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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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Class 1 RNaseIII endoribonucleases of PPIV (PPR3) and SPCSV (CSR3) suppress RNAi in leaves of Nicotiana benthamiana.(A) The RNaseIII signature motif (blue) and dsRNA-binding domain (red) conserved in Class 1 RNaseIII endoribonucleases, and substitution of two conserved amino acid residues of the catalytic site are depicted (mutated proteins PPR3-Ala and CSR3-Ala). (B) Suppression of sense ssRNA—induced RNAi. The left and right side of leaves of gfp-transgenic N. benthamiana line 16c were infiltrated with Agrobacterium tumefaciens (Agro) strains expressing gfp to induce silencing of the constitutively expressed gfp transgene (note green fluorescence in leaf veins) and co-infiltrated with Agro strains for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, or GUS (β-glucuronidase, negative control) or mock-infiltrated with buffer (negative control). Leaves were photographed and analyzed 3 days post-infiltration. Accumulation of gfp mRNA and siRNA was analyzed by northern blotting. Ethidium bromide—stained gels of rRNA were used as loading controls. (C) Immunoblot of the RNaseIII proteins in the infiltrated tissues shown in (B). Control indicates purified recombinant CSR3 (positive control). (D) Suppression of dsRNA (hairpin RNA)-induced RNAi. Co-infiltration was carried out as in (B), except that an Agro strain expressing double-stranded (hairpin) gfp was used. Agro strains expressing GUS or the SPCSV p22 silencing suppressor were used as a negative and a positive control, respectively. Accumulation of gfp mRNA and siRNA was determined by northern blotting. (E) Reversion of RNAi. Suppression of the gfp transgene was achieved by sense-mediated silencing as in (B). After 24 h, the same leaf spots were infiltrated with an Agro strain for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, GUS (negative control), or the HCpro silencing suppressor of Potato virus A (positive control) and photographed 3 days later.
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ppat.1004711.g001: Class 1 RNaseIII endoribonucleases of PPIV (PPR3) and SPCSV (CSR3) suppress RNAi in leaves of Nicotiana benthamiana.(A) The RNaseIII signature motif (blue) and dsRNA-binding domain (red) conserved in Class 1 RNaseIII endoribonucleases, and substitution of two conserved amino acid residues of the catalytic site are depicted (mutated proteins PPR3-Ala and CSR3-Ala). (B) Suppression of sense ssRNA—induced RNAi. The left and right side of leaves of gfp-transgenic N. benthamiana line 16c were infiltrated with Agrobacterium tumefaciens (Agro) strains expressing gfp to induce silencing of the constitutively expressed gfp transgene (note green fluorescence in leaf veins) and co-infiltrated with Agro strains for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, or GUS (β-glucuronidase, negative control) or mock-infiltrated with buffer (negative control). Leaves were photographed and analyzed 3 days post-infiltration. Accumulation of gfp mRNA and siRNA was analyzed by northern blotting. Ethidium bromide—stained gels of rRNA were used as loading controls. (C) Immunoblot of the RNaseIII proteins in the infiltrated tissues shown in (B). Control indicates purified recombinant CSR3 (positive control). (D) Suppression of dsRNA (hairpin RNA)-induced RNAi. Co-infiltration was carried out as in (B), except that an Agro strain expressing double-stranded (hairpin) gfp was used. Agro strains expressing GUS or the SPCSV p22 silencing suppressor were used as a negative and a positive control, respectively. Accumulation of gfp mRNA and siRNA was determined by northern blotting. (E) Reversion of RNAi. Suppression of the gfp transgene was achieved by sense-mediated silencing as in (B). After 24 h, the same leaf spots were infiltrated with an Agro strain for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, GUS (negative control), or the HCpro silencing suppressor of Potato virus A (positive control) and photographed 3 days later.

Mentions: Compared with Dicers, Class 1 RNaseIIIs have a simple structure. Similar to the Class 1 RNaseIII of Escherichia coli [23], CSR3 contains a single catalytic domain and a dsRNA-binding domain and cleaves long dsRNA molecules in an Mg2+-dependent manner [21]. CSR3 cleaves ds-siRNA, suppresses sense-mediated RNAi, and counteracts antiviral RNAi in plants [24]. The RNAseIII of HvAV-3e also cleaves ds-siRNA [22]. However, it is not known whether the iridovirus RNaseIII can suppress RNAi, and therefore we compared RNAi suppression potential between the Pike-perch iridovirus (PPIV) Class 1 RNaseIII (PPR3) and CSR3 in plant and animal tissues (Fig. 1A). We were also interested to find out whether these proteins have broad spectrum of activity allowing suppression of RNAi in both animal and plant kingdoms. Our results reveal that the viral Class 1 RNaseIII enzymes have conserved functions in RNAi suppression, making it possible to identify this class of RNA suppressors using bioinformatics approaches, but the spectrum of unrelated organisms in which they are active differs.


