A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants

  1. Mingming Chen
  2. Naoyoshi Kumakura
  3. Hironori Saito
  4. Ryan Muller
  5. Madoka Nishimoto
  6. Mari Mito
  7. Pamela Gan
  8. Nicholas T Ingolia
  9. Ken Shirasu
  10. Takuhiro Ito
  11. Yuichi Shichino
  12. Shintaro Iwasaki  Is a corresponding author
  1. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Japan
  2. RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Japan
  3. Plant Immunity Research Group, RIKEN Center for Sustainable Resource Science, Japan
  4. Department of Molecular and Cell Biology, University of California, Berkeley, United States
  5. Laboratory for Translation Structural Biology, RIKEN Center for Biosystems Dynamics Research, Japan
  6. Department of Biological Science, Graduate School of Science, The University of Tokyo, Japan

Abstract

Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier presented by antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocaglates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a eukaryotic translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to the Ophiocordyceps genus and that its eIF4A, a molecular target of rocaglates, harbors an amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered C. orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant–fungus tug-of-war centered on secondary metabolites produced by host plants.

Editor's evaluation

In this important paper, Chen and colleagues identify a species of fungus, Ophiocordyceps sp. BRM1, that is able to grow on Aglaia sp. plants despite their production of rocaglate inhibitors of the eIF4A translation initiation factor. Through a series of convincing experiments, the authors identify an amino acid substitution encoded in the fungal eIF4A gene that preserves eIF4A activity in the presence of these compounds. The authors conclude the substitution evolved to bypass this defense mechanism, similar to the way in which the plant itself bypasses it. The work will be of interest to fungal biologists and colleagues studying plant-microbe interactions.

https://doi.org/10.7554/eLife.81302.sa0

eLife digest

Although plants may seem like passive creatures, they are in fact engaged in a constant battle against the parasitic fungi that attack them. To combat these fungal foes, plants produce small molecules that act like chemical weapons and kill the parasite. However, the fungi sometimes fight back, often by developing enzymes that can break down the deadly chemicals into harmless products.

One class of anti-fungal molecules that has drawn great interest is rocaglates, as they show promise as treatments for cancer and COVID-19. Rocaglates are produced by plants in the Aglaia family and work by targeting the fungal molecule eIF4A which is fundamental for synthesizing proteins. Since proteins perform most of the chemistry necessary for life, one might think that rocaglates could ward off any fungus. But Chen et al. discovered there is in fact a species of fungi that can evade this powerful defense mechanism.

After seeing this new-found fungal species successfully growing on Aglaia plants, Chen et al. set out to find how it is able to protect itself from rocoglates. Genetic analysis of the fungus revealed that its eIF4A contained a single mutation that ‘blocked’ rocaglates from interacting with it. Chen et al. confirmed this effect by engineering a second fungal species (which infects cucumber plants) so that its elF4A protein contained the mutation found in the new fungus. Fungi with the mutated eIF4A thrived on cucumber leaves treated with a chemical derived from rocaglates, whereas fungi with the non-mutated version were less successful.

These results shed new light on the constant ‘arms race’ between plants and their fungal parasites, with each side evolving more sophisticated ways to overcome the other’s defenses. Chen et al. hope that identifying the new rocaglate-resistant eIF4A mutation will help guide the development and use of any therapies based on rocaglates. Further work investigating how often the mutation occurs in humans will also be important for determining how effective these therapies will be.

Introduction

Fungi that infect plants are of great economic relevance because they cause severe crop losses (~10%) worldwide (Oerke, 2006). Therefore, the mechanisms underlying plant–fungus interactions have attracted great interest and have been extensively studied (Lo Presti et al., 2015). Secondary metabolites with antimicrobial activities are among the means naturally developed by plants for the control of fungal infections (Collemare et al., 2019). For example, tomatine, a glycoalkaloid secreted from the leaves and stems of tomato, has both fungicidal properties and insecticidal activities (Vance et al., 1987). Camalexin, an indole alkaloid produced by Brassicaceae plants, including the model plant Arabidopsis thaliana, also has antifungal properties (Nafisi et al., 2007).

