USE OF THE E. COLI α-HEMOLYSIN SECRETION SYSTEM IN BACTERIA DESIGNED FOR SYMBIOTIC CONTROL OF PIERCE’S DISEASE IN GRAPEVINES AND SHARPSHOOTERS Project Leader:

Strains of Alcaligenes xylosoxidans denitrificans (Axd) that secrete anti-Xylella factors are being developed for use in a strategy to prevent the spread of Xf or possibly to cure infected grapevines. We built constructs that fused the last 60 amino acids of the autotransporter α-hemolysin from E. coli to test proteins. These were two different forms of anti-BSA single chain antibodies (scFvs) which are surrogates for anti-Xylella effector proteins. These proteins were efficiently secreted from E. coli when co-expressed with the proteins HlyB and HlyD. HlyB, HlyD, and TolC together form the membrane structure used by α-hemolysin to cross both the inner and outer membrane of the cell. We report here on efforts to move this system into Axd, a species not closely-related to E. coli, but that can survive in both grapevine and sharpshooters. INTRODUCTION The glassy-winged sharpshooter (GWSS) is the principal vector of the xylem-limited bacterium Xylella fastidiosa (Xf), which causes Pierce’s disease (PD) in grapes. Limiting the spread of this pathogen by rendering GWSS incapable of pathogen transmission or by interfereing with the replication of Xf in the plant may stop the spread of PD. These endpoints can be accomplished by genetically modifying bacteria that live in the sharpshooter, the plant, or both in a method called symbiotic control. Symbiotic control seeks to modify the phenotype of an organism indirectly by modifying its symbiotic bacteria. Symbiotic control approaches to disrupt pathogen infection of humans are being developed by several groups. These include interference with the ability of triatomid bugs to transmit pathogens causing Chagas’ disease (Beard et al., 2001), interference with HIV attachment to its target cells in the reproductive tracts of humans (Chang et al., 2003; Rao et al., 2005), and the elimination of persistent Candida infections from biofilms in chronically infected human patients (Beninati et al., 2000). Symbiotic control has also been applied to deliver cytokines mammalian guts to relieve colitis (Steidler et al., 2000; Steidler, 2001). Thus, the method has wide applicability. Alcaligenes xylosoxidans denitrificans (Axd) is Gram negative pseudomonad-like species that can colonize the GWSS foregut and cibarium, as well as various plant tissues, including grape xylem. It is non-pathogenic in insects, plants and healthy humans. Given these characteristics, Axd has become the focus of our symbiotic control efforts to control PD in grapes. Over the past several years we developed the technology to stably modify Axd by inserting genes into its chromosome via transposition, have developed methods to suppress horizontal gene transfer, and have isolated a single chain antibody that recognizes an epitope on the surface of the PD strain of Xf. (Bextine et al., 2004). We are currently engaged in combining these systems in order to produce strains of Axd that are suitable for environmental release in a practical strategy symbiotic control strategy for PD. One way to deliver anti-Xylella protein factors from Axd in grapevine is by secretion. Secreted anti-Xylella factors might circulate throughout the plant, reaching foci of infection across physical xylem boundaries. Secretion from Gram-negative bacteria, however, is complicated by the fact that these species have two membranes that a protein must cross before appearing outside the cell. Gram negatives contain at least 6 identified types of secretion systems. Unfortunately, many of these systems are unpredictable when expressed heterologously. One system that seems to have wide applicability is the ahemolysin autotransporter from E. coli (Fernandez et al., 2000). This protein is secreted in a single energy-dependent step across both membranes of Gram negative bacteria when the other components of the system are also present (the proteins HlyB, HlyD, and TolC). Fusion of the last 60 amino acids of the protein is sufficient to target any N-terminal passenger protein for secretion. We report here the evaluation of the E. coli α-hemolysin system for use in Axd to secrete soluble anti-Xylella protein effectors in grapevine and insects. OBJECTIVES 1. Test the E. coli α-hemolysin secretion system in E. coli and Axd.