Role of Type I Secretion in Pierce ’ S Disease

In previous work, we discovered that: 1) tolC was absolutely required not only for pathogenicity, but also for survival of Xylella fastidiosa (Xf) strain Temecula in Vitis vinifera grapevines; 2) that the loss of multi-drug resistance (MDR) efflux through the Type I secretion system was the primary reason that tolC Temecula could not survive in grapevines, and 3) that gene knockouts of Type I system components associated with offensive Type I effector secretion, including knockouts of associated with colicin V secretion and all three colicin effectors also resulted in significant loss of pathogenicity. This raised the possibilities that 1) colonization and pathogenicity of grapevines by Xf involves the exclusion of other bacteria from the xylem niche by way of these effectors, and/or 2) that these Type I effectors might directly affect plant cells. To test these ideas, PCR primers were designed to amplify all 8 hemolysins and all 3 colicins from Xf Temecula and used to amplify full length genes of all 11 effectors from Temecula. Surprisingly, these same primers were used to amplify nine equivalent homologs from EB92.1 and the predicted amino acid sequences of all 9 EB92.1 homologs were identical to the corresponding Temecula homologs. This result was confirmed by the results of the draft EB92.1 genomic DNA sequence obtained through funding of a separate project, and extended to include the remaining 2 effectors, which were 100% identical to Temecula. Furthermore, no additional hemolysins or colicins have been found to date in the draft EB92.1 genome that were not found in Temecula. These results strongly indicate that although these Type I effectors are important for plant colonization and subsequent development of PD, they are not directly responsible for the overt disease symptoms caused by Temecula (and not caused by biocontrol strain EB92.1, which has all 11 identical effectors). In addition, one hemolysin, one calcium binding hemolysin , all three colicins and uidA (GUS, as a control gene) were separately cloned into plant transient expression vectors and inoculated into tobacco and Vitis vinifera grape plants. GUS expression confirmed that the expression system worked, but no obvious plant responses were observed due to expression of the individual hemolysins or colicins inside the plant cell, further casting doubt on a direct role for these effectors in symptom elicitation. LAYPERSON SUMMARY Previously funded work has shown that the bacterial pathogen Xylella fastidiosa (Xf) relies upon a specific protein secretion device to both attack grape plants, causing Pierce’s disease (PD), and to defend itself from natural grape plant defenses against bacterial pathogens. Previous work also demonstrated that the proteins secreted by Xf using the attack mechanism played a role in the development of PD symptoms. It is now shown that the proteins secreted by Xf do not directly elicit PD symptoms in grape, but instead play an indirect, and as yet unknown, role in allowing the pathogen to grow within grape plants. It is also shown that these proteins are widely conserved in Xf strains attacking almond, coffee and citrus, in addition to grape, making any technology that is effective in disabling these proteins in grape widely applicable to other crops as well. INTRODUCTION In Gram-negative bacteria, multidrug resistance (MDR) efflux pumps are composed of three protein components, two of which are localized in the inner membrane, and one, TolC, that traverses both the periplasm and outer membrane (Koronakis et al. 2004). At least five characterized families of MDR efflux pumps exist in bacteria: the ATP-binding cassette (ABC) family (Davidson and Chen 2004), the major facilitator (MF) family (Pao et al. 1998), the small multidrug resistance (SMR) family (Paulsen et al 1997), the resistance-nodulation-cell division (RND) family (Tseng et al. 1999), and the multidrug and toxic compound extrusion (MATE) family (Brown et al. 1999). All utilize TolC as a common periplasm/outer membrane protein component (Figure 1). In addition to (defensive) MDR efflux, TolC is also essential for type-I dependent secretion of a variety of degradative enzymes and offensive effectors, some of which are antibiotic and others involved in plant or animal pathogenicity (Figure 1). These include a variety of hydrolases (proteases, phosphatases, esterases, nucleases and glucanases) and protein toxins, including hemolysins and bacteriocins (Koronakis et al. 2004). Orthologs of tolC are highly conserved among diverse Gram-negative pathogenic bacteria, and strains typically carry multiple homologues per strain (Sharff et al. 2001), including all sequenced strains of Xanthomonas, Pseudomonas and Ralstonia. 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 Pierce’s disease (PD) of grape and citrus variegated chlorosis (CVC). Analyses of the CVC and PD published genomes showed that there was no type III secretion (hrp) system, but there were at least two complete type I secretion systems present, together with multiple genes encoding