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

Both xylem sap and stem tissue are key components in the Xylella fastidiosa (Xf)-grapevine interaction. In this research we investigate protein expression in xylem sap and stem tissue of highly tolerant and susceptible grape genotypes. Ten sequential samplings of stem tissues have been conducted from infected and non-infected grapevines ranging from 1 day to 12 weeks post inoculation. Protein extraction and analyses on these tissues has recently been initiated. Plants for xylem sap extraction are currently being grown in the greenhouse. Xylem sap will be extracted multiple times post inoculation from Xf and water-inoculated plants. Differentially expressed proteins in both stem tissue and xylem sap will be identified and investigated in more detail in the coming months. Results obtained will deepen our understanding of host-pathogen interactions, a key component in fighting Pierce’s disease (PD). INTRODUCTION Xylem sap is very important for Xf growth in planta. Be it as individual cells at the beginning of an infection or later in biofilms, Xf not only obtain their nutrients from xylem sap but also are in constant contact with it. Andersen et al. (2004a) and Toscano et al. (2004) reported that the source of xylem sap affects Xf growth rates and growth characteristics. Results from bioassays conducted in our lab also indicate that xylem sap collected from various PD resistant Vitis genotypes has dramatically different effects on Xf growth in comparison with controls consisting of artificial media (PW) and xylem sap from ‘Chardonnay’ (V. vinifera). To date, numerous studies have investigated inorganic and organic solutes in grape xylem sap showing that xylem sap chemistry is a function of temperature and fertilization, and changes over time (Andersen and Brodbeck, 1989a, 1989b, 1991; Andersen et al., 1995, 2004b). Although some xylem sap compounds have been suggested to be related to the susceptibility, i.e. [P]*[citrate] to [Ca]*[Mg] ratio (Andersen et al. 2004a), a complete understanding of the influence of xylem sap chemistry on the host pathogen interaction is missing. Specifically, as of now, the protein composition of the xylem sap has not been investigated in detail. The direct contact between sap and Xf makes xylem sap a promising venue to interfere with a successful pathogen colonization of the host. In other host plant pathogen systems, extracellular/apoplastic proteins were found to be responsive to disease pressure and in some instances important in suppressing 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). Combined with the evidence for xylem sap effects on Xf growth, these examples suggest that the analysis of the xylem sap proteome is likely to result in the identification of protein(s) influencing the interaction of grapevines and Xf. Identified proteins may provide information to develop approaches and/or be part of strategies to improve grapevine tolerance or resistance to Xf. In addition, the identification of PD-specific proteins would allow the production of specific antibodies which may potentially be used for serological diagnostic tests for PD. Recent findings of genotypic differences in symptomology and Xf populations in stems (canes) of resistant and susceptible grapevine genotypes highlight the importance of this tissue in the host-pathogen interaction. Krivanek et al. (2005) developed a cane maturation index (CMI) to quantify the uneven cane maturation manifested in the green-island symptoms that arise in PD infected plants. They found “green-island” formation as measured by the CMI to correlate better with PD resistance then other phenotypic symptoms. The irregular nature of the symptoms suggests that localized, rather than systemic signals are responsible for the spatial patterns observed. Thus, signal and signal recognition as well as signal transduction events appear to occur localized in stem tissue since a systemic signal transaction and recognition is unlikely to result in the characteristic green-island symptomology. Furthermore, in a companion study Krivanek and Walker (2005) found that, in resistant genotypes, Xf populations in stem internodes were significantly smaller than in leaves. In contrast, corresponding samples from susceptible genotypes were not significantly different. The two studies highlight the importance