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Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

Weinheimer I, Jiu Y, Rajamäki ML, Matilainen O, Kallijärvi J, Cuellar WJ, Lu R, Saarma M, Holmberg CI, Jäntti J, Valkonen JP - PLoS Pathog. (2015)

Bottom Line: Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues.However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans.These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland.

ABSTRACT
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

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Sense-mediated silencing of gfp expression in transgenic C. elegans.(A) Normalized GFP fluorescence intensity (FI) over time after feeding four different gfp-transgenic strains of C. elegans with E. coli expressing gfp mRNA. GFP fluorescence intensity was set at 100% at the beginning of each experiment (0 h), and relative changes in fluorescence intensity were followed over time. Each GFP fluorescence curve corresponds to a different tissue type. gfp expression was driven by promoters specifically active in (i) anterior and posterior intestine (C. elegans strain RT476), (ii) body muscle (strain SJ4157), (iii) nerve cord and ring (strain NM2415), or (iv) head muscle (strain NP738). Introgression of a deletion in rrf-3, a gene encoding a cellular RNA—dependent RNA polymerase homolog that interferes with RNAi in C. elegans [74, 75], to the transgenic strain RT476 resulted in no further enhancement of gfp silencing. At least 25 individual nematodes were assessed from each transgenic line at each time point in three independent experiments. Bars indicate S.E.M. (B) Morphology of examined body parts in C. elegans.
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ppat.1004711.g003: Sense-mediated silencing of gfp expression in transgenic C. elegans.(A) Normalized GFP fluorescence intensity (FI) over time after feeding four different gfp-transgenic strains of C. elegans with E. coli expressing gfp mRNA. GFP fluorescence intensity was set at 100% at the beginning of each experiment (0 h), and relative changes in fluorescence intensity were followed over time. Each GFP fluorescence curve corresponds to a different tissue type. gfp expression was driven by promoters specifically active in (i) anterior and posterior intestine (C. elegans strain RT476), (ii) body muscle (strain SJ4157), (iii) nerve cord and ring (strain NM2415), or (iv) head muscle (strain NP738). Introgression of a deletion in rrf-3, a gene encoding a cellular RNA—dependent RNA polymerase homolog that interferes with RNAi in C. elegans [74, 75], to the transgenic strain RT476 resulted in no further enhancement of gfp silencing. At least 25 individual nematodes were assessed from each transgenic line at each time point in three independent experiments. Bars indicate S.E.M. (B) Morphology of examined body parts in C. elegans.

Mentions: Owing to systemic spread of silencing in C. elegans, large numbers of the nematodes actively performing RNAi can be obtained by feeding them bacteria engineered to express high levels of a specific dsRNA [6]. Because CSR3 suppressed only sense-mediated RNAi in N. benthamiana, however, an E. coli strain engineered to express high levels of gfp mRNA was used as an RNAi inducer in C. elegans. These bacteria were fed to four transgenic strains of C. elegans expressing gfp under different tissue-specific promoters (Fig. 3A, S1 Table). gfp silencing was observed in all four strains, whereas no detectable reduction of GFP fluorescence was observed in nematodes fed bacteria transformed with an empty (no insert) plasmid or with a promoterless plasmid as tested with strain RT476 (S1A,B Fig.). The greatest (ca. 5-fold) reduction of GFP fluorescence was observed in strain RT476 (Fig. 3A, S1B Fig.) expressing gfp under the intestine-specific promoter vha-6 [27]. Strand-specific reverse transcription—PCR (RT-PCR) detected exclusively gfp sense transcripts (mRNA) in the gfp-transformed bacteria, and no antisense gfp transcripts were detected (S1C Fig.), suggesting that efficient sense-mediated silencing of gfp could be achieved in intestine tissue of C. elegans. Hence, strain RT476 was chosen for use in the RNAi suppression experiments.


Suppression of RNAi by dsRNA-degrading RNaseIII enzymes of viruses in animals and plants.

