PLASMID ADDICTION AS A NOVEL APPROACH TO DEVELOPING A STABLE PLASMID VECTOR FOR XYLELLA FASTIDIOSA

• Author(s): Igo, Michele; Young, Glenn; Kirkpatrick, Bruce;
• Abstract: Current approaches to understanding the progression of Pierces disease are limited by the lack of genetic techniques that can be used to study the biology of Xylella fastidiosa (Xf). In particular, extrachromosomal elements, such as plasmids, having long-term stability in Xf when grown in lab cultures or en planta, have not yet been satisfactorily developed. We will develop vectors that exhibit stable maintenance by Xf by adapting previously described genetic and microbiological techniques. Our particular research efforts will focus on taking advantage of a well-studied bacteriological phenomenon called plasmid addiction (2, 4, 10). The major mechanistic principle of plasmid addiction is that the plasmid carries a genetic trait that the host bacterium requires for viability. The trait does not affect the metabolic properties of the bacterium nor does it affect reproduction. However, loss of the plasmid-encoded trait is a lethal event, so by definition plasmid addiction ensures vector stability. In addition, we will systematically evaluate other genetic mechanisms for increasing plasmid stability including multimer resolution and active partitioning systems. Finally, we will examine the stability of each of the newly developed vectors for Xf in vitro and en planta. The results of this analysis will allow us to construct one or more stable plasmid vectors that can be used by all researchers using genetic approaches to develop methods that limit Xf-related diseases. Xylella fastidiosa is a Gram-negative, endophytic bacterium, which is responsible for a number of economically important plant diseases (for recent reviews, see (5, 7, 8)). Diseases that are important to the California agricultural economy include Pierces disease of grapevine, almond leaf scorch, alfalfa dwarf, and oleander leaf scorch. Some strains of Xf, such as the Pierces disease strains, have very wide host ranges and are capable of colonizing the xylem of widely divergent plant species. In many plant species, infection by Xf does not provoke symptoms or noticeable distress. However, the colonization of certain plants, such as grapevines, leads to the development of disease symptoms and of plant decline. Although the specific details of the disease process are not fully understood, it is known that Xf forms a biofilm within xylem vessels that has a major impact on the movement of sap within the xylem tissue. Disease symptoms seem to be dependent on the rate and extent of colonization of the xylem tissue by Xf. Some of the symptoms observed in infected grapevines include leaf marginal necrosis, severe leaf scorch, and dieback. Another important aspect of the disease cycle involves the insect vector. Xf is transmitted from plant to plant by xylemfeeding insects including the glassy-winged sharpshooter (5, 7, 8). The insect vectors acquire the bacterium by feeding on infected plants. Since the Pierces disease strain can colonize numerous plant species, the source of inoculum can be infected grapevines or symptomless plants present in the riparian habitats surrounding the vineyard. In vectors showing the highest transmission efficiencies, Xf is present as a polar biofilm in the insect foregut and is transmitted to uninfected plants during subsequent feeding events. In susceptible plants, efficient transmission of Xf occurs at low bacterial cell numbers (<100 cultivable cells per insect head). Thus, an important feature of the Xf infectious cycle is the ability of this pathogen to colonize and interact with the xylem tissue of plants and the foregut of insect vectors. Successful colonization of these hosts is dependent on the ability of Xf to subvert host defense networks and to acquire essential nutrients. To better understand how Xf survives in and interacts with its hosts, many research laboratories have been working to identify genes important for virulence and nutrient acquisition. However, rapid progress in this area is affected by the lack of genetic and molecular tools necessary to investigate the contribution of Xf genes to the infection process. One extremely important tool that is needed to advance these studies is a plasmid that is maintained by Xf throughout the infectious cycle. The goal of our project is to develop this type of plasmid. Plasmid-addiction systems consist of a pair of genes that specify two components: a stable toxin and an unstable antidote (for recent reviews, see (2, 4, 10). When a bacterium looses the plasmid harboring one of these addiction systems, the cured cells loose the ability to produce the unstable antidote and, as a result, the lethal effect of the stable toxin kills the bacterium. Thus, to remain alive each living bacterium in a sample must retain the plasmid to continue producing antidote. We will test the two different types of addiction modules that have been identified in bacteria. The first type of addiction system consists of a toxin that is encoded by a stable mRNA, but expression of the toxin is limited by the antidote, which is a small unstable antisense RNA molecule that blocks mRNA translation. The antisense mRNA antidote is produced as long as the plasmid is retained. Both the hok/sok system of plasmid R1 and the pnd locus of plasmid R483 utilize this mechanism of establishing addiction. Inclusion of the hok/sok system has been shown to successfully stabilize engineered plasmids in divergent species of bacteria including Escherichia coli, Salmonella typhi, Pseudomonas putida, and Serratia marcescens (3). The second type of addition system consists of a stable protein toxin and an unstable antitoxin protein. Similar to the previous example, antitoxin is produced as long as the plasmid is retained. One of the best characterized of this type of addiction system is the parDE system from the broad-host range plasmid RK2 (also called RP4). Addition of a region of RK2, which includes the parDE system, to a poorly maintained plasmid has been shown to enhance stability of a wide range of bacteria such as Alcaligenes eutrophus, Alcaligenes latus, Azotobacter chroococcum, Klebsiella pneumoniae, Pseudomonas aeruginosa, P. putida, and E. coli (1, 9). Interestingly, placing more than one type of plasmid addiction module onto the same plasmid provides an additive effect on plasmid stability (6). Thus we will also evaluate whether placing the two different types of plasmid addition system leads to additional plasmid stability in Xf.
• Publication Date: Dec 2004
• Journal: 2004 Pierce's Disease Research Symposium