Abstract
A biofilm lifestyle is critical for bacterial pathogens to colonize and protect themselves from host immunity and antimicrobial chemicals in plants and animals. The formation and regulation mechanisms of phytobacterial biofilm are still obscure. Here we found that the protein Ralstonia solanacearum resistance to ultraviolet C (RuvC) is highly abundant in biofilm and positively regulates pathogenicity by controlling systemic movement in tomato xylem. RuvC protein accumulates at the later stage of biofilm development and specifically targets Holliday junction (HJ)-like structures to disrupt the biofilm extracellular DNA (eDNA) lattice, thus facilitating biofilm dispersal. Recombinant RuvC protein can resolve extracellular HJ to prevent bacterial biofilm formation. Heterologous expression of R. solanacearum or Xanthomonas oryzae pv. oryzae RuvC with plant secretion signal in tomato or rice confers resistance to bacterial wilt or bacterial blight disease, respectively. Plant chloroplast-localized HJ resolvase monokaryotic chloroplast 1 (MOC1), which shares structural similarity with bacterial RuvC, shows a strong inhibitory effect on bacterial biofilm formation. Relocalization of SlMOC1 to apoplast in tomato roots leads to increased resistance to bacterial wilt. Our novel finding reveals a critical pathogenesis mechanism of R. solanacearum and provides an efficient biotechnology strategy to improve plant resistance to bacterial vascular disease.
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Data availability
Sequence data in this article can be found in the R. solanacearum database, NCBI website and Sol Genomics Network website under the following accession numbers: RuvC (RSc0503), GlyD (RSc3206), GspD (RSc3114), HrcC (RSp0874), TraG (RSc2586), GyrB (RSc3440), SlACTIN2 (Solyc11g005330), SlPR1b (Solyc00g174340), SlPR1a (Solyc00g174330), SlWRKY3 (Solyc02g088340), XooRuvC (PXO_01556), XooRuvX (PXO_02001), SlMOC1 (Solyc01g008700), OsMOC1 (Os01g16340). The raw data for RNA-seq are deposited in the Gene Expression Omnibus (GSE270737) at the National Center for Biotechnology Information. Source data are provided with this paper.
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Acknowledgements
We thank P. Yin, J. Yan and G. Li for sharing the plasmids and bacterial strains; F. Li for the assistance in tobacco transformation; the Center for Protein Research Huazhong Agricultural University for technical support; J. Zhou and P. He for discussion and suggestions on the manuscript. This research was supported by the National Key Research and Development Program of China (Grant No. 2022YFA1304400 to D.J.), the National Natural Science Foundation of China (Grant No. 32272556 to B.L.), the Fundamental Research Funds for the Central Universities (Grant No. 2662023PY006 and 2662023ZKPY001 to B.L.) and Funds from the National Key Laboratory of Agricultural Microbiology (Grant No. AML2023C01 to B.L.).
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B.L. and D.J. conceived the research. X.D., X.Y. and B.L. designed the experiments. X.D. performed the biofilm-related assays, disease infection, bacterial mutants generation, transgenic tomato and in vitro protein purification. C.F. generated tobacco transgenic plants. P.L. and Y.L. helped with bacterial wilt disease assay and protein expression in E. coli. J.T. performed rice leaf blight disease assay. J.X., Y.F., J.C., M.Y. and D.J. analysed data and provided critical feedback. X.Y., K.T. and B.L. wrote the paper with input from all authors.
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Extended data
Extended Data Fig. 1 Holliday junction resolvase RuvC was identified in the R. solanacearum biofilm proteomics.
a, Peptide coverage profile on RuvC protein sequence from LC-MS/MS analysis. Different colors highlight the peptides identified from the proteomic data. R. solanacearum GMI1000 was grown in cell culture plates for 48 h at 28°C and the biofilm was collected in PBS buffer. The suspension was sonicated and filtrated through a bacterial filter followed by biofilm protein extraction for LC-MS/MS analysis. b, The spectrum of four peptides that match RuvC by LC-MS/MS. The spectrum was generated using pLabel software.
Extended Data Fig. 2 Verification and transcriptomic characterization of ΔruvC mutants.
a, Verification of the ΔruvC knockout mutant. Determination of the genotype by PCR using three pairs of primers that amplify the full-length RuvC gene, the RuvC upstream fragment with spectinomycin (Spec) gene, and the partial fragment of fliC gene which encodes R. solanacearum flagellin, respectively. b, Detection of RuvC-HA expression level in three RuvC complementary strains (ComRuvC) by western blotting. Total proteins were analyzed by an α-HA antibody and the loading was shown by Coomassie brilliant blue (CBB) staining. c, Heatmap showing the correlation between each sample calculated by Pearson correlation coefficient. d, Scatter plots of whole-genome transcript fragments per kilobase of transcript per million mapped reads in GMI1000 versus ΔruvC knockout mutant. The x-axis indicates gene expression in GMI1000 and the y-axis indicates gene expression in ΔruvC knockout mutant.
