scholarly journals Coat protein mutations in an attenuated Cucumber mosaic virus encoding mutant 2b protein that lacks RNA silencing suppressor activity induces chlorosis with photosynthesis gene repression and chloroplast abnormalities in infected tobacco plants

Virology ◽  
2014 ◽  
Vol 456-457 ◽  
pp. 292-299 ◽  
Author(s):  
Tomofumi Mochizuki ◽  
Ryota Yamazaki ◽  
Tomoya Wada ◽  
Satoshi T. Ohki
2009 ◽  
Vol 90 (12) ◽  
pp. 3015-3021 ◽  
Author(s):  
Anurag Sunpapao ◽  
Takashi Nakai ◽  
Fang Dong ◽  
Tomofumi Mochizuki ◽  
Satoshi T. Ohki

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.


2010 ◽  
Vol 23 (3) ◽  
pp. 294-303 ◽  
Author(s):  
Inmaculada González ◽  
Llucia Martínez ◽  
Daria V. Rakitina ◽  
Mathew G. Lewsey ◽  
Félix A. Atencio ◽  
...  

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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Yingying Gao ◽  
Jinrui Yang ◽  
Xiaobei Zhang ◽  
Aizhong Chen ◽  
Zhouhang Gu ◽  
...  

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.


2013 ◽  
Vol 177 (1) ◽  
pp. 66-74 ◽  
Author(s):  
Liying Sun ◽  
Ida Bagus Andika ◽  
Jiangfeng Shen ◽  
Di Yang ◽  
Claudio Ratti ◽  
...  

2009 ◽  
Vol 75 (4) ◽  
pp. 297-306 ◽  
Author(s):  
Jun Ohnishi ◽  
Katsuyuki Hirai ◽  
Ayami Kanda ◽  
Tomio Usugi ◽  
Tetsuo Meshi ◽  
...  

2008 ◽  
Vol 82 (7) ◽  
pp. 3250-3260 ◽  
Author(s):  
Katsuyuki Hirai ◽  
Kenji Kubota ◽  
Tomofumi Mochizuki ◽  
Shinya Tsuda ◽  
Tetsuo Meshi

ABSTRACT Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue (∼0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.


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