UNDERSTANDING CONTROL OF XYLELLA FASTIDIOSA CELL AGGREGATION: IMPORTANCE IN COLONIZATION AND BIOFILM DEVELOPMENT IN GRAPEVINE AND SHARPSHOOTER FOREGUT Project Co-Leaders:

Our investigation focused on aggregation and ‘autoaggregation’of Xylella fastidiosa (Xf) cells. This study is particularly directed toward elucidating how Xf cells aggregate to cause PD symptoms. We have documented that both type IV and type I pili are important in the process. Furthermore, it was discerned that surface properties of WT cells change from being more hydrophobic to more hydrophilic as the cells age, and that cell surface charge does not prevent aggregation. In addition, all cells with pili were effectively transmitted by BGSS while the double mutant, fimX/pilO, was apparently not transmitted which may not be surprising since no pili are present on these mutants to adhere the cells to the insect foregut regions. INTRODUCTION The ability of Xylella fastidiosa (Xf) to cause symptoms of Pierce’s disease is generally assumed to be related to its colonization of grapevine xylem where the pathogen forms biofilms and cell aggregates. From a disease standpoint such aggregates and biofilms are important for several reasons, including possible direct blockage of sap flow through xylem vessels or indirect blockage through initiation of tylose formation. Cell aggregates may also facilitate pathogen spread from vessel element to vessel element via enzyme digested pit membranes (Newman et al., 2004) — individual cells likely lack sufficient ‘enzymatic power’ to breach pit membranes, but a compact aggregate of cells would be much more effective in this regard. Furthermore, enzyme production may not be expressed in individual cells, but be regulated in aggregates associated with quorum sensing. From the standpoint of the pathogen, cell aggregates and biofilms likely facilitate nutrient adsorption, protection from environmental stresses, and phytochemicals. Determining how Xylella fastidiosa is able to inhabit the xylem environment and block the transpiration stream through the production of biofilms and bacterial cell masses would be informative toward facilitating development of novel control approaches. Furthermore, insight into the selective acquisition, retention, and transmission of Xf by leafhopper vectors represents a priority area of interest. Earlier, we demonstrated several unique and important features of Xf biology not previously recognized, including the observation that the bacteria posses functional type IV pili that allow the cells to migrate via twitching motility upstream in grape xylem elements (http://www.nysaes.cornell.edu/pp/faculty/hoch/movies/; Meng et al., 2005), that they posses type I pili that function in adhering the cells to xylem (De La Fuente et al., 2007a; 2007b; Li et al., 2007), and more recently that at some as yet undefined time or condition individual bacteria that are separated by relatively great distances ‘autoaggregate’ into large masses. In our in vitro studies, this occurred after six or more days of growth (initiated from only a few cells) in PD2 media (http://www.nysaes.cornell.edu/pp/faculty/hoch/agg/). Aside from a slow population build up of cells in xylem vessels at or near sites of sap flow constrictions (pits, element end-wall openings) which we consider cell aggregates, it is possible that many individual cells normally distributed throughout xylem elements are able to quickly autoaggregate into large cell masses contributing to vessel blockage. This phenomenon may explain, in part, why PD symptom development (reddening and drying of leaf margins) often occurs within a short time span—from overnight to a few days. Figure 1. SEM of frozen-hydrated Xf colony edge exhibiting a peripheral fringe of twitchmigrating cells in ‘rafts.’