AF1, 5Ј-TCG GCC CCT GGG CNG YNG A-3Ј and ruses trigger, suppress, and are regulated by fungal RNA silenc- Dcl2-BR2, 5Ј-GGG CCC GGC CCC KNC KYT GDA T-3Ј) and ing pathways is anticipated to provide new insights into the (Dcl2-CF1, 5Ј-GGT AGA TGT GCC CCA AGA TGB TNG origin, biological functions, and molecular mechanisms of RNA CNG AYG T-3Ј and Dcl2-ER1, 5Ј-AGG ACG GCG TCG CCN silencing. ARR AAY TC-3Ј). The cloned PCR products were sequenced and used to screen a C. parasitica EP155 ␭-Bluestar phage Materials and Methods (Novagen, San Diego, CA) genomic library, which was con- Fungal Strains, Growth Conditions, and Transformation. C. parasitica structed from partial Sau3AI-digested genomic DNA according strains were maintained on Difco PDA as described (35). to the manufacturer’s instructions. Several independent genomic Cultures used for RNA or protein preparation were grown for 7 clones containing the dcl-1 and dcl-2 genes were sequenced by days at room temperature under ambient light on PDA or PDA using the ABI-Prism BigDye Terminator Ready-Reaction Cycle overlaid with cellophane to facilitate harvesting of mycelia. Sequencing kit (Applied Biosystems). The 5Ј end and 3Ј end of Preparation and transformation of C. parasitica spheroplasts was the dcl-1 and dcl-2 transcripts were determined by using the carried out essentially as described by Churchill et al. (36). FirstChoice RNA Ligase-Mediated Rapid Amplification of Hygromycin (40 ␮g/ml), benomyl (0.7 ␮g/ml), or blasticidin (300 cDNA Ends (RLM-RACE) kit (Ambion, Austin, TX) according ␮g/ml) was included in the growth medium to provide for to the manufacturer’s instructions. PCR fragments were ampli- selection of transformants. Transfection of fungal spheroplasts fied from oligo(dT)-primed cDNA with primers located at the with in vitro-transcribed hypovirus RNA transcripts was per- transcription start site and polyadenylation sites and sequenced formed as described (37). Reovirus MyRV1-Cp9B21-infected C. to determine the location of introns. Sequence data for C. parasitica strain EP155 was provided by Bradley Hillman (Rut- parasitica dcl-1 and dcl-2 have been submitted to the GenBank gers, The State University of New Jersey, New Brunswick, NJ). database under accession numbers DQ186989 and DQ186990, Mating assays were performed with deletion strain ⌬dcl-1 or respectively. ⌬dcl-2 derived from strain EP155 [mating type A or MAT1–2 (38)] as one parent and strain EP146 [mating type a or MAT1–1 This study was supported in part by Public Health Service Grant (39)] as the second parent, grown on autoclaved American GM55981 (to D.L.N.).

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