Domains of the cucumber mosaic virus 2b silencing suppressor protein affecting inhibition of salicylic acid-induced resistance and priming of salicylic acid accumulation during infection

The cucumber mosaic virus (CMV) 2b silencing suppressor protein allows the virus to overcome resistance to replication and local movement in inoculated leaves of plants treated with salicylic acid (SA), a resistance-inducing plant hormone. In Arabidopsis thaliana plants systemically infected with CMV, the 2b protein also primes the induction of SA biosynthesis during this compatible interaction. We found that CMV infection of susceptible tobacco (Nicotiana tabacum) also induced SA accumulation. Utilization of mutant 2b proteins expressed during infection of tobacco showed that the N- and C-terminal domains, which had previously been implicated in regulation of symptom induction, were both required for subversion of SA-induced resistance, while all mutants tested except those affecting the putative phosphorylation domain had lost the ability to prime SA accumulation and expression of the SA-induced marker gene PR-1.

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Functional analysis of the Cucumber mosaic virus 2b protein: pathogenicity and nuclear localization

Journal of General Virology ◽

10.1099/vir.0.80250-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 3135-3147 ◽

Cited By ~ 54

Author(s):

Yongzeng Wang ◽

Tzvi Tzfira ◽

Victor Gaba ◽

Vitaly Citovsky ◽

Peter Palukaitis ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Nuclear Localization ◽

Zucchini Yellow Mosaic Virus ◽

Yellow Mosaic Virus ◽

Silencing Suppressor ◽

Nuclear Localization Signals ◽

Systemic Movement ◽

2B Protein ◽

Function Expression

The 2b protein encoded by Cucumber mosaic virus (CMV) has been shown to be a silencing suppressor and pathogenicity determinant in solanaceous hosts, but a movement determinant in cucumber. In addition, synergistic interactions between CMV and Zucchini yellow mosaic virus (ZYMV) have been described in several cucurbit species. Here, it was shown that deletion of the 2b gene from CMV prevented extensive systemic movement of the virus in zucchini squash, which could not be complemented by co-infection with ZYMV. Thus, ZYMV expressing a silencing suppressor with a different target could not complement the CMV 2b-specific movement function. Expression of the 2b protein from an attenuated ZYMV vector resulted in a synergistic response, largely restoring infection symptoms of wild-type ZYMV in several cucurbit species. Deletion or alteration of either of two nuclear localization signals (NLSs) did not affect nuclear localization in two assays, but did affect pathogenicity in several cucurbit species, whilst deletion of both NLSs led to loss of nuclear localization. The 2b protein interacted with an Arabidopsis thaliana karyopherin α protein (AtKAPα) in the yeast two-hybrid system, as did each of the two single NLS-deletion mutants. However, 2b protein containing a deletion of both NLSs was unable to interact with AtKAPα. These data suggest that the 2b protein localizes to the nucleus by using the karyopherin α-mediated system, but demonstrate that nuclear localization was insufficient for enhancement of the 2b-mediated pathogenic response in cucurbit hosts. Thus, the sequences corresponding to the two NLSs must have another role leading to pathogenicity enhancement.

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Salicylic Acid-Induced Resistance to Cucumber mosaic virus in Squash and Arabidopsis thaliana: Contrasting Mechanisms of Induction and Antiviral Action

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0428 ◽

2005 ◽

Vol 18 (5) ◽

pp. 428-434 ◽

Cited By ~ 74

Author(s):

Carl N. Mayers ◽

Kian-Chung Lee ◽

Catherine A. Moore ◽

Sek-Man Wong ◽

John P. Carr

Keyword(s):

Arabidopsis Thaliana ◽

Salicylic Acid ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Induced Resistance ◽

Host Species ◽

Cell Movement ◽

Signal Transduction Pathway ◽

Model Systems ◽

Antimycin A

Salicylic acid (SA)-induced resistance to Cucumber mosaic virus (CMV) in tobacco (Nicotiana tabacum) results from inhibition of systemic virus movement and is induced via a signal transduction pathway that also can be triggered by antimycin A, an inducer of the mitochondrial enzyme alternative oxidase (AOX). In Arabidopsis thaliana, inhibition of CMV systemic movement also is induced by SA and antimycin A. These results indicate that the mechanisms underlying induced resistance to CMV in tobacco and A. thaliana are very similar. In contrast to the situation in tobacco and A. thaliana, in squash (Cucurbita pepo), SA-induced resistance to CMV results from inhibited virus accumulation in directly inoculated tissue, most likely through inhibition of cell-to-cell movement. Furthermore, neither of the AOX inducers antimycin A or KCN induced resistance to CMV in squash. Additionally, AOX inhibitors that compromise SAinduced resistance to CMV in tobacco did not inhibit SAinduced resistance to the virus in squash. The results show that different host species may use significantly different approaches to resist infection by the same virus. These findings also imply that caution is required when attempting to apply findings on plant-virus interactions from model systems to a wider range of host species.

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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.

