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Differential expression of cucumber RNA-dependent RNA polymerase 1 genes during antiviral defence and resistance.

ABSTRACT: RNA-dependent RNA polymerase 1 (RDR1) plays a crucial role in plant defence against viruses. In this study, it was observed that cucumber, Cucumis sativus, uniquely encodes a small gene family of four RDR1 genes. The cucumber RDR1 genes (CsRDR1a, CsRDR1b and duplicated CsRDR1c1/c2) shared 55%-60% homology in their encoded amino acid sequences. In healthy cucumber plants, RDR1a and RDR1b transcripts were expressed at higher levels than transcripts of RDR1c1/c2, which were barely detectable. The expression of all four CsRDR1 genes was induced by virus infection, after which the expression level of CsRDR1b increased 10-20-fold in several virus-resistant cucumber cultivars and in a broad virus-resistant transgenic cucumber line expressing a high level of transgene small RNAs, all without alteration in salicylic acid (SA) levels. By comparison, CsRDR1c1/c2 genes were highly induced (25-1300-fold) in susceptible cucumber cultivars infected with RNA or DNA viruses. Inhibition of RDR1c1/c2 expression led to increased virus accumulation. Ectopic application of SA induced the expression of cucumber RDR1a, RDR1b and RDRc1/c2 genes. A constitutive high level of RDR1b gene expression independent of SA was found to be associated with broad virus resistance. These findings show that multiple RDR1 genes are involved in virus resistance in cucumber and are regulated in a coordinated fashion with different expression profiles.

SUBMITTER: Leibman D

PROVIDER: S-EPMC6637986 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

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Differential expression of cucumber RNA-dependent RNA polymerase 1 genes during antiviral defence and resistance.

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