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Appressorium-mediated plant infection by Magnaporthe oryzae is regulated by a Pmk1-dependent hierarchical transcriptional network

Abstract

Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development—including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis—as well as controlling a additional set of virulence-associated transcription factor–encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention.

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Fig. 1: Global comparative transcriptional profile analysis to define the response of M. oryzae to surface hydrophobicity and the presence/absence of the Pmk1 MAPK.
Fig. 2: Functional analysis and comparative global transcriptional profile analysis in response to the presence/absence of the M. oryzae TF Mst12.
Fig. 3: Defining the hierarchy of transcriptional control during appressorium development by M. oryzae.
Fig. 4: Characterization of the Hox7 homeobox TF and its role in the regulation of gene expression during appressorium development by M. oryzae.
Fig. 5: Phosphoproteomic analysis reveals Pmk1-dependent phosphorylation of Hox7 in M. oryzae.
Fig. 6: Purified recombinant GST–Pmk1 phosphorylates MBP–Hox7 and SUMO–Mst12 in vitro.
Fig. 7: Pmk1-dependent phosphorylation of Hox7 is required for appressorium development.

Data availability

The RNA-seq data described in this study have been submitted to the European Nucleotide Archive (ENA): appressorial RNA-seq data under the accession number PRJEB36580 and mycelial RNA-seq data under the accession number PRJEB44745. The ChIP–seq data described in this study have been submitted to Gene Expression Omnibus under the accession number GSE182534. The proteomic data described in this study have been deposited into the ProteomeXchange Consortium via PRIDE (Perez-Riverol et al., 2019) partner repository with the dataset identifier PXD025700. The PRM data have been made publicly available through PanoramaWeb (https://panoramaweb.org/84ne1U.url) and the corresponding ProteomeXchange ID for the data is PXD028052. The M. oryzae genome database used in this study was http://fungi.ensembl.org/Magnaporthe_oryzae/Info/Index. All M. oryzae strains generated in this study are freely available on request from the corresponding authors. Source data are provided with this paper.

Code availability

Scripts for the analysis and prediction of the peaks of ChIP–seq experiments have been publicly deposited in GitHub at https://github.com/threadmapper/sequence-under-peaks.

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Acknowledgements

This project was supported by a European Research Council Advanced Investigator award (to N.J.T.) under the European Union’s Seventh Framework Programme FP7/2007-2013/ERC Grant Agreement 294702 GENBLAST, BBSRC grant BB/N009959/1, and by the Gatsby Charitable Foundation. We thank D. MacLean for help with statistical analysis and C. Dean (John Innes Centre) for her group’s guidance with the ChIP–seq analysis.

Author information

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Authors

Contributions

M.O.-R. and N.J.T. conceptualized the project. Experimental analyses were carried out by M.O.-R., N.C-M., M.M-U., A.B.E., M.N., I.E., X.Y., M.J.K., X.Y., C.M. and G.R.L. F.L.H.M designed, and P.D. and F.L.H.M. carried out, the phosphoproteomic analysis. G.V.-P. and B.V. generated the Δrpp3 mutant. Bioinformatic analysis was performed by M.O.-R., D.M.S., B.T., J.C. and V.W. The paper was written by M.O.-R. and N.J.T., with contributions from all authors.

Corresponding authors

Correspondence to Míriam Osés-Ruiz or Nicholas J. Talbot.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks Lászlo Nagy, Antonio Di Pietro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The Pmk1 MAP kinase signalling pathway regulates appressorium development in response to surface hydrophobicity.

a. Micrographs to show development of a wild type M. oryzae strain Guy11 expressing histone H1–RFP nuclear marker and septin Sep3–GFP, inoculated on a hydrophobic (HP) or hydrophilic (HL) surface (Scale bar = 10 µm). b. Bar chart to show proportion of Guy11 germlings containing, 1, 2, 3, 4 or more nuclei following incubation for 24 h on HP or HL surfaces. (Grey: HP; White: HL; Circles represent replicates for HP surface; Triangles represent replicates for HL surface) (n = 135 conidia examined in 3 biological replicates; each biological replicate is colour coded; data is presented as Mean ±SEM; Multiple unpaired t-test; HP versus HL; 1 nucleus P = 0.014037). c. Live-cell imaging to show nuclear number and the presence/absence of conidial autophagic cell death of Guy11 on HP, ∆pmk1 mutant on HP, and Guy11 on HL surfaces respectively. Each strain expressed the H1–RFP nuclear marker. d. Rice blast disease symptoms of Guy11 and the ∆pmk1 mutant. Rice seedlings of cultivar CO-39 were spray-inoculated with M. oryzae conidial suspensions of equal concentrations and incubated for 5 days.