However, some fungi can overcome these toxic compounds to infect plants. The best-known strategy is the detoxification of antifungal compounds by the secretion of specific enzymes (Crombie et al., 1986; Osbourn et al., 1995; Pareja-Jaime et al., 2008). Thus, plants and infectious fungi are engaged in an arms race during the course of evolution. However, other than detoxification, the mechanistic diversity of the plant–fungus competition centered on plant secondary metabolites is largely unknown.

Rocaglates, small molecules synthesized in plants of the genus Aglaia, exemplify antifungal secondary metabolites (Engelmeier et al., 2000; Iyer et al., 2020). In addition to its antifungal properties, this group of compounds is of particular interest because of its antitumor activities (Alachkar et al., 2013; Bordeleau et al., 2008; Cencic et al., 2009; Chan et al., 2019; Ernst et al., 2020; Lucas et al., 2009; Manier et al., 2017; Nishida et al., 2021; Santagata et al., 2013; Skofler et al., 2021; Thompson et al., 2021; Thompson et al., 2017; Wilmore et al., 2021; Wolfe et al., 2014). Moreover, recent studies have suggested potency against viruses such as SARS-CoV-2 (Müller et al., 2021; Müller et al., 2020) and hepatitis E virus (Praditya et al., 2022). Rocaglates target translation initiation factor (eIF) 4A, a DEAD-box RNA binding protein, and function as potent translation inhibitors with a unique mechanism: rocaglate treatment does not phenocopy the loss of function of eIF4A but instead leads to gain of function. Although eIF4A activates the translation of cellular mRNA through ATP-dependent RNA binding, rocaglates impose polypurine (A and G repeated) sequence selectivity on eIF4A, bypassing the ATP requirements and evoking mRNA-selective translational repression (Chen et al., 2021; Chu et al., 2020; Chu et al., 2019; Iwasaki et al., 2019; Iwasaki et al., 2016; Rubio et al., 2014; Wolfe et al., 2014). The artificial anchoring of eIF4A (1) becomes a steric hindrance to scanning 40S ribosomes (Iwasaki et al., 2019; Iwasaki et al., 2016), (2) masks cap structure of mRNA by tethering eIF4F (Chu et al., 2020), and (3) reduces the available pool of eIF4A for active translation initiation events by the sequestration of eIF4A on mRNAs (Chu et al., 2020).

Since eIF4A is an essential gene for all eukaryotes, Aglaia plants must have a mechanism to evade the cytotoxicity of the rocaglates they produce. This self-resistance is achieved by the unique amino acid substitutions at the sites in eIF4A proteins where rocaglates directly associate (Iwasaki et al., 2019). Given the high evolutionary conservation of eIF4A and thus its rocaglate binding pocket (Iwasaki et al., 2019), these compounds may target a wide array of natural fungi.

Irrespective of the antifungal nature of rocaglates, we found a parasitic fungus able to grow on Aglaia plants. De novo transcriptome analysis from the fungus revealed that this species belongs to the Ophiocordyceps genus, whose members infect ants and cause a 'zombie' phenotype (Andersen et al., 2009; Araújo and Hughes, 2019; de Bekker et al., 2017), but constitutes a distinct branch in the taxon. Strikingly, eIF4A from this fungus possessed an amino acid substitution in the rocaglate binding site and thus showed resistance to the compound. Using Colletotrichum orbiculare, a cucumber-infecting fungus, as a model, we demonstrated that the genetically engineered fungus with the substitution showed insensitivity to the translational repression induced by rocaglates, facilitating its infection of plants even in the presence of this compound. Our results indicate fungal resistance to plant secondary metabolites independent of detoxification enzymes and a unique contest between plants and fungi centered on secondary metabolites synthesized in the host plant.

