Comparison of in vivo and in vitro translation of Cowpea Mosaic Virus RNAs

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

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Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection

Plants ◽

10.3390/plants10050963 ◽

2021 ◽

Vol 10 (5) ◽

pp. 963

Author(s):

Maria C. Holeva ◽

Athanasios Sklavounos ◽

Rajendran Rajeswaran ◽

Mikhail M. Pooggin ◽

Andreas E. Voloudakis

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Topical Application ◽

Plant Virus ◽

Plant Protection ◽

Post Inoculation ◽

Tobacco Plants ◽

Double Stranded Rna

Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.

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Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Journal of Virology ◽

10.1128/jvi.00540-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8329-8344 ◽

Cited By ~ 67

Author(s):

Jamie Ashby ◽

Emmanuel Boutant ◽

Mark Seemanpillai ◽

Adrian Sambade ◽

Christophe Ritzenthaler ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Cell Extract ◽

Transport Processes ◽

In Vitro Assays ◽

Protein Functions ◽

Intercellular Spread

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

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In vitro and in vivo aggregation of the defective PM2 tobacco mosaic virus protein

Journal of Molecular Biology ◽

10.1016/s0022-2836(66)80056-6 ◽

1966 ◽

Vol 19 (1) ◽

pp. 140-IN8 ◽

Cited By ~ 21

Author(s):

Albert Siegel ◽

G.J. Hills ◽

Roy Markham

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Virus Protein

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Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5433-5441.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5433-5441

Author(s):

B Y Ahn ◽

P D Gershon ◽

E V Jones ◽

B Moss

Keyword(s):

Rna Polymerase ◽

Vaccinia Virus ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Viral Rna ◽

In Vitro Translation ◽

Virus Protein ◽

Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

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