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. 2000 Feb 29;97(5):2247-52.
doi: 10.1073/pnas.040570097.

The gene coding for the Hrp pilus structural protein is required for type III secretion of Hrp and Avr proteins in Pseudomonas syringae pv. tomato

W Wei  1 , A Plovanich-Jones, W L Deng, Q L Jin, A Collmer, H C Huang, S Y He
Affiliations

The gene coding for the Hrp pilus structural protein is required for type III secretion of Hrp and Avr proteins in Pseudomonas syringae pv. tomato

W Wei et al. Proc Natl Acad Sci U S A. .

Abstract

Bacterial surface appendages called pili often are associated with DNA and/or protein transfer between cells. The exact function of pili in the transfer process is not understood and is a matter of considerable debate. The Hrp pilus is assembled by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato (Pst) strain DC3000. In this study, we show that the hrpA gene, which encodes the major subunit of the Hrp pilus, is required for secretion of putative virulence proteins, such as HrpW and AvrPto. In addition, the hrpA gene is required for full expression of genes that encode regulatory, secretion, and effector proteins of the type III secretion system. hrpA-mediated gene regulation apparently is through effect on the mRNA level of two previously characterized regulatory genes, hrpR and hrpS. Ectopic expression of the hrpRS gene operon restored gene expression, but not protein secretion, in the hrpA mutant. Three single amino acid mutations at the HrpA carboxyl terminus were identified that affect the secretion or regulatory function of the HrpA protein. These results define an essential role of the Hrp pilus structural gene in protein secretion and coordinate regulation of the type III secretion system in Pst DC3000.

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Figures

Figure 1
Figure 1
Immunoblot analysis of HrpW, HrcJ, and HrcC proteins in Pst DC3000 and hrp mutants in hrp-inducing medium (A) and in planta (B). Conditions for bacterial growth and immunoblot analysis are described in Materials and Methods. For analysis of HrpW expression in hrp-inducing cultures, the levels of HrpW in cell-associated (C) and supernatant (S) fractions were examined.
Figure 2
Figure 2
RNA blot analysis of hrp and avr transcripts in Pst DC3000 and hrp mutants. hrp gene operons, named by their first genes, are diagrammed at the top. The directions of transcription are indicated by arrows. The hrpW gene is linked to the core hrp cluster, whereas avrPto is not. Conditions for bacterial growth and RNA analysis are described in Materials and Methods. The genes indicated on the left were used as probes. The 23S rRNA visualized after ethidium bromide staining was used as loading control. RNA was isolated from bacteria grown in hrp-repressing LB or hrp-inducing minimal medium (MM). *, The hrcC and hrcZ genes are located within the hrpF and hrpA gene operons, respectively. See ref. for a more detailed description of the P. syringae hrp gene cluster.
Figure 3
Figure 3
(A) A diagram of the P. syringae pv. tomato DC3000 HrpA protein (113 aa in length). Six amino acid residues (*) conserved among HrpA proteins of P. syringae pvs. tomato, syringae, and glycinea and E. amylovora were mutated by site-directed mutagenesis. The E94-to-K94 mutation, indicated by an arrowhead, was obtained by random mutagenesis. Amino acid residue substitutions that did not affect the HrpA function are indicated by hatched bars. Those substitutions that eliminated the ability of pHRPA to complement the genomic hrpA mutation are indicated by solid bars. The HrpA function was assayed by the ability (+) or inability (−) of the corresponding pHRPA derivatives to complement the genomic hrpA deletion mutation for HR elicitation in tobacco leaves and disease causation in Arabidopsis thaliana leaves. (B) Immunoblot analysis of the effect of single amino acid mutations of HrpA on the production of HrpW, HrcJ, and HrcC in hrp-inducing medium. Both cell-associated (C) and supernatant (S) fractions were analyzed for HrpW. Conditions for bacterial growth and immunoblot analysis are described in Materials and Methods.
Figure 4
Figure 4
(A) Effect of the hrpA mutation on the steady-state message abundance of hrpRS. The procedures for bacterial growth and RNA preparation and blotting are the same as in the legend to Fig. 2, except that the RNA gel blot was hybridized to the hrpRS gene probe. (B) Effects of ectopic expression of hrpRS on the accumulation of Hrp/Hrc proteins in the hrpA mutant. Bacteria were grown in LB supplemented with (+) or without (−) 35 μM salicylic acid (SA). The original culture was used directly for analysis by SDS/PAGE followed by immunoblot analysis with antibodies against HrpW and HrcC, respectively, without further fractionation. (C) Effects of SA-induced expression of hrpRS from pHRPRS1 on the accumulation of Hrp/Hrc proteins in the hrpA mutant in planta. Bacteria were supplemented with 35 μM SA before infiltration. Constitutive expression of Pst DC3000 hrpRS genes from pHRPRS2 also restored the expression of HrpW in the hrpA mutant (see Fig. 5).
Figure 5
Figure 5
Effect of ectopic expression of the hrpRS operon in the hrpA mutant on HrpW and AvrPto secretion. Bacteria were grown in hrp-inducing minimal medium. Bacterial supernatant (S) and cell (C) fractions were analyzed by SDS/PAGE followed by immunoblotting with HrpW (A) or AvrPto antibody (B).
Figure 6
Figure 6
A hypothetical model depicting hrpA-mediated coupling of protein secretion and gene regulation. Arrow 1 indicates that hrpA directly affects the transcription or RNA stability of the hrpRS operon independent of its effects on secretion of Hrp and Avr proteins. Arrow 2 indicates that hrpA indirectly regulates the hrpRS operon through its involvement in secretion of a negative regulator (HrpV?) as well as other Hrp and Avr proteins. The question marks indicate uncertainties in the model regarding whether (i) hrpA affects hrpRS expression directly and/or indirectly through its effects on secretion, (ii) Hrp and Avr proteins are secreted through the pilus, and (iii) a negative regulator (e.g., HrpV) is secreted.
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