ISOLATION, CHARACTERIZATION AND GENETIC MANIPULATION OF XYLELLA FASTIDIOSA HEMAGGLUTININ GENES Project Leader:

Xylella fastidiosa (Xf) possesses genes for hemagglutinins (HAs), large adhesion proteins involved in cell-cell aggregation and biofilm formation. Mutations in either one of the functional HAs, HxfA (PD2118) or HxfB (PD1792), result in hypervirulent strains that move faster and cause more severe disease in grapevines. Computer analyses of the HA proteins identified several regions that might be possible adhesion domains (ADs) responsible for cell-cell and/or cell-surface binding. We cloned 6 Xf HA fragments that may contain potential ADs into protein expression vectors and to date have prepared antibodies against 1 AD protein fragment that is conserved in both HxfA and B. Recombinant proteins from the other 5 ADs are being purified and prepared for injection. Western blot analyses of using Xf proteins extracted from Xf cells grown in liquid culture showed a very faint reaction with an Xf protein of approximately 220kd but this result needs to be confirmed using a higher quality antibody. Recent discoveries in the Bruening lab using phage technology indicate that HA are more abundant in cells grown on solid medium compared to liquid medium and we are now repeating our Western analyses using cells grown on solid medium. Once Xf HA cell-cell binding domains are identified they will be expressed in transgenic tobacco and grapevines where we hope the proteins will act as a “molecular glue” to aggregate insect-inoculated Xf cells, retard their ability to systemically colonize plants and potentially provide a unique form of resistance against PD. INTRODUCTION Xylella fastidiosa (Xf) hemagglutinins (HAs) are large secreted proteins (200-300kD) that play important roles in mediating cell-cell contact and plant pathogenicity. Mutations were made in both Xf HA genes, HxfA (PD2118) and HxfB (PD1792), by transposon mutagenesis and the resulting mutants did not form aggregates in liquid culture and they had reduced biofilm formation in vitro and in planta (Guilhabert and Kirkpatrick 2005). When inoculated into grapevines the mutant cells showed hypervirulence and more rapid colonization of xylem vessels (Guilhabert and Kirkpatrick 2005). The premise of this research is to determine whether over-expressing Xf HA adhesion domains in the xylem, either by transformation of grapevines or inoculation of grapevines with HA expressing endophytes, the HA will act as a “molecular glue” which clumps the Xf cells and retards their ability to systemically colonize grapevine and cause Pierce’s disease (PD). Because of the large size of the HA genes (10kb), we cannot transform grapevines with the whole HA gene. Therefore we are trying to identify the active adhesion domains (ADs) responsible for cell-cell aggregation by dividing the HA genes into several smaller fragments that we believe will contain the cell-cell AD. Recombinant proteins derived from these fragments are being expressed in E. coli, purified and injected into rabbits to produce AD specific antisera. The resulting antisera will be used in ELISA, Western blot analysis, immunolocalization studies and cell-cell clumping experiments to determine which of the HA fragment(s) contain functional ADs that can later be transformed into plants. OBJECTIVES 1a. Use antibodies we have prepared against a conserved, putative binding domain (AD2) that is present in both Xf hemagglutinins (HA), which we have named HxfA and HxfB, to determine the native size and location of Xf HA in cultured Xf cells and PD-affected grapevines. b. Determine if these antibodies (Fab fragments) can prevent cell-cell clumping in liquid Xf cultures.