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. 2017 Aug;18(6):768-782.
doi: 10.1111/mpp.12435. Epub 2016 Aug 21.

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Lijuan Liu  1 , Yanping Wang  1 , Fuhao Cui  1 , Anfei Fang  1 , Shanzhi Wang  1 , Jiyang Wang  1 , Chao Wei  1 , Shuai Li  1 , Wenxian Sun  1
Affiliations

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Lijuan Liu et al. Mol Plant Pathol. 2017 Aug.

Abstract

Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system-dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N-myristoylation motif is essential for its localization. Chemical-induced expression of AvrXccB suppresses flg22-triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S-adenosyl-l-methionine-dependent methyltransferases SAM-MT1 and SAM-MT2. Interestingly, SAM-MT1 is not only self-associated, but also associated with SAM-MT2 in vivo. SAM-MT1 and SAM-MT2 expression is significantly induced upon stimulation of microbe-associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.

Keywords: AvrXccB; S-adenosyl-l-methionine-dependent methyltransferase; Xanthomonas campestris pv. campestris; plant immunity; type III effector.

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Figures

Figure 1
Figure 1
AvrXccB is secreted in a type III secretion system‐dependent manner in Xanthomonas campestris pv. campestris (Xcc). Western blotting using anti‐haemagglutinin (HA) antibody showed that AvrXccB‐HA was present in hrp (hypersensitive response and pathogenicity)‐inducing culture medium of the Xcc wild‐type (WT) strain B186 transformed with the avrXccB‐HA construct, but not in that of the B186ΔhrcC mutant carrying avrXccB‐HA. AvrXccB‐HA was detected in cell lysates of the avrXccB‐HA‐transformed wild‐type and ΔhrcC strains. No non‐specific cross‐reaction was detected in the EV‐transformed wild‐type and ΔhrcC strains. EV, empty vector; WT, Xcc B186; ΔhrcC, B186ΔhrcC; α‐HA, anti‐HA antibody. Lower panel shows the loading control stained with Coomassie brilliant blue (CBB).
Figure 2
Figure 2
The subcellular localization of AvrXccB to the plasma membrane in transgenic Arabidopsis plants depends on its putative N‐myristoylation motif. (A) The putative N‐myristoylation motif at the N‐terminus of AvrXccB, as revealed by sequence alignment of multiple N‐myristoylated proteins, including AvrXccE1 (AAM40923.1), XopE1 (CAJ21925.1), XopE2 (CAJ23957.1), XopJ (CAJ23833.1) and HopZ1a (ABK13740.1) in Xanthomonas and Pseudomonas spp. The protein sequences were all downloaded from the National Center for Biotechnology Information (NCBI) website. The second residue Gly (G), marked by an asterisk, is highly conserved and is required for protein myristoylation. (B, C) Subcellular localization of AvrXccB‐GFP. (D, E) Subcellular localization of AvrXccBG2A‐GFP with a point mutation in the second residue Gly in transgenic Arabidopsis plants. The left panels show green fluorescence, the central panels show red fluorescence after staining with the membrane‐binding lipophilic dye FM4‐64, and the right panels depict the overlay images of the two fluorescence signals. The yellow colour in overlay images indicates overlap between green and red fluorescence. Broken squares in (B, D) were enlarged to show details in (C, E). (F–I) The plasmolysed epidermal cells expressing AvrXccB‐GFP (F, G) or AvrXccBG2A‐GFP (H, I). Broken squares in (F, H) were enlarged to show details in (G, I). Asterisks indicate areas between the cell wall and plasma membrane in plasmolysed cells; Hechtian strands indicated by arrows are noticeable in (F, G), but not in (H, I). Scale bar, 10 µm. GFP, green fluorescent protein. Green and red fluorescence were observed using confocal laser scanning microscopy.
Figure 3
Figure 3
Heterologous expression of AvrXccB suppresses microbe‐associated molecular pattern‐triggered immunity in Arabidopsis. (A) flg22‐induced expression of luciferase driven under the NHO1 promoter was dramatically inhibited by transiently expressed AvrXccB. The inhibition was attenuated in AvrXccBC239A‐transfected protoplasts and was completely lost in AvrXccBG2A‐ and AvrXccBH182A‐transfected protoplasts. Protoplasts isolated from NHO1::LUC transgenic Arabidopsis plants were transfected either with the empty vector (EV) or the indicated effector constructs, and the relative luciferase activity was measured at 12 h after flg22 treatment. EV+ represents EV‐transfected protoplasts treated with 1 μm flg22 and EV– indicates EV‐transfected protoplasts with mock treatment, which was used as a control for basal NHO1‐LUC expression. Each data point [mean ± standard error (SE)] consists of five replicates. Asterisks indicate statistically significant difference between EV+ and each indicated construct (Mann–Whitney test, P < 0.05). (B) Ectopic expression of FLAG‐tagged AvrXccB and its variants AvrXccBG2A, AvrXccBH182A and AvrXccBC239A in transgenic Arabidopsis seedlings at 24 h after dexamethasone (DEX) treatment, as detected