ROLE OF TYPE I SECRETION IN PIERCES DISEASE

• Author(s): Gabriel, Dean; Lee, Richard;
• Abstract: Xylella fastidiosa (Xf) is a xylem-inhabiting Gram-negative bacterium that causes serious diseases in a wide range of plant species (Purcell and Hopkins 1996). Two of the most serious of these are Pierces disease (PD) of grape and Citrus Variegated Chlorosis (CVC). The entire genomes of both PD and CVC have been sequenced (Simpson et al. 2000). Availability of the complete genomic DNA sequence of both a PD and a CVC strain of Xf should allow rapid determination of the roles played by genes suspected of conditioning pathogenicity of CVC and/or PD. For example, analyses of the CVC and PD genomes showed that there was no type III secretion system, but there were at least two complete type I secretion systems present, together with multiple genes encoding type I effectors in the RTX (repeats in toxin) family of protein toxins, including bacteriocins and hemolysins. RTX proteins form pores in lipid bilayers of many prokaryotic and eukaryotic species and cell types; at least one is associated with pathogenicity in plants. However, lack of useful DNA cloning vectors and/or techniques for working with either CVC or PD strains have impeded progress in functional genomics analyses. There have been only three reports of transformation of Xf; one with a commercial transposase-transposon complex (Guilhabert et al. 2002) and two others with narrow host range plasmids that utilize origins of replication derived from Xf. One plasmid is an integrative vector that carries the CVC chromosomal origin of replication that provides a brief period of unstable replication in Xf (Monteiro et al. 2001). The second is a replicative shuttle vector that carries the pUC origin for replication in Escherichia coli and a rolling circle replicon derived from a cryptic CVC plasmid (Quin and Hartung 2001). However, this plasmid proved unstable in the absence of antibiotic selection. We describe here the transformation of two Xf /PD strains using the small, stable, broad host range shuttle vector, pUFR047 (De Feyter et al. 1993). This vector is one of a series of well characterized conjugational shuttle vectors based on repW and is widely used to shuttle DNA fragments from E. coli to various species and strains of Xanthomonas, where the vector is stabilized in the absence of antibiotic selection by the parA locus (De Feyter et al.1990). This is the first report of stable transformation of any Xf strain using a broad host range cloning vector.
• Publication Date: Dec 2002
• Journal: 2002 Pierce's Disease Research Symposium