Characterization Of The Pseudomonas Syringae Pathovar Tomato Dc3000 Rets Hybrid Two Component Sensor For Induction Of The Type Three Secretion System And Motility

RetS is a unique, hybrid two component sensor highly conserved among sequenced Pseudomonas species. RetS is best characterized in P. aeruginosa where it modulates chronic versus acute infection with its antagonist LadS via signaling through GacS/A and small regulatory RNAs. We conducted a transposon mutagenesis screen of P. syringae DC3000 carrying a hrp box promoter fused to a GUS reporter plasmid to investigate regulation of the type three secretion system. We isolated several transposon insertion events in PSPTO_4868 that showed reduced reporter activity in hrp inducing medium. The predicted coding sequence of PSPTO_4868 shows 58.6% identity over 889 residues to P. aeruginosa RetS (PA4856) and 94.0% identity over 929 residues to P. syringae B728a RetS (Psyr_4408). A DC3000 PSPTO_4868 pKnockout strain (hereafter [INCREMENT]retS) showed reduced type three secretion (T3SS) activity in vitro as measured by an avrPtoB::Lux reporter plasmid in hrp minimal medium supplemented with fructose or mannitol, but not when supplemented with sucrose, succinate, glutamate, or GABA as a carbon source. qRT-PCR confirmed that the [INCREMENT]retS is unable to fully activate the T3SS, and this response is modulated at least in part through RsmZ similarly to P. aeruginosa. The mutant was able to swarm in the presence of NaCl where WT is immotile. [INCREMENT]retS had no HR defect in tobacco. These results indicate that RetS in DC3000 is potentially involved in pathogenesis, but its actual role in planta remains to be determined.