THE ROLE OF LIPOPOLYSACCHARIDE IN VIRULENCE, BIOFILM FORMATION, AND HOST SPECIFICITY OF XYLELLA FASTIDIOSA Principal Investigator:

This project aims to further elucidate the molecular mechanisms Xylella fastidiosa (Xf) employs during the infection of its host. We are focusing on the lipopolysaccharide (LPS) component of the outer membrane of Xf. LPS contains a conserved lipid A and core portion and a variable O-antigen portion. In particular, we are examining the variable portion of the LPS molecule, the O-antigen. O-antigen as been implicated in virulence in many bacterial species and we hypothesize it may also be involved in Xf virulence. More specifically, we are investigating if this particular portion of the LPS molecule contributes to Xf surface attachment and biofilm formation, two critical steps for successful infection of the xylem of the host. Additionally, we will determine if LPS contributes to the high level of host specificity observed for this pathogen. LAYPERSON SUMMARY Xylella fastidiosa (Xf) is a bacterium that has the ability to infect and colonize many different plant species, causing significant damage in some. In grapevine, this disease is known as Pierce’s disease (PD), which has caused major losses to the California grape industry. Xf also infects other economically important crops such as almond, oleander and citrus. Interestingly, while all Xf isolates belong to the same group or species, some isolates can cause disease in one host but not another. For example, oleander isolates cannot cause disease in grapevine and vice versa. One major goal of this project is to understand the bacterial mechanisms that dictate this high level of host specificity. This research project is specifically focused on understanding the role of the Xf cell surface component lipopolysaccharide (LPS) in the pathogenic interaction between the grapevine, almond, and oleander hosts. LPS plays an important role in virulence for many bacterial pathogens. We are investigating the involvement of LPS in Xf colonization of its host and other key aspects of the disease process, like attachment to the plant cell wall. Most importantly, should LPS prove to be an important factor during Xf plant infection, its abundance on the bacterial cell surface makes it a logical target for disease control. Furthermore, antimicrobial compounds exist that disrupt LPS synthesis or weaken the LPS layer, making the bacterium more sensitive to other stresses. Therefore, compounds targeted towards LPS synthesis could increase the efficacy of other anti-Xf compounds currently being developed by other researchers when both are used in conjunction. INTRODUCTION Xylella fastidiosa (Xf) is a gram negative, xylem-limited bacterium with a broad host range. Xf causes disease in economically important hosts such as grape, almond, citrus, coffee, peach, plum and alfalfa as well as several tree and ornamental hosts such as oleander. Additionally, Xf colonizes many plant species that never develop any visible symptoms or stresses. (Hopkins and Purcell, 2002). The molecular mechanisms that determine this host specificity are poorly understood. We are currently exploring the role of lipopolysaccharides (LPS) as both a virulence factor and host specificity determinant of Xf. We are focusing on the O-antigen portion of the LPS molecule in 3 isolates of Xf that colonize different hosts: Fetzer, a Pierce’s Disease (PD) isolate; Dixon, an almond leaf scorch (ALS) isolate; and Ann-1, an oleander leaf scorch (OLS) isolate. While the grape and almond isolates are considered to be separate subspecies or pathovars, both ALS and PD isolates can cause disease in grapevine (Almeida and Purcell, 2003). However, Xf isolated from grapevines cannot cause disease in almonds indicating a fundamental difference between ALS and PD isolates. Furthermore, the oleander strain cannot infect grape or almond and both the almond and grape strains cannot infect oleander. This provides an opportunity to study the role of the O-antigen moiety of the LPS molecule as a potential host specificity determinant for Xf. LPS is primarily displayed on the cell surface, thereby mediating interactions between the bacterial cell and its environment by way of initial adhesion of the bacterial cell to a surface or host cell (Genevaux et al. 1999, Nesper et al. 2001). LPS (sometimes called “endotoxin”) has also been implicated as a major virulence factor in animal and plant and pathogens, such as Escherichia coli, Xanthomonas campestris pv. campestris, and Ralstonia solanacearum (Muhldhorfer and Hacker 1994, Dow et al. 1995; Hendrick et al. 1984). Additionally, host perception of LPS is well documented and occurs in both plants and animals (Newman et al. 2000). The immune system can recognize several regions of the LPS structure and can mount a defense response against bacterial invasion based on this recognition. Bacteria can also circumvent the host’s immune system by altering the structure of their LPS molecule or by masking it with capsular or exopolysaccharides. LPS is composed of 3 parts: 1) lipid A, 2) core oligosaccharide and 3) O-antigen polysaccharide (see Figure 1). Lipid A is anchored in the membrane and the core oligosaccharides are attached on the preformed lipid A molecule. O-antigen is