Spermine-mediated tight sealing of the Magnaporthe oryzae appressorial pore–rice leaf surface interface

Abstract

Cellular adhesion mediates many important plant–microbe interactions. In the devastating blast fungus Magnaporthe oryzae1, powerful glycoprotein-rich mucilage adhesives2 cement melanized and pressurized dome-shaped infection cells—appressoria—to host rice leaf surfaces. Enormous internal turgor pressure is directed onto a penetration peg emerging from the unmelanized, thin-walled pore at the appressorial base1,2,3,4, forcing it through the leaf cuticle where it elongates invasive hyphae in underlying epidermal cells5. Mucilage sealing around the appressorial pore facilitates turgor build-up2, but the molecular underpinnings of mucilage secretion and appressorial adhesion are unknown. Here, we discovered an unanticipated and sole role for spermine in facilitating mucilage production by mitigating endoplasmic reticulum (ER) stress in the developing appressorium. Mutant strains lacking the spermine synthase-encoding gene SPS1 progressed through all stages of appressorial development, including penetration peg formation, but cuticle penetration was unsuccessful due to reduced appressorial adhesion, which led to solute leakage. Mechanistically, spermine neutralized off-target oxygen free radicals produced by NADPH oxidase-1 (Nox1)3,6 that otherwise elicited ER stress and the unfolded protein response, thereby critically reducing mucilage secretion. Our study reveals that spermine metabolism via redox buffering of the ER underpins appressorial adhesion and rice cell invasion and provides insights into a process that is fundamental to host plant infection.

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Fig. 1: SPS1 is required for appressorial penetration of rice leaf cuticles.
Fig. 2: SPS1 is required for appressorial mucilage production and adhesion.
Fig. 3: Spermine is required for appressorial mucilage production, adhesion and penetration.
Fig. 4: Spermine buffers Nox1-generated ROS to mitigate ER stress and promote mucilage secretion.

Data availability

The M. oryzae SPS1 gene sequence is available at NCBI under the accession MGG_01296. Data supporting the findings of this study are available from the corresponding author upon request. Mutant strains generated during the course of this study are available from the corresponding author upon request and with an appropriate APHIS permit. Numerical and statistical source data that underlie the graphs in figures and extended data are provided with the paper.

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Acknowledgements

We thank Y. Zhou and J. Russ of the UNL Microscopy Core facility for SEM and TEM imaging. We thank J. Seravalli, UNL Redox Biology Center, for LC–MRM mass spectrometry. We thank H. Appeah, UNL, for technical assistance. We thank D. S. Carvalho and A. Góes-Neto, Federal University of Minas Gerais, Brazil, for assistance with the phylogenetic analysis. We thank N. J. Talbot, The Sainsbury Laboratory, UK, for the gift of Δnox1. This work was supported by a National Science Foundation award to R.A.W. through the Plant Biotic Interactions program (IOS-1758805). R.O.R. was supported by a Capes scholarship/Science without Borders/Process number 99999.009138/2013-07 award. N.T.T.P. was supported by a UNL UCARE scholarship.

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Contributions

R.A.W. conceptualized the study. R.A.W. acquired funding for the experiments. R.O.R. and R.A.W. conceived, designed and guided the study. R.O.R., C.E. and N.T.T.P. acquired and analysed the data. Specifically, R.O.R. generated the mutant strains and characterized them together with N.T.T.P; confocal microscopy was performed by R.O.R. and C.E.; and SEM and TEM analyses were performed by R.O.R. The appressorial adhesion assays were performed by R.O.R. R.O.R. and R.A.W. interpreted the data. R.O.R. and R.A.W. wrote the first draft of the paper. R.A.W. wrote the final draft of the paper.

Corresponding author

Correspondence to Richard A. Wilson.

