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. 2007 Jul 31;104(31):12902-6.
doi: 10.1073/pnas.0702500104. Epub 2007 Jul 23.

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

Gert C Segers  1 , Xuemin Zhang, Fuyou Deng, Qihong Sun, Donald L Nuss
Affiliations

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

Gert C Segers et al. Proc Natl Acad Sci U S A. .

Abstract

The role of RNA silencing as an antiviral defense mechanism in fungi was examined by testing the effect of dicer gene disruptions on mycovirus infection of the chestnut blight fungus Cryphonectria parasitica. C. parasitica dicer-like genes dcl-1 and dcl-2 were cloned and shown to share a high level of predicted amino acid sequence identity with the corresponding dicer-like genes from Neurospora crassa [Ncdcl-1 (50.5%); Ncdcl-2 (38.0%)] and Magnaporthe oryzae [MDL-1 (45.6%); MDL-2 (38.0%)], respectively. Disruption of dcl-1 and dcl-2 resulted in no observable phenotypic changes relative to wild-type C. parasitica. Infection of Deltadcl-1 strains with hypovirus CHV1-EP713 or reovirus MyRV1-Cp9B21 resulted in phenotypic changes that were indistinguishable from that exhibited by wild-type strain C. parasitica EP155 infected with these same viruses. In stark contrast, the Deltadcl-2 and Deltadcl-1/Deltadcl-2 mutant strains were highly susceptible to mycovirus infection, with CHV1-EP713-infected mutant strains becoming severely debilitated. Increased viral RNA levels were observed in the Deltadcl-2 mutant strains for a hypovirus CHV1-EP713 mutant lacking the suppressor of RNA silencing p29 and for wild-type reovirus MyRV1-Cp9B21. Complementation of the Deltadcl-2 strain with the wild-type dcl-2 gene resulted in reversion to the wild-type response to virus infection. These results provide direct evidence that a fungal dicer-like gene functions to regulate virus infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Conserved polypeptide domains for C. parasitica dicer-like proteins DCL-1 and DCL-2. The location of each domain along the predicted amino acid sequence is indicated above the corresponding box representing the domain. The percentage amino acid identity relative to N. crassa DCL-1 and M. oryzae 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 domain; DUF238, Domain of Unknown Function 283; RNIIIa and RNIIIb, RNase III a and b domains; dsrm, dsRNA-binding domain.
Fig. 2.
Fig. 2.
Disruption of C. parasitica dicer-like genes dcl-1 and dcl-2. (A) Genomic organization and disruption constructs for C. parasitica dicer-like genes. Disruption of dcl-1 was performed with the PCR-based strategy of Davidson et al. (44) as described by Deng et al. (45). The PCR fragment used for disruption transformation extended 1,020 bp upstream and 1,422 bp downstream of the dcl-1 coding region and contained the hygromycin resistance cassette substituted for a region of the coding region extending from nucleotide 2018 to nucleotide 2024 (construct 1). Double mutants (Δdcl-1/Δdcl-2) were constructed by disrupting dcl-1 in a Δdcl-2 background by using a PCR disruption fragment that contained the benomyl resistance cassette (construct 2). The gene-replacement plasmid construct for C. parasitica dcl-2 was made by using genomic clone dcl-2, which contains the complete dcl-2 ORF with 5′ and 3′ flanking regions of ≈5 and 2 kb, respectively. The 2,690-bp MfeI–NdeI fragment that contains 910 bp of upstream sequence and 1,780 bp of dcl-2 coding sequence was replaced with a cassette conferring hygromycin resistance (46). The resulting dcl-2 gene disruption construct was digested with NotI to release the insert before transformation of wild-type strain EP155. At least two independent deletion mutants were generated and characterized for each dicer-like gene and the double dicer knockout. (B) Southern blotting analysis of C. parasitica dicer-like gene disruption mutants with a probe specific for dcl-1. Genomic