EVALUATION OF GENES ISOLATED BY A FUNCTIONAL GENETIC SCREEN FOR SUPPRESSION OF BACTERIAL GROWTH OR SYMPTOMS IN PIERCE’S DISEASE Project Leaders:

Our overall objective is to identify genes from cDNA libraries of either grape or heterologous plants that, when up regulated in grape, will disrupt infection, spread or symptom development by the xylem-limited bacteria, Xylella fastidiosa (Xf). Hence, we are interested in the effect of the genetic disruption of Pierce’s disease (PD) symptoms on the movement or establishment of the bacterium in the xylem of susceptible grape plants. Recent published information from our laboratory established that specific transgenes from homologous or heterologous hosts that block programmed cell death (PCD) during plant disease development, can arrest both symptom development and microbial growth in planta in a range of plant-microbe interactions. A functional screen was used to evaluate cDNA libraries of grape and tomato for genes that, when overexpressed in tissues stimulated to undergo PCD, would block the death and therefore represent potential anti-PCD (antidisease symptom) genes. Collectively, more than 500,000 cDNAs were screened and 12 genes were cloned that when overexpressed as transgenes in tomato or grape blocked PCD. Three of these genes when overexpressed as transgenes blocked PCD triggered by a verified ceramide-derived inducer of plant PCD. One of these gene, designated as PR1A in grape, was chosen for further direct characterization. This gene has high sequence homology to a gene family from humans, nematodes, hookworms and several plant species, wherein its expression is correlated with situations in which PCD is blocked in both animal and plant diseases. When overexpressed as a transgene in grape, the PCD sensitive grape tissues is now insensitive to microbial inducers of PCD. We believe that examination of the molecular basis of cell death in presymptomatic and symptomatic tissues, along with the immediate assessment of the effect of expressing anti-apoptotic transgenes in PD infected tissues on the development of death-related symptoms in grape, will be very informative in the short run in terms of PD biology and physiology. In a longer time frame these data will likely yield genetic strategies for protection of grape against infection by Xf in years not decades. INTRODUCTION Published information from our laboratory confirms that specific transgenes from homologous or heterologous plants, that block PCD during plant disease development (4), as well as chemical inhibitors of apoptotic proteases (3), can arrest both symptom development and microbial growth in planta in a range of plant-microbe interactions (3, 4, 5). The conserved genetically determined PCD process can be studied by biochemical, cytological and genetic techniques and can be transgenically manipulated by techniques developed in our laboratory (3, 4). ). PCD is now well established as a key pathway involving many gene products in numerous diseases of animals and plants. We further established that expression of the antiapoptotic p35 gene in transgenic grape tissue blocked cell death (PD) symptoms in Xf infected tissue. This demonstrates that the anti-apototic genes to be recovered from the cDNA library screens have excellent potential to provide protection in grape against PD. Based on previous results we tested the effect of the p35 transgene from baculovirus on viability of roots, produced on Xf infected chardonnay and observed protection of the roots against death in the presence of Xf. This indicates a role for PCD in PD and provides optimism that novel genetic determinants of resistance can be identified using this screen. Given the strategies used it is likely the genes will function in grape by altering the effect of Xf infection in grape through suppression of symptoms either directly on cell death or indirectly by modifying the behavior of the bacterial in the xylem. It should be emphasized that the effect of anti-apoptotic transgenes on plants is not to induce so-called systemic acquired resistance (SAR) as no markers of SAR are induced in the presence of anti-apoptotic genes such as the p35 gene (4). We believe that the effect of expressing anti-apoptotic transgenes in PD infected tissues on the development of death-related symptoms in grape will contribute significant information in terms of PD biology and physiology. In a longer time frame these data will likely yield genetic or chemical-based signaling strategies for protection of grape against infection by Xf in years not decades, perhaps similar to the effects we reported previously in tomato (4). OBJECTIVES 1. Create grape transgenic plants over-expressing candidate anti-apoptotic plant genes obtained through cDNA library screens. 2. Evaluate these specific anti-apoptotic plant genes in grape for effect on Xf and PD symptoms. 3. Apply signal molecule discovery tactics to elucidation of the molecular basis of susceptibility, focusing first on grape PR1A.