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Graft transmission of RNA silencing to non-transgenic scions for conferring virus resistance in tobacco.

ABSTRACT: RNA silencing is a mechanism of gene regulation by sequence specific RNA degradation and is involved in controlling endogenous gene expression and defense against invasive nucleic acids such as viruses. RNA silencing has been proven to be transmitted between scions and rootstocks through grafting, mostly using transgenic plants. It has been reported that RNA silencing of tobacco endogenous genes, NtTOM1 and NtTOM3, that are required for tobamovirus multiplication, resulted in high resistance against several tobamoviruses. In the present study, we examined the graft transmission of RNA silencing for conferring virus resistance to non-transgenic scions of the same and different Nicotiana species grafted onto rootstocks in which both NtTOM1 and NtTOM3 were silenced. Non-transgenic Nicotiana tabacum (cvs. Samsun and Xanthi nc) and N. benthamiana were used as scions for grafting onto the rootstocks silenced with both genes. Short interfering RNA (siRNA) of NtTOM1 and NtTOM3 was detected in both the scions and the rootstocks eight weeks after grafting. The leaves were detached from the scions and inoculated with several tobamoviruses. The virus accumulation was tested by ELISA and northern blot analysis. The viruses were detected in grafted scions at extremely low levels, showing that virus resistance was conferred. These results suggest that RNA silencing was induced in and virus resistance was conferred to the non-transgenic scions by grafting onto silenced rootstocks. The effect of low temperature on siRNA accumulation and virus resistance was not significantly observed in the scions.

SUBMITTER: Md Ali E

PROVIDER: S-EPMC3661558 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Graft transmission of RNA silencing to non-transgenic scions for conferring virus resistance in tobacco.

Md Ali Emran E Kobayashi Kappei K Yamaoka Naoto N Ishikawa Masayuki M Nishiguchi Masamichi M

PloS one 20130522 5

RNA silencing is a mechanism of gene regulation by sequence specific RNA degradation and is involved in controlling endogenous gene expression and defense against invasive nucleic acids such as viruses. RNA silencing has been proven to be transmitted between scions and rootstocks through grafting, mostly using transgenic plants. It has been reported that RNA silencing of tobacco endogenous genes, NtTOM1 and NtTOM3, that are required for tobamovirus multiplication, resulted in high resistance aga ...[more]

PMID: 23717405

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Project description:BackgroundRNA-silencing is a conserved gene regulation and surveillance machinery, which in plants, is also used as major defence mechanism against viruses. Various virus-specific dsRNA structures are recognized by the silencing machinery leading to degradation of the viral RNAs or, as in case of begomoviruses, to methylation of their DNA genomes. Viruses produce specific RNA silencing suppressor (RSS) proteins to prevent these host defence mechanisms, and as these interfere with the silencing machinery they also disturb the endogenous silencing reactions. In this paper, we describe how expression of AC2 RSS, derived from African cassava mosaic geminivirus changes transcription profile in tobacco (Nicotiana tabacum) leaves and in flowers.ResultsExpression of AC2 RSS in transgenic tobacco plants induced clear phenotypic changes both in leaves and in flowers. Transcriptomes of these plants were strongly altered, with total of 1118 and 251 differentially expressed genes in leaves and flowers, respectively. The three most up-regulated transcript groups were related to stress, cell wall modifications and signalling, whereas the three most down-regulated groups were related to translation, photosynthesis and transcription. It appears that many of the gene expression alterations appeared to be related to enhanced biosynthesis of jasmonate and ethylene, and consequent enhancement of the genes and pathways that are regulated by these hormones, or to the retrograde signalling caused by the reduced photosynthetic activity and sugar metabolism. Comparison of these results to a previous transcriptional profiling of HC-Pro RSS-expressing plants revealed that some of same genes were induced by both RSSs, but their expression levels were typically higher in AC2 than in HC-Pro RSS expressing plants. All in all, a large number of transcript alterations were found to be specific to each of the RSS expressing transgenic plants.ConclusionsAC2 RSS in transgenic tobacco plants interferes with the silencing machinery. It causes stress and defence reactions for instance via induction of the jasmonate and ethylene biosynthesis, and by consequent gene expression alteration regulated by these hormones. The changed sugar metabolism may cause significant down-regulation of genes encoding ribosomal proteins, thus reducing the general translation level.

Project description:The enzyme AHAS (acetohydroxy acid synthase), which is involved in the biosynthesis of valine, leucine and isoleucine, is the target of several classes of herbicides. A model of tobacco AHAS was generated based on the X-ray structure of yeast AHAS. Well conserved residues at the herbicide-binding site were identified, and the roles of three of these residues (Phe-205, Val-570 and Phe-577) were determined by site-directed mutagenesis. The Phe-205 mutants F205A, F205H, F205W and F205Y showed markedly decreased levels of catalytic efficiency, and cross-resistance to two or three classes of herbicides, i.e. Londax (a sulphonylurea herbicide), Cadre (an imidazolinone herbicide) and TP (a triazolopyrimidine derivative). None of the mutations caused significant changes in the secondary or tertiary structure of the enzyme. Four mutants of Phe-577, i.e. F577D, F577E, F577K and F577R, showed unaltered V(max) values, but substantially decreased catalytic efficiency. However, these mutants were highly resistant to two or three of the tested herbicides. The three mutants F577D, F577E and F577R had a similar secondary structure to that of wild-type AHAS. Conservative mutations of Phe-577, i.e. F577W and F577Y, did not affect the kinetic properties of the enzyme or its inhibition by herbicides. The mutation Val-570 to Asn abolished the binding affinity of the enzyme for FAD as well as its activity, and also caused a change in the tertiary structure of AHAS. However, the mutant V570Q was active, but resistant to two classes of herbicides, i.e. Londax and TP. The conservative mutant V570I was substantially reduced in catalytic efficiency and moderately resistant to the three herbicides. The results of this study suggest that residues Phe-205, Val-570 and Phe-577 in tobacco AHAS are located at or near the binding site that is common for the three classes of herbicides. In addition, Phe-205 and Val-570 are probably located at the herbicide-binding site that may overlap partially with the active site. Selected mutants of Phe-577 are expected to be utilized to construct herbicide-resistant transgenic plants.

Dataset Information

Graft transmission of RNA silencing to non-transgenic scions for conferring virus resistance in tobacco.

Publications

Graft transmission of RNA silencing to non-transgenic scions for conferring virus resistance in tobacco.

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