Fig 1.
HpaB and its C-terminal domain are involved in virulence and HR induction in Xcc.
(A) Schematic presentation of generation of hpaB deletion mutants. aa = amino acid residues. (B) The average lesion lengths of Chinese radish (Raphanus sativus var. radiculus cv. Manshenhong) leaves caused by Xcc strains (wild type strain 8004, hpaB-deletion mutants, and hpaB-deletion mutants carrying hpaB-recombinant plasmid) with a cell density of OD600 = 0.001 were measured 10 days post-inoculation. Values are the mean ± standard deviation from three repeats, each with at least 60 leaves. (C) Analysis of HR induced by Xcc strains. Bacteria of Xcc with a cell density of OD600 = 0.1 were inoculated into the leaves of pepper plant (Capsicum annuum cv. ECW-10R). Leaves were photographed at 24 h post-inoculation.
Fig 2.
The translocation of T3Es is dependent on HpaB in Xcc.
The mutants ΔavrBs1, ΔhpaB, and ΔhrpF carrying plasmids that respectively encode N-terminal signals of XC0052, XC0241, XC1210, XC1553, XC2602, and XC3176, in-frame fused with AvrBs159-445−3×FLAG were infiltrated into the expanded leaves of pepper plant (Capsicum annuum cv. ECW-10R) at an OD600 of 0.1. The phenotypes were photographed at 24 h post-inoculation. The wild type strain 8004 was used here as the positive control and the deletion mutants ΔavrBs1 and ΔhrpF as the negative control.
Fig 3.
The C-terminal domain of HpaB is required for the secretion and translocation of T3Es in Xcc.
(A-C) Xcc strains were incubated in hrp-inducing medium MMX. Total cell extracts (TE) and culture supernatants (S) were analyzed by immunoblotting with an ant-FLAG antibody or anti-RNPβ antibody. T3Es AvrBs1 (A) and XC3176 (B) tagged with 3×FLAG epitope tag were introduced into wild type strain 8004, T3S-defective ΔhrcV, ΔhpaB, and hpaB C-terminal deletion mutant Δ137–160, respectively. (C) Non-effector Hpa1 tagged with 3×FLAG epitope tag was introduced to wild type strain 8004, T3S-defective ΔhrcV, ΔhpaB, Δ45–160, and hpaB C-terminal deletion mutant Δ137–160, respectively. (D) Translocation assays were performed based AvrBs1 reporter. Xcc strains harboring or not with plasmid pJAG1553 expressing AvrAC1-102-AvrBs159-445 fusion were infiltrated into the expanded leaves of pepper (Capsicum annuum cv. ECW-10R) at an OD600 of 0.1, respectively. The phenotypes were photographed at 24 h post-inoculation. Wild type strain 8004 and ΔhpaB were used here as positive and negative control, respectively. (E) Translocation assays were performed with Cya reporter. Wild type strain 8004, T3S-defective ΔhrcV, ΔhpaB, Δ45–160, and Δ137–160, with plasmid pJAA1553 expressing AvrAC1-102-Cya2-400−3×FLAG fusion were infiltrated into Chinese cabbage (Brassica oleracea cv. Jingfeng NO. 1) leaves at an OD600 of 0.1. The cAMP level found in infected leaves was tested at 8 and 24 h post-inoculation, respectively. Values are the mean ± standard deviation from three repeats, each with 3 leaves.
Fig 4.
The C-terminal domain of HpaB is dispensable for interaction with T3Es in Xcc.
(A-B) GST alone and GST-fused HpaB derivates were immobilized on MagneGSTTM particles and incubated with cell lysates containing 6×His-AvrBs1 (A) and 6×His-HpaA (B), respectively. Samples were analyzed using immunoblotting with hexahistidine-specific antibody and GST-specific antibody.
Fig 5.
The C-terminal domain of HpaB is dispensable for self-interaction in Xcc.
