OF THE LIPOPOLYSACCHARIDE-MEDIATED RESPONSE TO XYLELLA FASTIDIOSA INFECTION IN GRAPEVINE . REPORT TYPE : Final Report for CDFA agreement number 11-0148-SA PRINCIPAL INVESTIGATOR

Xylella fastidiosa (Xf) is a gram-negative, xylem-limited bacterium that causes serious diseases in economically important crops, such as Pierce’s Disease (PD) of grapevine. Lipopolysaccharide (LPS) is the dominant macromolecule displayed on the bacterial cell surface. LPS acts as a selective barrier, preventing entry of toxic substances into the cell, and as an anchor for superficial structures. Finally, LPS is a well-described pathogenassociated molecular pattern (PAMP) and is known to elicit host basal defense responses in model plant systems. LPS is composed of a conserved lipid A-core oligosaccharide component and a variable O-antigen. Through mutations made in wzy (XP0836), which encodes an O-antigen polymerase, we have demonstrated that the Xf O-antigen contributes to plant and insect colonization, and depletion of the O-antigen causes a severe reduction in overall virulence in planta. The goal of this project was to determine the role of the Xf O-antigen in modulation of basal defense responses in grapevine. Specifically, we demonstrated that specific alterations to the LPS structure caused a change in the elicitation of the grapevine response to Xf, therefore affecting critical, early stages of Xf establishment in planta. We also demonstrated that Xf LPS and O-antigen structural variants have the potential to be used as a preventative treatment for the control of PD. LAYPERSON SUMMARY: Xylella fastidiosa (Xf), a bacterial pathogen, is the causal agent of Pierce's disease (PD) of grapevine and poses a serious threat to the viticulture industry. We have demonstrated that truncation of the O-antigen portion of the lipopolysaccharide (LPS) entity alters the adhesive properties of the cell, leading to a defect in mature biofilm formation. Furthermore, depletion of the O-antigen results in a significantly less virulent pathogen that is severely compromised in host colonization. Additionally, LPS is a Pathogen-Associated Molecular Pattern (PAMP) that potentially triggers the basal immune response. We hypothesize that the long chain O-antigen also allows Xf to circumvent the innate immune system by masking the conserved core and lipid A portions of the LPS chain from the host immune system. The goal of the proposed work is to further explore the role of LPS, specifically focusing on the O-antigen moiety, in the interaction between Xf and the grapevine host and to use this information to develop and evaluate an environmentally sound preventative application for PD. INTRODUCTION: Xylella fastidiosa (Xf), a gram-negative, fastidious bacterium, is the causal agent of Pierce’s Disease (PD) of grapevine (Vitis vinifera) and several other economically important diseases (Chatterjee et al., 2008; Varela, 2001). PD has devastated some viticulture areas in California, and there are currently no effective control measures available to growers targeted towards the bacterium itself. Lipopolysaccharide (LPS) is a tripartite glycolipid molecule that is an integral part of the Gram-negative bacterial outer membrane. It is prominently displayed on the outer surface of the cell, thereby mediating interactions between the bacterial cell wall and its environment. LPS plays diverse roles for the bacterial cell. It provides structural integrity to the cells and can act as a permeability barrier to toxic antimicrobial substances (Raetz & Whitfield, 2002). Because of is location in the outer membrane, it is also a key contributor to the initial adhesion to a surface or host cell (Walker et al., 2004). We have been exploring the roles of LPS in the Pierce’s Disease cycle and in the Plant-MicrobeInsect (PMI) interactions of Xf. We targeted our studies towards the outermost exposed region of the LPS molecule, the O-antigen. By mutating a key O-antigen polymerase, wzy (XP0836), in the Temecula1 (Pierce’s