Results

Identification of a fungal parasite on the rocaglate-producing plant Aglaia

Considering that Aglaia plants possess antifungal rocaglates (Engelmeier et al., 2000; Iyer et al., 2020), parasitic fungi should have difficulty infecting rocaglate-producing plants. In contrast to this idea, we identified a fungus growing on the surface of the stem of the Aglaia odorata plant with tremendous vitality (Figure 1A). To characterize this fungus, we isolated the RNA, conducted RNA sequencing (RNA-Seq), reconstructed the transcriptome, and annotated the functionality of each gene (Supplementary file 1).

Figure 1 with 5 supplements see all
Identification of Aglaia-parasitic Ophiocordyceps sp. BRM1.

(A) Image of a parasite fungus growing on Aglaia odorata. (B) Multilocus phylogenetic tree of Ophiocordyceps species generated from maximum likelihood phylogenetic analysis of ITS, SSU, LSU, RPB1, and TEF1α sequences. Tolypocladium species were used as outgroups. The best DNA substitution models of ITS, LSU, SSU, RPB1, and TEF1α were calculated as TIM3ef + G4, TIM1 + I + G4, TIM3ef + I + G4, TrN + I + G4, and TIM1 + I + G4, respectively. Numbers on branches are percent support values out of 1000 bootstrap replicates. Only bootstrap values greater than 50% support are shown. Endophytes are highlighted with green dots.

Figure 1—source data 1

Files for the full and unedited pictures corresponding to Figure 1A.

https://cdn.elifesciences.org/articles/81302/elife-81302-fig1-data1-v1.zip

This Aglaia-infecting fungus belonged to the Ophiocordyceps genus, which is known as zombie-ant fungus (Andersen et al., 2009; Araújo and Hughes, 2019; de Bekker et al., 2017). Ophiocordyceps spp. are members of the phylum Ascomycota and constitute the taxonomic groups with the highest number of entomopathogenic species among all fungal genera. In most cases, each Ophiocordyceps spp. has a specific host insect species, develops fruiting bodies from the remains of host insects, and produces spores. In addition to insect infection, a moth parasite, Ophiocordyceps sinensis, has been found to reside on many plant species, suggesting that Ophiocordyceps also has an endophytic lifestyle (Wang et al., 2020; Zhong et al., 2014). We performed a BLASTn search (Camacho et al., 2009) using the internal transcribed spacer (ITS) between rRNAs as a query and found that, among all the deposited nucleotide sequences in the database, 29 of the top 30 hits were from Ophiocordyceps species (Supplementary file 2).

To identify the species-level taxon of the Aglaia-infecting fungus, we conducted a multilocus phylogenetic analysis for comparison with currently accepted species in the Ophiocordyceps genus (Supplementary file 3). For this purpose, sequences of the ITS, small subunit ribosomal RNA (SSU), large subunit rRNA (LSU), translation elongation factor 1-alpha (TEF1α), and RNA polymerase II largest subunit (RPB1) were used as previously reported for the classification of Ophiocordyceps species (Xiao et al., 2017; Supplementary file 3). These sequences from 68 isolates were aligned, trimmed, and concatenated, resulting in a multiple sequence alignment comprising 3910 nucleotide positions, including gaps (gene boundaries ITS, 1–463; LSU, 464–1363; SSU, 1364–2248; RPB1, 2249–2922; TEF1α, 2923–3910). Then, the best-scoring maximum likelihood (ML) tree was generated from the concatenated sequence alignment using the selected DNA substitution models for each sequence (Figure 1B). This analysis indicated that the Aglaia-infecting fungus was distinct from the other species of Ophiocordyceps (Figure 1B). In particular, the strain isolated from Aglaia was positioned on a long branch separated from the most closely related strain, O. coccidiicola NBRC 100682, as supported by the 95% bootstrap value. The separation of Aglaia-infecting fungus from other Ophiocordyceps species was also confirmed by single-locus alignments (Figure 1—figure supplements 15), although the positions of the Aglaia-infecting fungus in the tree were different. We note that RPB1-locus alignment was an exception since the de novo-assembled transcriptome from the Aglaia-infecting fungus lacked the sequence of the homolog.

