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Functional replacement of ancestral antibacterial secretion system in a bacterial plant pathogen

Nature Ecology & Evolution volume 9, pages 1393–1404 (2025)Cite this article

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Abstract

Two distinct molecular machineries, T4SS and T6SS, have been found within the order Xanthomonadales to be involved in outcompeting other bacterial species through the secretion of toxic effector proteins. However, the ecological and evolutionary basis leading xanthomonads to evolve two secretion systems with such similar functions remain unclear. Here we show that Xanthomonas translucens (Xt) lineages have switched from an X-T4SS-mediated to T6SS-i4-mediated bacterial killing strategy. T6SS-i4 was only found in Xt strains lacking X-T4SS and vice versa, resulting in a patchy distribution of the two nanoweapons along the Xt phylogeny. Using genetic and fluorescence-based methods, we demonstrated that X-T4SS and T6SS-i4 are crucial for interbacterial competition in Xt, but not Xt T6SS-i3. Combined comparative genetic and phylogenetic analyses further revealed that the X-T4SS gene clusters have been subject to degradation and had several loss events, while T6SS-i4 was inserted through independent gain events. Overall, this research supports the ancestral state of X-T4SS and provides new insights into the mechanisms promoting Xt survival within their ecological niches.

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Fig. 1: X-T4SS and T6SS-i4 show non-overlapping distribution patterns within Xt strains.
Fig. 2: Xt interbacterial competition relies on T6SS-i4, but not T6SS-i3.
Fig. 3: Xt X-T4SS is crucial for interbacterial competition.
Fig. 4: X-T4SS gene phylogeny is congruent with the species tree.
Fig. 5: T6SS-i4 gene phylogeny is not congruent with the species tree.
Fig. 6: Evolutionary scenarios explaining X-T4SS loss, T6SS-i4 gain and resulting mutual exclusion profiles in xanthomonads.

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Data availability

All data supporting the findings of this study are available in the paper and Supplementary Information and as source data files via Zenodo at https://doi.org/10.5281/zenodo.15367606 (ref. 69). Further information and requests for resources and reagents should be directed to C.B. (claude.bragard@uclouvain.be). Bacterial strains and plasmids used in this study can be made available on request following the signing of a material transfer agreement.

Code availability

Code for the data statistical analyses is available via Zenodo at https://doi.org/10.5281/zenodo.15367606 (ref. 69).

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Acknowledgements

We thank The Ohio Supercomputer Center for providing high-performance computing resources and microscope and imaging specialist D. Ignacio from Hunt Optics and Imaging Inc. Olympus for his valuable help in designing and optimizing laser scanning confocal microscope parameters for time-lapse videos. We would like to thank J. Mahillon (UCLouvain, Louvain-la-Neuve, Belgium) for his precious advice and for enriching intellectual discussions and L. M. Rodriguez-R (University of Innsbruck, Austria) for fruitful discussions. C.P. was supported by the Foundation for Training in Industrial and Agricultural Research (FRIA, FNRS) (grant no. 40021596). Part of this article is based on work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology) (C.P., R.K. and C.B.). We are grateful for support from a Fulbright Scholar Award to Uruguay to J.M.J.

Author information

Authors and Affiliations

  1. Applied Microbiology-Plant health, Earth & Life Institute, UCLouvain, Louvain-la-Neuve, Belgium

    Chloé Peduzzi, François Nikis & Claude Bragard

  2. Department of Plant Pathology, The Ohio State University, Columbus, OH, USA

    Jules Butchacas, Marcus V. Merfa & Jonathan M. Jacobs

  3. Infectious Diseases Institute, The Ohio State University, Columbus, OH, USA

    Jules Butchacas, Marcus V. Merfa & Jonathan M. Jacobs

  4. Levo International, Bloomfield, CT, USA

    Nathaniel Heiden

  5. Louvain Institute of Biomolecular Science and Technology (LIBST), UCLouvain, Louvain-la-Neuve, Belgium

    Manon Martin

  6. Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park, PA, USA

    Veronica Roman-Reyna

  7. PHIM Plant Health Institute, University of Montpellier, IRD, Institut Agro, Montpellier, France

    Ralf Koebnik

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Contributions

C.P., C.B., R.K. and J.M.J. designed the study and C.B., R.K. and J.M.J. supervised the conducted research. C.P., J.B. and F.N. performed the experiments. C.P. performed bioinformatic analyses. C.P. and M.M. performed statistical analyses. J.B., N.H. and M.V.M. assisted with the experiments and V.R.-R. and R.K. assisted with the bioinformatic analyses. C.P. wrote the manuscript and all authors discussed the results and edited the manuscript.

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Correspondence to Claude Bragard.

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Extended data

Extended Data Fig. 1 Xt (a) T6SS-i4 and (b) X-T4SS are required for inter-bacterial competition.

Representative NA plates with tenfold dilution series of the quantitative assay showing E. coli survival using Amersham Typhoon laser scanner.

Extended Data Fig. 2 Xt growth is not impacted by (a) T6SS and (b) X-T4SS mutations during bacterial competition assays against E. coli.

Quantitative competition assay with E. coli mixed in a 10:1 ratio (Xt:E. coli) for 10 h of co-cultivation. Boxplot represents Xt growth log CFU/ml after 0 h and 10 h of co-cultivation. All boxplots show 1st and 3rd quartiles around the median. Whiskers represent the smallest and largest value no further than 1.5 * IQR (inter-quartile range) from the hinge. Dots indicate independent biological replicates (n = 2 at 0 h; n = 6 at 10 h). Significance was tested by two-sided Student’s T-test. Significance codes: *** (P < 0.001), ** (P < 0.01), * (P < 0.05), only significant comparisons are shown (ΔT6SS-i3: P = 1, ΔT6SS-i4: P = 1, ΔT6SS-i3 ΔT6SS-i4: P = 1, Δhcpi3: P = 1, Δhcpi4: P = 1, Δhcpi3 Δhcpi4: P = 1, and Δhcpi4::hcpi4: P = 1; ΔvirD4: P = 1, and ΔvirD4::virD4: P = 1).

