SCREENING OF GRAPE CDNA LIBRARIES AND FUNCTIONAL TESTING OF GENES CONFERRING RESISTANCE TO PIERCES DISEASE

• Author(s): Gilchrist, David; Lincoln, James; Lindow, Steven; Walker, Andrew;
• Abstract: Our overall objective is to identify genes from cDNA libraries of either grape or heterologous plants that, when induced in grape, will disrupt infection, spread or symptom development by the xylem-limited bacteria, Xylella fastidiosa (Xf). We are interested in the effect of the genetic disruption of PD symptoms on the movement or establishment of the bacterium in the xylem of susceptible grape plants. Specific objectives are to: a) create cDNA libraries from several different grape backgrounds, including three with PD resistance; b) develop a functional A. rhizogenes-based cDNA screen in grape; and c) investigate the potential of blocking PD symptom expression and disease impact with anti-PCD (anti-apoptotic) transgenes. To these ends we have created full-length cDNA libraries from resistant and susceptible grape and developed an Agrobacterium rhizogenes-based transformation procedure that provides a functional screen for genes that alter the disease phenotype. Transformation of grape explants with A. rhizogenes results in the emergence of a transformed root containing a single new DNA insert, from which the transgene can be re-isolated for characterization. The identified genes will be those that directly affect the ability of the pathogen to cause disease and is not dependent on DNA sequence relationships. Pathogenicity tests with any isolated disease-disrupting cDNA will first involve a transient expression system using micropropagated (MP) plants that are vegetative clones of sterile grape plants in small plastic boxes that can be infected with Xf under sterile conditions. This ensures that these plants will have uniform physiology without confounding by stress inductions as would likely occur in the field or greenhouse grown plants. The MP plants show foliar symptoms typical of infected plants under field and greenhouse conditions. Transient assays with test genes involve infiltration of A. tumefaciens containing the gene of interest into MP leaf tissue. The bacteria transfer the test gene into leaf cells that are presymptomatic will determine if the expression of the transgene in the leaf can block PD symptoms.
• Publication Date: Dec 2004
• Journal: 2004 Pierce's Disease Research Symposium