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Alanine Scanning of Cucumber Mosaic Virus (CMV) 2B Protein Identifies Different Positions for Cell-To-Cell Movement and Gene Silencing Suppressor Activity

PLoS ONE ◽

10.1371/journal.pone.0112095 ◽

2014 ◽

Vol 9 (11) ◽

pp. e112095 ◽

Cited By ~ 8

Author(s):

Katalin Nemes ◽

Ákos Gellért ◽

Ervin Balázs ◽

Katalin Salánki

Keyword(s):

Gene Silencing ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Cell Movement ◽

Silencing Suppressor ◽

Suppressor Activity ◽

Alanine Scanning ◽

2B Protein

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The Rate of Cell-to-Cell Movement in Squash of Cucumber Mosaic Virus Is Affected by Sequences of the Capsid Protein

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.7.628 ◽

1999 ◽

Vol 12 (7) ◽

pp. 628-632 ◽

Cited By ~ 34

Author(s):

Sek-Man Wong ◽

Sharon Swee-Chin Thio ◽

Michael H. Shintaku ◽

Peter Palukaitis

Keyword(s):

Amino Acids ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Capsid Protein ◽

Cell Movement ◽

Systemic Infection ◽

Long Distance ◽

Local Movement ◽

Long Distance Movement

The M strain of cucumber mosaic virus (CMV) does not infect squash plants systemically and moves very slowly in inoculated cotyledons. Systemic infection and an increase in the rate of local movement were observed when amino acids 129 or 214 of the M-CMV capsid protein (CP) were altered to those present in the Fny strain of CMV. While the opposite alterations to the CP of Fny-CMV inhibited systemic infection of squash, they did not show the same effects on the rates of both cell-to-cell and long-distance movement. However, the ability of CMV to infect squash systemically was affected by the rate of cell-to-cell movement.

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Enhanced Glutathione Metabolism Is Correlated with Sulfur-Induced Resistance in Tobacco mosaic virus–Infected Genetically Susceptible Nicotiana tabacum Plants

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-10-0117 ◽

2010 ◽

Vol 23 (11) ◽

pp. 1448-1459 ◽

Cited By ~ 46

Author(s):

Kerstin Höller ◽

Lóránt Király ◽

András Künstler ◽

Maria Müller ◽

Gábor Gullner ◽

...

Keyword(s):

Nicotiana Tabacum ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Induced Resistance ◽

Marker Gene ◽

General Mechanism ◽

Glutathione Metabolism ◽

Tobacco Plants ◽

Glutathione Biosynthesis

Sulfur-induced resistance, also known as sulfur-enhanced defense (SIR/SED) was investigated in Nicotiana tabacum cv. Samsun nn during compatible interaction with Tobacco mosaic virus (TMV) in correlation with glutathione metabolism. To evaluate the influence of sulfur nutritional status on virus infection, tobacco plants were treated with nutrient solutions containing either sufficient sulfate (+S) or no sulfate (−S). Sufficient sulfate supply resulted in a suppressed and delayed symptom development and diminished virus accumulation over a period of 14 days after inoculation as compared with −S conditions. Expression of the defense marker gene PR-1a was markedly upregulated in sulfate-treated plants during the first day after TMV inoculation. The occurrence of SIR/SED correlated with a higher level of activity of sulfate assimilation, cysteine, and glutathione metabolism in plants treated with sulfate. Additionally, two key genes involved in cysteine and glutathione biosynthesis (encoding adenosine 5′-phosphosulfate reductase and γ-glutamylcysteine synthetase, respectively) were upregulated within the first day after TMV inoculation under +S conditions. Sulfate withdrawal from the soil was accelerated at the beginning of the infection, whereas it declined in the long term, leading to an accumulation of sulfur in the soil of plants grown with sulfate. This observation could be correlated with a decrease in sulfur contents in TMV-infected leaves in the long term. In summary, this is the first study that demonstrates a link between the activation of cysteine and glutathione metabolism and the induction of SIR/SED during a compatible plant-virus interaction in tobacco plants, indicating a general mechanism behind SIR/SED.

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Cucumber mosaic virus and its 2b RNA silencing suppressor modify plant-aphid interactions in tobacco

Scientific Reports ◽

10.1038/srep00187 ◽

2011 ◽

Vol 1 (1) ◽

Cited By ~ 73

Author(s):

Heiko Ziebell ◽

Alex M. Murphy ◽

Simon C. Groen ◽

Trisna Tungadi ◽

Jack H. Westwood ◽

...

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Rna Silencing ◽

Silencing Suppressor ◽

Rna Silencing Suppressor

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The Suppressor of Transgene RNA Silencing Encoded by Cucumber mosaic virus Interferes with Salicylic Acid-Mediated Virus Resistance

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.6.715 ◽

2001 ◽

Vol 14 (6) ◽

pp. 715-724 ◽

Cited By ~ 158

Author(s):

Liang-Hui Ji ◽

Shou-Wei Ding

Keyword(s):

Salicylic Acid ◽

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Virus Resistance ◽

Rna Silencing ◽

Defense Mechanism ◽

Antiviral Defense ◽

Oxidase Gene ◽

Systemic Spread ◽

Systemic Infections

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pre-treated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.

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