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

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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Figure 1: Isolation, identification and antifungal activity of endophytes.
Figure 2: Confocal imaging of M6–Fusarium interactions in finger millet roots.
Figure 3: Behaviour and interactions of endophyte M6 and F. graminearum in vitro on microscope slides.
Figure 4: Characterization of phenazine mutant ewpR-5D7::Tn5 and FA resistance mutant ewfR-7D5::Tn5 and their interactions.
Figure 5: Characterization of di-guanylate cyclase mutant ewgS-10A8::Tn5 and colicin V mutant ewvC-4B9::Tn5.
Figure 6: Microscopy imaging of other virulence traits associated with wild-type M6 (W) and each mutant (M).

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Acknowledgements

The authors thank M. Struder-Kypke (Department of Molecular and Cellular Biology, University of Guelph) for assistance with confocal microscopy and for her comments. The authors thank A. Schaafsma and L. Tamburic-Ilincic (Ridgetown College, University of Guelph) for providing hybrid maize and wheat seeds, respectively. The authors also thank M. Atalla for assistance with disease scoring. W.K.M. was supported by generous scholarships from the Government of Egypt and the University of Guelph (International Graduate Student Scholarships, 2012, 2014). The authors thank L. Smith (University of Guelph) for graphics. This research was supported by grants to M.N.R. by the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), Grain Farmers of Ontario (GFO), the Natural Sciences and Engineering Research Council of Canada (NSERC) and the CIFSRF programme, jointly funded by the International Development Research Centre (IDRC, Ottawa) and Global Affairs Canada.

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Authors

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.

Corresponding author

Correspondence to Manish N. Raizada.

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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).

Supplementary information

Supplementary information

Supplementary Figures 1–8, Supplementary Tables 1–7, 8 Supplementary Video legends 1–2 (PDF 11972 kb)

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. (MP4 7491 kb)

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 (MP4 3269 kb)

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Mousa, W., Shearer, C., Limay-Rios, V. et al. Root-hair endophyte stacking in finger millet creates a physicochemical barrier to trap the fungal pathogen Fusarium graminearum. Nat Microbiol 1, 16167 (2016). https://doi.org/10.1038/nmicrobiol.2016.167

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