THE ROLES THAT DIFFERENT PILI CLASSES IN XYLELLA FASTIDIOSA PLAY IN COLONIZATION OF GRAPEVINES AND PIERCE’S DISEASE PATHOGENESIS Project Leaders:

Type I and type IV pili of Xylella fastidiosa play different roles in twitching motility, biofilm formation, and cell-cell aggregation. Thirty twitching mutants were generated with an EZ::TN transposome system and type IV pilus-associated genes were identified, including fimT, pilX, pilY1, pilO, and pilR. Mutations in all resulted in a twitch-minus phenotype except that pilY1 mutant was twitching-reduced. A fimA mutant lacked type I pili and altered biofilm development and twitching. A fimA/pilO double mutant lacked both classes of pili, was twitch-minus and produced almost no visible biofilm. The gene for the type IV pilin (pilA), was cloned and expressed (predicted 15 KDa protein). The pilin sequence is 38% and 55% identical to that of the type IV pilin from Pseudomonas aeruginosa and P. syringae pv. tomato, respectively. A monoclonal antibody against the pilA gene product (prepilin) is being prepared. INTRODUCTION Xylella fastidiosa (Xf) has both type I pili and type IV pili located at one pole of the cells, and exhibits twitching motility and biofilm formation (Meng et al, 2005). Twitching functions in host colonization of many gram-negative bacteria. Approximately 40 genes have been identified that are involved in the biogenesis and function of type IV pili in P. aeruginosa (Mattick, 2002), including those encoding structural and regulatory proteins. In several bacteria, type IV pili are known to function in attachment and biofilm formation (Hélaine et al., 2005, Schilling et al., 2001); known virulence factors. The main structural protein of IV pili, pilin, is encoded by pilA and is essential for the twitching motility in P. aeruginosa and P. stutzeri (Mattick et al., 2002; Graupner et al., 2000). Our recent study revealed that type I pili play a central role in cell attachment and biofilm formation, and that type IV pili mediate twitching motility against a flowing current in microfluidic chambers. A fimA mutant (no type I pili) was capable of twitching motility. In contrast pilB and pilQ mutants (no type IV pili) did not twitch and were greatly impaired in their ability to migrate downward in grapevine shoots (Meng et al., 2005). We have identified several previously undescribed genes in Xf that are associated with pili development and their associated phenotypes. We have cloned and expressed pilA and its product, pilin, the primary structural protein of type IV pili. Antibodies to PilA and other surface proteins will be used for development of diagnostic tests and eventually in the development of novel controls for Pierce’s Disease. OBJECTIVES 1. Characterize the putative type I pili gene cluster and phenotpes associated with genes. 2. Characterize two additional gene clusters that are likely to be involved in regulation of type IV pili and related functions. 3. Development of monoclonal antibodies to Xf. RESULTS Characterization of gene clusters associated with pil genes. Thirty twitching-defective mutants, representing 12 different open reading frames, were obtained from approximately 3000 Kan insertion mutants generated via the EZ::TN system. Insertions occurred in homologs of pilus-related genes of P. aeruginosa, including PD0019 (fimT), PD0022 (pilX), PD0023 (pilY1), PD1693 (pilO), and PD1928 (pilR) that reside in four different gene clusters (Table 1; Figure 1). Open reading frame PD0062 corresponds to the fimA gene of E. coli (precursor for type I pili). A second round of mutagenesis in mutant 6E11 (lacks type I pili) was performed with EZ::TN system to select mutants that lacked both pilus types. Six non-twitching mutants were obtained having insertions in PD1923 (pilC in DM11, DM15), PD1693 (pilO in DM12), PD1671 (DM13), PD0609 (DM14) and PD0022 (pilX in DM16), respectively. DM12 (fimA/ pilO) was selected for further study. pilO resides in operon pilMNOPQ (Van Sluys et al., 2003). Homologs in P. aeruginosa are required for type IV pilus assembly (Mattick, 2002).