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)

Class 1 RNaseIII endoribonucleases of PPIV (PPR3) and SPCSV (CSR3) suppress RNAi in leaves of Nicotiana benthamiana.(A) The RNaseIII signature motif (blue) and dsRNA-binding domain (red) conserved in Class 1 RNaseIII endoribonucleases, and substitution of two conserved amino acid residues of the catalytic site are depicted (mutated proteins PPR3-Ala and CSR3-Ala). (B) Suppression of sense ssRNA—induced RNAi. The left and right side of leaves of gfp-transgenic N. benthamiana line 16c were infiltrated with Agrobacterium tumefaciens (Agro) strains expressing gfp to induce silencing of the constitutively expressed gfp transgene (note green fluorescence in leaf veins) and co-infiltrated with Agro strains for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, or GUS (β-glucuronidase, negative control) or mock-infiltrated with buffer (negative control). Leaves were photographed and analyzed 3 days post-infiltration. Accumulation of gfp mRNA and siRNA was analyzed by northern blotting. Ethidium bromide—stained gels of rRNA were used as loading controls. (C) Immunoblot of the RNaseIII proteins in the infiltrated tissues shown in (B). Control indicates purified recombinant CSR3 (positive control). (D) Suppression of dsRNA (hairpin RNA)-induced RNAi. Co-infiltration was carried out as in (B), except that an Agro strain expressing double-stranded (hairpin) gfp was used. Agro strains expressing GUS or the SPCSV p22 silencing suppressor were used as a negative and a positive control, respectively. Accumulation of gfp mRNA and siRNA was determined by northern blotting. (E) Reversion of RNAi. Suppression of the gfp transgene was achieved by sense-mediated silencing as in (B). After 24 h, the same leaf spots were infiltrated with an Agro strain for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, GUS (negative control), or the HCpro silencing suppressor of Potato virus A (positive control) and photographed 3 days later.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352025&req=5

ppat.1004711.g001: Class 1 RNaseIII endoribonucleases of PPIV (PPR3) and SPCSV (CSR3) suppress RNAi in leaves of Nicotiana benthamiana.(A) The RNaseIII signature motif (blue) and dsRNA-binding domain (red) conserved in Class 1 RNaseIII endoribonucleases, and substitution of two conserved amino acid residues of the catalytic site are depicted (mutated proteins PPR3-Ala and CSR3-Ala). (B) Suppression of sense ssRNA—induced RNAi. The left and right side of leaves of gfp-transgenic N. benthamiana line 16c were infiltrated with Agrobacterium tumefaciens (Agro) strains expressing gfp to induce silencing of the constitutively expressed gfp transgene (note green fluorescence in leaf veins) and co-infiltrated with Agro strains for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, or GUS (β-glucuronidase, negative control) or mock-infiltrated with buffer (negative control). Leaves were photographed and analyzed 3 days post-infiltration. Accumulation of gfp mRNA and siRNA was analyzed by northern blotting. Ethidium bromide—stained gels of rRNA were used as loading controls. (C) Immunoblot of the RNaseIII proteins in the infiltrated tissues shown in (B). Control indicates purified recombinant CSR3 (positive control). (D) Suppression of dsRNA (hairpin RNA)-induced RNAi. Co-infiltration was carried out as in (B), except that an Agro strain expressing double-stranded (hairpin) gfp was used. Agro strains expressing GUS or the SPCSV p22 silencing suppressor were used as a negative and a positive control, respectively. Accumulation of gfp mRNA and siRNA was determined by northern blotting. (E) Reversion of RNAi. Suppression of the gfp transgene was achieved by sense-mediated silencing as in (B). After 24 h, the same leaf spots were infiltrated with an Agro strain for expression of PPR3, PPR3-Ala, CSR3, CSR3-Ala, GUS (negative control), or the HCpro silencing suppressor of Potato virus A (positive control) and photographed 3 days later.
Mentions: Compared with Dicers, Class 1 RNaseIIIs have a simple structure. Similar to the Class 1 RNaseIII of Escherichia coli [23], CSR3 contains a single catalytic domain and a dsRNA-binding domain and cleaves long dsRNA molecules in an Mg2+-dependent manner [21]. CSR3 cleaves ds-siRNA, suppresses sense-mediated RNAi, and counteracts antiviral RNAi in plants [24]. The RNAseIII of HvAV-3e also cleaves ds-siRNA [22]. However, it is not known whether the iridovirus RNaseIII can suppress RNAi, and therefore we compared RNAi suppression potential between the Pike-perch iridovirus (PPIV) Class 1 RNaseIII (PPR3) and CSR3 in plant and animal tissues (Fig. 1A). We were also interested to find out whether these proteins have broad spectrum of activity allowing suppression of RNAi in both animal and plant kingdoms. Our results reveal that the viral Class 1 RNaseIII enzymes have conserved functions in RNAi suppression, making it possible to identify this class of RNA suppressors using bioinformatics approaches, but the spectrum of unrelated organisms in which they are active differs.

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