Extended Data Fig. 3 RuvC contributes to R. solanacearum vertical spread in the xylem.
a, Detection of green fluorescent protein (GFP) expression in transgenic GMI1000, ΔruvC, and ComRuvC carrying psbA:GFP by western blotting. The plasmid pBBR1-psbA:GFP was transformed into different strain backgrounds by electroporation. b, Quantitative data of GFP fluorescent intensity in the xylem at 9 dpi based on the staining in the Fig. 2a. Data are presented as mean values ± SD (n=3 individual plants). The uninfected plants were the blank control group. c, RuvC is not involved in R. solanacearum colonization in roots. Eight days after the soil-drenching inoculation, the bacterial growth in roots of tomato susceptible variety Moneymaker was determined by serial dilution. Data are presented as mean values ± SD (n=7 individual plants). Different letters represent significant differences (P < 0.05) according to one-way ANOVA analysis. d, The distribution and movement of WT, ΔruvC, and ComRuvC post petiole inoculation. Four-week-old Moneymaker was inoculated with indicated GFP-labeled strains. Stem cross-sections were taken at the inoculation site (IS), 2 cm above (A-2), 4 cm above (A-4), 2 cm below (B-2), and 4 cm below (B-4) the inoculation site. e, Quantitative data of GFP fluorescent intensity in the xylem at 6 dpi based on the staining in d. Data are presented as mean values ± SD (n=3 individual plants). The uninfected plants were the blank control group. Statistically significant differences in b and e are indicated by different lowercase letters (Two-way ANOVA, P < 0.05). The above experiments were repeated three times with similar results.
Extended Data Fig. 4 Secretion, enzymatic activity, and protein structure of RuvC.
a, Verification of α-RuvC antibody specificity. Total proteins from the bacterial culture of GMI1000, ΔruvC, and ComRuvC strains were subjected to WB with α-RuvC antibody. b, The secretion of RuvC is partially dependent on the type II and IV secretion system. The bacterial culture (Total), supernatant preparations (SPs) and cell pellets (CPs) were subjected to western blotting. β-tubulin was detected as a control. c, RuvC exists in extracellular membrane vesicles (MVs). The extracellular membrane vesicles were precipitated by ultracentrifuge and analyzed. d, Schematic diagram of the common Holliday junction substrate HJ98. e, His-RuvC fusion proteins were purified from E. coli and separated by SDS-PAGE. Protein expression was induced by IPTG and purified by affinity chromatography with Ni-NTA agarose beads. f, The cleavage activity of RuvC towards cruciform DNA HJ98 was positively correlated with the concentration. His-RuvC protein with increasing concentrations was applied to the reaction. The untreated samples were the blank control group. The band intensity was quantified by ImageJ and the percentage of cleaved substrate relative to the input DNA is shown at the bottom. g, The enzymatic efficiency of RuvC to cruciform DNA HJ98. His-RuvC protein (200 ng/μL) was incubated with HJ98 DNA (100 nmol/μL) in a 20 μL reaction mix. h, Topological diagram of the RuvC secondary structure. Predicted regions required for the enzyme activity are shaded in gray. i, Two perpendicular views of the overall 3D structure of RuvC and its mutation variant. Three conserved acidic residues in the enzymatic region were mutated to alanine (A) as shown on the right. j, RuvC does not affect the cell viability of R. solanacearum. Data are presented as mean values ± SD (n=3 biological replicates). Different letters represent significant differences according to one-way ANOVA analysis with two-sided test. The above experiments were repeated three times with similar results.
Extended Data Fig. 5 The enzymatic activity is required for RuvC function in regulating the virulence of R. solanacearum.
a, Detection of RuvC expression level in three RuvCD7A complementary strains (ComRuvCD7A) by western blotting. Total proteins were analyzed by an α-HA antibody and the loading was shown by Coomassie brilliant blue (CBB) staining. Wild-type strain GMI1000 and ComRuvC were used as controls, respectively. b, Disease symptoms of tomato susceptible variety Moneymaker upon ComRuvCD7A infection by soil-drenching inoculation. The images were taken at 12 dpi. c, Disease index of tomato plans inoculated with R. solanacearum strains. Data are presented as mean values ± SEM (n=12 individual plants). d, Percentage of surviving plants to total plants after inoculation. Statistical analysis was performed using a Log-rank (Mantel-Cox) test. The above experiments were repeated three times with similar results.
