IDENTIFICATION AND UTILIZATION OF COLD TEMPERATURE INDUCED GRAPEVINE METABOLITES TO MANAGE PIERCE'S DISEASE Principal Investigator:

This work builds on discoveries made in the past seven years of research on better understanding the mechanism(s) responsible for the Pierce’s disease (PD)-cold curing phenomenon. A thaumatin-like (TLP) grape protein was found in elevated levels in the xylem sap from cold-exposed vines and we cloned and expressed TLP in E.coli. We found that TLP protein possesses higher levels of Xylella fastidiosa (Xf) toxicity in vitro compared to E. coli protein extracts from cells containing the cloning vector but no TLP gene. Greater amounts of total phenolics were measured in xylem sap extracted from cold-exposed vines. In collaboration with the Waterhouse lab we are characterizing these phenolic compounds and assessing their potential anti-Xf activity in vitro. One phenolic compound, trans-resveratrol, which only occurred in Pinot noir grapevines exposed to cold temperatures, and was shown by two different labs to be toxic to Xf cells grown in vitro Previously, greenhouse grown Pinot Noir and Cabernet Sauvignon vines treated with commercial abscisic acid (ABA) were shown to have higher levels of recovery from PD than non-treated vines, as well as producing higher levels of polyphenolic compounds. In fall 2010 we treated Riesling vines growing in a vineyard in Napa that had light to moderate PD symptoms vines with a foliar spray or a soil drench treatment of ABA. The severity of PD symptoms in the treated vines will be compared with the severity of symptoms in non-treated PD-affected vines in fall, 2010. LAYPERSON SUMMARY We have succeeded in producing recombinant thaumatiin-like grape protein (TLP) in E. coli. We have also observed that grapevine TLP produced by E. coli has a deleterious effect on Xylella fastidiosa (Xf) when it is grown in vitro in the laboratory. This supports our rationale for overexpressing TLP in grapevines as a potentially promising approach to decreasing the size of Xf populations in Pierce’s disease (PD)-affected grapevines. We have been characterizing the phenolic compounds in cold xylem sap and comparing these to warm xylem sap. We have noticed a number of differences, specifically the presence of the phenolic compound trans-resveratrol in cold sap and its absence in warm sap in some grape varieties. This suggests that trans-resveratrol may play a role in the cold curing process. In previously described research we added trans-resveratrol to solid media used to grow Xf and observed that the Temecula strain is inhibited at trans-resveratrol concentrations lower than those concentrations that inhibit the Fetzer strain. This further suggests that phenolic compounds play a role in the cold curing process. Ongoing field trials are examining if root or foliar applications of the plant hormone ABA could stimulate the synthesis of phenolic compounds in field grown vines infected with Xf and possibly decrease the severity of PD symptoms in the field vines. INTRODUCTION Previous research conducted in the Purcell laboratory at UC Berkeley definitively demonstrated that Vitis vinifera grapevines that were infected with Xylella fastidiosa (Xf) the bacterial pathogen that causes Pierce’s disease (PD) could often be cured of the infection if exposed to freezing temperatures for some period of time. This “cold curing” phenomenon likely explains why PD is restricted to areas that have mild winter temperatures. Research conducted in our laboratory by Dr. Melody Meyer confirmed and expanded the work performed by Purcell, et. al. She found that grapevines exposed to cold temperatures had elevated levels of a thaumatin-like protein (TLP) that has been shown to have antimicrobial properties in other plant host/pathogen interactions. We cloned and expressed the grapevine TLP in E. coli and showed that incubation of the cloned TLP with cultured Xf cells considerably decreased the viability of the Xf cells compared to incubating the Xf cells with other appropriate controls. We are now cloning the TLP gene in an Agrobacterium binary vector with the intention of over-expressing the TLP in transgenic grapevines. Once TLP-transgenic grapevines are obtained and characterized they will be inoculated with Xf using mechanical and insect inoculation. The vines will then be rated for symptom development and compared to non-transgenic Xf-inoculated vines.