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

INTRODUCTION Current approaches to understanding the progression of Pierce’s 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.