A highly-conserved single-stranded DNA-binding protein in Xanthomonas functions as a harpin-like protein to trigger plant immunity

Abstract

Harpins are produced by gram-negative phytopathogenic bacteria and typically elicit hypersensitive response (HR) in non-host plants. The characterization of harpins in Xanthomonas species is largely unexplored. Here we demonstrate that Xanthomonas produce a highly conserved single-stranded DNA-binding protein (SSB(X)) that elicits HR in tobacco as by harpin Hpa1. SSB(X), like Hpa1, is an acidic, glycine-rich, heat-stable protein that lacks cysteine residues. SSB(X)-triggered HR in tobacco, as by Hpa1, is characterized by the oxidative burst, the expression of HR markers (HIN1, HSR203J), pathogenesis-related genes, and callose deposition. Both SSB(X)- and Hpa1-induced HRs can be inhibited by general metabolism inhibitors actinomycin D, cycloheximide, and lanthanum chloride. Furthermore, those HRs activate the expression of BAK1 and BIK1 genes that are essential for induction of mitogen-activated protein kinase (MAPK) and salicylic acid pathways. Once applied to plants, SSB(X) induces resistance to the fungal pathogen Alternaria alternata and enhances plant growth. When ssb(X)was deleted in X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak in rice, the resulting ssb(Xoc)mutant was reduced in virulence and bacterial growth in planta, but retained its ability to trigger HR in tobacco. Interestingly, ssb(Xoc)contains an imperfect PIP-box (plant-inducible promoter) and the expression of ssb(Xoc)is regulated by HrpX, which belongs to the AraC family of transcriptional activators. Immunoblotting evidence showed that SSB(x) secretion requires a functional type-III secretion system as Hpa1 does. This is the first report demonstrating that Xanthomonas produce a highly-conserved SSB(X) that functions as a harpin-like protein for plant immunity.

Figure 1. Expression of ssb_Xoc is induced in rice suspension cells.

Real-time quantitative PCR analysis of ssb_Xoc transcript levels in X. oryzae pv. oryzicola wild-type RS105 and mutants RΔhrpG and RΔhrpX. Strains were grown in NB or rice suspension cells, and designated as (−) and (+), respectively. The ratios (shown in units of log₂) reflect ssb_Xoc transcript levels between different strains in two different growth conditions. 1. +RS105/−RS105; 2. −RΔhrpG/−RS105; 3. −RΔhrpX/−RS105; 4. +RΔhrpG/+RS105; 5. +RΔhrpX/+RS105. Data represent the means ± standard deviations (SD) from three replicates.

Figure 2. A highly conserved single-stranded DNA-binding protein (SSB) triggers a HR in tobacco.

