The overall goal of this research is to characterize resistance to root-knot nematodes (RKN) in grapevine rootstocks. These nematodes are an important grape pest, and strategies to minimize nematode damage are an important component of grape production in many grapegrowing regions worldwide. Numerous genes that confer resistance against plant parasitic nematodes have been described, and several of these have now been cloned. The best studied of these genes is the tomato gene Mi-1, which confers resistance against several species of RKN including Meloidogyne incognita, M. javanica, and M. arenaria. Response to RKN in resistant grape rootstocks resembles the Mi-1-mediated resistance in tomato. For example, root tips of the resistant grape rootstock Harmony develop a hypersensitive response when penetrated by juveniles from M. incognita Race 3; in addition, the resistance phenotype in both tomato and grape are effective only at temperatures below 32 °C. Since no nematode resistance gene from grape has yet been cloned, we are interested in applying molecular tools and knowledge developed from studying the tomato Mi-1 gene to grapevine-nematode interactions.
From previous studies, we have identified 77 unique cDNA candidate clones whose mRNAs may be expressed more abundantly preceding Mi-1 associated cell death by suppression subtractive hybridization. Here we propose to use an established set of assays to test those cDNA clones to identify signaling pathway components leading to nematode resistance and their relationship to cell death. These assays include determining the effect of altered expression levels of a candidate cDNA on cell death in a leaf infiltration assay and determination of the nematode resistance phenotype in transformed roots. To examine the biological function of these candidate genes relative to cell death, we employed virus-induced gene silencing (VIGS) using the potato virus X (PVX) vector as a gene knockout system. These cDNA clones have been amplified by PCR to sub-clone into the PVX binary vector pGr106. The cell death relevant genes will be identified using VIGS as a functional assay to be followed by northern blot analysis to confirm induction of selected clones.
Research progress in non-model plants like grapevine is contingent upon the effectiveness of plant transformation technology. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. To further test nematode resistance in transgenic roots harboring genes of interest, we have adapted an ex vitro protocol to evaluate the potential of this technique to produce transgenic composite grapevine plants. We need to alter and improve this technique to generate transgenic grapevine roots for future study. In addition, we found that kanamycin is not a suitable selection marker for composite plant regeneration because too many putative transformants are not affected and escape detection. Efforts to optimize A. rhizogenes-mediated transformation using embryogenic tissue of various grape cultivars is underway in our laboratory.