Characterization of the hrpC and hrpRS Operons of Pseudomonas syringae Pathovars Syringae, Tomato, and Glycinea and Analysis of the Ability of hrpF , hrpG , hrcC , hrpT , and hrpV Mutants To Elicit the Hypersensitive Response and Disease in Plants

Abstract

ABSTRACT The species Pseudomonas syringae encompasses plant pathogens with differing host specificities and corresponding pathovar designations. P. syringae requires the Hrp (type III protein secretion) system, encoded by a 25-kb cluster of hrp and hrc genes, in order to elicit the hypersensitive response (HR) in nonhosts or to be pathogenic in hosts. DNA sequence analysis of the hrpC and hrpRS operons of P. syringae pv. syringae 61 (brown spot of beans), P. syringae pv. glycinea U1 (bacterial blight of soybeans), and P. syringae pv. tomato DC3000 (bacterial speck of tomatos) revealed that the 13 genes comprising the right half of the hrp cluster (including those in the previously sequenced hrpZ operon) are conserved and identically arranged. The hrpC operon is comprised of hrpF , hrpG , hrcC , hrpT , and hrpV. hrcC encodes a putative outer membrane protein that is conserved in all type III secretion systems. The other four genes appear to be characteristic of group I Hrp systems, such as those possessed by P. syringae and Erwinia amylovora . The predicted products of these four genes in P. syringae pv. syringae 61 are HrpF (8 kDa), HrpG (15.4 kDa), HrpT (7.5 kDa), and HrpV (13.4 kDa). HrpT is a putative outer membrane lipoprotein. HrpF, HrpG, and HrpV are all hydrophilic proteins lacking N-terminal signal peptides. The HrpG, HrcC, HrpT, and HrpV proteins of P. syringae pathovars syringae and tomato (the two most divergent pathovars) had at least 76% amino acid identity with each other, whereas the HrpF proteins of these two pathovars had only 36% amino acid identity. The HrpF proteins of P. syringae pathovars syringae and glycinea also showed significant similarity to the HrpA pilin protein of P. syringae pathovar tomato. Functionally nonpolar mutations were introduced into each of the genes in the hrpC operon of P. syringae pv. syringae 61 by insertion of an nptII cartridge lacking a transcription terminator. The mutants were assayed for their ability to elicit the HR in nonhost tobacco leaves or to multiply and cause disease in host bean leaves. Mutations in hrpF , hrcC , and hrpT abolished or greatly reduced the ability of P. syringae pv. syringae 61 to elicit the HR in tobacco. The hrpG mutant had only weakly reduced HR activity, and the activity of the hrpV mutant was indistinguishable from that of the wild type. Each of the mutations could be complemented, but surprisingly, the hrpV subclone caused a reduction in the HR elicitation ability of the Δ hrpV :: nptII mutant. The hrpF and hrcC mutants caused no disease in beans, whereas the hrpG , hrpT , and hrpV mutants had reduced virulence. Similarly, the hrcC mutant grew little in beans, whereas the other mutants grew to intermediate levels in comparison with the wild type. These results indicate that HrpC and HrpF have essential functions in the Hrp system, that HrpG and HrpT contribute quantitatively but are not essential, and that HrpV is a candidate negative regulator of the Hrp system.