PLASMID ADDICTION AS A NOVEL APPROACH FOR DEVELOPING A STABLE PLASMID VECTOR FOR XYLELLA FASTIDIOSA Project Leaders:

The lack of genetic and molecular tools that can be used to study the biology of Xylella fastidiosa (Xf) has made it extremely difficult for researchers to use genetic methods to establish the importance of a particular gene in the development of Pierce’s disease (PD). During the period under review, we have focused on developing plasmid vectors that are stably maintained in Xf throughout the infection cycle. To increase the stability of autonomously replicating plasmid vectors, we have introduced two different types of stabilizing elements into plasmid vectors pXF004, pRL1342, and pBBR1MCS-5. These stabilizing elements include the plasmid addiction systems, hok/sok and parDE, and the active partitioning system, parA. We are currently examining how addition of these stability elements affects plasmid maintenance both in vitro and en planta. We have also developed two integration vectors, which will allow researchers to introduce genes into two different nonessential regions of the Xf chromosome. We are currently evaluating the properties of the insertion strains en planta to make sure that these strains still exhibit the normal PD infectious cycle and have begun to examine the usefulness of both of these vectors for complementation analysis in Xf. INTRODUCTION Xylella fastidiosa (Xf) is the causative agent of numerous economically important plant diseases, including Pierce’s disease (PD) of grapevine (Hopkins and Purcell 2002). 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. The virulence determinants of Xf include proteins involved in adhesion and biofilm formation, extracellular enzymes, and toxins. A fundamental strategy for investigating virulence in bacterial pathogens is to generate mutations and examine the impact of the absence of these gene products on pathogenicity. Over the past five years, many research laboratories have been generating insertion mutations in specific Xf genes and examining the impact of these mutations on the development of PD (Guilhabert and Kirkpatrick 2003, Feil et al. 2003, Reddy et al. 2004, Roper et al. 2004, Meng et al. 2005, HernandezMartinez et al. 2006). These studies have led to the identification of a number of mutant strains that do not show the normal PD infection cycle. Although the simplest explanation for these phenotypes is that the gene containing the insertion mutation is required for the normal development of PD, it is also possible that a secondary mutation was acquired during the construction of the original mutation and that the secondary mutation is responsible for the phenotype. The classic approach to overcoming this type of objection is to perform complementation analysis. If the reintroduction of a wild-type copy of the gene into the mutant strain restores the normal PD infection cycle en planta, the researcher can conclude that the specific gene is important for the development of PD. One common strategy used to reintroduce the wildtype copy of a gene in Gram-negative bacteria involves the use of autonomously replicating plasmid vectors that carry antibiotic resistance genes and multiple cloning sites. Plasmid vectors with these features have been developed that are capable of replicating in Xf and that are stably maintained in the presence of antibiotics. These plasmids have been extremely useful for introducing genes into Xf and for in vitro complementation studies. Unfortunately, most of these plasmids are quickly lost from Xf in the absence of selective pressure, which limits the usefulness of these plasmids for studies en planta. Therefore, a major goal of this study is to develop a set of plasmid vectors that will allow researchers to perform complementation analysis en planta. OBJECTIVES 1. Develop a stable plasmid vector for Xf a. Evaluate the potential of various plasmid addiction systems for ability to convert plasmids known to replicate in Xf into stable vectors. b. Evaluate how plasmid maintenance by Xf is affected by other genetic mechanisms known to affect plasmid stability, such as systems for multimer resolution and active partitioning systems. 2. Evaluate the stability of the newly development plasmid vectors when propagate in Xf en planta.