Abstract
Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.
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Acknowledgements
We thank H. Pan and X. Zhang from Jilin University for their generous help over B. maydis transformation. We also thank W. Zhang from Nanjing Agricultural University for providing the PHPLCδ1 and PHFAPP1 plasmids. This work was supported by grants from: the National Natural Science Foundation of China (NSFC) (grant nos. 31825022 and 31772153), National Key Research and Development Plan of China (grant no. 2016YFD0100600) and Tencent Exploration Award to X.W.C.; NSFC (grant no. 31871920), Open Funds of the State Key Laboratory of Crop Genetics and Germplasm Enhancement (grant no. ZW201803), International Cooperation and Exchange Program of Sichuan Science and Technology Department (grant no. 2020YFH0137) and Science Foundation of Education Department of Sichuan Province (grant no. 17ZA0313) to M.H.; NSFC (grant no. 31772152) to W.T.L.; NSFC (grant no. 31401351) to J.W.; NSFC (grant no. 31601290) to J.J.Y.; and NSFC (grant no. 31701779) to X.B.Z.. M.C. was supported by NSF (grant no. 1237975), NIH (grant no. GM59962) and USDA NIFA (grant no. 2017-67013-26590). Research in the laboratory of N.J.T. was supported by the Biotechnology and Biological Sciences Research Council Institute Strategic Grant in Plant Health (grant no. BB/N009959/1) and the Gatsby Charitable Foundation.
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Contributions
M.H. and X.W.C. conceived and designed the study. M.H., J.S., Y.P.X., J.H.C., M.L.L., T.Q., K.K.Z., Y.Y.C., Y.L., W.W.L., J.W., Z.K.W., L.S.R., B.Z.T., S.M.L., G.X.P. and X.F.S. performed experiments with phenotypical and biochemical assays. M.O.R., I.E., X.Y., J.J.Y., X.B.Z., B.T.M., J.C.W., J.L.L., H.Q., L.S., L.W., Q.Q.H., P.Q., Y.L., J.F., D.Q.L., Y.P.W., X.W., L.J., G.S.G., F.H., G.H.S., Y.X.X., W.M.W., X.J.W., P.L., L.H.Z. and S.G.L. contributed to reagents, plant and fungal materials. M.H., J.S., Y.P.X., J.H.C., L.M.L., T.Q., Z.K.W. and X.W.C. collected data. M.H., M.S.C., N.J.T. and X.W.C. analysed the data and wrote the manuscript.
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Extended data
Extended Data Fig. 1 Sep3 associates with PIPs in M. oryzae.
a, Pulldown analysis on the interaction between Sep3 and PIPs. Total extracts of transformants of M. oryzae expressing Sep3-GFP or GFP only were immunoprecipitated by anti-PIP antibody and then subjected to immunoblot analysis by anti-GFP antibody. n.s. represents non-specific protein. The experiment was repeated twice with similar results. b, Role of PB domain for Sep3 binding PIPs determined by the protein lipid overlay assay. Bacterially expressed GST-Sep3 and GST-Sep36Q was respectively incubated with the commercial lipid strip. GST antibody was then used to detect proteins bound on the lipid strip. The experiment was repeated twice with similar results.
Extended Data Fig. 2 Generation of RNAi transformants for Knocking down the PI-3K encoding gene VPS34 of M. oryzae.
a, Schematic representation of VPS34 RNAi constructs pSilent-Vps34. Promoter of gene RP27 was used to drive the transcription of a self-complementary hairpin RNA targeting VPS34. IT represents the intron 2 of M. oryzae cutinase encoding gene (MGG_01943). b, RT-qPCR analysis on expression levels of VPS34 RNAi transformants. pSilent1 represents the empty RNAi vector transformant, whereas pSilent1-Vps34.1 and pSilent1-Vps34.2 represent two independent VPS34 RNAi transformants. Data from n = 3 biological replicates are depicted as bar plots showing the mean ± s.d. overlaid with the individual data points. Two-sided Student’s t-test was used to calculate statistical significance.
Extended Data Fig. 3 Phylogenetic analysis of Elo homologues and generation of targeted ELO1 gene deletion mutants.
a, Phylogenetic tree of Elo homologues. b, Transmembrane domain analysis of M. oryzae Elo1 and yeast (Sc) Elo proteins by the TMHMM software. c, ClustalOmega alignment of Elo homologues. d, Schematic representation of the targeted deletion of ELO1 in M. oryzae. e, PCR analysis of the targeted deletion in the Δelo1 mutants. Genomic DNA was analyzed by PCR with primer pairs indicated in panel (d). f, RT-PCR analysis of the Δelo1 mutants.
