Identification of a Plant-Encoded Analog of PKR, the Mammalian Double-Stranded RNA-Dependent Protein Kinase

Langland, Jeffrey; Jin, Shaowei; Jacobs, Bertram L.; Roth, Don A.

doi:10.1104/pp.108.3.1259

Cited by 60 publications

(37 citation statements)

References 39 publications

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“…A putative candidate is the dsRNA-dependent protein kinase (PKR), which functions in dsRNA signalling and host defence against virus infection in mammals (Williams, 1999) and has also been described as having a role in pathogenesis in plants (Hiddinga et al, 1988;Hu & Roth, 1991;Langland et al, 1995). There are many questions that have to be addressed but the results presented in this work further support the idea that RNA silencing reported as an antiviral defence mechanism in plants most likely also plays an antiviral role in mammalian cells.…”

supporting

confidence: 56%

Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants

Delgadillo

Sáenz

Salvador

et al. 2004

Journal of General Virology

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RNA silencing has a well-established function as an antiviral defence mechanism in plants and insects. Using an Agrobacterium-mediated transient assay, we report here that NS1 protein from human influenza A virus suppresses RNA silencing in plants in a manner similar to P1/HC-Pro protein of Tobacco etch potyvirus, a well-characterized plant virus silencing suppressor. Moreover, we have shown that NS1 protein expression strongly enhances the symptoms of Potato virus X in three different plant hosts, suggesting that NS1 protein could be inhibiting defence mechanisms activated in the plant on infection. These data provide further evidence that an RNA silencing pathway could also be activated as a defence response in mammals.Higher eukaryotes are involved in a continuing battle against viruses. To minimize the effects of viral infection, a number of defence mechanisms have been developed based on the recognition of specific molecular patterns produced only in infected cells (Plasterk, 2002). This is the case with double-stranded RNA (dsRNA) molecules, which are not normally found in eukaryotic cells but are generated as an intermediate molecule during virus replication (Hutvagner & Zamore, 2002). In mammalian cells, dsRNA appears to play a major role in the induction of the interferon (IFN) response following viral infection (Stark et al., 1998). In recent years, another dsRNA-mediated defence mechanism known as RNA silencing has been described (Dougherty & Parks, 1995;Montgomery & Fire, 1998).RNA silencing is a sequence-specific RNA degradation process that leads to elimination of the targeted RNA mediated by cytoplasmic nucleases and plays a natural antiviral role in plants (for reviews, see Baulcombe, 2002;Carrington et al., 2001;Vance & Vaucheret, 2001;Vazquez Rovere et al., 2002;Waterhouse et al., 2001). Recently, this mechanism of antiviral defence has also been reported in insect cells (Li et al., 2002).In response to these types of host antiviral defences, it is not unexpected that viruses have devised counteracting mechanisms that interfere with them at different levels. Thus, many animal viruses are known to prevent or inhibit IFN-mediated defence (García-Sastre, 2001). The same is true for RNA silencing: many plant viruses (Carrington & Whitham, 1998;Li & Ding, 2001;Voinnet et al., 1999) and one insect virus (Li et al., 2002) have been shown to encode silencing suppressor proteins. Nearly 20 silencing suppressors described so far show no sequence similarity and hence appear to have evolved independently to overcome silencing-mediated defence. Thus, it is not easy to predict which protein is going to behave as an RNA silencing suppressor based on sequence analysis.Since this RNA silencing has been described in species from different kingdoms (fungi, animals and plants), it has been proposed that it can also play an antiviral role in mammalian cells (Cullen, 2002;Gitlin et al., 2002). Here, we have investigated whether the NS1 protein, a multifunctional protein of human influenza A virus, could act as an RN...

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supporting

confidence: 56%

Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants

Delgadillo

Sáenz

Salvador

et al. 2004

Journal of General Virology

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“…Third, MIR VIGS may have lower side effects. The traditional VIGS vector produces siRNAs and long double-stranded RNAs, and the latter may activate RNA-dependent protein kinase pathways and cause nonspecific cell death in plants, a phenomenon well documented in mammalian cells (Langland et al, 1995;Davis and Watson, 1996;Bridge et al, 2003;Sledz et al, 2003;Tang and Galili, 2004). By contrast, MIR VIGS uses DNA virus vector to express miRNA, and this may avoid triggering the protein kinase pathway.…”

