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Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts

Nature Microbiology volume 7, pages 831–843 (2022)Cite this article

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Abstract

Plant-pathogenic fungi form intimate interactions with their associated bacterial microbiota during their entire life cycle. However, little is known about the structure, functions and interaction mechanisms of bacterial communities associated with fungal fruiting bodies (perithecia). Here we examined the bacterial microbiome of perithecia formed by Fusarium graminearum, the major pathogenic fungus causing Fusarium head blight in cereals. A total of 111 shared bacterial taxa were identified in the microbiome of 65 perithecium samples collected from 13 geographic locations. Within a representative culture collection, 113 isolates exhibited antagonistic activity against F. graminearum, with Pantoea agglomerans ZJU23 being the most efficient in reducing fungal growth and infectivity. Herbicolin A was identified as the key antifungal compound secreted by ZJU23. Genetic and chemical approaches led to the discovery of its biosynthetic gene cluster. Herbicolin A showed potent in vitro and in planta efficacy towards various fungal pathogens and fungicide-resistant isolates, and exerted a fungus-specific mode of action by directly binding and disrupting ergosterol-containing lipid rafts. Furthermore, herbicolin A exhibited substantially higher activity (between 5- and 141-fold higher) against the human opportunistic fungal pathogens Aspergillus fumigatus and Candida albicans in comparison with the clinically used fungicides amphotericin B and fluconazole. Its mode of action, which is distinct from that of other antifungal drugs, and its efficacy make herbicolin A a promising antifungal drug to combat devastating fungal pathogens, both in agricultural and clinical settings.

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Fig. 1: Assessment of perithecium-associated bacterial communities.
Fig. 2: Isolation and identification of the F. graminearum antagonist ZJU23.
Fig. 3: Herbicolin A is the major antifungal compound produced by ZJU23.
Fig. 4: Ergosterol biosynthesis is involved in the fungal susceptibility to HA.
Fig. 5: HA interacts with ergosterol and targets fungal lipid rafts.
Fig. 6: HA exhibits broad-spectrum antifungal activity.

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Data availability

The genome sequence of ZJU23, Pa58 has been deposited in the NCBI BioProject database with accession codes PRJNA707237 and PRJNA795028. Raw data of amplicon sequencing, genome sequences of herbicolin A-resistant S. cerevisiae mutants and transposon mutants of P. agglomeans ZJU23 are deposited in the Genome Sequence Archive of the Beijing Institute of Genomics (BIG) Data Center with accession numbers CRA003916, CRA006594 and CRA006602 in bioproject PRJCA003858 (https://bigd.big.ac.cn/gsa). Other data supporting the findings of the present study are available within this article, in Extended Data and the Supplementary Information. Source data are provided with this paper.

Code availability

Scripts employed in the microbiome analysis are available at https://github.com/YongxinLiu/WheatFHB.

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Acknowledgements

We thank J. Blodgett (Washington University, St. Louis, USA) and C. Liu (Zhejiang University, China) for herbicolin A biosynthetic gene cluster analysis; Y. Xiao (Chinese Academy of Sciences, China) and F. Xu (Zhejiang University, China) for structural identification of compounds; Q. Wang (Northwest A&F University, China) for analysing transposon insertion sites in the ZJU23 and mutated sites in yeast; and W. Fu (Zhejiang University, China) for the molecular docking analysis. This work was supported by grants from: the Key Technology R&D Program of Zhejiang Province (grant no. 2019C02034) to Z.M.; the National Science Fund for Excellent Young Scholars (grant no. 31922074) to Y.C.; the China Agriculture Research System (grant no. CARS-3-29) to Z.M.; the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (grant no. XDA24020104) to Y.B.; and the Youth Innovation Promotion Association CAS (grant no. 2021092) to Y.-X.L.

Author information

Author notes

  1. These authors contributed equally: Sunde Xu, Yong-Xin Liu, Tomislav Cernava.

Authors and Affiliations

  1. State Key Laboratory of Rice Biology, Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Department of Plant Protection, Zhejiang University, Hangzhou, China

    Sunde Xu, Hongkai Wang, Yaqi Zhou, Xing-Xing Shen, Ziyue Wen, Xueping Zhou, Zhonghua Ma, Yunlong Yu & Yun Chen

  2. State Key Laboratory of Plant Genomics, CAS-JIC Centre of Excellence for Plant and Microbial Science, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Yong-Xin Liu, Baoyuan Qu & Yang Bai