Extended Data Fig. 4 ELO1 encodes an ER-membrane associated protein required for VLCFAs biosynthesis.
a, Subcellular localization of Elo1-GFP on ER membrane as shown by co-localization with the ER maker fusion protein mRFP-HDEL. White dotted lines show scans used to generate corresponding GFP and mRFP fluorescence intensity distribution in the linescan graph. Scale bar, 10 μm. The experiment was repeated twice with similar results. b, RT-qPCR analysis on the expression profiles of ELO1 in Guy11 at different developmental processes (n = 3 biological replicates, mean ± s.d., two-sided Student’s t-test). c, Quantitation of fatty acids of different carbon lengths in M. oryzae. Fatty acids were extracted from Guy11 and a Δelo1 mutant, and subjected to UPLC-MS/MS analysis (n = 3 biologically independent samples, mean ± s.d., two-sided Student’s t-test). d, Quantitation of PIP by UPLC-MS/MS on M. oryzae (n = 3 biologically independent samples, mean ± s.d., two-sided Student’s t-test). e, Quantitation of PIP2 by UPLC-MS/MS on M. oryzae (n = 3 biologically independent samples, mean ± s.d., two-sided Student’s t-test).
Extended Data Fig. 5 VLCFA biosynthesis is crucial for host penetration by M. oryzae.
a, Infection of detached rice leaves with spores in spotting inoculation assay. Mean length of blast lesions was calculated after five-days infection (n = 15 independent lesions, mean ± s.d., two-sided Student’s t-test). Fungal biomass in infected leaves was measured based on qPCR analysis of POT2 (n = 3 biological replicates, mean ± s.d., two-sided Student’s t-test). b, Infection structure development on onion epidermis at 24 hpi. Black arrows and arrowheads indicate conidium and appressorium respectively; white arrowheads indicate invasive hypha. Scale bar, 10 μm. Appressorium and penetration peg formation were quantitated from at least 100 spores (n = 3 biologically independent samples, mean ± s.d., two-sided Student’s t-test). c, Infection structure development on rice leaf sheath at 24 hpi. Scale bar, 10 μm. The experiment was repeated three times with similar results. d, Detached leaves infected with Guy11 in spotting inoculation assay with 500 μM VLCFAs biosynthesis inhibitors. The solvent DMSO was used as a negative control. Length of blast lesion was calculated five days after infection (n = 15 independent lesions, mean ± s.d., two-sided Student’s t-test). e, Rice leaves infected with Guy11 in spore spraying inoculation in the presence of 500 μM inhibitor. Lesion density was calculated from infected leaf segments of 5 cm in length (n = 21 independent leaves for DMSO, n = 28 for metazachlor, n = 37 for cafenstrole, n = 33 for diallate; mean ± s.d., two-sided Student’s t-test). f, TEM analysis on the penetration of onion epidermis by M. oryzae in the presence or absence of an inhibitor at 24 hpi. Scale bar, 2 μm. The experiment was repeated twice with similar results.
Extended Data Fig. 6 Effects on inhibition of M. oryzae pathogenicity are proportional to the concentrations of the VLCFA biosynthesis inhibitors, and combination of VLCFAs inhibitors improves efficacy of carbendazim in reducing pathogenicity.
Three VLCFA biosynthesis inhibitors and DMSO control were individually applied in a field trial. Rice leaves were sprayed with Guy11 spore suspension supplemented with an inhibitor at concentrations of 0, 20, 50, 100, 200, 500, 700 and 1000 μΜ, respectively. Blast lesion density was quantitated five-days post infection. a, Metazachlor (n = 27 independent leaves for 0 μΜ, n = 29 for 20 μΜ, n = 29 for 50 μΜ, n = 30 for 100 μΜ, n = 29 for 200 μΜ, n = 29 for 500 μΜ, n = 30 for 700 μΜ, n = 31 for 1000 μΜ; mean ± s.d.). b, Cafenstrole (n = 29 independent leaves for 0 μΜ, n = 29 for 20 μΜ, n = 28 for 50 μΜ, n = 29 for 100 μΜ, n = 29 for 200 μΜ, n = 28 for 500 μΜ, n = 29 for 700 μΜ, n = 29 for 1000 μΜ; mean ± s.d.). c, Diallate (n = 32 independent leaves for 0 μΜ, n = 30 for 20 μΜ, n = 29 for 50 μΜ, n = 30 for 100 μΜ, n = 31 for 200 μΜ, n = 31 for 500 μΜ, n = 31 for 700 μΜ, n = 30 for 1000 μΜ; mean ± s.d.). d, Effects of combination of three VLCFAs inhibitors on pathogenicity of M. oryzae. The three inhibitors were combined in equal amount to a final concentration of 500 μM in Guy11 spore suspension. Then the spore suspension containing combined inhibitors or the three separate inhibitors was inoculated onto rice leaves. DMSO was used as solvent control. Blast lesion density was quantitated after five days of infection (n = 30 independent leaves for DMSO, n = 30 for metazachlor, n = 31 for cafenstrole, n = 30 for diallate, n = 32 for combination of three inhibitors; mean ± s.d.). e, Effects of combination of each VLCFAs inhibitor with carbendazim on pathogenicity of M. oryzae. The three inhibitors were separately combined with carbendazim to produce Guy11 spore suspension which contains 500 μM VLCFAs inhibitor and 8 mM carbendazim. The spore suspension was inoculated onto rice leaves. Carbendazim, metazachlor, cafenstrole and diallate is respectively abbreviated as Car, Met, Caf and Dia. Blast lesion density was quantitated after five-days of infection (n = 31 independent leaves for DMSO, n = 32 for carbendazim, n = 31 for metazachlor, n = 31 for cafenstrole, n = 30 for diallate, n = 29 for combination of carbendazim with metazachlor, n = 31 for combination of carbendazim with cafenstrole, n = 32 for combination of carbendazim with diallate; mean ± s.d.).
