Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato. Plant Biotechnol J 9:797-806

Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK, USA.
Plant Biotechnology Journal (Impact Factor: 5.75). 09/2011; 9(7):797-806. DOI: 10.1111/j.1467-7652.2011.00589.x
Source: PubMed


Virus-induced gene silencing (VIGS) is one of the commonly used RNA silencing methods in plant functional genomics. It is widely known that VIGS can occur for about 3 weeks. A few reports show that duration of VIGS can be prolonged for up to 3 months. Increasing the duration of endogenous gene silencing and developing a method for nonintegration-based persistent VIGS in progeny seedlings will widen the application of VIGS. We used three marker genes that provoke visible phenotypes in plants upon silencing to study persistence and transmittance of VIGS to progeny in two plant species, Nicotiana benthamiana and tomato. We used a Tobacco rattle virus (TRV)-based VIGS vector and showed that the duration of gene silencing by VIGS can occur for more than 2 years and that TRV is necessary for longer duration VIGS. Also, inoculation of TRV-VIGS constructs by both Agrodrench and leaf infiltration greatly increased the effectiveness and duration of VIGS. Our results also showed transmittance of VIGS to progeny seedlings via seeds. A longer silencing period will facilitate detailed study of target genes in plant development and stress tolerance. Further, the transmittance of VIGS to progeny will be useful in studying the effect of gene silencing in young seedlings. Our results provide a new dimension for the application of VIGS in plants.

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Available from: Senthil-Kumar Muthappa,
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    • "VIGS has been shown to occur for a shorter period of approximately 3 weeks and the efficiency decreases after a month resulting in partial or complete recovery of plants from the silencing (Ratcliff et al., 2001; Hiriart et al., 2003; Ryu et al., 2004) (Supplementary Figure 2A). However, recent evidences suggest that some VIGS vectors can be used to maintain the gene silencing for several months by suitably modifying plant growth conditions that favor viral multiplication (Fu et al., 2006; Tuttle et al., 2008; Senthil-Kumar and Mysore, 2011b, 2014) (Supplementary Figure 2B) and can transmit to next generation (Senthil-Kumar and Mysore, 2011b) behaving like stable transgenic plants (Supplementary Figure 2C). "
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    ABSTRACT: Virus-induced gene silencing (VIGS) is an effective tool for gene function analysis in plants. Over the last decade, VIGS has been successfully used as both a forward and reverse genetics technique for gene function analysis in various model plants, as well as crop plants. With the increased identification of differentially expressed genes under various abiotic stresses through high-throughput transcript profiling, the application of VIGS is expected to be important in the future for functional characterization of a large number of genes. In the recent past, VIGS was proven to be an elegant tool for functional characterization of genes associated with abiotic stress responses. In this review, we provide an overview of how VIGS is used in different crop species to characterize genes associated with drought-, salt-, oxidative- and nutrient-deficiency-stresses. We describe the examples from studies where abiotic stress related genes are characterized using VIGS. In addition, we describe the major advantages of VIGS over other currently available functional genomics tools. We also summarize the recent improvements, limitations and future prospects of using VIGS as a tool for studying plant responses to abiotic stresses.
    Frontiers in Plant Science 07/2014; 5:323. DOI:10.3389/fpls.2014.00323 · 3.95 Impact Factor
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    • "In total, 488 seeds were germinated and none of them became bleached (Figure S2B in File S1), thus confirming the non-heritability of the PfPDS silencing effect in the TRV-VIGS system in Physalis. However, under strictly controlled temperature conditions, viral vectors can remain in seeds for future generations, and silencing effects have been maintained for more than two years in Nicotiana [38]. Despite this report, the efficacy of VIGS is generally held to be transient. "
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    ABSTRACT: The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the 'Chinese lantern'). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS). Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution and development of the morphological diversities within the Solanaceae.
    PLoS ONE 01/2014; 9(1):e85534. DOI:10.1371/journal.pone.0085534 · 3.23 Impact Factor
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    • "We, therefore, expected to see a silencing phenotype two weeks after TRV inoculation. Silencing of NbPDS and NbChlH genes in plants produces photobleaching and yellowing of leaves, respectively [28]. At 8 dpi, all the leaf disks were green, and the expected silencing phenotype started from 14 dpi and continued for few weeks (Figure 1A). "
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    ABSTRACT: Understanding the function of a particular gene under various stresses is important for engineering plants for broad-spectrum stress tolerance. Although virus-induced gene silencing (VIGS) has been used to characterize genes involved in abiotic stress tolerance, currently available gene silencing and stress imposition methodology at the whole plant level is not suitable for high-throughput functional analyses of genes. This demands a robust and reliable methodology for characterizing genes involved in abiotic and multi-stress tolerance. Our methodology employs VIGS-based gene silencing in leaf disks combined with simple stress imposition and effect quantification methodologies for easy and faster characterization of genes involved in abiotic and multi-stress tolerance. By subjecting leaf disks from gene-silenced plants to various abiotic stresses and inoculating silenced plants with various pathogens, we show the involvement of several genes for multi-stress tolerance. In addition, we demonstrate that VIGS can be used to characterize genes involved in thermotolerance. Our results also showed the functional relevance of NtEDS1 in abiotic stress, NbRBX1 and NbCTR1 in oxidative stress; NtRAR1 and NtNPR1 in salinity stress; NbSOS1 and NbHSP101 in biotic stress; and NtEDS1, NbETR1, NbWRKY2 and NbMYC2 in thermotolerance. In addition to widening the application of VIGS, we developed a robust, easy and high-throughput methodology for functional characterization of genes involved in multi-stress tolerance.
    BMC Plant Biology 12/2013; 13(1):193. DOI:10.1186/1471-2229-13-193 · 3.81 Impact Factor
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