Figure 2. HpaM is essential for secretion of T3SS effectors in Xcc.

Type III secretion signal sequence-gusA fusion reporter plasmids pGUSavrAC and pGUSxopN were introduced into Xcc strains. The resulting recombinant strains were cultured in XVM2 medium for 12 h and the β-glucuronidase (GUS) activities were determined. Values are the means ± standard deviation from three repeats. (A) GUS activities in the cultural supernatant (Secreted) and the total culture (Total) produced by pGUSavrAC and pGUSxopN in different background strains. (B) Western blot assay. The recombinant plasmid pRavrACH6, which contains the T3E AvrAC encoding sequence fused with 6×His tag in its C-terminus, was introduced into Xcc strains. The resulting recombinant strains were cultured in XVM2 medium for 12 h and proteins in cultural supernatant (secreted protein) were collected by ultra-filtration using Amicon Ultra-15 centrifugal filter (Millipore Corporation, Billerica, MA, USA) and the total proteins in Xcc cells were prepared as previously described⁶². 30 μg of secreted or cell protein was electrophoresed in SDS-PAGE gel and transferred to a PVDF membrane. The presence of AvrAC was detected by anti-His₆ monoclonal antibody. (C) Cya protein translocation assay. The pLavrAC₁₀₂::CyaA fusion construct was transferred into Xcc strains and the resulting recombinant strains were then used to inoculate Chinese radish (Raphanus sativus) leaves. The cAMP level was determined 24 h post-inoculation. Values given are the means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. 8004, wild type strain; ∆hpaM, hpaM deletion mutant; ∆hrcV, hrcV deletion mutant.