Cucumber mosaic virus 2b protein inhibits RNA silencing pathways in green alga Chlamydomonas reinhardtii

The 2b proteins encoded by cucumber mosaic virus (CMV) subgroup I strains suppress RNA silencing primarily by competitively binding small RNAs (sRNAs) in the host cell cytoplasm. Interestingly, 2b proteins encoded by CMV subgroup II strains accumulate predominantly in nuclei. Here we determined that whereas the 2b protein (Fny2b) of subgroup IA strain Fny-CMV is highly effective in suppressing both sense RNA-induced and inverted repeat-induced posttranscriptional gene silencing, the 2b protein (LS2b) of the subgroup II strain LS-CMV was not as effective. Reducing nuclear accumulation of LS2b by mutating a residue in its nuclear localization sequence had no effect on RNA silencing suppressor activity, while attenuated viral symptoms. Electrophoretic mobility shift assays showed that the sRNA binding of LS2b was weaker and more selective than that of Fny2b. The domain determining the differential sRNA-binding ability was delimited to the putative helix α1 region. Moreover, LS2b mutants that completely lost suppressor activity still retained their weak sRNA-binding ability, suggesting that sRNA binding is not sufficient for LS2b to suppress RNA silencing. Considering the subgroup I strain-encoded 2b proteins that require sRNA-binding ability for the suppression of RNA silencing, we suggest that in addition to binding sRNA, the 2b proteins of subgroup II CMV strains would require extra biological activities to achieve RNA silencing inhibition.

Download Full-text

Reprogramming of RNA silencing triggered by cucumber mosaic virus infection in Arabidopsis

Genome Biology ◽

10.1186/s13059-021-02564-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Maria Luz Annacondia ◽

German Martinez

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Virus Resistance ◽

Rna Silencing ◽

High Throughput Sequencing ◽

Small Interfering Rnas ◽

Genome Wide ◽

Endogenous Genes ◽

Silencing Pathways ◽

Relationship Of

Abstract Background RNA silencing has an important role mediating sequence-specific virus resistance in plants. The complex interaction of viruses with RNA silencing involves the loading of viral small interfering RNAs (vsiRNAs) into its host ARGONAUTE (AGO) proteins. As a side effect of their antiviral activity, vsiRNAs loading into AGO proteins can also mediate the silencing of endogenous genes. Here, we analyze at the genome-wide level both aspects of the interference of cucumber mosaic virus (CMV) with the RNA silencing machinery of Arabidopsis thaliana. Results We observe CMV-derived vsiRNAs affect the levels of endogenous sRNA classes. Furthermore, we analyze the incorporation of vsiRNAs into AGO proteins with a described antiviral role and the viral suppressor of RNA silencing (VSR) 2b, by combining protein immunoprecipitation with sRNA high-throughput sequencing. Interestingly, vsiRNAs represent a substantial percentage of AGO-loaded sRNAs and displace other endogenous sRNAs. As a countermeasure, the VSR 2b loaded vsiRNAs and mRNA-derived siRNAs, which affect the expression of the genes they derive from. Additionally, we analyze how vsiRNAs incorporate into the endogenous RNA silencing pathways by exploring their target mRNAs using parallel analysis of RNA end (PARE) sequencing, which allow us to identify vsiRNA-targeted genes genome-wide. Conclusions This work exemplifies the complex relationship of RNA viruses with the endogenous RNA silencing machinery and the multiple aspects of virus resistance and virulence that this interaction induces.

Download Full-text

Self-interaction of the cucumber mosaic virus 2b protein plays a vital role in the suppression of RNA silencing and the induction of viral symptoms

Molecular Plant Pathology ◽

10.1111/mpp.12051 ◽

2013 ◽

Vol 14 (8) ◽

pp. 803-812 ◽

Cited By ~ 21

Author(s):

Aixia Xu ◽

Zhijing Zhao ◽

Wenhu Chen ◽

Hengmu Zhang ◽

Qiansheng Liao ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

Vital Role ◽

2B Protein

Download Full-text

The 2b protein of cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants

Journal of General Virology ◽

10.1099/vir.0.013219-0 ◽

2009 ◽

Vol 90 (12) ◽

pp. 3015-3021 ◽

Cited By ~ 23

Author(s):

Anurag Sunpapao ◽

Takashi Nakai ◽

Fang Dong ◽

Tomofumi Mochizuki ◽

Satoshi T. Ohki

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Shoot Apical Meristem ◽

Rna Silencing ◽

Apical Meristem ◽

Leaf Primordia ◽

Shoot Meristem ◽

Tobacco Plants ◽

2B Protein ◽

Infected Tobacco

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Δ2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Δ2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Δ2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Δ2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Δ2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Δ2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Δ2b, whereas accumulation of Δ2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.

