A strategy for screening an inhibitor of viral silencing suppressors, which attenuates symptom development of plant viruses

Shimura, Hanako; Fukagawa, Takako; Meguro, Ayano; Yamada, Hideaki; Oh-hira, Mahito; Sano, Shinsuke; Masuta, Chikara

doi:10.1016/j.febslet.2008.10.046

Cited by 25 publications

(25 citation statements)

References 23 publications

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“…Because rgs-CaM was reported to interact with TEV HC-Pro (13), we tested for in vitro proteinprotein interactions between purified rgs-CaM and RSS proteins using surface plasmon resonance (SPR) analysis (14). We found that rgs-CaM interacted with the HC-Pro protein encoded by the potyvirus, turnip mosaic virus (TuMV) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

Nakahara

Masuta

Yamada

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM–mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

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Section: Resultsmentioning

confidence: 99%

“…The siRNA-binding activity of RSSs is important for their antisilencing activities (14,19,32). Therefore, by interfering with siRNA binding, rgs-CaM might attenuate RSS antisilencing activity.…”

Section: Resultsmentioning

confidence: 99%

Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

Nakahara

Masuta

Yamada

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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209

224

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“…We hypothesized that CAT3 may inactivate the RSS activity of 2b by binding with 2b, thus inhibiting CMV multiplication. To investigate the effect of CAT3 on the RSS activity of 2b, we used a protoplast assay that we have previously developed to measure viral RSS activities on the cellular level (Shimura et al, 2008). As shown in Figure 9B, CAT3 expression actually decreased the RSS activity of 2b by 30% to 40%, but it did not affect another virus RSS, P19.…”

Section: Cat3 Expression Compromises Cell Death Induced By 2b In Protmentioning

confidence: 99%

“…Protoplasts were prepared from leaves of N. benthamiana and used to analyze RSS activity essentially as described by Shimura et al (2008). For this assay, we used the Dual-Luciferase Reporter Assay System (Promega) using firefly luciferase (Fluc) and Renilla luciferase (Rluc).…”

Section: Protoplast Assay For Rss Activitymentioning

confidence: 99%

Virus-Induced Necrosis Is a Consequence of Direct Protein-Protein Interaction between a Viral RNA-Silencing Suppressor and a Host Catalase

et al. 2011

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Many plant host factors are known to interact with viral proteins during pathogenesis, but how a plant virus induces a specific disease symptom still needs further research. A lily strain of Cucumber mosaic virus (CMV-HL) can induce discrete necrotic spots on infected Arabidopsis (Arabidopsis thaliana) plants; other CMV strains can induce similar spots, but they are not as distinct as those induced by CMV-HL. The CMV 2b protein (2b), a known RNA-silencing suppressor, is involved in viral movement and symptom induction. Using in situ proximity ligation assay immunostaining and the protoplast assays, we report here that CMV 2b interacts directly with Catalase3 (CAT3) in infected tissues, a key enzyme in the breakdown of toxic hydrogen peroxide. Interestingly, CAT3, normally localized in the cytoplasm (glyoxysome), was recruited to the nucleus by an interaction between 2b and CAT3. Although overexpression of CAT3 in transgenic plants decreased the accumulation of CMV and delayed viral symptom development to some extent, 2b seems to neutralize the cellular catalase contributing to the host defense response, thus favoring viral infection. Our results thus provide evidence that, in addition to altering the type of symptom by disturbing microRNA pathways, 2b can directly bind to a host factor that is important in scavenging cellular hydrogen peroxide and thus interfere specifically with that host factor, leading to the induction of a specific necrosis.Cucumber mosaic virus (CMV) belongs to the genus Cucumovirus and contains tripartite, single-stranded sense RNAs designated RNA1 to RNA3 in decreasing order of molecular mass. RNA1 and RNA2 encode replicase proteins 1a and 2a, respectively, and RNA3 encodes the movement protein 3a. A subgenomic RNA,

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“…Kinetic parameters of the binding in vitro of the 2b protein and eight mutant variants to three types of sRNAs and a viral RNA The ability of the CMV 2b protein to bind to sRNAs in vitro had been demonstrated previously (Goto et al 2007;Rashid et al 2008;Shimura et al 2008), and the affinity of an Escherichia coli-expressed 2b protein tagged with maltose-binding protein (MBP-2b) for a 39 biotinylated siRNA in vitro was also determined (Shimura et al 2008). Furthermore, using a battery of six 2b protein mutants a correlation between ability to bind to siRNAs in vitro and suppressor activity had been found (González et al 2010).…”

Section: In Vivo Characterization Of 2b Protein Mutantsmentioning

confidence: 99%

RNA binding is more critical to the suppression of silencing function ofCucumber mosaic virus2b protein than nuclear localization

González¹,

Rakitina²,

Semashko³

et al. 2012

RNA

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Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.

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A strategy for screening an inhibitor of viral silencing suppressors, which attenuates symptom development of plant viruses

Cited by 25 publications

References 23 publications

Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

Virus-Induced Necrosis Is a Consequence of Direct Protein-Protein Interaction between a Viral RNA-Silencing Suppressor and a Host Catalase

RNA binding is more critical to the suppression of silencing function ofCucumber mosaic virus2b protein than nuclear localization

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