Extended Data Fig. 7 Analysis of septin ring formed by Sep4-GFP and Sep5-GFP in M. oryzae.
Microscopic analysis and quantitation of septin ring formation at 24 hpi in the presence or absence of 500 μM inhibitor. DMSO was used as solvent control. Percentage of septin ring formation was calculated from 100 spores at 24 hpi (n = 3 biologically independent samples, mean ± s.d., two-sided Student’s t-test). Scale bar, 10 μm.
Extended Data Fig. 8 Inhibitors of VLCFA biosynthesis have no obvious effects on autophagic progression and conidial cell death during appressorium development of M. oryzae.
a, Microscopic images and quantitation of autophagosomes during appressorium development. Spore suspension of Guy11 expressing GFP-Atg8 was allowed to undergo appressorium development on hydrophobic surface in exposure to each of the three inhibitors at a concentration 500 μΜ or the solvent control DMSO. Mean autophagosome numbers was quantitated in conidium, germ tube and appressorium (n = 20 independent spores, mean ± s.d.). Scale bar, 10 μm. b, Autophagic conidial cell death of Guy11 at 24 hpi of appressorium development in the presence of inhibitors or solvent DMSO. Autophagic clearance of GFP in the conidial three cells of spores was quantitated from 100 spores (n = 3 independent samples, mean ± s.d.). Scale bar, 10 μm.
Extended Data Fig. 9 Inhibitors of VLCFAs biosynthesis impaired septin organization and septin-dependent pathogenicity of B. maydis.
a, Microscopic analysis and quantitation of septin puncta detected by BmSep3-GFP at hyphal tips infecting maize leaf at 24 hpi. B. maydis spore suspension was supplemented with 500 μM of each VLCFAs inhibitor and then used for inoculation of maize leaf. DMSO was the solvent control. Percentage of septin dot formation was calculated from 50 hypal tip at 24 hpi (n = 3 independent samples, mean ± s.d., two-sided Student’s t-test). Scale bar, 10 μm. b, Microscopic analysis and quantitation of septin dot detected by BmSep4-GFP from 50 hyphal tips infecting maize leaf at 24 hpi (n = 3 independent samples, mean ± s.d., two-sided Student’s t-test). Scale bar, 10 μm. c, Schematic representation of the targeted deletion of BmSEP3 and BmSEP4 in B. maydis. d, PCR analysis of the targeted deletion in the ΔBmsep3 and ΔBmsep4 mutants. Genomic DNA was analyzed by PCR with primer pairs indicated in panel C. e, Maize leaf infected with B. maydis by a spore suspension spraying assay (n = 7 independent leaves, mean ± s.d., two-sided Student’s t-test).
Extended Data Fig. 10 Inhibitors of VLCFA biosynthesis have no observable effects on host growth or health.
a, Seedlings of rice after seven days of spray with 500 μM inhibitor. b, Yields of rice plants sprayed with inhibitor. Plants were sprayed with 500 μM inhibitor for the first time at the heading stage, and sprayed for the second time at the booting stage. Agronomic traits including seed setting rate, 1000-seed-weight and grain weight per plant were collected from reprehensive plants (n = 6 independent plants, mean ± s.d.). c, Wheat seedlings after seven days of spray with 500 μM inhibitor. d, Maize after seven days of spray with 500 μM inhibitor. e, Locust after seven days of spray with 500 μM inhibitor. f, Quantitative analysis on daily food consumption and feces production behavior of locust after inoculation with 500 μM inhibitor (n = 3 biological replicates, mean ± s.d.).
Supplementary information
Supplementary Table 1
Supplementary Tables 1 and 2.
Supplementary Video 1
Three-dimensional reconstruction of co-localization between Sep3–GFP and a pool of PHFAPP1/PHPLCδ1–mRFP in Guy11 appressorium.
Supplementary Video 2
Three-dimensional reconstruction of the septin ring at the base of appressorium in Guy11 and Δelo1.
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He, M., Su, J., Xu, Y. et al. Discovery of broad-spectrum fungicides that block septin-dependent infection processes of pathogenic fungi. Nat Microbiol 5, 1565–1575 (2020). https://doi.org/10.1038/s41564-020-00790-y
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DOI: https://doi.org/10.1038/s41564-020-00790-y
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