Section: Discussionmentioning

confidence: 99%

Virus-Based MicroRNA Expression for Gene Functional Analysis in Plants

et al. 2010

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Traditional virus-induced gene silencing (VIGS) is a powerful virus-based short interfering RNA-mediated RNA silencing technique for plant functional genomics. Besides short interfering RNAs, microRNAs (miRNAs) have also been shown to regulate gene expression by RNA silencing in various organisms. However, plant virus-based miRNA silencing has not been reported. In addition, a number of plant miRNAs have been identified or predicted, while their functions are largely unknown. Thus, there is an urgent need for the development of new technologies to study miRNA function. Here, we report that a modified cabbage leaf-curl geminivirus vector can be used to express artificial and endogenous miRNAs in plants. Using this viral miRNA expression system, we demonstrate that VIGS using artificial miRNAs, dubbed as "MIR VIGS," was effective to silence the expression of endogenous genes, including PDS, Su, CLA1, and SGT1, in Nicotiana benthamiana. Silencing of SGT1 led to the loss of N-mediated resistance to Tobacco mosaic virus. Furthermore, using this viral miRNA expression system, we found that viral ectopic expression of endogenous miR156 and miR165 but not their mutants in N. benthamiana resulted in earlier abnormal developmental phenotypes, and expression of miR165 induced abnormal chlorotic spots on leaves. These results demonstrate that the cabbage leaf-curl geminivirus-based miRNA expression system can be utilized not only to specifically silence genes involved in general metabolism and defense but also to investigate the function of endogenous miRNAs in plants.

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“…Technical barriers for high-throughput functional genomics have recently been lowered considerably by the development of pHELLSGATE vectors that utilize the Gateway recombination system and give the possibility of making hpRNA constructs for large sets of genes (Helliwell and Waterhouse, 2003). Tang and Galili (2004) raised the hypothesis that next generation RNAi vectors should contain characteristics of micro-RNA structures, because microRNAs do not trigger the PKR pathway, the RNA-dependent protein kinase pathway that causes nonspecific cell death in mammalian cells and could function as part of the plant stress response (Langland et al, 1995). Fast and efficient systems of transformation and regeneration of transgenic plants are necessary to successfully use RNAi constructs for plant functional genomics.…”

Section: Discussionmentioning

confidence: 99%

RNA Interference-Based Gene Silencing as an Efficient Tool for Functional Genomics in Hexaploid Bread Wheat

2006

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Insertional mutagenesis and gene silencing are efficient tools for the determination of gene function. In contrast to gain-or lossof-function approaches, RNA interference (RNAi)-induced gene silencing can possibly silence multigene families and homoeologous genes in polyploids. This is of great importance for functional studies in hexaploid wheat (Triticum aestivum), where most of the genes are present in at least three homoeologous copies and conventional insertional mutagenesis is not effective. We have introduced into bread wheat double-stranded RNA-expressing constructs containing fragments of genes encoding Phytoene Desaturase (PDS) or the signal transducer of ethylene, Ethylene Insensitive 2 (EIN2). Transformed plants showed phenotypic changes that were stably inherited over at least two generations. These changes were very similar to mutant phenotypes of the two genes in diploid model plants. Quantitative real-time polymerase chain reaction revealed a good correlation between decreasing mRNA levels and increasingly severe phenotypes. RNAi silencing had the same quantitative effect on all three homoeologous genes. The most severe phenotypes were observed in homozygous plants that showed the strongest mRNA reduction and, interestingly, produced around 2-fold the amount of small RNAs compared to heterozygous plants. This suggests that the effect of RNAi in hexaploid wheat is gene-dosage dependent. Wheat seedlings with low mRNA levels for EIN2 were ethylene insensitive. Thus, EIN2 is a positive regulator of the ethylene-signaling pathway in wheat, very similar to its homologs in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Our data show that RNAi results in stably inherited phenotypes and therefore represents an efficient tool for functional genomic studies in polyploid wheat.

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Identification of a Plant-Encoded Analog of PKR, the Mammalian Double-Stranded RNA-Dependent Protein Kinase

Cited by 60 publications

References 39 publications

Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants

Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants

Virus-Based MicroRNA Expression for Gene Functional Analysis in Plants

RNA Interference-Based Gene Silencing as an Efficient Tool for Functional Genomics in Hexaploid Bread Wheat

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