Extended Data Fig. 6 Transient expression of secreted RuvC in tomato roots enhances the resistance to bacterial wilt.
a, Expression of secreted RsRuvCPR1-HA protein in Moneymaker roots. RuvC protein fused with the signal peptide of SlPR1 driven by 35S promoter was transformed to Moneymaker roots by Agrobacterium-mediated hairy root transformation (OxRsRuvCPR1). Total proteins were analyzed by western blotting with an α-HA antibody. Plants transformed with an empty binary vector were used as controls. b, Disease phenotype of the GMI1000-infected tomato plants. Photos were taken at 7 dpi. c, Disease index of OxRsRuvCPR1 and the control plants post GMI1000 inoculation. The results are represented by the average according to the disease index scale. Data are presented as mean values ± SEM (n=7 individual plants). The plants expressing the empty vector (EV) were the control group. d, The survival percentage was analyzed by comparing the number of survival plants to total plants from the data in c. Statistical analysis was performed using a Log-rank (Mantel-Cox) test. The above experiments were repeated three times with similar results.
Extended Data Fig. 7 Transgenic tomato expressing secreted RuvC exhibit enhanced resistance to bacterial wilt.
a, Expression of RsRuvCPR1-HA in Moneymaker transgenic lines. Stable transgenic plants carrying 35S::RsRuvCPR1-HA (OxRsRuvCPR1) were generated and total protein extracts from the transformed tomato roots were analyzed by western blotting with an α-HA antibody. Wild type Moneymaker was used as a control. b, Growth phenotype of OxRsRuvCPR1 transgenic tomato plants. Eight-week-old plants were photographed. Scale bars indicate 30 mm. c, Ten-week-old seedling height of OxRsRuvCPR1 lines and WT (n=15 individual plants). d, Mature fruit weight of OxRsRuvCPR1 lines and WT in the experimental field (n=28 individual fruits). e, The movement of GFP-labeled GMI1000 in OxRuvCPR1 and Moneymaker plants. Four-week-old plants were inoculated with GFP-labeled GMI1000 by petiole inoculation. Stem cross-sections were taken at the inoculation site (IS), 2 cm above (A-2), 4 cm above (A-4), 2 cm below (B-2), and 4 cm below (B-4) the inoculation site. f, Quantitative data of GFP signal intensity in the xylem at 6 dpi based on the images of (e). Data are presented as mean values ± SD (n=3 individual plants). Statistically significant differences are indicated by different lowercase letters (Two-way ANOVA, P < 0.05). g, The expression of defense genes was not induced in OxRsRuvCPR1 plant. Data were normalized to the expression of SlACTIN2 in RT-qPCR analysis. Data are presented as mean values ± SD (n=3 technical repeats from one independent experiment). The wild type plant Moneymaker was used as the control group. The results in c and d are represented by a box plot of the maximum to minimum values of the original data points. Centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to 1.5× the interquartile range from the 25th and 75th percentiles. Different letters in c, d and g indicate significant difference by one-way ANOVA (P < 0.05). The above experiments were repeated three times with similar results.
Extended Data Fig. 8 Ectopic expression of the secreted RsRuvC in tobacco.
a, Verification of the RsRuvCPR1 expression in Nicotiana tabacum transgenic lines. Total protein extracts from transgenic lines were analyzed by western blotting with an α-HA antibody. Wild-type N. tabacum SR1 was used as a control. b, Growth phenotype of OxRsRuvCPR1 transgenic tobacco plants. four-week-old plants were photographed. Scale bars indicate 3 cm. The wild type tobacco SR1 was used as the control group. The above experiments were repeated three times with similar results.