Source data

Extended Data Fig. 2 Expression of melanin biosynthesis pathway genes during appressorium development by M. oryzae.

a. Micrograph to show appressorium development by Guy11 and ∆mst12 mutant after incubation for 24 h on HP surface (Bar = 10 µm). b. Rice blast disease symptoms caused by Guy11 and ∆mst12 mutant. Rice seedlings of cultivar CO-39 were spray-inoculated with M. oryzae conidial suspensions of equal concentrations and incubated for 5 days. c. Table to show names and accession numbers of melanin biosynthesis enzymes of M. oryzae. d. Line graph to show mean of normalized counts of gene expression of melanin biosynthesis enzyme-encoding genes during a time course appressorium development for Guy11, ∆pmk1 and ∆mst12 mutants incubated on HP surface between 0 h and 24 h. e. Heatmap to show levels of transcript abundance of genes involved in melanin biosynthesis in a ∆pmk1 mutant compared to Guy11 in conidia germinated on HP surfaces between 0 h and 24 h. f. Heatmap to show levels of transcript abundance of genes involved in melanin biosynthesis in a ∆mst12 mutant compared to Guy11 incubated on HP surface between 0 h and 24 h. Levels of expression are represented as moderated logarithmic fold change (mod_lfc) (blue= downregulated in mutants versus Guy11; red= upregulated in mutants versus Guy11).

Extended Data Fig. 3 Mst12-dependent gene expression and sub-cellular localization of a subset of M. oryzae effectors during appressorium development.

a. Heatmap showing temporal pattern of transcript abundance of 436 Mst12-dependent genes predicted to encode secreted proteins during appressorium development between 0 h and 24 h on a HP surface. b. Heatmap showing levels of transcript abundance of 7 known effector-encoding genes2 in a ∆mst12 mutant during appressorium development. (blue= downregulated in ∆mst12 mutant versus Guy11; red= upregulated in ∆mst12 mutant versus Guy11). Levels of expression are represented as moderated logarithmic fold change (mod_lfc) c. Table to show name and accession number of 7 known effector genes differentially regulated in ∆mst12 compared to Guy11. d. Live-cell imaging of Septin5-GFP expression in appressoria of ∆mst12 and pmk1as mutants 24 h after conidial germination on a HP surface. The pmk1AS mutant was incubated ± 5 µM NA-PP1 added at 6-8 h, once appressorium development was underway. 1NA-PP1 is a specific inhibitor of the analogue-sensitive allele Pmk1 expressed by this mutant3 (Bar = 10 µm). e. Micrographs to show expression of effector Bas2pGFP in a pmk1AS mutant during appressorium formation, 24 h after conidia were inoculated on rice leaf sheath of blast-susceptible rice cultivar Mokoto, in the presence or absence (control) of 5 µM 1Na-PP1 (Bar = 10 µm). f. Micrographs to show cellular localization of exocyst and polarity proteins Exo70-GFP, Sec5-GFP, Sec6-GFP, Spa2-GFP and Mlc1-GFP in the appressorium pore of Guy11 and ∆mst12 following incubation on HP surface for 24 h (Bar = 5 µm). (g-k). Bar charts to show defects in localization of exocyst components during appressorium development of ∆mst12 compared to Guy11 on HP surface at 24 h (n = 180 conidia examined in 3 biological replicates; each biological replicate is colour coded; data are presented as Mean ±SEM).