Given that the most closely related Ophiocordyceps species is sufficiently distinct from the Aglaia-infecting fungus in sequence and that no similar fungi grown in Aglaia plants were reported before, we named the fungus Ophiocordyceps sp. BRM1 (Berkeley, Ryan Muller, strain 1). Consistent with its isolation from the Aglaia plant, this fungus was closely related to known endophytic Ophiocordyceps spp. (Figure 1B and Supplementary file 3; Wang et al., 2020; Zhong et al., 2014).

Transcriptome assembly uncovers the unique mutation in eIF4A of the Aglaia-infecting fungus

The parasitic nature of Ophiocordyceps sp. BRM1 on plants producing the antifungal rocaglate led us to hypothesize that the fungus may have a mechanism to evade the toxicity of the compounds. Indeed, the host plant Aglaia achieves this task by introducing an amino acid substitution in eIF4A, a target of rocaglates (Iwasaki et al., 2019). The substituted amino acid (Phe163, amino acid position in human eIF4A1) lies at the critical interface for rocaglate interaction (Figure 2A and D; Iwasaki et al., 2019). Accordingly, we investigated possible amino acid conversions in eIF4As of the Ophiocordyceps sp. BRM1. Among the de novo-assembled transcriptome, ~60 DEAD-box RNA binding protein genes, including 4 transcript isoforms of eIF4A, were found (Supplementary file 1).

Figure 2 with 3 supplements see all
The effect of an amino acid substitution found in Ophiocordyceps sp. BRM1 eIF4A on RocA-mediated polypurine RNA clamping.

(A, B) Alignments of eIF4A protein sequences from higher eukaryotes (A) and fungal species (B), including the de novo-assembled Ophiocordyceps sp. BRM1 eIF4A gene with four transcript isoforms (iso). (C) The summary of Kd determined by fluorescence polarization assay in Figure 2—figure supplement 2A–H is depicted. WT and mutated eIF4A proteins from the indicated species were used. To measure ATP-independent RNA clamping induced by RocA (50 µM), ADP and Pi (1 mM each) were included in the reaction. The data are presented as the mean and s.d. values. (D) RocA (sphere model with light pink-colored carbons), the modeled His, Gly, and Leu residues (surface model with cyan-colored carbons) at the Phe163 residue in human eIF4A1 (surface model with green-colored carbons), and RNA (surface model with yellow-colored carbons) in the complex of human eIF4A1•RocA•AMP-PNP•polypurine RNA (PDB: 5ZC9) (Iwasaki et al., 2019).

Remarkably, we observed an amino acid conversion in Ophiocordyceps sp. BRM1 eIF4A at the same residue as in the Aglaia plant eIF4A. A Gly residue replaced Phe163 (human position) in all four transcript isoforms (from the same eIF4A gene) in Ophiocordyceps sp. BRM1 (Figure 2B, Figure 2—figure supplement 1A), whereas His residues prevailed in the close kin of Ophiocordyceps species and other fungi.

Gly153 in Ophiocordyceps sp. BRM1 eIF4A eliminated rocaglate-mediated polypurine RNA clamping