Download Full-text

Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-3-0294 ◽

2010 ◽

Vol 23 (3) ◽

pp. 294-303 ◽

Cited By ~ 125

Author(s):

Inmaculada González ◽

Llucia Martínez ◽

Daria V. Rakitina ◽

Mathew G. Lewsey ◽

Félix A. Atencio ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Small Rnas ◽

Rna Silencing ◽

Silencing Suppressor ◽

Suppressor Activity ◽

Rna Silencing Suppressor ◽

2B Protein

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.

Download Full-text

Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein

The Plant Cell ◽

10.1105/tpc.111.092718 ◽

2012 ◽

Vol 24 (1) ◽

pp. 259-274 ◽

Cited By ~ 110

Author(s):

Cheng-Guo Duan ◽

Yuan-Yuan Fang ◽

Bang-Jun Zhou ◽

Jian-Hua Zhao ◽

Wei-Na Hou ◽

...

Keyword(s):

Dna Methylation ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

2B Protein

Download Full-text

Analysis of Mechanisms Involved in the Cucumber mosaic virus Satellite RNA-mediated Transgenic Resistance in Tomato Plants

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.1.98 ◽

2004 ◽

Vol 17 (1) ◽

pp. 98-108 ◽

Cited By ~ 29

Author(s):

Fabrizio Cillo ◽

Mariella M. Finetti-Sialer ◽

Maria A. Papanice ◽

Donato Gallitelli

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

Resistance Mechanism ◽

Degradation Process ◽

Transgenic Tomato ◽

Satellite Rna ◽

Total Recovery ◽

Tomato Plants ◽

Free Strain

Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CMV-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.

Download Full-text

Rice Stripe Mosaic Virus–Encoded P4 Is a Weak Suppressor of Viral RNA Silencing and Is Required for Disease Symptom Development

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-19-0239-ia ◽

2020 ◽

Vol 33 (3) ◽

pp. 412-422

Author(s):

Chao Zhang ◽

Dong Chen ◽

Guoyi Yang ◽

Xiyuan Yu ◽

Jianguo Wu

Keyword(s):

Mosaic Virus ◽

Rna Silencing ◽

Matrix Protein ◽

Expression System ◽

Potato Virus X ◽

Bimolecular Fluorescence Complementation ◽

Developmental Defects ◽

The Matrix ◽

Silencing Pathways ◽

Viral Suppressors

Viral suppressors of RNA silencing (VSRs) are a cluster of viral proteins that have evolved to counteract eukaryotic antiviral RNA silencing pathways, thereby contributing to viral pathogenicity. In this study, we revealed that the matrix protein P4 encoded by rice stripe mosaic virus (RSMV), which is an emerging cytoplasmic rhabdovirus, is a weak RNA silencing suppressor. By conducting yeast two-hybrid, bimolecular fluorescence complementation, and subcellular colocalization assays, we proved that P4 interacts with the rice endogenous suppressor of gene silencing 3 (OsSGS3). We also determined that P4 overexpression has no effect on OsSGS3 transcription. However, P4 can promote the degradation of OsSGS3 via ubiquitination and autophagy. Additionally, a potato virus X–based expression system was used to confirm that P4 enhances the development of mosaic symptoms on Nicotiana benthamiana leaves by promoting hydrogen peroxide accumulation but not cell death. To verify whether P4 is a pathogenicity factor in host plants, we generated transgenic P4-overexpressing rice plants that exhibited disease-related developmental defects including decreased plant height and excessive tillering. Our data suggest that RSMV-encoded P4 serves as a weak VSR that inhibits antiviral RNA silencing by targeting OsSGS3.

Download Full-text