Source data

Extended Data Fig. 4 ChIP–seq analysis of the Mst12 TF.

a. Micrographs to show cellular localization of Mst12–GFP during appressorium development following incubation on HP surface for 24 h (Bar = 10 µm). b. Rice blast disease symptoms of Guy11, ∆mst12 mutant and complemented strain ∆mst12: Mst12–GFP (T3 and T7 are two independent transformants). Rice seedlings of cultivar CO-39 were spray-inoculated with conidial suspensions of equal concentrations of each M. oryzae strain and incubated for 5 days. c. Table to show number of differentially regulated genes (padj <0.01, mod_lfc>1 or mod_lfc < -1) Guy11 and the ∆mst12 mutant during mycelium growth determined by RNA-seq analysis. d. Table to show over-represented motifs in peaks determined in Mst12 ChIP–seq experiment by Find Individual Motif Occurrence (FIMO – see https://meme-suite.org/meme/doc/fimo.html). Over-representation of each motif in specific sets of DEGs (up and downregulated in mycelium, or expressed during appressorium formation) was analysed using Fisher’s exact test and P values are indicated. The percentage value represents the proportion of the total pool of DEGs represented by that each set of motif-defined gene sets.

Extended Data Fig. 5 Appressorium development and pathogenicity assay of clade 4-associated TF null mutants.

a. Box and Whisker plots to show the number of rice blast disease lesions per 5 m of leaf tissue. Twenty-one-day-old rice seedlings of rice cultivar Co-39 were inoculated with uniform conidial suspensions (5 × 104 conidia ml-1) of ∆rpp1, ∆rpp2, ∆rpp4 and ∆rpp5 mutants and the isogenic wild type M. oryzae strain Guy11 or ∆ku70. Data points are shown in whisker plots which show 25th/75th percentiles, the median and the minimum and maximum values by the ends of the whiskers. A two-tailed non-parametric Mann–Whitney statistical test was conducted to determine significant differences. The plots show the results from 3 biological replications of the experiment and data points are colour coded for each replicate. Error bars show the standard deviation. b. Light micrographs showing appressorium development of Guy11, ∆aclR, ∆rpp1, ∆rpp2, ∆rpp4, ∆rpp5, ∆rpp3, ∆rpp3∆pig1 and ∆hox7 mutants germinated on HP surfaces and observed 24 h following conidial germination (Bar = 10 µm).

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Extended Data Fig. 6 Hox7 operates downstream of Pmk1 MAP kinase to regulate a subset of cellular pathways required for appressorium development and plant infection.

a. Bar chart to show the proportion of germlings of Guy11 and ∆hox7 that develop appressoria (round data points), re-germinated aberrant appressorial swellings (squares) and undifferentiated germlings (triangles) following conidial germination on an HP surface for 24 h (n = 371 conidia examined over 3 biological replicates; biological replicates are colour coded; bars represent Mean ± SEM). b. Table to show the total number of DEGs, upregulated genes (padj <0.01; mod-lfc >1) and downregulated genes (padj <0.01; mod-lfc < -1) of ∆pmk1 versus Guy11, ∆mst12 versus Guy11 and ∆hox7 versus Guy11 at 14 h during appressorium development. c. Heatmap showing levels of relative transcript abundance between ∆pmk1 versus Guy11, ∆mst12 versus Guy11 and ∆hox7 versus Guy11 of a common set of 709 genes. For all heatmaps, the levels of expression are represented as moderated logarithmic fold change (mod_lfc) (blue= downregulated in mutants versus Guy11; red= upregulated in mutants versus Guy11. d. Heatmap showing levels of relative transcript abundance between ∆pmk1 versus Guy11 and ∆hox7 versus Guy11 of 1942 M. oryzae genes. e. Table to show the most representative cellular pathways found among 1942 genes with overlapping expression profiles in ∆pmk1 and ∆hox7. f. Bar charts to show mean normalized counts of gene expression of cyclin, CDK-related, and DNA damage response (DDR) pathway-related genes during appressorium development in Guy11, ∆pmk1, ∆mst12 and ∆hox7 mutants. g. Heatmap to show relative transcript abundance of autophagy-related genes in Δpmk1, ∆mst12 and ∆hox7 mutants compared to Guy11.