DNA, prepared from strain EP155 and mutants Δdcl-1, Δdcl-2, and Δdcl-1/Δdcl-2, was digested with XhoI and hybridized with the dcl-1-specific probe shown in A. Note that the 0.9-kb XhoI fragment in the lanes containing DNA from the wild-type strain EP155 (lane 1) and the Δdcl-2 mutant (lane 3) was replaced by a 3.9-kb band in the Δdcl-1 mutant (lane 2) and two bands of 2.1 and 1.3 kb predicted for disruption of dcl-1 in the double dicer mutant (lane 4) with the benomyl-cassette-containing disruption construct. (C) Southern blotting analysis of disruption mutants with a probe specific for dcl-2. Genomic DNA for strains indicated in B was digested with NsiI and hybridized with the dcl-2-specific probe shown in A. Note that the 4-kb NsiI band present in the lanes containing DNA from strain EP155 (lane 1) and the Δdcl-1 mutant (lane 2) was replaced by a 2-kb band in the lanes containing DNA from the Δdcl-2 (lane 3) and double dicer (lane 4) mutants because of the presence of a NsiI site in the hph cassette of the integrated disruption construct.
Fig. 3.
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 Δdcl-1, Δdcl-2, and double mutant Δdcl-1/Δdcl-2 are shown. (Middle) Corresponding strains and a complemented Δdcl-2 strain, Δdcl-2C, infected with hypovirus CHV1-EP713 (marked at left as CHV1 infected) are shown. The Δdcl-2 deletion mutant was complemented with a genomic DNA clone of the dcl-2 coding region cloned into plasmid pCPXNBn1 to generate the complementation plasmid pCDCL2, which contains the benomyl resistance cassette and the C. parasitica glyceraldehyde-3-phosphate dehydrogenase promoter (24) to drive expression of the inserted dcl-2 coding region. Cultures were grown for 7 days on PDA. (Bottom) Corresponding strains infected with reovirus MyRV1–9B21 (marked at left as MyRV1 infected) are shown. Cultures were grown for 9 days on PDA.
Fig. 4.
Fig. 4.
Quantitation of mycovirus RNA in dicer mutant C. parasitica strains. (A) Agarose gel analysis of relative dsRNA accumulation for hypovirus CHV1-EP713 and mutant strain Δp29, which lacks suppressor of RNA silencing p29, in wild-type strain EP155 and the Δdcl-2 deletion mutant strain. Lane M, 1-kb DNA ladder size markers; lane 1, CHV1-EP713-infected control strain EP155; lane 2, Δp29-infected strain EP155; lane 3, Δp29-infected Δdcl-2 mutant strain. The migration positions of hypovirus dsRNAs are shown at the right. The slowest migrating band represents full-length viral dsRNA, whereas the faster migrating bands represent internally deleted viral dsRNAs commonly generated by hypoviruses (47). The migration positions of rRNAs are indicated by asterisks. Results of semiquantitative RT-PCR analysis of total viral RNA in the corresponding CHV1-EP713 and Δp29-infected strains relative to 18S rRNA sequences are shown in the chart below the agarose gel. The values are normalized to the viral RNA accumulation in Δp29-infected strain EP155 (set to a value of 1), with the standard deviation based on three independent measurements of two independent RNA preparations indicated by the error bars. (B) Agarose gel analysis of viral dsRNA accumulation in reovirus MyRV1-Cp9B21-infected strain EP155 (lane 1), infected mutant Δdcl-1 (lane 2), infected mutant Δdcl-1 (lane 3), and infected double mutant Δdcl-1/Δdcl-2 (lane 4). Lane M contains 1-kb DNA ladder size markers. The migration position of the three largest MyRV1-Cp9B21 dsRNA segments are shown at the right. Results of semiquantitative RT-PCR analysis of MyRV1-Cp9B21 RNA in the corresponding strains are shown in the chart below the agarose gel. In this case, the relative amount of total viral RNA was estimated by measuring the 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 1), with the standard deviation based on three independent measurements of two independent RNA preparations indicated by the error bars.
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