EFFECTS OF FIMBRIAL (FIMA, FIMF) AND AFIMBRIAL (XADA, HXFB) ADHESINS ON THE ADHESION OF XYLELLA FASTIDIOSA TO SURFACES Project Leader:

We investigated the role of fimbrial and afimbrial adhesions in the attachment of Xylella fastidiosa (Xf) to grape. We have individually disrupted FimA, FimF, XadA, and HxfB to assess their role in adhesion to plants and in the disease process. We performed adhesion assays using each mutant and wild-type separately as well as a combination of two mutants at one time to observe the phenotypes of these mutants using fluorescence or confocal microscopy. The fimbrial mutants FimAor FimFdid not aggregate nor did they attach to the glass surface whereas the adhesion mutants XadAor HxfBdid not attach to glass but did form aggregates and attached to cells that had adhered to a surface. All mutants had fewer single cells or aggregates that remained attached to glass than wild-type cells did after washing steps. We observed that afimbrial mutant cells (i.e. XadAor HxfB-) were clumped on top of fimbrial mutant cells (i.e. FimAor FimF-). Both afimbrial and fimbrial proteins thus apparently play a role in attachment of cells to glass in the early phases of adhesion while fimbrial proteins appear more important in cell-to-cell aggregations than afimbrial proteins. To determine if these adhesions are important in virulence, rooted grapevine cuttings were inoculated with FimA-, FimF-, XadA-, HxfB-, and wild-type Xf ‘Temecula’ or ‘STL’. A higher incidence and severity of disease was observed in vines inoculated with the wild-type Xf strain compared with FimA-, FimF-, XadAor HxfBmutant strains. Similarly, wild-type strain Xf ‘STL’ resulted in more vines with symptoms than FimA-, FimFor XadAmutants of this strain indicating that the process of attachment appears to involve similar genes in both the ‘Temecula’ and ‘STL’ strains. It thus appears that successful colonization of plants by Xf requires both cell-to-cell and cell-to-surface attachment. INTRODUCTION Attachment is the first step in the colonization process of bacterial pathogens. Attachment of Xf to xylem vessels and insect vectors may be required for both virulence and transmission. We therefore investigated the role of various fimbrial and afimbrial adhesions produced by Xf. Amongst the afimbrial adhesions, Xf has a homolog to XadA, an adhesion shown to be important in virulence of Xanthomonas oryzae pv. oryzae to rice. Since Xf is also a xylem inhabiting plant pathogen we hypothesized that XadA would also be a virulent determinant for Xf in grape. Similarly HecA was shown to be a virulent factor for Erwinia chrysanthemi to tobacco seedlings. Xf has four homologs to the HecA adhesion, among them are HxfA (PD2118) and HxfB (PD1792). These hemagglutins are the largest genes of the Xf genome and we hypothesized that these adhesions are important in the colonization process. Previous studies showed that HxfA and HxfB caused early grapevine death (hypervirulence) and mediated contact between Xf cells, which resulted in colony formation and biofilm maturation within the xylem vessels (Guilhabert and Kirkpatrick 2005). In the present study, inoculations were performed several times, and to ensure virulence was not diminished due to a high number of passage in the laboratory, we recreated the mutants multiple times using a low passage wild-type strain of Xf ‘Temecula’. Inoculations were repeated several times with either low passage wild-type cells or each of the recreated mutants (FimA-, FimF, XadA-, and HxfB-). OBJECTIVES 1. Determine the role of adhesions, in particular of the adhesion XadA and hemagglutinin HxfB in the attachment and virulence of Xf in grape. 2. Develop adhesion assays to characterize the behavior of the fimbrial and adhesion mutants of Xf. RESULTS Objective 1 XadA and HxfB (PD1792) mutants of Xf grape strains ‘Temecula’ and ‘STL’ were produced using the method described previously (Feil et al. 2003). Because the hemagglutinin HxfB is a large gene (10 kb), we constructed several vectors to disrupt this gene to maximize our chance to disrupt an important domain in the HxfB protein. Characterization of HxfB mutants was done by PCR and sequencing. To assess the virulence of adhesion mutants we have infected grape with each of these mutants FimA, FimF, XadA, and HxfB (mutants were derived from both grape strains of Xf ‘Temecula’ or ‘STL’) and wild-type cells of the ‘Temecula’ or ‘STL’ grape strain and recorded the number of diseased plants over time. We created the mutants two separate times in a low-passage ‘Temecula’ background and repeated the inoculations twice for a total of three separate experiments. All these experiments gave the same results. Specifically, HxfBmutants were always less virulent than wild-type cells. This result contrasts with previous studies on HxfBmutant by Guilhabert and Kirkpatrick (2005). One reason could be that the site of