Subcellular distribution analysis of the cucumber mosaic virus 2b protein

The cucumoviral 2b protein is a viral counterdefence factor that interferes with the establishment of virus-induced gene silencing in plants. Synthetic peptides were used to generate an antibody to the 2b protein encoded by the Fny strain of cucumber mosaic virus (Fny-CMV). This polyclonal antibody was able to recognize the Fny-CMV 2b protein in a 10000 g pellet fraction of infected tobacco. No protein of equivalent size was detected in mock-inoculated or tobacco mosaic virus-infected samples. This represents the first demonstration of 2b protein expression by a subgroup I strain of CMV. Subcellular fractionation experiments on CMV-infected tobacco leaf tissue showed that the Fny-CMV 2b protein accumulated within a fraction that sedimented at forces of less than 5000 g and that the 2b protein was solubilized only by treatment with urea or SDS. These results suggested that the 2b protein associates either with the nucleus or cytoskeleton of the host cell. Further analysis showed that the 2b protein was enriched in a fraction that sedimented through a 2·2 M sucrose cushion. This fraction was also enriched in histones, suggesting that the CMV 2b protein associates preferentially with the host cell nucleus.

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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.

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Cucumber mosaic virus-induced gene silencing in banana

Scientific Reports ◽

10.1038/s41598-019-47962-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Yuh Tzean ◽

Ming-Chi Lee ◽

Hsiao-Hsuan Jan ◽

Yi-Shu Chiu ◽

Tsui-Chin Tu ◽

...

Keyword(s):

Gene Silencing ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Virus Induced Gene Silencing

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Symptom induction and RNA silencing suppression by the cucumber mosaic virus 2b protein

Plant Signaling & Behavior ◽

10.4161/psb.5.6.11643 ◽

2010 ◽

Vol 5 (6) ◽

pp. 705-708 ◽

Cited By ~ 17

Author(s):

Mathew G. Lewsey ◽

Inmaculada González ◽

Natalia O. Kalinina ◽

Peter Palukaitis ◽

Tomas Canto ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

2B Protein ◽

Silencing Suppression

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The Weak Small RNA-Binding Activity of the 2b Proteins of Subgroup II Cucumber Mosaic Virus Strains Is Insufficient for RNA Silencing Suppression

Frontiers in Microbiology ◽

10.3389/fmicb.2021.760937 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yingying Gao ◽

Jinrui Yang ◽

Xiaobei Zhang ◽

Aizhong Chen ◽

Zhouhang Gu ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

Rna Binding ◽

Nuclear Accumulation ◽

Nuclear Localization Sequence ◽

Binding Ability ◽

Suppressor Activity ◽

Subgroup Ii ◽

2B Protein

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.

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Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize

Plant Physiology ◽

10.1093/plphys/kiab443 ◽

2021 ◽

Author(s):

Huangai Li ◽

Danfeng Zhang ◽

Ke Xie ◽

Yan Wang ◽

Qiansheng Liao ◽

...

Keyword(s):

Gene Silencing ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

High Throughput ◽

Large Scale ◽

Growth Period ◽

Post Inoculation ◽

Virus Induced Gene Silencing ◽

Combination Strategy ◽

Gene Characterization

Abstract Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

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