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

This project focuses on the lipopolysaccharide (LPS) component of the outer membrane of Xylella fastidiosa (Xf). In particular, we are investigating if the O-antigen portion of this molecule contributes to Xf surface attachment and biofilm formation. More importantly, by targeting genes involved in O-antigen biosynthesis, we will determine if LPS is an important virulence factor for Xf infection of grape. 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 many different plant species. In some plants, this bacterium can cause serious disease. In grapevine, this disease is known as Pierce’s disease (PD) and has caused millions of dollars of damage to the California grape industry alone. Xf species also infect 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 strains 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. We are focusing on a key component of the bacterial cell membrane, called lipopolysaccharide (LPS), and how certain parts of this molecule may be important in dictating host specificity. We are also investigating how the LPS molecule is related to bacterial virulence and other key aspects of the disease process, like attachment to the plant cell wall. This molecule makes up more than 70% of the bacterial membrane and if LPS does prove to be an important factor during Xf plant infection, its abundance in the bacterial cell membrane makes it a logical target for disease control. Furthermore, antimicrobial compounds that weaken the LPS molecule generally make the bacterium more sensitive to other stresses. Therefore, compounds targeted towards LPS synthesis could increase the efficacy of other anti-Xylella compounds currently being developed when both are used in conjunction. INTRODUCTION Xylella fastidiosa (Xf) is a gram negative, xylem-limited bacteria with a broad host range encompassing at least 28 families of monocots and dicots, and causes disease in hosts such as grape, almond, peach, plum, alfalfa, elm, sycamore, coffee, oleander, maple, and citrus (Hopkins and Purcell, 2002). The molecular mechanisms that determine this host specificity are poorly understood. This project aims to explore the role of lipopolysaccharides (LPS) as both a virulence factor and host specificity determinant of Xf. We will focus on the O-antigen portion of the LPS molecule in three strains of Xf, the PD strain (Temecula 1), the almond leaf scorch strain (Dixon-ALS), and oleander leaf scorch strain (Ann-1). These three strains were chosen because either full or draft sequences of the genome are available. This will allow us to easily identify genes that are putatively involved in O-antigen biosynthesis and to construct targeted deletion knockouts in these genes. Additionally, these three strains infect different hosts, grape, almond and oleander. Interestingly, while grape and almond isolates are considered to be separate subspecies or pathovars, grape strains can cross colonize and cause both PD and ALS symptoms. Whereas almond strains do not cause PD symptoms in grape but can sometimes colonize grape, albeit at low titer (Almeida and Purcell, 2003). Furthermore, the oleander strain cannot infect grape or almond and both the almond and grape strains cannot infect oleander (Almeida and Purcell, 2003). 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 comprises approximately 70% of the outer membrane of gram-negative bacteria and is therefore essential for growth and viability (Figure 1A). Because LPS is what is largely displayed on the cell surface it mediates interactions between the bacterial cell and its surrounding environment. LPS (sometimes called “endotoxin”) has been implicated as a major virulence factor in both plant and animal pathogens, such as Escherichia coli, Xanthomonas campestris pv. campestris, and Ralstonia solanacearum just to name a few (Muhldhorfer and Hacker 1994, Dow et al. 1995; Hendrick et al. 1984). Because of its location in the outer membrane, LPS can also contribute to the initial adhesion of the bacterial cell to a surface or host cell (Genevaux et al. 1999, Nesper et al. 2001). 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 in response to 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.