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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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Characterization of a genomic hrcUP265G mutant.A. The P265G mutation in HrcU abolishes bacterial pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUP265G (hrcUP265G) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or HrcU-c-Myc (HrcU) as indicated were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 7 dpi. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines indicate the infiltrated areas.B. T3S of early and late substrates is abolished in the presence of hrcUP265G. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) were incubated in secretion medium and total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for HrpF, HrpB2, AvrBs3 and the c-Myc epitope respectively. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.C. HrpB2 oversecretion in the hpaC deletion mutant is suppressed by the genomic hrcUP265G mutation. X. campestris pv. vesicatoria strains 85* (wt), 85*hrcUP265G (P265G), 85*hrcUP265GΔhpaC (hrcUP265GΔhpaC) and 85*ΔhpaC (ΔhpaC) were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies.D. HrpF secretion by strain 85*hrcUP265G is restored upon ectopic expression of hrcU-c-myc. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or encoding HrcU-c-Myc (HrcU) as indicated were incubated in secretion medium. TE and SN were analysed by immunoblotting using a HrpF-specific antibody.
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fig03: Characterization of a genomic hrcUP265G mutant.A. The P265G mutation in HrcU abolishes bacterial pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUP265G (hrcUP265G) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or HrcU-c-Myc (HrcU) as indicated were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 7 dpi. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines indicate the infiltrated areas.B. T3S of early and late substrates is abolished in the presence of hrcUP265G. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) were incubated in secretion medium and total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for HrpF, HrpB2, AvrBs3 and the c-Myc epitope respectively. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.C. HrpB2 oversecretion in the hpaC deletion mutant is suppressed by the genomic hrcUP265G mutation. X. campestris pv. vesicatoria strains 85* (wt), 85*hrcUP265G (P265G), 85*hrcUP265GΔhpaC (hrcUP265GΔhpaC) and 85*ΔhpaC (ΔhpaC) were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies.D. HrpF secretion by strain 85*hrcUP265G is restored upon ectopic expression of hrcU-c-myc. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or encoding HrcU-c-Myc (HrcU) as indicated were incubated in secretion medium. TE and SN were analysed by immunoblotting using a HrpF-specific antibody.

Mentions: To confirm these results we introduced the hrcUP265G mutation into the genome of X. campestris pv. vesicatoria strains 85-10 and 85* respectively. The resulting mutant strains 85-10hrcUP265G and 85*hrcUP265G did not elicit visible disease symptoms and the HR when inoculated into leaves of susceptible and resistant pepper plants respectively (Fig. 3A). Furthermore, T3S of the translocon protein HrpF, the effector proteins AvrBs3, XopJ-c-Myc and XopE2-c-Myc (ectopically expressed from corresponding expression constructs) and HrpB2 was abolished in strain 85*hrcUP265G, which supports the finding that the P265G mutation in HrcU leads to a loss of protein function (Fig. 3B). Loss of efficient HrpB2 secretion was also observed in strain 85*hrcUP265GΔhpaC, suggesting that HrpB2 oversecretion in the hpaC deletion mutant is suppressed in the presence of HrcUP265G (Fig. 3C). The hrcUP265G mutant phenotype was restored with respect to virulence and T3S (shown for HrpF secretion) upon ectopic expression of hrcU-c-myc (Fig. 3A and D).


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)

Characterization of a genomic hrcUP265G mutant.A. The P265G mutation in HrcU abolishes bacterial pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUP265G (hrcUP265G) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or HrcU-c-Myc (HrcU) as indicated were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 7 dpi. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines indicate the infiltrated areas.B. T3S of early and late substrates is abolished in the presence of hrcUP265G. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) were incubated in secretion medium and total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for HrpF, HrpB2, AvrBs3 and the c-Myc epitope respectively. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.C. HrpB2 oversecretion in the hpaC deletion mutant is suppressed by the genomic hrcUP265G mutation. X. campestris pv. vesicatoria strains 85* (wt), 85*hrcUP265G (P265G), 85*hrcUP265GΔhpaC (hrcUP265GΔhpaC) and 85*ΔhpaC (ΔhpaC) were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies.D. HrpF secretion by strain 85*hrcUP265G is restored upon ectopic expression of hrcU-c-myc. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or encoding HrcU-c-Myc (HrcU) as indicated were incubated in secretion medium. TE and SN were analysed by immunoblotting using a HrpF-specific antibody.
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fig03: Characterization of a genomic hrcUP265G mutant.A. The P265G mutation in HrcU abolishes bacterial pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10hrcUP265G (hrcUP265G) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or HrcU-c-Myc (HrcU) as indicated were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 7 dpi. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines indicate the infiltrated areas.B. T3S of early and late substrates is abolished in the presence of hrcUP265G. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) were incubated in secretion medium and total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for HrpF, HrpB2, AvrBs3 and the c-Myc epitope respectively. AvrBs3, XopJ-c-Myc and XopE2-c-Myc were encoded by corresponding expression constructs.C. HrpB2 oversecretion in the hpaC deletion mutant is suppressed by the genomic hrcUP265G mutation. X. campestris pv. vesicatoria strains 85* (wt), 85*hrcUP265G (P265G), 85*hrcUP265GΔhpaC (hrcUP265GΔhpaC) and 85*ΔhpaC (ΔhpaC) were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies.D. HrpF secretion by strain 85*hrcUP265G is restored upon ectopic expression of hrcU-c-myc. X. campestris pv. vesicatoria strains 85* (wt) and 85*hrcUP265G (hrcUP265G) carrying the empty vector (−) or encoding HrcU-c-Myc (HrcU) as indicated were incubated in secretion medium. TE and SN were analysed by immunoblotting using a HrpF-specific antibody.
Mentions: To confirm these results we introduced the hrcUP265G mutation into the genome of X. campestris pv. vesicatoria strains 85-10 and 85* respectively. The resulting mutant strains 85-10hrcUP265G and 85*hrcUP265G did not elicit visible disease symptoms and the HR when inoculated into leaves of susceptible and resistant pepper plants respectively (Fig. 3A). Furthermore, T3S of the translocon protein HrpF, the effector proteins AvrBs3, XopJ-c-Myc and XopE2-c-Myc (ectopically expressed from corresponding expression constructs) and HrpB2 was abolished in strain 85*hrcUP265G, which supports the finding that the P265G mutation in HrcU leads to a loss of protein function (Fig. 3B). Loss of efficient HrpB2 secretion was also observed in strain 85*hrcUP265GΔhpaC, suggesting that HrpB2 oversecretion in the hpaC deletion mutant is suppressed in the presence of HrcUP265G (Fig. 3C). The hrcUP265G mutant phenotype was restored with respect to virulence and T3S (shown for HrpF secretion) upon ectopic expression of hrcU-c-myc (Fig. 3A and D).

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