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Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU.

Lorenz C, Büttner D - Mol. Microbiol. (2010)

Bottom Line: T3S substrate specificity is controlled by HpaC, which promotes secretion of translocon and effector proteins but prevents efficient secretion of the early substrate HrpB2.The results of mutant studies showed that cleavage of HrcU contributes to pathogenicity and secretion of late substrates but is dispensable for secretion of HrpB2, which is presumably secreted prior to HrcU cleavage.As HrcU(Y318D) did not interact with HrpB2 and HpaC, we propose that the substrate specificity switch leads to the release of HrcU(C) -bound HrpB2 and HpaC.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.

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The Y318D mutation in HrcU suppresses the hpaC mutant phenotype and affects HrcU cleavage.A. Infection studies with hrcU wild-type and hrcUY318D mutant strains. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUY318D (hrcUY318D), 85*hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC), 85*ΔhpaC (ΔhpaC), 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 5 and 6 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. In planta growth of hrcUY318D mutants. X. campestris pv. vesicatoria strains 85-10 (wt), 85-10hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC) and 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW pepper plants and bacterial growth was analysed over a period of 8 days. Values are the mean of three samples from three plants. Error bars represent standard deviations. The asterisk indicates a significant difference to the wild-type strain with P < 0.005 based on the results of an unpaired Student's t-test.C. T3S assays with hrcUY318D mutants. Strains 85* (wt), 85*hrcUY318D (hrcUY318D), 85*ΔhpaC (ΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the putative translocon proteins HrpF and XopA, the effector protein AvrBs3, the pilus assembly protein HrpB2 and the c-Myc epitope. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.D. The Y318D mutation in HrcU affects proteolytic cleavage. Equal amounts of total-cell extracts from X. campestris pv. vesicatoria strain 85*ΔhrcU (ΔhrcU) and E. coli carrying the empty vector (−) or encoding HrcU-c-Myc (wt) and HrcUY318D-c-Myc (Y318D), respectively, as indicated were analysed by immunoblotting using a c-Myc epitope-specific antibody.
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fig07: The Y318D mutation in HrcU suppresses the hpaC mutant phenotype and affects HrcU cleavage.A. Infection studies with hrcU wild-type and hrcUY318D mutant strains. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUY318D (hrcUY318D), 85*hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC), 85*ΔhpaC (ΔhpaC), 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 5 and 6 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. In planta growth of hrcUY318D mutants. X. campestris pv. vesicatoria strains 85-10 (wt), 85-10hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC) and 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW pepper plants and bacterial growth was analysed over a period of 8 days. Values are the mean of three samples from three plants. Error bars represent standard deviations. The asterisk indicates a significant difference to the wild-type strain with P < 0.005 based on the results of an unpaired Student's t-test.C. T3S assays with hrcUY318D mutants. Strains 85* (wt), 85*hrcUY318D (hrcUY318D), 85*ΔhpaC (ΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the putative translocon proteins HrpF and XopA, the effector protein AvrBs3, the pilus assembly protein HrpB2 and the c-Myc epitope. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.D. The Y318D mutation in HrcU affects proteolytic cleavage. Equal amounts of total-cell extracts from X. campestris pv. vesicatoria strain 85*ΔhrcU (ΔhrcU) and E. coli carrying the empty vector (−) or encoding HrcU-c-Myc (wt) and HrcUY318D-c-Myc (Y318D), respectively, as indicated were analysed by immunoblotting using a c-Myc epitope-specific antibody.

Mentions: It was previously reported that the phenotype of T3S4 mutants from animal pathogenic bacteria can be suppressed upon introduction of point mutations into the C-terminal domain of FlhB/YscU family members (Kutsukake et al., 1994; Williams et al., 1996; Edqvist et al., 2003; Wood et al., 2008; Zarivach et al., 2008). To test this for X. campestris pv. vesicatoria, we introduced a point mutation (Y318D) into the chromosomal hrcU genes of strains 85-10 and 85-10ΔhpaC, respectively, which led to an exchange of the tyrosine residue at amino acid position 318 of HrcU by aspartic acid. Equivalent mutations in the C-terminal domains of YscU (YscUY317D) and FlhB (FlhBY323D) were shown to suppress the phenotypes of mutants deleted in the T3S4 genes yscP and fliK respectively (Kutsukake et al., 1994; Minamino and MacNab, 2000a; Edqvist et al., 2003; Wood et al., 2008). When X. campestris pv. vesicatoria hrcU wild-type and hrcUY318D mutant strains were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants, respectively, strain 85-10hrcUY318D induced disease symptoms and the HR similarly to the wild type whereas strain 85-10ΔhpaC led to significantly reduced symptoms as expected (Fig. 7A; Büttner et al., 2006). The double mutant 85-10hrcUY318DΔhpaC induced wild-type disease symptoms, suggesting that HrcUY318D suppresses the hpaC mutant phenotype in susceptible plants (Fig. 7A). Furthermore, HrcUY318D partially restored the HR induction by strain 85-10hrcUY318DΔhpaC in resistant ECW-10R plants. However, a wild-type HR was observed for the hrpG* derivative 85*hrcUY318DΔhpaC (Fig. 7A).


Secretion of early and late substrates of the type III secretion system from Xanthomonas is controlled by HpaC and the C-terminal domain of HrcU.

