COMPARATIVE PROTEOMIC ANALYSIS OF STEM TISSUE AND XYLEM SAP FROM PIERCE’S DISEASE RESISTANT AND SUSCEPTIBLE GRAPEVINES Project Leaders:

Analyses of host plant resistance / susceptibility mechanisms to Xylella fastidiosa (Xf) infection are critical for understanding host-pathogen interactions. Proteomic analyses of stem tissue and xylem sap samples were initiated to complement genomic approaches employed in elucidating Pierce’s disease resistance mechanisms. Samples from one highly resistant and two susceptible grape genotypes were collected at multiple time points post-inoculation from control and Xf-inoculated plants. Two-dimensional gel electrophoresis revealed numerous proteins that were differentially expressed and dependent on plant genotype and/or inoculation treatment. Proteins identified by oMALDI-TOF comprised a wide range of functional types. The importance of these proteins with respect to host-pathogen interactions will be investigated further. INTRODUCTION While numerous factors including temperature, fertilization and time are known to affect xylem sap chemistry (Andersen and Brodbeck, 1989a, 1989b, 1991; Andersen et al., 1995, 2004b), the protein composition of grape xylem sap in response to Xylella fastidiosa (Xf) infection has not been investigated in detail to date. In other host plant-pathogen systems, xylem sap proteins were shown to be important in suppression of disease development (Ceccardi et al., 1998; Guo et al., 1993; Nemec, 1995; Reimers and Leach, 1991; Reimers et al., 1992; Rep et al., 2002; Young et al., 1995). Without a doubt, due to its xylem limited growth habit, Xf ‘s growth and development are influenced by xylem sap characteristics. Thus manipulation of the xylem sap composition presents a promising venue to interfere with Xf infections. Disease expression in stems of grape vines results in blocking of water flow to the shoot and, thus, is critical to the lethal nature of Pierce’s disease (PD). Xf infection results in uneven cane maturation which expresses itself in the formation of green-islands. The irregular nature of green-islands suggests the involvement of localized rather than systemic signals in the formation of the observed spatial symptomology. The importance of Xf populations in stems in respect to PD resistance of grape genotypes was also illustrated in recent studies (Krivanek et al., 2005; Krivanek and Walker, 2005). Thus, examination of stem tissue provides an opportunity to identify important aspects of plant-pathogen interactions. Examination of xylem sap and stem protein makeup is a new approach that allows us to complement our genomic studies conducted on the same susceptible and resistant sibling genotypes employed in this study. OBJECTIVES 1. Discover xylem sap and stem proteins differentially expressed in PD resistant and susceptible grapes in response to Xf infection. 2. Identify differentially expressed proteins from xylem sap and stem induced by Xf. RESULTS Objective 1. Discovery of differentially expressed proteins. PD resistant (9621-67) and PD susceptible (9621-94) genetic lines selected from a segregating population of V. rupestris x V. arizonica as well as vinifera grape, Chardonnay were used in this comparative study. We completed expression experiment conducted in the greenhouse where treatment and control grapevines were mechanically inoculated with Xf suspension respectively. Leaf and stem tissues were then collected at ten time points from as early as day one post inoculation up to three months when PD symptom was fully expressed in susceptible grapes. A separate set of grapes (same genotypes and treatments as above) was grown in the greenhouse for xylem sap protein extraction at 2 time points post inoculation. The xylem sap was extracted using a pressure chamber following the same sampling scheme as above. Samples (stem, leaf and