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Extended data

Extended Data Fig. 1 Δsps1 physiology compared to WT.

a, Sporulation rates of WT and the Δsps1 mutant strains were not significantly different (unpaired two-tailed t-test, t = 0.1346 df = 4, p = 0.8994) following 14 days of growth on CM. Value are the mean of spores liberated from three independent plates. Bars are standard deviation. b, WT and Δsps1 strains after ten days of growth on 88 mm petri-dishes containing complete media (CM) or 1 % (w/v) glucose minimal media (GMM), as indicated. Three independent plate test experiments gave similar results. c, Appressorium formation rates on detached rice leaf sheath surfaces. Values are means obtained by determining how many of n=50 germinating conidia had formed appressoria by 24 h.p.i., repeated in triplicate. Bars are standard deviation. Bars with different letters are significantly different (unpaired two-tailed t-test, t = 24.04, df = 4, p < 0.0001). d, Appressorial penetration rates on detached rice leaf sheaths. Values are means obtained by determining how many of n=50 appressoria had penetrated rice cuticles by 30 h.p.i., as determined by observing primary hyphae or early IH in underlying rice cells, repeated in triplicate. Bars are standard deviation. Bars with different letters are significantly different (unpaired two-tailed t-test, t = 94.75 df = 4, p < 0.0001). c, d, Leaf sheaths were inoculated with 1 x 105 spores per mL of the indicated strain. Source data

Extended Data Fig. 2 Appressorial developmental processes prior to penetration are not affected by SPS1 deletion.

a, Superoxide detection by nitrotetrazolium blue chloride (NBT) staining shows Δsps1 appressoria generated reactive oxygen species (ROS) at the appressorial cell wall like WT. Samples were stained with 0.3 mM NBT at 24 h.p.i. for one hour before imaging. Images are representative of three experiments performed on plastic cover slips. b, Live-cell imaging of detached rice leaf sheaths by bright field microscopy shows unmelanized patches covering the pore at the base of both WT and Δsps1 appressoria. Scale bar is 10 μm. Images are representative of three experiments. c, Δsps1 appressoria on cellophane were dislodged at 24 h.p.i. by sonication for 30 min. In this example, the liberated appressorium has been flipped upside down, revealing a perfectly formed appressorial pore. Scale bar is 5 μm. d, Penetration rates of Δsps1 and WT appressoria at 24 h.p.i. after treatment with 2.5 mM spermine at the indicated time points. Values are the mean penetration rates determined for n=50 appressoria, repeated in triplicate. Bars are standard deviation. Significant differences of the means comparing WT and Δsps1 at a given treatment are denoted by different lowercase letters (two-way ANOVA, F2, 12 = 1126, p < 0.0001). Significant differences of the means within WT (one-way ANOVA, F2, 6 = 1261, p < 0.0001) or within Δsps1 (one-way ANOVA, F2, 6 = 1.00, p = 0.42) at different treatments are denoted by different uppercase letters. Source data

Extended Data Fig. 3 SPS1 deletion affects appressorial cell wall shape and thickness and mucilage production.

a, Transmission electron microscopy (TEM) of Δsps1 and WT appressoria on plastic coverslips. Scale bar is 400 nm. b, Measurements of appressorial cell wall thickness after TEM. Values are the mean ± standard deviation of measurements from appressoria (n=144 for WT and n=174 for Δsps1) . Significant differences of means comparing WT and Δsps1 at the given treatment are denoted by different letters (unpaired two-tailed t-test, t = 2.580 df = 316, p = 0.0103). c, TEM of Δsps1 and WT appressoria on plastic coverslips at 24 h.p.i. showing examples of extracellular secretions, likely mucilage, that are sparser in Δsps1 than WT samples. Scale bar is 600 nm. Source data

Extended Data Fig. 4 SPS1 homologues are present in the genomes of other filamentous fungi including those that form appressoria or appressoria-like infection structures.

Unrooted maximum likelihood phylogenetic tree shows two main clusters of species carrying Sps1 homologues (light gray and dark gray). Branches of M. oryzae (syn. Pyricularia oryzae) and related species of Pyricularia (magnaporthe-like sexual morphs) are shown in blue. Values represent the support percentage for each branch. Branch length scale is shown in the bottom left. Asterisks indicate species that form appressoria or appressoria-like structures.

Extended Data Fig. 5 Loss of spermine induces the unfolded protein response (UPR) and cell wall integrity (CWI) response.

