CONTINUED ASSESSMENT OF XYLELLA FASTIDIOSA FIMBRIAL ADHESINS AS IMPORTANT VIRULENCE FACTORS IN PIERCE’S DISEASE: INFLUENCE OF XYLEM SAP Principal Investigator:

Specific biological characteristics of Xylella fastidiosa (Xf) Temecula were investigated in microfluidic flow chambers in vitro by examining the effect of xylem sap from Pierce’s disease (PD) susceptible V. vinifera and resistant V. smalliana grapevines on Xf cell growth, aggregation, biofilm formation, and motility. Growth of Xf was observed in both V. smalliana and V. vinifera xylem saps in microfluidic flow chambers. While Xf cell density increased in V. smalliana sap, the cells exhibited a reduction in aggregation and biofilm formation compared to that observed in V. vinifera sap. In addition, motility via pilus twitching activity was reduced in V. smalliana sap when compared to similar activities in V. vinifera sap, indicating V. smalliana sap may inhibit the function of type IV pili. Normal twitching motility of Xf was restored once V. smalliana sap was exchanged with V. vinifera sap, indicating that chemical components of V. vinifera sap possibly influence the function of type IV pili of Xf cells, that in turn may affect aggregation. LAYPERSON SUMMARY Cells of Xylella fastidiosa (Xf) aggregate, form biofilms, and occlude the host’s vascular (xylem) system, resulting in Pierce’s disease symptoms in grapevine. Colonization of grapevine xylem by Xf involves migration of individual cells through a process of twitching motility by which hair-like type IV pili are repeatedly extended from the cell, attach to the xylem surface, and are retracted, pulling the cell forward. Using microfluidic ‘artificial’ chambers through which xylem sap from highly susceptible and resistant grapevines is flowing, the biological behavior of Xf in these saps was assessed. Toward this we have observed reduced motility in sap from a resistant grapevine, V. smalliana. Also reduction in formation of cell aggregates and biofilms. INTRODUCTION This project continues efforts toward understanding the biological relationship between Xylella fastidiosa (Xf) cells and the xylem environment, and specifically the roles of fimbrial adhesins (type I and type IV pili, and associated proteins) in Xf virulence, motility, aggregation and autoaggregation, and biofilm development. The research targets the functional biology of Xf in xylem sap. It tests and explores traits of sap and xylem vessels from resistant and susceptible grapevines, and eventually that of citrus, that may inhibit or promote Xf cell activities associated with pil and fim gene products. Previous observations describing roles for fimbrial adhesins (type I and type IV pili) in Xf virulence, motility, aggregation, and biofilm development have provided insight into their genetic mechanisms and regulation (De La Fuente, 2007; 2008). Studies on Xf motility and biofilm formation under natural conditions viz., in planta, have been hindered in part by the optical inaccessibility of vascular tissue. Recent studies have shown the importance of xylem sap chemistry on growth, aggregation, and attachment of Xf cells, highlighting the establishment of stable cultures in 100% xylem sap (Andersen, 2007; Zaini, 2009). Studies with V. riparia and V. vinifera cv. Chardonnay sap (100%) in either microfluidic chambers or in culture tubes have shown that the pathogen responds to this more natural chemical environment differently than it does in rich artificial media such as PD2 (Zaini, 2009). Aggregation and biofilm development are enhanced (Zaini, 2009), and early indication is that twitching motility is also greater—in both the number of Xf cells and in rate of movement. It was reported that xylem sap from Pierce’s disease (PD) resistant V. rotundifolia maintained Xf in a planktonic state, whereas the bacterium was more likely to form aggregates when incubated in xylem sap from susceptible V. vinifera cultivars (Liete, 2004). Those directed the attention to a more natural environmental system for Xf—one that will greatly enhance the value and significance of information generated in studying Xf in an in vitro system: the inclusion of xylem sap and xylem vessel tissue. Previous work that both type I and type IV pili are involved in aggregation and biofilm development (Li, 2007), type IV pili of Xf are involved in twitching motility within the xylem vessels of grapevine (Meng, 2005). Citrus is often grown adjacent to vineyards in California and may be considered a potential reservoir for PD Xf (Bi, 2007). Xylem sap from commercial citrus plantings in Temecula (grapefruit, orange, lemon) did not support Xf biofilm development while at the same time