PIERCE’S DISEASE CONTROL AND BACTERIAL POPULATION DYNAMICS IN WINEGRAPE VARIETIES GRAFTED TO ROOTSTOCKS EXPRESSING ANTI-APOPTOTIC SEQUENCES. Principal Investigator:

Previous research in our lab first established a determinative role for a genetically regulated process of programmed cell death (PCD), in the leaf scorch and cane death symptoms in Pierce’s disease (PD) and then developed a functional screen for PCD-suppressing plant genes from a cDNA library of grape genes. The functional screen identified six candidate DNA sequences potentially able to suppressing Xylella fastidiosa (Xf)induced PCD when expressed as transgenes. Two of the sequences (VvPR1 and UT456) were selected to test for ability to suppress the PCD-dependent symptoms of PD. Greenhouse experiments over several years confirmed that these two different anti-PCD DNA sequences prevented PD symptoms in the PD-susceptible cultivar Thompson Seedless and the commercial rootstock Freedom. Furthermore, the bacterial titer in the transgenic plants was reduced four to six orders of magnitude below that reached in untransformed control vines. All untransformed control plants died within 2-3 months after inoculation while the transgenic plants were asymptomatic for 12 months. The net effect of these transgenes is to limit bacterial titer but not distribution of bacteria in the asymptomatic plants. From the perspective of the grape-bacterial interaction, it appears that the anti-PCD genes suppress PD symptoms and functionally confine Xf to an endophytic ecology in the xylem equivalent to that seen in the related asymptomatic host Vitis californica. Clonal copies of the transgenic and control plants were moved to two field locations under an APHIS permit secured by PIPRA and were inoculated July 21, 2011. Greenhouse data obtained from grafting experiments indicate the protective effect of these genes may be transferred across a graft union to protect a susceptible untransformed scion. Grafted plants expressing VvPR1 and UT456 in the rootstock, but not the scion, have been moved to the field site for inoculation in the spring of 2012. LAYPERSON SUMMARY Xylella fastidiosa induces Pierce’s disease (PD) symptoms that are the result of the activation of a genetically regulated process of programmed cell death(PCD). We identified six novel anti-PCD genes from a grape cDNA library functional screen for ability to suppress PCD. Two of these grape sequences, VvPR1 and UT456, when expressed as transgenes in the PD susceptible Thompson Seedless plants, suppressed PD symptoms and dramatically reduced bacterial levels in inoculated plants. The remaining four genes were tested this year, along with VvPR1 and UT456; each of the four provided substantial suppression of both PD symptoms and bacterial titer. However, none were as effective as VvPR1 and UT456. Currently in progress are a series of experiments designed to evaluate whether the protective effect of these two sequences can protect untransformed susceptible winegrape scions across a graft union. Preliminary data suggest that 50% or more of the susceptible scions grafted to either VvPR1 or UT456 showed less PD symptoms and had lower bacterial titers than the unprotected control plants. While these results are encouraging, they are not complete or definitive and the experiment is continuing. The relative susceptibility of the suite of eight commercial winegrape varieties was tested under controlled greenhouse conditions prior to field testing these varieties as scions on the transgenic rootstocks. Mechanism of action experiments initiated recently suggests a genetically conserved basis for suppression of PCD and the protection against PD. This project is now moving from the proof-of-concept to potential application and characterization of these plants under field conditions with appropriate APHIS permits: initial field plantings were begun in July 2010 with additional plantings to be made in Solano and Riverside Counties in 201