ER stress induces the UPR and CWI. Relative transcript abundances of UPR-associated genes (LHS1, KAR2, SCJ1, SIL1, PDI1)29 and a cell wall integrity-associated gene (CCW12)30 were upregulated in the Δsps1 strain grown in liquid complete media without spermine supplementation compared to WT. Levels of the indicated genes were reduced in Δsps1 mycelia when grown in the presence of 1 mM spermine. Expression levels were normalized against M. oryzae β-tubulin gene expression. Quantitative real-time PCR assays were carried out in triplicate. Template controls did not show amplification. Error bars indicate standard deviation. Significant differences of the means comparing WT and Δsps1 at the indicated treatment are denoted by different lowercase letters (two-way ANOVA, LHS1 F1, 8 = 432.9. NT, p < 0.0001. + Spermine, p = 0.0327; KAR2 F1, 8 = 22.10. NT, p < 0.0001. + Spermine, p = 0.0096; SIL1 F1, 8 = 42.12. NT, p < 0.0001. + Spermine, p = 0.2416; PDI1 F1, 8 = 45.40. NT, p < 0.0001. + Spermine, p = 0.2443; SCJ1 F1, 8 = 26.42. NT, p < 0.0001. + Spermine, p = 0.7101; CCW12 F1, 8 = 49.28. NT, p < 0.0001. + Spermine, p = 0.0151). Significant differences of the means within WT or within Δsps1 are denoted by different uppercase letters (two-way ANOVA, LHS1 F1, 8 = 432.9. WT, p = 0.9781. Δsps1, p < 0.0001; KAR2 F1, 8 = 22.10. WT, p = 0.0055. Δsps1, p < 0.0001; SIL2 F1, 8 = 42.12. WT, p = 0.0328. Δsps1, p < 0.0001; PDI1 F1, 8 = 45.40. WT, p = 0.0129. Δsps1, p < 0.0001; SCJ1 F1, 8 = 26.42. WT, p = 0.7503. Δsps1, p < 0.0001; CCW12 F1, 8 = 49.28. WT, p = 0.9996. Δsps1, p < 0.0001). Source data

Extended Data Fig. 6 Tunicamycin and ascorbic acid treatments affect appressorial penetration and adhesion.

a, Penetration rates at 30 h.p.i. for appressoria formed on rice leaf sheath surfaces from germinating WT spores treated or untreated (NT) with Tunicamycin (5 µg/mL) at the indicated time points. Values are the mean penetration rates determined for n=50 appressoria, repeated in triplicate. Bars are standard deviation. Significant differences of the means at a given time are denoted by different lowercase letters (one-way ANOVA, F4,10 = 173.00, p < 0.0001). b, Penetration rates at 30 h.p.i. for appressoria formed on rice leaf sheath surfaces from spores treated with 0.25 µM ascorbic acid. NT, no treatment. Values are the mean penetration rates determined for n=50 appressoria, repeated in triplicate. Bars are standard deviation. Significant differences of the means comparing WT and Δsps1 at a given treatment are denoted by different lowercase letters (two-way ANOVA, F1, 8 = 74.30. NT, p < 0.0001; + Ascorbic acid p < 0.0001). Significant differences of the means within WT or within Δsps1 are denoted by different uppercase letters (letters (two-way ANOVA, F1, 8 = 74.30. WT, p = 0.9450; Δsps1, p < 0.0001). c, Rates of WT appressorial adhesion in 96-well plates at 24 h.p.i. after germinating spores were treated at 6 h.p.i. with 2.5 μM ascorbic acid (asc) and/ or 5 μg/ mL Tunicamycin (Tun). This was repeated in triplicate for each treatment. Significant differences of the means are denoted by different lowercase letters (one-way ANOVA, F2,6 = 80.35, p < 0.0001). Error bars indicate standard deviation. Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Video 1

A H1:RFP appressorium on the rice leaf sheath surface, and IH nuclei, at 36 h.p.i. with CFW staining.

Supplementary Video 2

Δsps1 H1:RFP appressoria on the rice leaf sheath surface at 36 h.p.i. with CFW staining.

Source data

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Source Data Extended Data Fig. 1

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Rocha, R.O., Elowsky, C., Pham, N.T.T. et al. Spermine-mediated tight sealing of the Magnaporthe oryzae appressorial pore–rice leaf surface interface. Nat Microbiol (2020). https://doi.org/10.1038/s41564-020-0786-x

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