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Extended Data Fig. 7 Complementation of Δhox7 mutant and ChIP–seq analysis.

a. Micrographs showing the extent of ∆hox7 complementation upon re-introduction of HOX7 under control of three native promoter lengths (2 kb, 1.5 kb and 1 kb), as shown in the schematic diagram. Conidia were inoculated on HP surface and incubated for 24 h (Bar = 10 µm). b. Bar chart to show the frequency of appressorium formation and range of aberrant infection structures formed by ∆hox7 mutants expressing HOX7 under control of 1 kb promoter (∆hox7:M11, ∆hox7:M13), 1.5 kb promoter (∆hox7:M24) and 2 kb promoter fragment (∆hox7:M32, ∆hox7:M35), n = 813 conidia examined in 3 biological replicates; each biological replicate is colour coded; the data are presented as Mean ± SEM) (legend X-axis- Re-G.App.N.M.: Re-germinated appressoria, non-melanised; B.M.App.: Branched melanised appressoria; App.: Appressoria). c. Micrographs to show appressorium development by Guy11, ∆hox7 and ∆hox7:Hox7-3xFLAG (expressed under control of 2 kb native promoter fragment). Bar = 10 µm. d. Table to show the total number of DEGs, upregulated genes (padj <0.01; mod-lfc >1) and downregulated genes (padj <0.01; mod-lfc < -1) of ∆pmk1 versus Guy11, ∆mst12 versus Guy11 and ∆hox7 versus Guy11 during mycelium development determined by RNA-seq analysis. e. Table to show over-represented motifs in peaks determined in Hox7 ChIP–seq experiment by FIMO (https://meme-suite.org/meme/doc/fimo.html). Over-representation of each motif in specific sets of DEGs (up and downregulated in mycelium, or expressed during appressorium formation) was analysed using Fisher’s exact test and P values are indicated. The percentage value represents the proportion of the total pool of DEGs represented by that each set of motif-defined gene sets.

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Extended Data Fig. 8 Hox7 conservation across fungi.

a. Diagram to show phosphorylated serine residues identified by phosphoproteomic analysis in Hox7 corresponding to serine 126, 158 and 254. KN represents the PFAM domain homeobox KN (PF05920). b. Phylogenetic neighbour-joining tree to show conservation across fungal and oomycete species Aspergillus nidulans, Phythopthora sojae, Phythopthora ramorum, Neurospora crassa, Cryptococcus neoformans, Saccharomyces cerevisiae, Fusarium graminearun, Magnaporthe oryzae and Podospora anserina. c. Amino acid alignment of HOX7 homologues identified using the Homeobox TFs InterPro term IPR001356 in the Fungal Transcription Factor Database http://ftfd.snu.ac.kr of Aspergillus nidulans, Phythopthora sojae, Phythopthora ramorum, Neurospora crassa, Cryptococcus neoformans, Saccharomyces cerevisiae, Fusarium graminearun, Magnaporthe oryzae and Podospora anserina.

Extended Data Fig. 9 Phosphoproteomic analysis reveals Pmk1-dependent phosphorylation of Hox7 in M. oryzae.

a. Diagram to show experimental design for PRM analysis to measure relative normalized intensities (R. N. I.) of peptides associated with serine 126, serine 158 and serine 254 residues of Hox7. The pmk1as conditional mutant was incubated on HP and germinated for 4 h (Baseline), 6 h and 8 h in the presence (+1NA-PP1 crimson bars) or absence (-1NA-PP1 green bars) of the ATP analogue Naphthyl-PP1. Falcon tubes represent sample collection. b. PRM showing R. N. I. of peptide associated with phosphorylated serine at 126 of Hox7 during appressorium development in pmk1as conditional mutant in the presence or absence of 1NA-PP1. Differences were assessed by a two-tailed unpaired t-test, using 2 biological replicates and 2 technical replicates per biological replicate, Mean ± SD. c. PRM showing R. N. I. of peptide associated with phosphorylated serine at 158 of Hox7 during appressorium development in pmk1as conditional mutant in the presence and absence of NA-PP1. Differences were assessed by a two-tailed unpaired t-test, 8 h (P = 0.0316), 2 biological replicates and 2 technical replicates per biological replicate, Mean ±SD. d. PRM showing R. N. I. of peptide associated with phosphorylated serine at 254 of Hox7 during appressorium development in pmk1as conditional mutant in the presence and absence of NA-PP1. Differences were assessed by a two-tailed unpaired t-test, 2 biological replicates and 2 technical replicates per biological replicate, Mean ±SD. Black asterisks below bar charts indicate that some peptides could not be detected in a biological or technical replicate. Red asterisks correspond to the significance of the P value.

