Effector-assisted breeding against powdery mildew in grapes.

In last three decades, a great deal of effort and resources have been placed by the grape breeding community on the identification of resistance (R) loci against the grape powdery mildew (GPM) pathogen Erysiphe necator, and their introgression in grapevine varieties of interest. However, the success of these breeding programs can be put at risk, if R-loci that the pathogen can overcome are deployed in the field. Moreover, breeders currently lack the tools that would enable them to swiftly identify new sources of resistance, and predict their durability under field conditions. Effector- assisted breeding has proven a potent contributor to modern breeding for the identification, functional characterization, and deployment of R-genes. The basis of the effector-assisted breeding lays on the principle that dominant R-genes typically mediate resistance through the recognition of matching effector proteins from the pathogen. During the first year of the project, we took the first steps towards the development of an effector-assisted breeding program in grape. Specifically, efforts were made to develop an Agrobacterium tumefaciens-based assay for the transient expression of GPM effectors in select grapevine lines that are currently used at the UC Davis GPM resistance breeding program, thus enabling the functional profiling of matching effector and R- gene pairs. Using as bioreporter the gene encoding for the enhanced green fluorescent protein (eGFP), different combinations of binary plasmid vectors and A. tumefaciens strains were tested for their efficacy to transiently express eGFP in Nicotiana benthamiana, as this is a more amendable and easier to work with system. The best binary vector/A. tumefaciens strains combinations were then tested for their ability to transiently express eGFP in select grapevine lines. A protocol for vacuum infiltrating detached grapevine leaves instead of entire plantlets was developed in parallel, thereby increasing the assay’s practicality and capacity. To this end, we were able to work out the conditions for achieving good levels of agroinfiltration into detached grapevine leaves but the expression level of the transgenes in agroinfiltrated tissue remains low and currently undergoes further optimization. Nonetheless, a set of 35 effector-encoding genes were cloned in one of our select binary vectors that we further modified it to add a 6x-His tag at the C-terminus of each transiently effector and their transient expression was tested in N. benthamaina. For 30 of these effectors we were able to confirm expression in this plant species either by means of a western blot analysis using an anti-6x-His tag antibody or by visual inspection of the plants, as at least six of these effectors triggered cell death in the agroinfiltrated tissue. We are currently completing the cloning of the 35 effectors in a second binary vector and we soon test their transient expression in our select grapevine lines as well.