Developing TRV-mediated Virus Induced Gene Silencing (VIGS) assay in potato roots for functional analysis of genes involved in nematode resistance and susceptibility
Plant viruses are able to elicit a form of posttranscriptional gene silencing and are commonly used as a reverse genetics tool for functional characterization of plant genes. The Virus Induced Gene Silencing proved to be very efficient in above-ground parts of the plant, while the silencing in roots requires often further optimization and is species-dependent.
The application of a TRV vector, which carries a 2b helper protein required for transmission by nematodes and successful gene silencing in plant roots was previously described for the Mi-1 gene (Valentine et al, 2004, Plant Physiology) and other tomato genes required for Mi-1 resistance signaling (Bhattarai et al 2007, Plant Physiology). Other methods, using TRV vectors without the 2b gene, was used to silence the Mi-9 gene (Jablonska et al, 2007) and is described in details by Kaloshian (2004). Additionally, in 2004 Ryu et al. published an improvement of the TRV-induced silencing method in Solanaceous species by using Agrodrench and later on they showed that the combined application of Agrobacterium carrying TRV vectors on the leaves and roots results in a more efficient and prolonged silencing in the whole plant (Senthil-Kumar et al, 2011, PBJ).
The aim of the project is to develop a successful and efficient VIGS protocol in potato roots using the existing vectors and a combination of the available methods. Different genotypes (cultivars) of potato will be tested and as a proof-of-concept candidates for the H1 resistance gene will be silenced to determine in a nematode resistance assay which one is the functional gene. The work involves molecular biology, including the cloning of H1 candidate genes in TRV constructs and setting up a VIGS assay in the greenhouse to assess the silencing effect by QPCR and classical nematode resistance assay.