by Western blot analyses. Top panel, the blot was probed with an anti‐FLAG antibody; bottom panel, the same blot was stained with Ponceau S to show protein loading. α‐FLAG, anti‐FLAG antibody. (C) flg22‐induced callose deposition in the wild‐type and the different transgenic Arabidopsis lines depicted in (B). Callose deposits per leaf were quantified after the seedlings had been incubated in 10 µm DEX or mock solution for 24 h, followed by 1 µm flg22 or water for another 24 h. Data are means ± SE. *Significant difference in flg22‐induced callose deposits in avrXccB transgenic plants with or without DEX treatment (Student's t‐test, P < 0.05). (D) flg22‐induced reactive oxygen species (ROS) production in wild‐type plants and the different transgenic Arabidopsis lines depicted in (B). ROS generation in the leaves sampled from the wild‐type and different transgenic plants with or without DEX treatment was induced by 1 µm flg22. The area under the curve for a 40‐min oxidative burst (Fig. S5, see Supporting Information) was calculated for each sample and then normalized to the mean value for the wild‐type plants with mock treatment within the same experiment. Data are means ± SE; n = 8 independent T1 lines for each construct. *Significant difference in flg22‐induced ROS generation in avrXccB transgenic plants with or without DEX treatment (Student's t‐test, P < 0.05).
Figure 4
Figure 4
Ectopic expression of AvrXccB in Arabidopsis promotes bacterial infection. (A) Growth of Xanthomonas campestris pv. campestris (Xcc) B186 in Col‐0 and the different transgenic plants depicted in Fig. 3B with or without dexamethasone (DEX) treatment. Leaves were pressure inoculated with Xcc B186 at an optical density at 600 nm (OD600) of 0.0005. (B) Growth of Pseudomonas syringae pv. tomato (Pst) DC3000 in Col‐0 and transgenic plants. Leaves were pressure inoculated with Pst DC3000 at OD600 = 0.0002. In planta bacterial populations were assessed at 0 and 3 days post‐inoculation. cfu, colony‐forming units. Data are means ± standard error. Different letters a–c indicate statistically significant difference (Duncan's multiple range test, P < 0.05).
Figure 5
Figure 5
AvrXccB physically interacts with an S‐adenosyl‐l‐methionine‐dependent methyltransferase (SAM‐MT) complex in Arabidopsis. (A) AvrXccB interacts with Arabidopsis SAM‐MT1‐NoTM as indicated by yeast two‐hybrid assay. The pGBKT7‐avrXccB and pGADT7‐SAM‐MT1‐NoTM constructs were co‐transformed into yeast Gold strain. The growth of yeast colonies on the plates with quadruple dropout medium indicates a positive interaction. (B) In vitro pull‐down assay to detect the interaction of AvrXccB and SAM‐MT1. Glutathione S‐transferase (GST) pull‐down was conducted with GST‐binding resins after purified GST‐AvrXccB or GST had been mutually incubated with His‐SAM‐MT1‐NoTM or His‐SAM‐MT2‐NoTM. The input proteins and precipitates were subjected to immunoblotting with anti‐His and anti‐GST antibodies. α‐GST, anti‐GST antibody; α‐His, anti‐histidine antibody. (C) Co‐immunoprecipitation (Co‐IP) assays to show AvrXccB interaction with SAM‐MT1 and SAM‐MT2 in vivo. SAM‐MT1‐HA and SAM‐MT2‐HA were transiently expressed alone or together with AvrXccB‐FLAG in Arabidopsis protoplasts. (D) Co‐IP assays to show SAM‐MT1 self‐association and interaction with SAM‐MT2 in vivo. SAM‐MT1‐HA and SAM‐MT2‐HA were transiently expressed alone or together with SAM‐MT1‐FLAG in Arabidopsis protoplasts. Co‐IP was performed with anti‐FLAG M2 agarose beads and the immunoprecipitated complex was analysed with an anti‐HA antibody. α‐FLAG, anti‐FLAG antibody; α‐HA, anti‐haemagglutinin antibody; IP, immunoprecipitation; WB, Western blot.
Figure 6
Figure 6
SAM‐MT1 and SAM‐MT2 are transcriptionally induced after the treatment of microbe‐associated molecular patterns and pathogen challenge in Arabidopsis. (A, B) Expression patterns of SAM‐MT1 in Arabidopsis seedlings at the indicated time points after treatment with flg22 (A) or elf18 (B). (C, D) Expression patterns of SAM‐MT1 (C) and SAM‐MT2 (D) at the indicated time points after Pseudomonas syringae pv. tomato (Pst) DC3000 inoculation. Arabidopsis leaves were spray inoculated with Pst DC3000 at an optical density at 600 nm (OD600) of 0.2. SAM‐MT, S‐adenosyl‐l‐methionine‐dependent methyltransferase.
Figure 7
Figure 7
The S‐adenosyl‐l‐methionine‐dependent methyltransferase SAM‐MT1 mainly localizes to punctate structures on the plasma membrane. (A, B) Subcellular localization of SAM‐MT1‐GFP (A) and green fluorescent protein (GFP) (B) in Nicotiana benthamiana cells. SAM‐MT1‐GFP and GFP were transiently expressed in N. benthamiana leaves. Scale bar, 10 µm. (C) SAM‐MT1‐GFP was partially co‐localized with DsRed‐Talin, a membrane‐localized protein, in N. benthamiana leaves. Bottom panels, broken squares in the top panels were enlarged to show detail. Scale bar, 10 µm. The images were photographed at 48 h after agroinfiltration.
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