Indeed, we found that the Gly substitution confers rocaglate resistance on eIF4A. To investigate rocaglate-targetability, we harnessed the fluorescence polarization assay with fluorescein (FAM)-labeled short RNA and purified recombinant eIF4A proteins (Figure 2—figure supplement 1B). As observed previously (Chen et al., 2021; Chu et al., 2020; Chu et al., 2019; Iwasaki et al., 2019; Iwasaki et al., 2016; Naineni et al., 2021), rocaglamide A (RocA), a natural rocaglate derivative isolated from Aglaia plants (Figure 2—figure supplement 1C; Janprasert et al., 1992), clamped human eIF4A1 on polypurine RNA ([AG]10) in an ATP-independent manner (e.g., in the presence of ADP + Pi) (Kd = ~0.42 µM, Figure 2C, left, Figure 2—figure supplement 2A, and Table 1). Whereas a high affinity for polypurine RNA was observed for eIF4A from O. sinensis (CO18 GCA 000448365) (Kd = ~0.11 µM, Figure 2C middle; Figure 2—figure supplement 2D; and Table 1) — the closest relative among whole-genome-sequenced Ophiocordyceps species (Figure 1B; de Bekker et al., 2017), Ophiocordyceps sp. BRM1 eIF4A showed a fairly high Kd (~17 µM, Figure 2C, right, Figure 2—figure supplement 2H, and Table 1).

Table 1
Summary of Kd (µM) between eIF4A protein and RNAs.

A fluorescence polarization assay between FAM-labeled RNA ([AG]10) and the indicated recombinant proteins was conducted to measure Kd in the presence of DMSO, RocA, or aglafoline. ND, not determined.

[AG]10
ADP + PiAMP-PNP
ProteinDMSORocAAglafolineDMSORocA
H. sapiens WT Phe1630.42 ± 0.06111 ± 2.90.067 ± 0.023
H. sapiens Phe163His4.0 ± 0.7116 ± 2.70.11 ± 0.025
H. sapiens Phe163Gly14 ± 2.021 ± 6.70.58 ± 0.13
O.sinensis WT His1540.11 ± 0.02241 ± 110.090 ± 0.014
O.sinensis His154Gly0.85 ± 0.1227 ± 7.00.37 ± 0.046
Ophiocordyceps sp. BRM1 Gly172PheND0.27 ± 0.0500.11 ± 0.021110 ± 580.053 ± 0.023
Ophiocordyceps sp. BRM1 Gly172HisND3.9 ± 0.981.5 ± 0.143.3 ± 0.830.051 ± 0.0091
Ophiocordyceps sp. BRM1 WT Gly172ND17±7.42.6±0.407.1±2.30.23±0.050

Given the Gly substitution in Ophiocordyceps sp. BRM1 eIF4A (Figure 2A), we hypothesized that this amino acid substitution explains the differential sensitivity to RocA. Indeed, both the Gly-to-Phe (human) and Gly-to-His (O. sinensis) substitutions in Ophiocordyceps sp. BRM1 eIF4A (Gly172Phe and Gly172His, respectively) sensitized the protein to RocA (Figure 2—figure supplement 2F and G), significantly reducing the Kd (Figure 2C, right, and Table 1). Conversely, introduction of a Gly residue into human and O. sinensis eIF4As reduced the affinity for polypurine RNA (Figure 2C, left, middle; Figure 2—figure supplement 2C and E, and Table 1).

A similar rocaglate sensitivity in RNA binding was also observed for adenylyl-imidodiphosphate (AMP-PNP), a ground-state ATP analog (Figure 2—figure supplement 3A–H and Table 1). Unlike ADP + Pi, the nonhydrolyzable analog AMP-PNP allowed basal binding to polypurine RNAs in the absence of RocA. The affinity was further increased by RocA in eIF4A with a Phe or His residues at 163 (human position). In contrast, the proteins with the Gly residue showed the relatively small affinity changes (Figure 2—figure supplement 3I and Table 1).

Taking these biochemical data together, we concluded that the Ophiocordyceps sp. BRM1 eIF4A evades rocaglate targeting by substituting a critical amino acid involved in its binding. When Phe163 was replaced by Gly in the crystal structure of the human eIF4A1•RocA complex (Iwasaki et al., 2019), the π-π stacking with ring C of RocA was totally lost (Figure 2D and Figure 2—figure supplement 1C), likely leading to reduced affinity for RocA. This mechanism to desensitize eIF4A to rocaglates was distinct from the Leu substitution found in Aglaia, which fills the space of the rocaglate binding pocket and thus prevents the interaction (Iwasaki et al., 2019).