(A) HR induction by the SSB_Xoc protein of X. oryzae pv. oryzicola. A. tumefaciens GV3101 (OD₆₀₀ = 0.5) containing hpa1, ssb_X and bax genes in the PVX vector pgR107 was inoculated into N. benthamiana tobacco leaves with a needleless syringe. Hpa1 and Bax were used as positive controls, and A. tumefaciens containing the empty vector PVX was used as a negative control. (B) Concentration of SSB_Xoc required for HR induction in tobacco cv. Xanthi. Purified SSB_Xoc was diluted in PBS buffer and inoculated into tobacco with needleless syringes. Hpa1, which functions as a harpin of X. oryzae pv. oryzicola, was used as a positive control and EVP as a negative control. (C) HR assays in tobacco inoculated with SSB_X homologues obtained from various bacterial species. SSB proteins were overproduced in E. coli, purified (see Methods), and diluted in PBS buffer to different concentrations from 0.01 to 50 µM. A typical image of HRs on tobacco leaves caused by the proteins at 1 µM was taken in this report. Numbers represent sections of leaves inoculated with the following: 1, EVP; 2, SSB_Xoc from X. oryzae pv. oryzicola RS105; 3, SSB_Xoo from X. oryzae pv. oryzae PXO99^A; 4, SSB_Xac from X. axonopodis pv. citri 306; 5, SSB_Xcv from X. campestris pv. vesicatoria 85-10; 6, SSB_Xcc from X. campestris pv. campestris 8004; 7, SSB_Pf from P. fluorescens Pf-5; 8, Hpa1_Xoc, from X. oryzae pv. oryzicola RS105; 9, SSB_Ea from E. amylovora 0065; 10, SSB_Ec from E. coli BL21 (DES); 11, SSB_Rs from R. solanacearum ZJ2731; and 12, SSB_Pst from P. syringae pv. tomato DC3000. (D) Assays for SSBx and Hpa1-induced HR in response to various metabolic inhibitors. Tobacco plants were infiltrated with SSB_X (1 µM) or Hpa1 (0.5 µM), which was heat-treated or incubated (see methods) with one of the following: 1 µM LaCl₃, 0.71 µM actinomycin D, 0.1 µM cycloheximide or protease K (0.5 U/ml). Leaf panels: 1, Hpa1; 2, heat-treated Hpa1; 3, protease K-treated Hpa1; 4, Hpa1 plus 1 µM LaCl₃; 5, Hpa1 plus 0.71 µM actinomycin D; 6, Hpa1 plus 0.1 µM cycloheximide; 7, SSB_X; 8, heat-treated SSB_Xoc; 9, protease K-treated SSB_Xoc; 10, SSB_Xoc plus LaCl₃; 11, SSB_Xoc plus 0.1 µM actinomycin D; 12, SSB_Xoc plus cycloheximide; 13, distilled water; and 14, EVP. Leaves in panels A to D were photographed 24–48 h after infiltration. (E) Analysis of DNA laddering in SSB_Xoc-treated tobacco leaves. Total genomic DNA was isolated from tobacco leaves 3, 6, 12, 24, 36 and 48 hpi with Hpa1 (0.5 µM), SSB_Xoc (1 µM) and EVP. DNA laddering was evaluated in 2% agarose gels. (F) Northern blot analysis in tobacco inoculated with SSB_X, Hpa1, or EVP. The marker genes, HIN1, HSR203J, PR1a and PR1b, were chosen as the targets. Total RNAs were extracted from tobacco leaves infiltrated with SSB_Xoc (1 µM), Hpa1 (0.5 µM), or PBS buffer. Aliquots (10 µg each) of the extracted RNAs were separated in 1% agarose gels, transferred onto membranes, and analyzed by northern blotting. Blots were hybridized with digoxigenin-labeled HIN1, HSR203J, PR1a and PR1b cDNA. The experiment was conducted twice with similar results.

Figure 3. SSB_Xoc from X. oryzae pv. oryzicola may function as a PAMP and activates PTI in tobacco.

SSB_Xoc-triggered HR depends on the accumulation of salicylic acid (SA). X. oryzae pv. oryzicola strain RS105 (OD₆₀₀ = 0.5), SSB_Xoc (1.0 and 5.0 µM), Hpa1 (1.0 and 5.0 µM), and EVP were inoculated into a NahG tobacco leaves (A) or wild-type tobacco cv. Xanthi (B). Photographs were taken 48 hpi. (C) SSB_Xoc-triggered HR is accompanied by the oxidative burst. The production of H₂O₂ was evaluated in tobacco leaves by staining with 3,3′-diaminobenzidine tetrahydrochloride (DAB). The reaction mixture contained 200 ml of 0.5 mM DAB in 50 mM Tris acetate buffer (pH 6.0) with purified SSB_Xoc (1 µM) or Hpa1 (0.5 µM). Fully-expanded tobacco leaves were infiltrated with needleless syringe, incubated at room temperature for 0 and 8 h, and decolorized in 80% (v/v) ethanol for 10 min at 70°C. Leaves were examined with an OLYMPUS IX71 microscope. PBS buffer was included as a negative control. (D) SSB_Xoc elicits callose deposition in tobacco cell walls. Callose deposition in tobacco leaves was observed using fluorescence microscopy (OLYMPUS IX71) and staining with aniline blue at 0 and 8 hpi. Purified SSB_Xoc (1 µM) or Hpa1 (0.5 µM) was infiltrated into tobacco leaves (N. benthamiana) using needleless syringes, and EVP was used as a negative control. Inoculated epidermal peels were incubated with aniline blue for 0.5 h. Fluorescence images were captured using a 400 nm exposure for 510 nm absorbed light (UV), and bright-field (BF) images were captured using general bright light. Panels (E) and (F) show Northern blot analysis of PTI signaling pathways in tobacco treated with SSB_Xoc, Hpa1, and PBS buffer (control). (E) The expression of BAK1, BIK1 and MAP3K; (F) The expression of NPR1, EIN2, COl1, and PR4. Purified SSB_Xoc (1 µM) or Hpa1 (0.5 µM) was infiltrated into tobacco leaves using needleless syringe, and PBS buffer was used as a negative control. RNA was extracted 8 hpi and 10 µg aliquots were separated on 1% agarose gels and transferred to nylon membranes. Blots were hybridized with digoxigenin-labeled cDNA probes of the indicated genes.

