Durable powdery mildew disease resistance in Vitis vinifera: proof-of-function and discovery of genetic variation
For the past sixty years, mlo resistance to barley powdery mildew has remained durable and is now incorporated into over 50%of the European barley acreage. Related mlo genes in Arabidopsis and tomato have recently been discovered and also confer resistance to powdery mildew via the same penetration resistance mechanism. Previously, we identified four strong candidates for mlo resistance in grapevine. To determine whether this gene could confer durable resistance in grapevine, we are pursuing a transgenic gene silencing approach and are screening Vitis vinifera germplasm for natural and induced mutations. In 2009, we designed, cloned and confirmed 17 new artificial microRNA (amiRNA) silencing constructs. After showing that Agroinfiltration and protoplast transfection were inefficient transient assays for detecting gene silencing, we initiated a collaboration with Dr. Bruce Reisch to develop stably transformed Chardonnay grapevines. Plans are to silence four VvMlo candidate resistance genes individually, in pairs, and all at once in a thorough effort to confirm resistance gene function. In addition, we implemented a similar strategy to silence two candidates for a second resistance gene from Arabidopsis, Pmr6, individually and in pairs. Thus far, typical results for biolistic transformation experiments have yielded 1000 to 3000 transiently-transformed Chardonnay cells per Petri dish, with regeneration of in vitro plants scheduled for 2010. While the transgenic assays described above could efficiently result in the development of transgenic V. vinifera cultivars with powdery mildew resistance, the information obtained could also be harnessed to identify V. vinifera breeding germplasm with previously undetected resistance alleles. Having previously discovered a surprising absence of functional genetic variation in preliminary investigations of Mlo DNA sequences across diverse Vitis spp., we successfully adapted and tested capillary electrophoresis ecoTILLING for grapevine in 2009. However, we documented the inefficiency of the technology when searching for known mutations and finding that peaks for known mutations were often hidden by background noise and that most peaks were false positives – artifacts of the technology (86%). Therefore, we developed and implemented a strategy to sequence 9 Mb (million base pairs) of our Mlo and Pmr6 candidate genes in a Chardonnay M2 seedling population segregating for random, induced point mutations. This will allow us to identify V. vinifera seedlings with functional mutations for use in grape breeding programs along with molecular markers perfectly linked with the resistance gene.