Article

Root-hair endophyte stacking in finger millet creates a physicochemical barrier to trap the fungal pathogen Fusarium graminearum

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Abstract

The ancient African crop, finger millet, has broad resistance to pathogens including the toxigenic fungus Fusarium graminearum. Here, we report the discovery of a novel plant defence mechanism resulting from an unusual symbiosis between finger millet and a root-inhabiting bacterial endophyte, M6 (Enterobacter sp.). Seed-coated M6 swarms towards root-invading Fusarium and is associated with the growth of root hairs, which then bend parallel to the root axis, subsequently forming biofilm-mediated microcolonies, resulting in a remarkable, multilayer root-hair endophyte stack (RHESt). The RHESt results in a physical barrier that prevents entry and/or traps F. graminearum, which is then killed. M6 thus creates its own specialized killing microhabitat. Tn5-mutagenesis shows that M6 killing requires c-di-GMP-dependent signalling, diverse fungicides and resistance to a Fusarium-derived antibiotic. Further molecular evidence suggests long-term host–endophyte–pathogen co-evolution. The end result of this remarkable symbiosis is reduced deoxynivalenol mycotoxin, potentially benefiting millions of subsistence farmers and livestock. Further results suggest that the anti-Fusarium activity of M6 may be transferable to maize and wheat. RHESt demonstrates the value of exploring ancient, orphan crop microbiomes.

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Author information

Affiliations

  1. Department of Plant Agriculture, University of Guelph, Guelph, Ontario N1G 2W1, Canada

    • Walaa K. Mousa
    • , Charles Shearer
    •  & Manish N. Raizada
  2. Department of Pharmacognosy, Mansoura University, Mansoura, Egypt

    • Walaa K. Mousa
  3. Department of Plant Agriculture, University of Guelph, Ridgetown Campus, Ridgetown, Ontario N0P 2C0, Canada

    • Victor Limay-Rios
  4. University of California Davis Genome Center, Davis, California 95616, USA

    • Cassie L. Ettinger
    •  & Jonathan A. Eisen

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Contributions

W.K.M. designed and conducted all experiments, analysed all data and wrote the manuscript. C.S. assisted in greenhouse trials. V.L.-R. performed the DON quantification experiments. C.L.E. and J.A.E. sequenced the M6 genome and provided gene annotations. M.N.R. helped to design the experiments and edited the manuscript. All authors read and approved the manuscript.

Competing interests

The authors declare no competing financial interests. However, a provisional US patent has now been filed on the application of M6 to corn and wheat (US patent application no. 62/056,012).

Corresponding author

Correspondence to Manish N. Raizada.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Supplementary Figures 1–8, Supplementary Tables 1–7, 8 Supplementary Video legends 1–2

Videos

  1. 1.

    Supplementary Video 1

    3D video showing RHESt. A 62 μm confocal stack with 46 sections was imaged from a 12 day old finger millet root previously seed-coated with GFP-tagged endophyte M6 (green colour) then inoculated with F. graminearum (Fg) at a distance of 0.5 cm to the left-hand side of the image followed by a 72 h incubation. On the right side of the image is the root (purple red) oriented downward. M6 cells (green, right of the root) stack to form a deep physical barrier on the rhizoplane on the same side as Fg inoculation. Root hairs (purple) unusually elongate on the same side (left) as Fg inoculation, bend parallel to the rhizoplane and become intercalated with M6 cells, in contrast to the side of the root that is distal to Fg (right). In this video, few Fg mycelia (purple threads) are observed, perhaps because most mycelia had not yet reached the root system (mycelia were clearly visible ˜1 mm away), were obscured or due to earlier death by the RHESt complex.

  2. 2.

    Supplementary Video 2

    3D imaging of a biofilm associated with endophyte M6 in vitro. Shown is a 45 μm confocal stack rendered as a 3D video. The biofilm was grown on a microscopic slide immersed in LB liquid medium inoculated with M6 at 37 °C and 50 rpm for 5 days, then stained with Ruby Film Tracer