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From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-Pro.

ABSTRACT: RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.

SUBMITTER: Torres-Barcelo C

PROVIDER: S-EPMC2567354 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-Pro.

Torres-Barceló Clara C Martín Susana S Daròs José-Antonio JA Elena Santiago F SF

Genetics 20080909 2

RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid su ...[more]

PMID: 18780745

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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.

Project description:BackgroundThe genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. However, the interaction networks between viruses and host factors are poorly understood. The genus Potyvirus is the largest group of plus-strand RNA viruses infecting plants. Potyviral nuclear inclusion a (NIa) plays many roles during infection. NIa is a polyprotein consisting of two domains, viral protein genome-linked (VPg) and protease (NIaPro), separated by an inefficiently utilized self-proteolytic site. To gain insights about the interaction between potyviral NIa and the host cell during infection, we constructed Tobacco etch virus (TEV, genus Potyvirus) infectious clones in which the VPg or the NIaPro domains of NIa were tagged with the affinity polypeptide Twin-Strep-tag and identified the host proteins targeted by the viral proteins by affinity purification followed by mass spectrometry analysis (AP-MS).ResultsWe identified 232 different Arabidopsis thaliana proteins forming part of complexes in which TEV NIa products were also involved. VPg and NIaPro specifically targeted 89 and 76 of these proteins, respectively, whereas 67 proteins were targeted by both domains and considered full-length NIa targets. Taking advantage of the currently known A. thaliana interactome, we constructed a protein interaction network between TEV NIa domains and 516 host proteins. The most connected elements specifically targeted by VPg were G-box regulating factor 6 and mitochondrial ATP synthase δ subunit; those specifically targeted by NIaPro were plasma membrane aquaporin PIP2;7 and actin 7, whereas those targeted by full-length NIa were heat shock protein 70-1 and photosystem protein LHCA3. Moreover, a contextualization in the global A. thaliana interactome showed that NIa targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths. Further analysis of NIa-targeted host proteins revealed that they are mainly involved in response to stress, metabolism, photosynthesis, and localization. Many of these proteins are connected with the phytohormone ethylene.ConclusionsPotyviral NIa targets many host elements during infection, establishing a network in which information is efficiently transmitted.

Dataset Information

From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-Pro.

Publications

From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of the Tobacco etch potyvirus HC-Pro.

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