Our data showed that eIF4A with His at position 163 (human position) is also a target of rocaglate (Figure 2C, Figure 2—figure supplement 2, Figure 2—figure supplement 3, and Table 1). This is most likely due to the functional replacement of the aromatic ring in Phe by the imidazole ring in His for stacking with ring C of rocaglates (Figure 2D). Although compared to the Phe substitution, the His substitution in human and Ophiocordyceps sp. BRM1 eIF4As was accompanied by an attenuated potency of RocA (Figure 2C, Figure 2—figure supplement 2, Figure 2—figure supplement 3, and Table 1), our data suggested that a wide array of fungi that possess the His variant (Figure 2A), including C. orbiculare (see below for details), are also susceptible to rocaglates.

Gly153 found in Ophiocordyceps sp. BRM1 eIF4A confers resistance to rocaglate-induced translational repression

The reduced affinity to polypurine RNA gained in the Ophiocordyceps sp. BRM1 eIF4A by Gly substitution led us to investigate the impact on rocaglate-mediated translational repression. To test this, we applied a reconstituted translation system with human factors (Iwasaki et al., 2019; Machida et al., 2018; Yokoyama et al., 2019). As observed in an earlier study (Iwasaki et al., 2019), this system enabled the recapitulation of translation reduction from polypurine motif-possessing reporter mRNA but not from mRNA with control CAA repeats in a RocA dose-dependent manner (Figure 3A). In contrast, replacing wild-type human eIF4A1 with the Phe163Gly mutant prevented the translation repression mediated by RocA (Figure 3A), consistent with the affinity between the recombinant human eIF4A1 proteins and polypurine RNA (Figure 2C, Figure 2—figure supplement 2, Figure 2—figure supplement 3, and Table 1).

Figure 3 with 3 supplements see all
The amino acid substitution in the Ophiocordyceps sp. BRM1 eIF4A confers translational resistance to rocaglates in fungi.

(A) RocA-mediated translational repression recapitulated by an in vitro reconstitution system with human factors. Recombinant proteins of H. sapiens eIF4A1 WT or Phe163Gly were added to the reaction with RocA. Reporter mRNA with CAA repeats or polypurine motifs was translated in the reaction. The data are presented as the mean and s.d. values (n = 3). (B) Translation of complex-preformed mRNAs to test the RocA gain of function. Recombinant proteins of Ophiocordyceps sp. BRM1 eIF4A1 WT or the Gly172His mutant were preincubated with the reporter mRNA possessing polypurine motifs in the presence or absence of RocA. After removal of free RocA by gel filtration, the protein-mRNA complex was added to RRL to monitor protein synthesis. The data are presented as the mean and s.d. values (n = 3). (C) MA (M, log ratio; A, mean average) plot of the translation efficiency changes caused by 3 µM aglafoline treatment in C. orbiculare eIF4AWT conidia. Resistant and sensitive mRNAs (FDR < 0.05) are highlighted. (D) Cumulative distribution of the translation efficiency changes in aglafoline-sensitive mRNAs (defined in C) in C. orbiculare eIF4AWT conidia treated with 0.3 or 3 µM aglafoline. (E) Cumulative distribution of the translation efficiency changes in aglafoline-sensitive mRNAs (defined in C) induced by 3 µM aglafoline treatment in C. orbiculare eIF4AWT and eIF4AHis153Gly conidia. (F) Cumulative distribution of the global translation alterations, which are footprint changes normalized to mitochondrial footprints, in aglafoline-sensitive mRNAs (defined in C) induced by 3 µM aglafoline treatment in C. orbiculare eIF4AWT and eIF4AHis153Gly conidia. (G) Box plot of the translation efficiency changes caused by 3 µM aglafoline treatment in conidia across mRNAs with or without an [A/G]6 motif in the 5′ UTR. The p values in (D–G) were calculated by the Mann–Whitney U test.