Weinheimer I, Jiu Y, Rajamäki ML, Matilainen O, Kallijärvi J, Cuellar WJ, Lu R, Saarma M, Holmberg CI, Jäntti J, Valkonen JP - PLoS Pathog. (2015)

Sense-mediated silencing of gfp expression in transgenic C. elegans.(A) Normalized GFP fluorescence intensity (FI) over time after feeding four different gfp-transgenic strains of C. elegans with E. coli expressing gfp mRNA. GFP fluorescence intensity was set at 100% at the beginning of each experiment (0 h), and relative changes in fluorescence intensity were followed over time. Each GFP fluorescence curve corresponds to a different tissue type. gfp expression was driven by promoters specifically active in (i) anterior and posterior intestine (C. elegans strain RT476), (ii) body muscle (strain SJ4157), (iii) nerve cord and ring (strain NM2415), or (iv) head muscle (strain NP738). Introgression of a deletion in rrf-3, a gene encoding a cellular RNA—dependent RNA polymerase homolog that interferes with RNAi in C. elegans [74, 75], to the transgenic strain RT476 resulted in no further enhancement of gfp silencing. At least 25 individual nematodes were assessed from each transgenic line at each time point in three independent experiments. Bars indicate S.E.M. (B) Morphology of examined body parts in C. elegans.
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ppat.1004711.g003: Sense-mediated silencing of gfp expression in transgenic C. elegans.(A) Normalized GFP fluorescence intensity (FI) over time after feeding four different gfp-transgenic strains of C. elegans with E. coli expressing gfp mRNA. GFP fluorescence intensity was set at 100% at the beginning of each experiment (0 h), and relative changes in fluorescence intensity were followed over time. Each GFP fluorescence curve corresponds to a different tissue type. gfp expression was driven by promoters specifically active in (i) anterior and posterior intestine (C. elegans strain RT476), (ii) body muscle (strain SJ4157), (iii) nerve cord and ring (strain NM2415), or (iv) head muscle (strain NP738). Introgression of a deletion in rrf-3, a gene encoding a cellular RNA—dependent RNA polymerase homolog that interferes with RNAi in C. elegans [74, 75], to the transgenic strain RT476 resulted in no further enhancement of gfp silencing. At least 25 individual nematodes were assessed from each transgenic line at each time point in three independent experiments. Bars indicate S.E.M. (B) Morphology of examined body parts in C. elegans.
Mentions: Owing to systemic spread of silencing in C. elegans, large numbers of the nematodes actively performing RNAi can be obtained by feeding them bacteria engineered to express high levels of a specific dsRNA [6]. Because CSR3 suppressed only sense-mediated RNAi in N. benthamiana, however, an E. coli strain engineered to express high levels of gfp mRNA was used as an RNAi inducer in C. elegans. These bacteria were fed to four transgenic strains of C. elegans expressing gfp under different tissue-specific promoters (Fig. 3A, S1 Table). gfp silencing was observed in all four strains, whereas no detectable reduction of GFP fluorescence was observed in nematodes fed bacteria transformed with an empty (no insert) plasmid or with a promoterless plasmid as tested with strain RT476 (S1A,B Fig.). The greatest (ca. 5-fold) reduction of GFP fluorescence was observed in strain RT476 (Fig. 3A, S1B Fig.) expressing gfp under the intestine-specific promoter vha-6 [27]. Strand-specific reverse transcription—PCR (RT-PCR) detected exclusively gfp sense transcripts (mRNA) in the gfp-transformed bacteria, and no antisense gfp transcripts were detected (S1C Fig.), suggesting that efficient sense-mediated silencing of gfp could be achieved in intestine tissue of C. elegans. Hence, strain RT476 was chosen for use in the RNAi suppression experiments.

Bottom Line: Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues.However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans.These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland.

ABSTRACT
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

Show MeSH
Related in: MedlinePlus