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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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Complementation studies with HrcU mutant derivatives.A. The conserved asparagine residue of the NPTH motif of HrcU is essential for pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10ΔhrcU (ΔhrcU) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A), HrcUH267A-c-Myc (H267A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 8 and 11 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. The N264A mutation abolishes T3S of translocon and effector proteins but does not affect secretion of the pilus assembly protein HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the translocon protein HrpF, the effector protein AvrBs3 (ectopically expressed from construct pDSF300) and HrpB2.C. HrcUP265G does not promote secretion of HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−), HrcU-c-Myc (HrcU), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies respectively.
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fig02: Complementation studies with HrcU mutant derivatives.A. The conserved asparagine residue of the NPTH motif of HrcU is essential for pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10ΔhrcU (ΔhrcU) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A), HrcUH267A-c-Myc (H267A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 8 and 11 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. The N264A mutation abolishes T3S of translocon and effector proteins but does not affect secretion of the pilus assembly protein HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the translocon protein HrpF, the effector protein AvrBs3 (ectopically expressed from construct pDSF300) and HrpB2.C. HrcUP265G does not promote secretion of HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−), HrcU-c-Myc (HrcU), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies respectively.

Mentions: As expected, strain 85-10 induced water-soaked lesions in ECW and the HR in ECW-10R plants whereas no plant reactions were observed after inoculation of strain 85-10ΔhrcU (Fig. 2A). The hrcU mutant phenotype was complemented by construct pBRMhrcU, which encodes a C-terminally c-Myc epitope-tagged HrcU derivative under control of the lac promoter (Fig. 2A). Partial complementation was observed for HrcUT266A-c-Myc and HrcUH267A-c-Myc, whereas strain 85-10ΔhrcU carrying HrcUN264A-c-Myc, HrcUP265A-c-Myc and HrcUP265G-c-Myc, respectively, did not cause visible plant reactions (Fig. 2A). We also performed infection assays with hrpG* strains that carry a mutated version of the key regulator HrpG and thus constitutively express the T3S genes (Rossier et al., 1999; Wengelnik et al., 1999). Notably, we observed a partial complementation of the hrcU mutant phenotype by HrcUP265A-c-Myc but not by HrcUP265G-c-Myc in the presence of hrpG* (Fig. 2A). We have previously observed that constitutive expression of the T3S genes promotes in planta symptom formation (Büttner et al., 2004; 2007; Lorenz and Büttner, 2009). The partial complementation of the hrcU mutant phenotype by HrcUP265A-c-Myc is in agreement with the finding that this HrcU mutant derivative is partially cleaved (see Fig. 1C).


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)

Complementation studies with HrcU mutant derivatives.A. The conserved asparagine residue of the NPTH motif of HrcU is essential for pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10ΔhrcU (ΔhrcU) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A), HrcUH267A-c-Myc (H267A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 8 and 11 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. The N264A mutation abolishes T3S of translocon and effector proteins but does not affect secretion of the pilus assembly protein HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the translocon protein HrpF, the effector protein AvrBs3 (ectopically expressed from construct pDSF300) and HrpB2.C. HrcUP265G does not promote secretion of HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−), HrcU-c-Myc (HrcU), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies respectively.
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fig02: Complementation studies with HrcU mutant derivatives.A. The conserved asparagine residue of the NPTH motif of HrcU is essential for pathogenicity. X. campestris pv. vesicatoria strains 85-10 (wt), 85* (wt), 85-10ΔhrcU (ΔhrcU) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A), HrcUH267A-c-Myc (H267A) and HrcUP265G-c-Myc (P265G), respectively, from corresponding expression constructs were inoculated into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed 8 and 11 dpi as indicated. For the better visualization of the HR, leaves were bleached in ethanol 2 dpi. Dashed lines mark the infiltrated areas.B. The N264A mutation abolishes T3S of translocon and effector proteins but does not affect secretion of the pilus assembly protein HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−) or encoding HrcU-c-Myc (wt), HrcUN264A-c-Myc (N264A), HrcUP265A-c-Myc (P265A), HrcUT266A-c-Myc (T266A) and HrcUH267A-c-Myc (H267A), respectively, were incubated in secretion medium. Total-cell extracts (TE) and culture supernatants (SN) were analysed by immunoblotting using antibodies specific for the translocon protein HrpF, the effector protein AvrBs3 (ectopically expressed from construct pDSF300) and HrpB2.C. HrcUP265G does not promote secretion of HrpB2. X. campestris pv. vesicatoria strains 85* (wt) and 85*ΔhrcU (ΔhrcU) carrying the empty vector (−), HrcU-c-Myc (HrcU), HrcUP265A-c-Myc (P265A) and HrcUP265G-c-Myc (P265G), respectively, were incubated in secretion medium. TE and SN were analysed by immunoblotting using HrpF- and HrpB2-specific antibodies respectively.
Mentions: As expected, strain 85-10 induced water-soaked lesions in ECW and the HR in ECW-10R plants whereas no plant reactions were observed after inoculation of strain 85-10ΔhrcU (Fig. 2A). The hrcU mutant phenotype was complemented by construct pBRMhrcU, which encodes a C-terminally c-Myc epitope-tagged HrcU derivative under control of the lac promoter (Fig. 2A). Partial complementation was observed for HrcUT266A-c-Myc and HrcUH267A-c-Myc, whereas strain 85-10ΔhrcU carrying HrcUN264A-c-Myc, HrcUP265A-c-Myc and HrcUP265G-c-Myc, respectively, did not cause visible plant reactions (Fig. 2A). We also performed infection assays with hrpG* strains that carry a mutated version of the key regulator HrpG and thus constitutively express the T3S genes (Rossier et al., 1999; Wengelnik et al., 1999). Notably, we observed a partial complementation of the hrcU mutant phenotype by HrcUP265A-c-Myc but not by HrcUP265G-c-Myc in the presence of hrpG* (Fig. 2A). We have previously observed that constitutive expression of the T3S genes promotes in planta symptom formation (Büttner et al., 2004; 2007; Lorenz and Büttner, 2009). The partial complementation of the hrcU mutant phenotype by HrcUP265A-c-Myc is in agreement with the finding that this HrcU mutant derivative is partially cleaved (see Fig. 1C).

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