Extended Data Fig. 9 The RuvC sequence is conserved in several pathogenic bacteria and expression of the XooRuvCPR1 in rice.
a, Sequence alignment of RuvCs in different bacteria. The aspartic acid active site was marked by carmine, and the glutamic acid site was highlighted by dark blue. Pa, Pseudomonas aeruginosa; Pst, Pseudomonas syringae pv. tomato str. DC3000; Ec, Escherichia coli; Rs, Ralstonia solanacearum; Xoo, Xanthomonas oryzae pv. Oryzicola; Bs, Bacillus subtilis. b, Purified His-XooRuvC and His-XooRuvX fusion proteins from E. coli. Expression was induced by IPTG and purified by affinity chromatography. c, XooRuvX treatment did not affect Xoo biofilm. Biofilms were stained with crystal purple and imaged in the PCR tubes. Data are presented as mean values ± SD (n=6 biological replicates). Statistically significant differences in c are indicated by different lowercase letters (One-way ANOVA, P < 0.05). d, Verification of the XooRuvC expression in rice transgenic lines. Total RNA was extracted from the transgenic rice leaves. The XooRuvC gene expression level detected by RT-PCR and ACTIN1 used as an internal control. Band intensity was quantified by Image J. Wild-type Nipponbare was used as a control. e, Gross morphologies of OxXooRuvCPR1 lines and WT in the experimental greenhouse. Scale bars indicate 30 cm. f, Images of seeds harvested from the experimental field. Scale bars indicate 10 mm. g, Hundred-grain weight of seeds. Data are presented as mean values ± SD (n=10 individual plants). The wild type plant Nipponbare was used as the control group. Statistically significant differences are indicated by different lowercase letters (One-way ANOVA, P < 0.05). The above experiments were repeated three times with similar results.
Extended Data Fig. 10 Plant Holliday junction resolvase also can inhibit the biofilms of pathogenic bacteria.
a, The full-length amino acid sequences of Holliday junction resolvases from several pathogenic bacteria and plants were used to generate the phylogenetic tree by the Neighbor-Joining method. The MOC1 homologous proteins in different crops are highlighted in green and the RuvC homologous proteins in a variety of pathogenic bacteria are highlighted in blue. ComEA, a membrane-bound high-affinity DNA-binding receptor in Bacillus atrophaeus, was selected as an outgroup. The above experiments were repeated three times with similar results. b, Schematic ___domain structure of MOC1. Sl, Solanum lycopersicum; Os, Oryza sativa. The signal peptide is predicted by InterPro or SignalP-5.0. c, Purified His-SlMOC1 and His-OsMOC1 fusion proteins from E. coli were separated by SDS-PAGE. Expression was induced by IPTG and purified by affinity chromatography with Ni-NTA agarose beads. The chloroplast transit peptide was removed during protein expression. d and e The general biofilm-inhibitory function of plant MOC1 proteins towards pathogenic bacteria. The purified His-SlMOC1 inhibits the biofilm formed by PXO99A, and His-OsMOC1 disrupts the biofilm generated by GMI1000. Biofilms were stained with crystal purple and imaged in the PCR tubes. Data are presented as mean values ± SD (n=6 biological replicates). Statistically significant differences in d and e are indicated by different lowercase letters (One-way ANOVA, P < 0.05). The above experiments were repeated three times with similar results.
Supplementary information
Supplementary Table 1
List of candidate proteins in biofilm identified by mass spectrometry.
Supplementary Table 2
Primers used in this study.
Supplementary Table 3
Summary of statistical analysis.
Source data
Source Data Figs. 1–5 and Source Data Extended Data Figs 2–10
Fig. 1b,c,e,f,g statistical source data. Fig. 2b,d statistical source data. Fig, 3a,b,d,e,h–j,l,n statistical source data. Fig. 4a,c,d,k,m unprocessed western blots and native page gels. Fig. 4e,f,i,j,l statistical source data. Fig. 5b–d,f–h,k–m,o,p statistical source data. Extended Data Fig. 2a,b unprocessed gels and western blots. Extended Data Fig. 3a unprocessed gels and western blots. Extended Data Fig. 3b,c,e statistical source data. Extended Data Fig. 4 a–c,e–g unprocessed gels and western blots. Extended Data Fig. 4j statistical source data. Extended Data Fig. 5a unprocessed western blots. Extended Data Fig. 5c,d statistical source data. Extended Data Fig. 6a unprocessed western blots. Extended Data Fig. 6c,d statistical source data. Extended Data Fig. 7a unprocessed western blots. Extended Data Fig. 7c,d,f,g statistical source data. Extended Data Fig. 8a unprocessed western blots. Extended Data Fig. 9b,d unprocessed gels. Extended Data Fig. 9c,g statistical source data. Extended Data Fig. 10c unprocessed gels. Extended Data Fig. 10d,e statistical source data.
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Du, X., Li, P., Fan, C. et al. Holliday junction resolvase RuvC targets biofilm eDNA and confers plant resistance to vascular pathogens. Nat. Plants 10, 1710–1723 (2024). https://doi.org/10.1038/s41477-024-01817-6
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DOI: https://doi.org/10.1038/s41477-024-01817-6