Figure 4. SSB_Xoc induces resistance to tobacco brown spot disease caused by A. alternata.

Fully-expanded tobacco leaves (cv. Xanthi) were sprayed twice in three-day intervals with purified SSB_Xoc (1 µM), Hpa1 (0.5 µM) and EVP (negative control). Three days after the second application of SSB_Xoc, leaves were inoculated with A. alternata strain TBA28A. Diameters of brown spot lesions were measured and photographed 14 dpi. Lesion size (diameter) are shown ± SD of triplicate measurements. Different letters above columns indicate significant differences at P = 0.01 using the Student’s t test.

Figure 5. SSB_Xoc enhances plant growth.

(A) Phenotype of tobacco (cv. Xanthi) and Arabidopsis thaliana (Col-0) grown on MS medium, 14 days after seed treatment with Hpa1 (0.5 µM), SSB_Xoc (1 µM), EVP, or double distilled water (DDW). Upper panel, tobacco; lower panel, Arabidopsis. (B) Fresh weight and root length of treated plants. Upper panel, fresh weight; lower panel, root length. Data are means ± SD of 50 randomly selected plants. Different letters above columns represent significant differences between treatments (P = 0.01 by t test).

Figure 6. Secretion of SSB_Xoc depends on the T3SS and is required for full virulence and bacterial growth in rice.

(A) Symptoms and (B) lesions lengths were used to assess the virulence of X. oryzae pv. oryzicola RS105 and selected mutants. One half of a rice leaf (IR24, two-months old) was inoculated with wild-type RS105, and the remaining half was inoculated with one of the following deletion mutants: ssb_Xoc deletion mutant RΔssb_X, hpa1 mutant RΔhpa1, the double mutant RΔssb_XΔhpa1, the complemented mutant CRΔssb_X, and the T3SS mutant RΔhrcV. Ten leaves were inoculated with each strain (OD₆₀₀ = 0.3; approximately 3×10⁸ cfu/ml) by leaf-needling, and the assay was conducted in triplicate. Bacterial leaf streak symptoms were photographed 14 dpi, and representative symptoms are shown (A). The average lesion lengths formed by the wild-type and mutants were measured 14 dpi (B), and data represent means ± SD from three replicates. Different letters in each data column indicate significant differences at P = 0.01 (t test). (C) Bacterial growth assays in planta. Strains (OD₆₀₀ = 0.3) were infiltrated into leaves of rice seedlings (IR24, two-weeks old) with blunt-end plastic syringes, and the cfu/cm² of tissue was evaluated as described in Methods. Data represent means ± SD from three replications. (D) and (E) demonstrated the secretion of SSB_Xoc (D) and Hpa1 (E) are dependent on a functional T3SS of X. oryzae pv. oryzicola. This experiment utilized X. oryzae pv. oryzicola RS105 and strains containing mutations in the following genes: hrcV (RΔhrcV), hrcC (RΔhrcC), hrpE (RΔhrpE), hpaB (RΔhpaB), hpaP (RΔhpaP), hpa1 (RΔhpa1) and ssb_Xoc (RΔssb_X) to express ssb_Xoc-c-myc or hpa1-c-myc fusion (as a positive control). After incubation (8 h) in hrp-inducing medium XOM3, total cell extracts (TEs) and culture supernatants (SNs) were analyzed by SDS-PAGE and immunoblotted with an anti-c-Myc antibody. The immunoblotting assay was conducted twice, and similar results were obtained each time. For the detection of SSB_Xoc, the strain RΔssb_X with the empty vector pUFR034 was used as a negative control (D).