Extended Data Fig. 3 Phenotypic characterization of Xt UPB513 T6SS and X-T4SS mutants.

(a, b) In vitro growth curve of (a) Xt UPB513 WT and derived strains mutated in and complemented for T6SS, (b) Xt LMG728 WT and derived strains mutants in and complemented for T4SS, in NB medium at 28 °C, OD600 measured at 45-minute intervals. Dots represent mean and error bars show s.e.m (n = 8 replicates per strain). (c) Barley cv. Hercule leaves infiltrated with Xtu UPB513 WT or T6SS mutants show water-soaking and necrotic lesions at 6 dpi (n = 8 plants).

Extended Data Fig. 4 Xt T6SS-i4 and X-T4SS are required for inter-bacterial competition against Pseudomonas sp. and Pantoea sp.

Representative qualitative competition plate using Xt UPB513 WT, Δhcpi4, Xt LMG728 WT and ΔvirD4 as attacker cells and Pantoea agglomerans UPB1357 and Pseudomonas sivasensis UPB1354 Tn7::mNeonGreen as target cells after 16 h of competition on NA.

Extended Data Fig. 5 VirB8-based phylogeny reveals xanthomonads with atypical X-T4SS sequences.

(a) The phylogenetic tree was constructed using autoMLSA2, by extracting and aligning VirB8 amino acid sequences from each type/pathotype strain of each Xanthomonas species and pathovar possessing X-T4SS and using IQ-TREE to infer the phylogenetic tree. (b) Comparison of the T4SS gene clusters from X. campestris pv. incanae and Stenotrophomonas maltophilia. Most X-T4SS genes (except for virD4) of strain CFBP 1606 share higher sequence identity with the corresponding genes from strain X28 than with those in strain WHRI 8527.

Extended Data Fig. 6 Various decay and loss events occurred in the X-T4SS clusters.

The tree on the left side is based on average nucleotide identity (ANI) of Xt strains, whereas the right side provides an overview of the organization of the X-T4SS gene clusters and their neighboring genes uvrB and thrS. Letters and numbers above the genes correspond to the virD and virB genes. Scales for distance and percentage identity between genes are indicated below. Genes are color-coded according to homology. Pseudogenized genes are highlighted with an asterisk. Fragments of remnant X-T4SS genes are found within the corresponding genomic location in the X-T4SS-lacking strains. The three ANI clades Xt-I, Xt-II and Xt-III are indicated in the tree.

Extended Data Fig. 7 X-T4SS-lacking strains possess T4E-T4I pairs.

Comparison of candidate X-T4SS effector-immunity pairs across strains possessing either X-T4SS, T6SS-i4, or none of them. The asterisk indicates an in-frame STOP codon.

Extended Data Fig. 8 Incongruency between the T6SS-i4 and the pan-genome tree topology of strain Xt LW16 is caused by the tssM gene.

Comparison between species (left) and T6SS-i4 (right) phylogenies. The pan-genome SNP-based tree was constructed using kSNP3 (kmer of 23 bp). The T6SS-i4 tree was inferred by maximum likelihood using IQ-TREE based on concatenated alignment of tssA, tssB, tssC, tssD, tssE, tssF, tssG, tssH, tssK, tssL, (a) with or (b) without tssM genes. Amino acid sequences were aligned by ClustalW. The bootstrap supports were estimated based on 1000 replicates. Trees were visualized with iTOL.

Extended Data Fig. 9 T6SS-i4 and ICE in type-2 neighborhood are flanked by att sites in strains (a) Xcg T8, (b) Xt 01, Xt UPB886, Xt UPB820 and (c) Xce LPF602.

(Upper panel) Genes corresponding to the same ICE modules are represented in similar colors. Genes with unknown functions are indicated in gray. attL and attR sites flanking the GI and attP in the T6SS-i4 lacking strain are pointed with red bars. (Lower panel) attL, attR and attP sites sequences are shown, 16-bp direct repeat sequences are boxed with dashed lines and tRNA-Gly sequences are underlined.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9, Tables 1–5 and Legends for Videos 1–16.

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Supplementary Video 1

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 2

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 3

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 4

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 5

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 6

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 7

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 8

Real-time monitoring of T6SS bacterial killing activity in X. translucens WT. Time-lapse video of Xt UPB513 WT, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt: to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 9

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 10

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 11

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 12

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 13

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 14

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 15

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

Supplementary Video 16

Real-time monitoring of T6SS bacterial killing activity in X. translucens T6SS-i4 mutant. Time-lapse video of Xt UPB513 ΔT6SS-i4, expressing RFP in competition with E. coli DH10B expressing GFP, mixed in a 10:1 (Xt to E. coli) ratio, on solid agarose pad (25% LB). Bar representing 10 μm is shown on the right, while magnification and time are indicated on the left. Pictures were taken every 6 min for 4 h 48 min.

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Peduzzi, C., Butchacas, J., Nikis, F. et al. Functional replacement of ancestral antibacterial secretion system in a bacterial plant pathogen. Nat Ecol Evol 9, 1393–1404 (2025). https://doi.org/10.1038/s41559-025-02773-w

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