(A-B) GST and GST-fused HpaB derivates were immobilized on MagneGSTTM particles and incubated with cell lysate containing Trx-6×His-HpaB, respectively. Samples were analyzed by immunoblotting with hexahistidine-specific antibody and GST-specific antibody. (C) GST, GST-HpaB, and GST-HpaB1-136 were immobilized on MagneGSTTM particles and incubated with cell lysate containing Trx-6×His, as negative control. (D) GST and GST-fused HpaB derivates were respectively immobilized on MagneGSTTM particles and incubated with cell lysate containing 6×His-HpaB1-144.
Fig 6.
The C-terminal domain of HpaB is dispensable for interaction with T3SS components in Xcc.
GST and GST-fused HpaB derivates were immobilized on MagneGSTTM particles and incubated with cell lysates containing 6×His-HrcN, 6×His-HrcQ, and 6×His-HrcU, respectively. Samples were analyzed using immunoblotting with hexahistidine-specific antibody and GST-specific antibody. The captured 6×His-HrcQ and 6×His-HrcU at weak level were remarked with asterisks.
Fig 7.
HrcN, a T3SS-associated ATPase, is required for virulence and HR of Xcc.
(A) HrcN of Xcc exhibits ATP hydrolysis ability. ATP hydrolysis by 1 μg purified 6×His-HrcN was measured using a malachite green phosphatase assay in different concentrations of ATP. Values are the mean ± standard deviation from three repeats. (B) The lesion lengths of Chinese radish (Raphanus sativus var. radiculus cv. Manshenhong) leaves caused by Xcc strains (wild type strain 8004, hrcN deletion mutant ΔhrcN, and complementation of ΔhrcN CΔhrcN) 10 days post-inoculation. Xcc strains were diluted into 10 ml NYG medium at an OD600 of 0.001 and inoculated on Chinese radish by cutting-leaf method. (C) Xcc strains were inoculated into the leaves of pepper plant (Capsicum annuum cv. ECW-10R). Leaves were photographed at 8 h and 24 h post-inoculation respectively.
Fig 8.
The C-terminal domain of HpaB is involved in ATPase-dependent dissociation of chaperone-effector complex in Xcc.
(A-B) The T3E XC3176 binding to impaired chaperone is not released by purified 6×His-HrcN. Chaperone-effector complexes (GST-HpaB/6×His-XC3176, GST-HpaB1-136/6×His-XC3176, and GST-HpaB111-160/6×His-XC3176) and GST were absorbed on MagneGSTTM particles and incubated without (A) or with (B) 6×His-HrcN 1 h at room temperature in presence of 150 μM ATP, respectively. The released proteins in supernatant (S) and preyed proteins (P) on MagneGSTTM particles were separated by magnet. Samples were analyzed by immunoblotting with hexahistidine-specific antibody and GST-specific antibody. (C-D) The C-terminal domain of HpaB is crucial for liberating T3E AvrAC. (C) Chaperone-effector complexes of (GST-HpaB/6×His-AvrAC and GST-HpaB1-136/6×His-6×His-AvrAC and GST alone were absorbed on MagneGSTTM particles and incubated 1 h at room temperature in presence of 150 μM ATP, respectively. (D) One μg purified 6×His-HrcN was incubated with GST-HpaB/6×His-AvrAC and GST-HpaB1-136/6×His-AvrAC complex immobilized on MagneGSTTM particles, respectively, in presence of ATP. The released 6×His-AvrAC at a weak level was remarked with asterisk.
Fig 9.
A working model for HpaB-dependent effector translocation in Xcc.
In Xcc WT strain, HpaB binds to and promotes the secretion of various effector proteins (E) after the secretion of translocon proteins (T) via the T3SS. HpaB binding and secretion of effectors is facilitated by ATPase, HrcN. In the HpaB C-terminal deletion (right side), the truncated HpaB can bind various effector proteins (E) but appears to be unable to facilitate secretion. This indicates the C-terminal deletion of HpaB may not affect the recognition of chaperone-effector complex but may block secretion of effector proteins by forming a stable complex with these proteins, which blocks their dissociation and subsequent secretion of effectors.