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Apple latent spherical virus, Cucumber mosaic virus (CMV) and. Pea early ..... Figure S7 Transmittance of VIGS to progeny seedlings for two generations in N.
Plant Biotechnology Journal (2011) 9, pp. 797–806

doi: 10.1111/j.1467-7652.2011.00589.x

Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato Muthappa Senthil-Kumar and Kirankumar S. Mysore* Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK, USA

Received 20 July 2010; revised 3 December 2010; accepted 6 December 2010. *Correspondence (fax 580 224 6692; e-mail [email protected])

Keywords: functional genomics, RNA silencing, Nicotiana benthamiana, tomato, Solanaceae, TRV.

Summary 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.

Introduction Virus-induced gene silencing (VIGS) is widely used to silence the target gene of interest in certain plants by exploiting natural plant defence mechanisms (Lu et al., 2003; Robertson, 2004; Voinnet, 2001). VIGS involves delivery of a fragment of plant gene (intended to be silenced) into plant cells via a recombinant virus. The plant defence mechanism silences both the targeted endogenous plant gene and the virus through post-transcriptional gene silencing [PTGS, reviewed in (Robertson, 2004; Voinnet, 2001)]. VIGS entails generation of double-stranded RNA (dsRNA) during virus replication and production of small interfering RNA (siRNA) with a 21–23 nucleotide length through a plant DICER like nuclease. These siRNAs bind to RNA-induced silencing complex (RISC) and target this complex to find a homology containing mRNA for cleavage. Such fragmented mRNAs are later destroyed by cellular nucleases causing suppression of target gene expression [reviewed in (Robertson, 2004; Watson et al., 2005)]. The Tobacco rattle virus (TRV)-based VIGS, vector is most widely used for gene silencing in Solanaceae plant species [reviewed in (Senthil-Kumar et al., 2008)]. TRV is a positivestrand RNA virus with a bipartite genome [RNA1 and RNA2; (MacFarlane, 1999)]. RNA1 encodes an RNA-dependent RNA polymerase and a movement protein. RNA2 encodes a coat protein (CP) and two nonstructural proteins from the subgenomic RNAs. TRV can move systemically in many plants and, unlike some other viruses, does not encode a strong silencing suppressor (Ratcliff et al., 2001). Both RNA1 and RNA2 are needed for infection and production of virus particles (MacFarlane, 1999). TRV can infect roots and move to aerial

plant parts and provoke VIGS (Liu et al., 2002a; MacFarlane, 1999; Ryu et al., 2004). TRV is also known to infect meristem tissue (Liu et al., 2002b; Ratcliff et al., 2001). A 29-kDa protein from RNA2 is involved in cell-to-cell movement. Also, a cysteinerich protein encoded by TRV has been implicated in seed transmission (Liu et al., 2002a; MacFarlane, 1999). For VIGS, both RNA1 and RNA2 were modified and cloned individually between the T-DNA borders of an Agrobacterium binary vector (Liu et al., 2002b). These TRV-VIGS vectors are mainly delivered into plants by Agrobacterium-mediated transient expression using leaf infiltration and Agrodrench methods (Ryu et al., 2004). TRVmediated VIGS has been used to silence genes in all plant parts namely root, leaf, stem, flower and fruit in a wide range of plant species [reviewed in Senthil-Kumar et al. (2008)]. VIGS was shown to be a transient phenomenon (Liu et al., 2002b; Lu et al., 2003), and stable inheritance of virus-mediated PTGS is not reported. In fact one study showed that TRV-mediated VIGS is not transmitted to plant progenies (Wege et al., 2007). The ability to generate gene knockdown phenotypes without having to genetically manipulate the plant genome is one of the advantages of VIGS compared to other gene suppression methods like mutagenesis and RNA interference (RNAi). It is a common belief that effective VIGS occurs only for 3 weeks (Ryu et al., 2004). Efficiency of VIGS has been shown to decrease after 1 month and plants start to recover from silencing (Ratcliff et al., 2001). Such a recovery can be either transient or stable and the extent of recovery phenotype can be partial or complete (Hiriart et al., 2003; Ratcliff et al., 2001). Persistence of VIGS for longer duration is influenced by age of the plant, viral titre, and environmental conditions that favour virus multiplication (Fu et al., 2006; Tuttle et al., 2008).

ª 2011 The Authors Plant Biotechnology Journal ª 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd

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798 Muthappa Senthil-Kumar and Kirankumar S. Mysore However, the exact mechanism for persistence of VIGS has not been fully understood. Maintenance of effective VIGS for longer duration is desirable for both forward and reverse genetics studies. In this manuscript, we provide experimental evidence to show that TRV-mediated VIGS can be maintained for more than 2 years in N. benthamiana and tomato plants. Combined inoculation of VIGS vectors by both Agrodrench and leaf infiltration methods induce effective gene silencing for longer period. Further, we report the transmittance of VIGS to progeny seedlings. Results from this study will help researchers adapt VIGS as a tool to study gene function in plant development and plant responses to stress.

Results VIGS in N. benthamiana and tomato persisted for 24 months and beyond To assess the persistence of TRV-VIGS for longer duration, we targeted three different genes namely PDS, ChlH and CpTF-TuB. Agrobacterium strains containing these TRV-VIGS constructs were inoculated into N. benthamiana and tomato plants by Agrodrench and ⁄ or leaf infiltration. Silencing of NbPDS and NbChlH in N. benthamiana resulted in photo-bleaching and yellowing phenotypes, respectively, while the silencing of NbCpEFTuB resulted in pale yellow (mosaic) leaves (Figure 1a). Similarly, two tomato species Solanum lycopersicum and S. pennellii plants silenced with the SlPDS gene also showed clear photobleaching (Figure S1a). Various parameters including frequency, efficiency and effectiveness of gene silencing as defined earlier (Senthil-Kumar et al., 2007) were measured from the time of initiation of silencing till 24 months postinoculation (mpi).

Frequency of gene silencing Frequency of gene silencing was calculated by counting the number of plants that showed silencing phenotype as described earlier (Senthil-Kumar et al., 2007) and in Experimental procedures section. At 24 mpi, the frequency of gene silencing was more than 80% for all the three genes that were targeted in N. benthamiana (Figure 1a). Silencing was observed in all N. benthamiana plant parts ranging from leaf, stem, flower and capsule (Figure 1b,c). The silencing phenotype was observed more in leaves and stem with more than 70% primary and secondary branches showing visible silencing phenotype (Figure 1c). However, visible silencing phenotype in reproductive organs like flowers and capsules was relatively low (Figure 1b). In tomato, S. pennellii showed highest frequency of gene silencing (>90%) at 24 mpi compared to S. lycopersicum which showed only 78% (Figure S1a) and silencing phenotype was clearly visible on stem, flowers and fruits (Figure S1b).

Efficiency of gene silencing Silencing of genes used in this study showed varying levels of reduction in total chlorophyll content ranging from 30% to 95%. Not all leaves of a plant silenced for a gene showed visible silencing phenotype. Hence, as previously shown (SenthilKumar et al., 2007), we assessed efficiency of gene silencing by estimating chlorophyll content in both leaves that showed silencing phenotype and green leaves of N. benthamiana and tomato. At the end of 24 mpi, the leaves showing silenced phenotype for NbPDS and NbChlH had more than 95% efficiency of gene silencing. However, the green leaves from the same

plants showed efficiency of