Dataset Information

Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

ABSTRACT:

Background

To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR.

Methods

Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-Pro^Tu), zucchini yellow mosaic virus (ZYMV) (P1/HC-Pro^Zy), and tobacco etch virus (TEV) (P1/HC-Pro^Te) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency.

Results

Our results indicated that the P1/HC-Pro^Tu plant has slightly lower RISC activity than P1/HC-Pro^Zy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-Pro^Tu could directly interfere with RISC activity.

Conclusions

In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.

SUBMITTER: Hong SF

PROVIDER: S-EPMC9844029 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Publications

Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

Hong Syuan-Fei SF Fang Ru-Ying RY Wei Wei-Lun WL Jirawitchalert Supidcha S Pan Zhao-Jun ZJ Hung Yu-Ling YL Pham Thanh Ha TH Chiu Yen-Hsin YH Shen Tang-Long TL Huang Chien-Kang CK Lin Shih-Shun SS

Virology journal 20230117 1

<h4>Background</h4>To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR.<h4>Methods</h4>Developing an endoge ...[more]

PMID: 36650505

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Project description:BackgroundRNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs). These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA) mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter) the helper component-proteinase (HC-Pro) derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent).ResultsOver expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1) were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S-adenosyl-L-methionine (SAM) were also decreased in these plants, apparently leading to decreased transmethylation capacity. The proteome analysis using 2D-PAGE indicated significantly altered proteome profile, which may have been both due to altered transcript levels, decreased translation, and increased proteosomal/protease activity.ConclusionExpression of the HC-Pro RSS mimics transcriptional changes previously shown to occur in plants infected with intact viruses (e.g. Tobacco etch virus, TEV). The results indicate that the HC-Pro RSS contributes a significant part of virus-plant interactions by changing the levels of multiple cellular RNAs and proteins.

Dataset Information

Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

Background

Methods

Results

Conclusions

Publications

Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

Similar Datasets

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