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RNA Silencing Functions As an Antiviral Defense Mechanism in Fungi

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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 silenc- ing 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 Wildtype dcl 1- l ocus Xho I Xho I Xho I 600bp Probe 0 9. Kb Disrupted dcl-1 locus Xho I hph Xho I Xho I 1 3.9 Kb XhoI Xho I tub Xho I Xho I Xho I 2 Fig. 1. Conserved polypeptide domains for C. parasitica dicer-like proteins 2.1 Kb 1.3 Kb 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. Wildtype 2- l ocus 600bp The percentage amino acid identity relative to N. crassa DCL-1 and M. oryzae Nsi I Nde I N Iis Mfe I Probe 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 Kb domain; DUF238, Domain of Unknown Function 283; RNIIIa and RNIIIb, RNase Mfe I N Iis Ns Ii III a and b domains; dsrm, dsRNA-binding domain. hph .2 0 Kb Disrupted dcl-2 locus 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.
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