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.
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.
Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.Show MeSH
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Mentions: In addition to the NPTH motif, we studied the contribution of the C-terminal domain of HrcU (HrcUC, amino acids 265–357, which correspond to the predicted C-terminal HrcU cleavage product) to bacterial pathogenicity and T3S. For this, we deleted codons 265–357 of the chromosomal hrcU gene in X. campestris pv. vesicatoria strain 85-10. The resulting deletion mutant strain 85-10ΔhrcU265–357 did not elicit disease symptoms and the HR in susceptible and resistant pepper plants, respectively, suggesting that HrcUC is essential for pathogenicity (Fig. 4A). The mutant phenotype was complemented by HrcU-c-Myc whereas a partial complementation was observed when we provided a c-Myc epitope-tagged derivative of HrcUCin trans (HrcU265–357-c-Myc; Fig. 4A). However, HrcU265–357-c-Myc complemented the ΔhrcU265–357 mutant phenotype in the presence of hrpG* (Fig. 4A). Immunoblot analyses of total-cell extracts from X. campestris pv. vesicatoria confirmed that HrcU-c-Myc and HrcU265–357-c-Myc were synthesized (Fig. 4B). As described above, we did not detect the full-length HrcU-c-Myc protein in cell extracts of X. campestris pv. vesicatoria. Furthermore, the amounts of HrcU265–357-c-Myc were increased when compared with the amounts of the cleavage product of HrcU-c-Myc and presumably do not reflect native protein levels (Fig. 4B). The analysis of additional expression constructs encoding HrcU265–357-c-Myc under control of an alternative promoter (e.g. the native hrcU promoter) should clarify whether the expression level of hrcU265–357-c-myc influences its ability to complement the ΔhrcU265–357 mutant phenotype.
Affiliation: Institute of Biology, Department of Genetics, Martin-Luther University Halle-Wittenberg, D-06099 Halle (Saale), Germany.