Lorenz C, Büttner D - Mol. Microbiol. (2010)

The Y318D mutation in HrcU suppresses the hpaC mutant phenotype and affects HrcU cleavage.A. Infection studies with hrcU wild-type and hrcUY318D mutant strains. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUY318D (hrcUY318D), 85*hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC), 85*ΔhpaC (ΔhpaC), 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 5 and 6 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. In planta growth of hrcUY318D mutants. X. campestris pv. vesicatoria strains 85-10 (wt), 85-10hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC) and 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW pepper plants and bacterial growth was analysed over a period of 8 days. Values are the mean of three samples from three plants. Error bars represent standard deviations. The asterisk indicates a significant difference to the wild-type strain with P < 0.005 based on the results of an unpaired Student's t-test.C. T3S assays with hrcUY318D mutants. Strains 85* (wt), 85*hrcUY318D (hrcUY318D), 85*ΔhpaC (ΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the putative translocon proteins HrpF and XopA, the effector protein AvrBs3, the pilus assembly protein HrpB2 and the c-Myc epitope. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.D. The Y318D mutation in HrcU affects proteolytic cleavage. Equal amounts of total-cell extracts from X. campestris pv. vesicatoria strain 85*ΔhrcU (ΔhrcU) and E. coli carrying the empty vector (−) or encoding HrcU-c-Myc (wt) and HrcUY318D-c-Myc (Y318D), respectively, as indicated were analysed by immunoblotting using a c-Myc epitope-specific antibody.
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Related In: Results  -  Collection

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fig07: The Y318D mutation in HrcU suppresses the hpaC mutant phenotype and affects HrcU cleavage.A. Infection studies with hrcU wild-type and hrcUY318D mutant strains. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUY318D (hrcUY318D), 85*hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC), 85*ΔhpaC (ΔhpaC), 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 5 and 6 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. In planta growth of hrcUY318D mutants. X. campestris pv. vesicatoria strains 85-10 (wt), 85-10hrcUY318D (hrcUY318D), 85-10ΔhpaC (ΔhpaC) and 85-10hrcUY318DΔhpaC (hrcUY318DΔhpaC) were inoculated into leaves of susceptible ECW pepper plants and bacterial growth was analysed over a period of 8 days. Values are the mean of three samples from three plants. Error bars represent standard deviations. The asterisk indicates a significant difference to the wild-type strain with P < 0.005 based on the results of an unpaired Student's t-test.C. T3S assays with hrcUY318D mutants. Strains 85* (wt), 85*hrcUY318D (hrcUY318D), 85*ΔhpaC (ΔhpaC) and 85*hrcUY318DΔhpaC (hrcUY318DΔhpaC) were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the putative translocon proteins HrpF and XopA, the effector protein AvrBs3, the pilus assembly protein HrpB2 and the c-Myc epitope. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.D. The Y318D mutation in HrcU affects proteolytic cleavage. Equal amounts of total-cell extracts from X. campestris pv. vesicatoria strain 85*ΔhrcU (ΔhrcU) and E. coli carrying the empty vector (−) or encoding HrcU-c-Myc (wt) and HrcUY318D-c-Myc (Y318D), respectively, as indicated were analysed by immunoblotting using a c-Myc epitope-specific antibody.
Mentions: It was previously reported that the phenotype of T3S4 mutants from animal pathogenic bacteria can be suppressed upon introduction of point mutations into the C-terminal domain of FlhB/YscU family members (Kutsukake et al., 1994; Williams et al., 1996; Edqvist et al., 2003; Wood et al., 2008; Zarivach et al., 2008). To test this for X. campestris pv. vesicatoria, we introduced a point mutation (Y318D) into the chromosomal hrcU genes of strains 85-10 and 85-10ΔhpaC, respectively, which led to an exchange of the tyrosine residue at amino acid position 318 of HrcU by aspartic acid. Equivalent mutations in the C-terminal domains of YscU (YscUY317D) and FlhB (FlhBY323D) were shown to suppress the phenotypes of mutants deleted in the T3S4 genes yscP and fliK respectively (Kutsukake et al., 1994; Minamino and MacNab, 2000a; Edqvist et al., 2003; Wood et al., 2008). When X. campestris pv. vesicatoria hrcU wild-type and hrcUY318D mutant strains were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants, respectively, strain 85-10hrcUY318D induced disease symptoms and the HR similarly to the wild type whereas strain 85-10ΔhpaC led to significantly reduced symptoms as expected (Fig. 7A; Büttner et al., 2006). The double mutant 85-10hrcUY318DΔhpaC induced wild-type disease symptoms, suggesting that HrcUY318D suppresses the hpaC mutant phenotype in susceptible plants (Fig. 7A). Furthermore, HrcUY318D partially restored the HR induction by strain 85-10hrcUY318DΔhpaC in resistant ECW-10R plants. However, a wild-type HR was observed for the hrpG* derivative 85*hrcUY318DΔhpaC (Fig. 7A).

Bottom Line: T3S substrate specificity is controlled by HpaC, which promotes secretion of translocon and effector proteins but prevents efficient secretion of the early substrate HrpB2.The results of mutant studies showed that cleavage of HrcU contributes to pathogenicity and secretion of late substrates but is dispensable for secretion of HrpB2, which is presumably secreted prior to HrcU cleavage.As HrcU(Y318D) did not interact with HrpB2 and HpaC, we propose that the substrate specificity switch leads to the release of HrcU(C) -bound HrpB2 and HpaC.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.

Show MeSH
Related in: MedlinePlus