  3. Institute of Environmental Biotechnology, Graz University of Technology, Graz, Austria

    Tomislav Cernava & Gabriele Berg

  4. Institute for Immunology, Department of Basic Medical Sciences, Center for Life Sciences, Tsinghua University, Beijing, China

    Tie Xia, Hefei Ruan & Yan Shi

  5. Department of Pharmaceutical Sciences, University of Hawaii, Hilo, HI, USA

    Shugeng Cao & Chunshun Li

  6. Department of Biology, Northeastern University, Boston, MA, USA

    Yunrong Chai

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  1. Sunde Xu
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Contributions

Y. Chen and Z.M. initiated, coordinated and supervised the project. S.X., H.W., Y.Z. and Z.W. collected samples and isolated bacterial strains. Y.-X.L., B.Q. and Y.B. performed bacterial community profiling. S.X. and H.W. performed gene knockout experiments. S.X., C.L. and S.C. identified compounds. S.X., T.X., H.R. and Y.S. performed lipid raft experiments. X-X.S. phylogenetically analysed the HA biosynthetic gene cluster. Y. Chen, S.X., Z.M., Y.-X.L. and Y.B. collected and analysed the data. Y. Chen, Z.M., S.X., Y.B., T.C. and Y.Y. wrote the manuscript. Y. Chen, Y. Chai, G.B. and X.Z. revised the manuscript.

Corresponding authors

Correspondence to Yunlong Yu, Yang Bai or Yun Chen.

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Nature Microbiology thanks Marcio Rodrigues and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Experimental procedure for assessing perithecia-associated bacterial communities.

a, Collection of perithecia and microscopic visualization of bacteria on the surface of perithecia using a scanning electron microscope. The experiment was repeated five times with similar results. b, c, Experimental procedure used to analyse the detailed analysis of the perithecium-associated microbiome and the corresponding cultivable bacterial isolates. Bacterial identification procedure was modified from Zhang et al., 201963.

Extended Data Fig. 2 Diversity and differential abundance of OTUs in the perithecium, stubble and soil samples.

a, Rarefaction curves of detected bacterial OTUs in microbiomes from each compartment. The saturation stage with increasing numbers of sequencing reads indicates that bacterial diversity was sufficiently covered by the implemented approach. The richness index of perithecium, stubble and soil samples is shown separately (mean ± s.e.m.). b, Constrained principal coordinate analysis (CPCoA) of Bray-Curtis dissimilarity showing compartment effects on the microbial community structure. Ellipses cover 68% of the samples for each compartment. c, Manhattan plot showing OTUs that were enriched or depleted in perithecium versus soil and stubble. Each dot or triangle represents a single OTU. OTUs enriched or depleted in perithecium samples are represented by filled or empty triangles, respectively (FDR adjusted P < 0.001, Wilcoxon rank-sum test). OTUs are arranged in taxonomic order and colored according to their phylum and the proteobacteria class. CPM, counts per million. Numbers of replicated samples for each compartment, n = 65 biologically independent samples. d, Source analysis of average perithecium compartment attribution using SourceTracker trained on the stubble and soil microbiome datasets. Replicates for each compartment, n = 65 biologically independent samples.

Source data

Extended Data Fig. 3 ZJU23 inhibitory effects on the formation of Fg perithecia on rice straws and the length of wheat seedlings in each treatment with or without conidial of Fg.

a, Fg perithecium formation in different treatments on rice straws. Images were taken after 14 days post-inoculation (dpi) from n = 6 biologically independent samples (mean ± s.e.m.). Different letters indicate significantly different groups (P < 0.05, ANOVA, Tukey HSD). The non-antagonistic P. agglomerans Pa58 strain was used as a control. The experiment was repeated three times with similar results. b, The length of wheat seedlings with or without F. graminearum (Fg) inoculation in each treatment are shown at 7 dpi. The length of the wheat seedlings at 7 dpi was measured and statistically analyzed (n = 10 biologically independent seedlings for each treatment, mean ± s.e.m.). Different letters indicate significantly different groups (P < 0.05, ANOVA, Tukey HSD). Representative wheat seedlings without Fg inoculation in each treatment are shown. The experiment was repeated three times with similar results.

