EVALUATING THE ROLES OF PILI IN TWITCHING AND LONG DISTANCE MOVEMENT OF XYLELLA FASTIDIOSA IN GRAPE XYLEM AND IN THE COLONIZATION OF SHARPSHOOTER FOREGUT Project Leaders:

Xylella fastidiosa cells were shown to exhibit twitching motility ‘upstream’ in microfabricated ‘artificial xylem vessels’. Such motility is due to extension and retraction of type IV pili present on the poles of the bacteria. Importantly, such upstream migration was subsequently demonstrated in planta. A survey of isolates from California, Texas and South Carolina revealed that all possessed motility characteristics. A number of mutants deficient in genes associated with type IV pilus functions were created, many of which may be useful in exploring targets for slowing development of the bacterial mass in xylem vessels. Type IV and type I pili were shown to have pronounced effects on colony and biofilm development. INTRODUCTION Once Xylella fastidiosa is introduced into xylem vessels in leaf, petiole, or other susceptible green tissues, how does it move in xylem elements farther upstream, e.g., into petioles from the leaf or down shoots and canes? This has long been a particularly puzzling and important question since xylem sap flow during the growing season is nearly always down the pressure gradient, viz., toward the leaf. It is seldom stagnant. Since Xf are non-flagellated bacteria, the consensus (albeit unproven) for their appearing in previously non-invaded regions upstream has been through the slow expansion of the colony through repeated cell division along xylem vessel walls. Lateral movement, from xylem element to element, has been proposed through dissolution of border pit membranes (Newman et al., 2004); but again, this does not explain long distance upstream movement. Our investigations have focused on the effects of physical and chemical environments on attachment, colony development, and biofilm formation by Xf in microfludic chambers fabricated to mimic xylem elements. This has resulted in identifying unmistakable long distance migration of individual bacteria. Even more interesting was the observation that they were able to migrate against a strong current of flowing media (Meng et al., 2005; Hoch, 2005). The movement was characteristic of twitching motility that occurs in some gram-negative bacterial species (Mattick, 2002). There are several important implications of this observation: this is not only the first observation of twitching movement by a non-flagellated plant pathogenic bacterium (albeit, Ralstonia solanacearum, that sometimes has flagella has been shown to exhibit colony features characteristic of twitching (Liu et al., 2001; Roine et al., 1996)), it is also the first time that such movement by Xf has been observed. Such motile behavior may be important in explaining how the bacteria spread in the grapevine from an inoculation point to upstream locations. Type IV pili are filamentous appendages (fimbriae) located at either one or both poles, depending on the species (Bradley, 1980; Henrichsen, 1983), are generally 5-7 nm in diameter, and may be up to several micrometers in length. They are assembled primarily from single structural protein subunits, pilin (PilA) (Mattick, 2002). Twitching movements are generated as the pili are retracted and dissembled. Because the pili tips are attached to the substratum, the cell moves toward that point of contact as the pili shorten (Mattick, 2002; Skerker and Berg 2001; Wall and Kaiser, 1999; Wolfgang et al., 2000). Type IV pili function and biogenesis in Pseudomonas aeurginosa involves more than 35 genes with conserved homologs existing in other bacteria that express twitching via type IV pili (Mattick, 2002). Xf likely produces type IV pili as its genome carries at least 26 genes that are related to pili synthesis and function (Simpson et al., 2000). Xylella produces fimbriae that are thought to function in adhesion of the bacterium. Biofilm deficient mutants (e.g., 6E11), the result of a disruption of the fimA gene, continue to migrate since they still possess the type IV pili; whereas, mutants deficient in genes that code for type IV pili are migration deficient and develop robust biofilms (Meng et al., 2005).