Lon protease functions as a negative regulator of type III protein secretion in Pseudomonas syringae

Abstract

SummaryThe central conserved region of the Pseudomonas syringae hrp pathogenicity island encodes a type III protein secretion system (TTSS) that is required for pathogenicity in plants. Expression of the hrp TTSS is controlled by the alternative sigma factor, HrpL, whose expression, in turn, is positively controlled by two truncated enhancer binding proteins, HrpR and HrpS. Although a number of environmental conditions are known to modulate hrp TTSS expression, such as stringent conditions and pathogenesis, the mechanism by which the activities of these transcriptional factors are modulated had not been established. In this study, HrpR and HrpS were found to be constitutively expressed under conditions in which the hrpL promoter was inactive. To identify a postulated negative regulator of hrpL expression, transposome (Tz) mutagenesis was used to isolate hrp constitutive mutants. P. syringae Pss61 and DC3000 hrp constitutive mutants were identified that carried lon::Tz insertions and exhibited increased cell length and UV sensitivity typical of Dlon mutants. The P. syringae Lon protease retained structural features of its homologues found in other bacteria and was capable of complementing an Escherichia coli Dlon mutant. P. syringae lon::Tz mutants exhibited enhanced expression of the hrpL promoter, suggesting an effect on HrpR and/or HrpS. HrpR was observed to be unstable in wild-type P. syringae strains grown in non-inductive media. However, the apparent half-life increased more than 10-fold in the P. syringae lon::Tz mutants or upon transfer to an inductive medium. The P. syringae lon mutants elicited rapidly developing plant responses and were shown to hypersecrete effector proteins, such as AvrPto. These results indicate that expression of the hrp regulon and type III secretion are negatively regulated by Lon-mediated degradation of HrpR.

Negative Regulation of Pathogenesis in Pseudomonas syringae pv. tabaci 11528 by ATP-Dependent Lon Protease

“…tabaci 11528 From a previous report, we know that the Lon protease functions as a negative regulator of TTSS in P. syringae pv. syringae 61 by degrading HrpR under hrp-repressing conditions (Bretz et al, 2002). Therefore, we decided to verify that the Lon protease also acts as a negative regulator of TTSS in P. syringae pv.…”

“…Proteolysis by Lon has been implicated in the regulation of hrp gene expression and the activity of this broadly conserved protein secretion system. The ability of P. syringae to elicit defense responses in resistant plants and pathogenesis in susceptible plants has been linked to a TTSS and effectors encoded by the environmentally regulated hrp regulon (Bretz et al, 2002;Collmer et al, 2002). The induction of hrp regulon expression during pathogenesis had been shown previously to be dependent upon the unusual enhancer binding proteins HrpR and HrpS and the alternative ).…”

“…tabaci 11528 Since the domains of the Lon protease are conserved in P. syringae homologues (Bretz et al, 2002), we aligned the lon gene nucleotide sequences of four pathovars. We designed a primer set (Table 2), lon-F(KpnI) and lon-R(SacI), to amplify the complete lon gene.…”

“…In hrp-repressing environments, such as nutrient-rich media, the Lon protease has been found to degrade HrpR and repress the expression of the hrp regulon. Under hrp-inducing conditions, however, the hrp regulon is down-regulated by another negative regulator, HrpV (Bretz et al, 2002).…”

“…Environmental regulation of the hrp regulon occurs primarily at the level of the hrpL promoter and involves Lon protease-mediated degradation of HrpR. Lonassociated degradation of HrpR is reduced under conditions inducive to hrp regulon expression (Bretz et al, 2002).…”