Figure 7. ssb_X is expressed in hrp-inducing conditions and regulated by HrpG and HrpX.

(A) Schematic map of a transcriptional fusion where the ssb_Xoc promoter of X. oryzae pv. oryzicola RS105 is fused to the gusA reporter gene. Upper panel shows pPIPAGUS containing the ssb_Xoc promoter and an imperfect PIP-box (TTCGC-N₁₉-TTCGT) fused with a promoter-less gusA gene. Lower panel shows pPIPBGUS with a mutated ssb_Xoc promoter (the first TT nucleotides replaced with AA) fused with gusA. (B) â-glucuronidase (GUS) activity in the hrp-inducing medium XOM3. Plasmids pPIPAGUS and pPIPBGUS were transferred into the wild-type RS105 and mutants RΔhrpG and RΔhrpX. The recombinant strains were then grown in hrp-inducing medium XOM3 for 16 h. GUS activity was determined by measuring the OD at 415 nm using ρ-nitrophenyl-â-D-glucuronide as a substrate. Data represent the mean ± SD of triplicate measurements. The different letters above each horizontal column indicate significant differences at P = 0.01 (t test). (C) Expression of ssb_Xoc in hrp-inducing and nutrient-rich media. Real-time quantitative RT-PCR was used to compare relative expression of ssb_Xoc in X. oryzae pv. oryzicola strains RS105, RΔhrpG, and RΔhrpX. RNA was isolated from strains grown in a nutrient-rich medium (NB) and the hrp-inducing medium (XOM3) for 16 h. The relative mRNA levels of ssb_Xoc in the hrpG and hrpX mutants were calculated with respect to the wild-type strain. Values given are the means ± SD of triplicate measurements from a representative experiment, and similar results were obtained in two other experiments. Different letters above horizontal columns represent significant differences at P = 0.01 using the Student’s t test. (D) Real-time RT-PCR evaluation of ssb_X expression in Xanthomonas species. Strains were grown at 28°C for 16 h in NB or one of the following hrp-inducing media: XOM3 for X. oryzae pv. oryzicola RS105 and X. oryzae pv. oryzae PXO99^A (Xiao et al. 2007), XVM2 for X. axonopodis pv. citri 306 & X. campestris pv. vesicatoria 85-10, and MMX for X. campestris pv. campestris 8004 (see methods). Relative mRNA quantitative of ssb_X was calculated with respect to the levels observed for wild-type strains grown in NB. Genes encoding 16S rRNA were used as internal controls. Data represent means ± SD of triplicate measurements (P = 0.01, t test).

Figure 8. Working model of SSB_X function.

SSB_Xoc, like Hpa1, which are regulated at the transcriptional level by HrpG and HrpX, are potentially secreted into the plant apoplast via the T3SS. The grey circle and black triangle indicate SSB_X and Hpa1 proteins that may be secreted through the Hrp pilus (encoded by hrpE), but not translocated through the translocon as other T3SEs (different white shapes), and then are possibly recognized by an unidentified receptor (question mark) which associates with BAK1. This interaction may result in phosphorylation of BIK1 and subsequent phosphotransfer to the MAPK cascade to activate the expression of genes involving in SA-, JA- and Eth-signaling pathways that lead to induced resistance (SAR and/or ISR) accompanied by callose deposition on cell walls and enhanced plant growth. MAPK signaling regulates NADPH oxidase-dependent oxidative burst in the early stages of plant defense.