Source data

Extended Data Fig. 4 Purification and identification of herbicolin A.

a, Comparative total ion flow diagram profile between ZJU23 and deletion mutants in the HA biosynthetic gene cluster. b, High-resolution mass spectra (HRMS) of herbicolin A showing a peak at m/z 1300.7378, which corresponds to the [M+H] + ion and another at m/z 650.8722, which corresponds to the [M+2H]2+ ion. c, 1H NMR spectrum of HA. d, NOESY NMR spectrum. e, TOCSY NMR spectrum. f, 1H-13C HSQC NMR spectrum. g, 1H-13C HMBC NMR spectrum.

Extended Data Fig. 5 Stereochemistry of Thr (a), N-Methy-Thr (b), Glu (c), Leu (d), allo-Thr (e) and Arg (f) residues in herbicolin A determined by Marfey’s method.

Shown are extracted ion chromatograms of amino acid-FDAA adducts detected by HPLC-Qtof-MS of hydrolyzed, FDAA-derivatized herbicolin A and the amino acid standards. (i) herbicolin A-L-FDAA; (ii) L-AA-L-FDAA; (iii) D-AA-L-FDAA. FDAA, 1-fluoro-2-4-dinitrophenyl-5-L-alanine amide.

Extended Data Fig. 6 Structures of intermediates in the herbicolin A biosynthesis.

a, Electrospray ionization spectra of herbicolin A, herbicolin B, intermediate 1–4. b, Structures of herbicolin A, herbicolin B, intermediate 1–4.

Extended Data Fig. 7 Growth inhibition of HA in various concentrations towards Fg mycelia in agar plates.

a, Live/dead staining of F. graminearum cells expressing the plasma membrane marker GFP-StoA, after treatment with HA. Dead cells are indicated by either yellow or red staining. Scale bar, 5 µm. The experiment was repeated five times with similar results. b, Survival curves of F. graminearum cells grown in liquid medium supplemented or not (Control) with the indicated concentrations of HA. Data presented the mean ± s.e.m.. The experiment was repeated five times with similar results.

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Extended Data Fig. 8 Origin of the herbicolin A biosynthetic cluster in ZJU23.

a, Heatmap representation of the amino acid identities (%) of individual HA biosynthetic proteins shared between ZJU23 and other tested strains. b, Phylogenies of the 10 genes in the cluster correspond to six different patterns. Specific genes supporting each of the patterns are listed on top. c, Microsynteny and amino acid sequence conservation between the HA biosynthesis cluster in ZJU23 and a predicted gene cluster in Ca. Fukatsuia symbiotica. The hits between the two clusters are indicated with different shades of yellow, and the identity scale (45–100%) is included. d, Phylogenetic tree of tested bacterial species. γ-Protebacteria, β-Proteobacteria and other proteobacteria are indicated with red, green and black lines.

Extended Data Fig. 9 Synergistic effects between herbicolin A and two tested fungicides, polyoxin B and carbendazim.

Two-dimensional matrix of dose–response for relative mycelial growth inhibition of F. graminearum in the combinations of herbicolin A and polyoxin B (a), and herbicolin A and carbendazim (c). Synergyfinder was used to generate a topographic two-dimensional map of synergy scores for the combination of herbicolin A and polyoxin B (b), herbicolin A and carbendazim (d), respectively. Synergistic and antagonistic dosage zones are highlighted in red and green on the synergy map, respectively. The area with the greatest synergy score is indicated by a white box.

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Extended Data Fig. 10 Proposed model for the mode of action of herbicolin A, which is secreted by ZJU23 in the perithecium microbiome, on Fusarium graminearum.

The biocontrol bacterium P. agglomerans ZJU23 was isolated from F. graminearum perithecium microbiome. ZJU23 showed strong in vitro and in situ antagonism, which was later attributed to the secreted antifungal compound herbicolin A (HA). The HA biosynthetic gene cluster AcbA-J was obtained via horizontal gene transfer from Candidatus Fukatsuia symbiotica. HA binds ergosterol and disrupts the integrity of ergosterol-containing fungal lipid rafts, which subsequently suppresses fungal growth, substantially reducing perithecium formation and virulence of F. graminearum.

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Supplementary Figs. 1–10 and Methods.

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Supplementary Data 1

Supplementary Data 1–9.

Supplementary Video 1

Interaction patterns between herbicolin A and membranes without ergosterol in an in silico model.

Supplementary Video 2

Interaction patterns between herbicolin A and membranes with ergosterol in an in silico model.

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Xu, S., Liu, YX., Cernava, T. et al. Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts. Nat Microbiol 7, 831–843 (2022). https://doi.org/10.1038/s41564-022-01131-x

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