TESTING TRANSGENIC GRAPEVINES FOR RESISTANCE TO PIERCE’S DISEASE Project Leaders:

Numerous genes involved in plant disease defense have been isolated and when introduced and expressed in transgenic plants, fungal and bacterial diseases have been greatly reduced. This strategy is especially appropriate for grapevines, where the industry is rooted in traditional European grapes with strong name recognition and very high disease susceptibility. Our laboratory has developed a set of transformed grapevines harboring genes that produce anti-microbial peptides (AMPs). Seventy-six ‘Chardonnay’ lines transformed with the magainin-type genes, mag-2 and MSI99, and with a PGL class gene, were produced. Some are now growing in tissue culture, in the greenhouse, and in the field (Vidal et al. 2003). AMPs are particularly effective against bacteria, and act by disrupting the cell membranes. The primary goal of this project is to study the potential resistance to Pierce’s disease (PD) of magainin and PGL-producing vines. In doing so, it becomes important to characterize the expression of these genes in each line. We are also studying whether AMPs can move from a transgenic rootstock to a non-transgenic scion. If so, it might be possible for engineered rootstocks to be used as a means to affect disease development in a range of scion varieties. Our data to date show that between 1 and 4 copies of the foreign gene can be found in each line studied, and that the inserted genes were correctly transcribed in 46 of 76 lines tested (via RT-PCR). An antibody was produced that can detect the presence of both mag-2 and MSI99 peptides. Using this antibody, AMP production was verified in leaf extracts of in vitro vines harboring mag-2 and MSI99 genes. Studies of PD resistance and movement of transgenic proteins from rootstocks to scions are at a very early stage in this newly funded project. INTRODUCTION Numerous genes involved in plant disease defense have been isolated (Punja 2001; Mourges et al. 1998). When disease resistance genes are introduced and expressed in transgenic plants, fungal and bacterial diseases have been greatly reduced (Mourges et al. 1998; Punja 2001; Van der Biezen 2001). This disease control strategy is especially appropriate for clonallypropagated crops, such as grapevines, where the industry is rooted in traditional European grapes with strong name recognition and very high disease susceptibility. Moreover, cross-breeding cannot produce disease resistant forms of elite varieties because other characteristics would be altered and varietal identity would be lost. Our laboratory has developed a set of transformed grapevines in which we have determined that anti-microbial peptides (AMPs) are transcribed. Seventy-six ‘Chardonnay’ lines transformed with the magainin-type genes, mag-2 and MSI99, and with a PGL class gene, were produced (Vidal et al. 2003). These are now growing in tissue culture in the greenhouse, and in the field. Magainins are small peptides with strong inhibitory activity against numerous bacteria and fungi (DeGray et al. 2001; Zasloff et al. 1988; Smith et al. 1998; Smith et al. 2001). The MSI99 peptide expressed in tobacco and banana was recently shown to be highly effective against several pathogens (Chakrabarti et al. 2003). In studies conducted in 2002 and 2003, we determined that some AMP-transgenic lines of ‘Chardonnay’ are significantly more resistant to tumorigenic strains of crown gall (Agrobacterium vitis). It is logical to think that these plants might have improved resistance to other bacterial diseases as well. Some AMP producing genes such as Shiva-1 are effective against Pierce’s disease (PD), according to a recently issued patent (Scorza and Gray 2001) but the subject warrants further study. Scorza and Gray described a trial of two lines of ‘Thompson Seedless’ expressing the Shiva-1 peptide; both eventually succumbed to PD, but one showed milder symptoms, which did not include the typical signs of marginal leaf burn when compared to the non-transformed control plant. Based on this data, the