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      Nonsense-Mediated Decay Serves as a General Viral Restriction Mechanism in Plants

      , ,
      Cell Host & Microbe
      Elsevier BV

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          Abstract

          Summary (+)strand RNA viruses have to overcome various points of restriction in the host to establish successful infection. In plants, this includes RNA silencing. To uncover additional bottlenecks to RNA virus infection, we genetically attenuated the impact of RNA silencing on transgenically expressed Potato virus X (PVX), a (+)strand RNA virus that replicates in Arabidopsis. A genetic screen in this sensitized background uncovered how nonsense-mediated decay (NMD), a host RNA quality control mechanism, recognizes and eliminates PVX RNAs with internal termination codons and long 3′ UTRs. NMD also operates in natural infection contexts, and while some viruses have evolved genome expression strategies to overcome this process altogether, the virulence of NMD-activating viruses entails their ability to directly suppress NMD or to promote an NMD-unfavorable cellular state. These principles of induction, evasion, and suppression define NMD as a general viral restriction mechanism in plants that also likely operates in animals.

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          Most cited references30

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          An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.

          Transient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50-folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the time-consuming regeneration of stably transformed plants.
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            A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs.

            Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack.
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              Methylation protects miRNAs and siRNAs from a 3'-end uridylation activity in Arabidopsis.

              Small RNAs of 21-25 nucleotides (nt), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), act as guide RNAs to silence target-gene expression in a sequence-specific manner. In addition to a Dicer homolog, DCL1, the biogenesis of miRNAs in Arabidopsis requires another protein, HEN1. miRNAs are reduced in abundance and increased in size in hen1 mutants. We found that HEN1 is a miRNA methyltransferase that adds a methyl group to the 3'-most nucleotide of miRNAs, but the role of miRNA methylation was unknown. Here, we show that siRNAs from sense transgenes, hairpin transgenes, and transposons or repeat sequences, as well as a new class of siRNAs known as trans-acting siRNAs, are also methylated in vivo by HEN1. In addition, we show that the size increase of small RNAs in the hen1-1 mutant is due to the addition of one to five U residues to the 3' ends of the small RNAs. Therefore, a novel uridylation activity targets the 3' ends of unmethylated miRNAs and siRNAs in hen1 mutants. We conclude that 3'-end methylation is a common step in miRNA and siRNA metabolism and likely protects the 3' ends of the small RNAs from the uridylation activity.
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                Author and article information

                Journal
                Cell Host & Microbe
                Cell Host & Microbe
                Elsevier BV
                19313128
                September 2014
                September 2014
                : 16
                : 3
                : 391-402
                Article
                10.1016/j.chom.2014.08.001
                ad04c24f-59fb-44b8-9c89-09b9bf2cc55d
                © 2014

                https://www.elsevier.com/tdm/userlicense/1.0/

                https://www.elsevier.com/open-access/userlicense/1.0/

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