Virus-induced Gene Silencing Analysis Research Articles

Efficient gene silencing induction in tomato by a viral satellite DNA vector

Virus-induced gene silencing (VIGS) in tomato ( Lycopersicon esculentum Mill.) is currently routinely analysed using Tobacco rattle virus (TRV)-based vector. We recently reported a new vector system modified from DNAβ (DNAmβ) of Tomato yellow leaf curl China virus (TYLCCNV) for VIGS analysis in Solanaceous species including tomato. Here, we describe DNAmβ-induced gene silencing in tomato. We found that DNAmβ-induced gene silencing was initiated from vascular tissues, and later scattered to other tissues. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants, was expressed in a variety of tissues and organs including leaf, shoot, stem, flower and fruit, and could be achieved at any growth stage. It was insensitive to temperature as high as 32 °C and no symptoms were observed in silenced plants. The DNAmβ vector worked efficiently in at least seven tomato cultivars, indicating that this system has great potential as a versatile VIGS system for routine functional analysis of genes in tomato.

Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae).

BackgroundThe lower eudicot genus Aquilegia, commonly known as columbine, is currently the subject of extensive genetic and genomic research aimed at developing this taxon as a new model for the study of ecology and evolution. The ability to perform functional genetic analyses is a critical component of this development process and ultimately has the potential to provide insight into the genetic basis for the evolution of a wide array of traits that differentiate flowering plants. Aquilegia is of particular interest due to both its recent evolutionary history, which involves a rapid adaptive radiation, and its intermediate phylogenetic position between core eudicot (e.g., Arabidopsis) and grass (e.g., Oryza) model species.ResultsHere we demonstrate the effective use of a reverse genetic technique, virus-induced gene silencing (VIGS), to study gene function in this emerging model plant. Using Agrobacterium mediated transfer of tobacco rattle virus (TRV) based vectors, we induce silencing of PHYTOENE DESATURASE (AqPDS) in Aquilegia vulgaris seedlings, and ANTHOCYANIDIN SYNTHASE (AqANS) and the B-class floral organ identity gene PISTILLATA in A. vulgaris flowers. For all of these genes, silencing phenotypes are associated with consistent reduction in endogenous transcript levels. In addition, we show that silencing of AqANS has no effect on overall floral morphology and is therefore a suitable marker for the identification of silenced flowers in dual-locus silencing experiments.ConclusionOur results show that TRV-VIGS in Aquilegia vulgaris allows data to be rapidly obtained and can be reproduced with effective survival and silencing rates. Furthermore, this method can successfully be used to evaluate the function of early-acting developmental genes. In the future, data derived from VIGS analyses will be combined with large-scale sequencing and microarray experiments already underway in order to address both recent and ancient evolutionary questions.

Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus.

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.