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Extended Data Fig. 10 Pmk1 activation by MEK2DD, phenotypes of Δhox7 mutants expressing phosphomimetic alleles of Hox7 and temporal analysis of HOX7 gene expression.

a. Western blot analysis of in vitro phosphorylation experiment of Pmk1 MAPK (GST- Pmk1)- with Hox7 (6xHis-SUMO-Hox7t-342) and Mst12 (6xHis-MBP-Mst12) in the presence or absence of the activated MEKDD.. Proteins were immunoblotted with appropriate antisera (listed on the right). Arrows indicate expected band sizes. Phosphorylation of the Pmk1 MAPK was detected by α-pTEpY antibody in the presence of MEKDD. His-tagged proteins were detected with α-His antibody. b. Micrographs to show representative phenotypes of germlings of Δhox7 mutants expressing phosphomimetic alleles of Hox7 incubated on HP surfaces for 24 h, as quantified in Fig. 7b (Bar= 10 µm). c. Line graph fo show temporal analysis of HOX7 transcript abundance in wild type Guy11 (blue), and ∆pmk1 (green) and ∆mst12 (purple) mutants during a time course of appressorium development measured by RNA-seq analysis.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Source data for Supplementary Fig. 7. Supplementary Table 7. Gene name, accession number, reported mutant phenotype and reference of each Clade 4 TF. Supplementary Table 11. Peptides used in the PRM assay. Supplementary Table 13. Primers used in this study.

Reporting Summary

Supplementary Table 1

RNA-seq analysis of a time course of appressorium development in Guy11 on an HL versus HP surface.

Supplementary Table 2

RNA-seq analysis of a time course of appressorium development in Guy11 versus ∆pmk1 on an HP surface.

Supplementary Table 3

DEGs as classified in the Venn diagram in Fig. 1d.

Supplementary Table 4

RNA-seq analysis of a time course of appressorium development in Guy11 versus ∆mst12 on an HP surface

Supplementary Table 5

DEGs as classified in the Venn diagram in Fig. 2e.

Supplementary Table 6

RNA-seq and ChIP–seq analysis of mycelium of Guy11 versus ∆mst12.

Supplementary Table 8

RNA-seq analysis of appressorium development in Guy11 versus ∆hox7 on an HP surface at 14h.

Supplementary Table 9

DEGs as classified in the Venn diagram in Fig. 5c.

Supplementary Table 10

RNA-seq and ChIP–seq analysis of mycelium of Guy11 versus the ∆hox7 mutant.

Supplementary Table 12

Spectral count data for Pmk1 in vitro phosphorylation assay of Hox7 and Mst12.

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Source data

Source Data Fig. 3

Numerical data for the graphs in Fig. 3h

Source Data Fig. 5

Numerical data for the graphs in Fig. 5b,c,e–g.

Source Data Fig. 7

Numerical data for the graph in Fig. 7b.

Source Data Extended Data Fig. 1

Numerical data for the graphs in Extended Data Fig. 1b.

Source Data Extended Data Fig. 3

Numerical data for the graphs in Extended Data Fig. 3g–k.

Source Data Extended Data Fig. 5

Numerical data for the graphs in Extended Data Fig. 5a.

Source Data Extended Data Fig. 6

Numerical data for the graphs in Extended Data Fig. 6a.

Source Data Extended Data Fig. 7

Numerical data for the graphs in Extended Data Fig. 7b.

Source Data Extended Data Fig. 9

Numerical data for the graphs in Extended Data Fig. 9b–d.

Source Data Extended Data Fig. 10

Un-cropped gels and autoradiographs for Extended Data Fig. 10a.

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Osés-Ruiz, M., Cruz-Mireles, N., Martin-Urdiroz, M. et al. Appressorium-mediated plant infection by Magnaporthe oryzae is regulated by a Pmk1-dependent hierarchical transcriptional network. Nat Microbiol 6, 1383–1397 (2021). https://doi.org/10.1038/s41564-021-00978-w

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