Given that rocaglate-mediated translational repression is driven by a gain of function (Chen et al., 2021; Iwasaki et al., 2016; Iwasaki et al., 2019), Ophiocordyceps sp. BRM1 eIF4A should not have this mode. To investigate this possibility, we used a preformed RocA-eIF4A-mRNA complex for the translation reaction (Iwasaki et al., 2019; Iwasaki et al., 2016). We first preincubated recombinant Ophiocordyceps sp. BRM1 eIF4A or the corresponding Gly172His mutant protein with a reporter mRNA possessing polypurine motifs in the presence or absence of RocA. If RocA could target the eIF4A protein, eIF4A should be stably clamped on the polypurine tract, providing steric hindrance to scanning ribosomes and thus repressing protein synthesis in rabbit reticulocyte lysate (RRL). Whereas WT Ophiocordyceps sp. BRM1 eIF4A could not alter translation (Figure 3B, left) due to its weaker ability to clamp on polypurine RNAs, the Gly172His mutant could act as a translation repressor (Figure 3B, right). These data indicated that Gly172His in Ophiocordyceps sp. BRM1 eIF4A restores the gain-of-function mechanism of RocA.

We further tested the impact of the Gly conversion in eIF4A in a fungus. Due to the difficulty of culturing and manipulating the genetics of Ophiocordyceps sp. BRM1 (data not shown), we instead harnessed C. orbiculare, an anthracnose-causing fungus (Gan et al., 2019; Gan et al., 2013). Through homology-directed repair induced by CRISPR–Cas9-mediated genome cleavage, we replaced endogenous eIF4A with wild-type (WT) or Gly-mutated (His153Gly) C. orbiculare eIF4A (Figure 2—figure supplement 1A, Figure 3—figure supplement 1A and B). Notably, we did not find any significant growth defects resulting from these genetic manipulations (Figure 3—figure supplement 1C and D).

Since the culture of the isolated strains requires a significant amount of the compounds, we used aglafoline (methyl rocaglate) (Figure 2—figure supplement 1C), a less expensive, commercially available natural derivative of rocaglates (Ko et al., 1992), instead of RocA. The difference between RocA and aglafoline is the dimethylamide group versus the methoxycarbonyl group (Figure 2—figure supplement 1C), which do not contribute to the association with eIF4A or polypurine RNA (Iwasaki et al., 2019), suggesting that the compounds should have similar mechanisms of action. As expected, aglafoline resulted in essentially the same molecular phenotype of ATP-independent polypurine clamping of the Ophiocordyceps sp. BRM1 eIF4A (Figure 3—figure supplement 1E–H and Table 1) as RocA (Figure 2C, right, Figure 2—figure supplement 2F–H; and Table 1).

To understand the translational repression induced by aglafoline in a genome-wide manner, we applied ribosome profiling, a technique based on deep sequencing of ribosome-protected RNA fragments (i.e., ribosome footprints) generated by RNase treatment (Ingolia et al., 2019; Ingolia et al., 2009; Iwasaki and Ingolia, 2017), to the isolated fungus strains. The ribosome footprints obtained from C. orbiculare (in the culture of conidia and mycelia) showed the signatures of this experiment: two peaks of footprint length at ~22 nt and ~30 nt (Figure 3—figure supplement 2A), which respectively represent the absence or presence of A-site tRNA in the ribosome (Lareau et al., 2014; Wu et al., 2019), and 3-nt periodicity along the open reading frame (ORF) (Figure 3—figure supplement 2B and C). By normalizing the footprint reads by the RNA abundance as measured by RNA-Seq, we calculated the translation efficiency and quantified its change induced by aglafoline treatment.

Strikingly, this genome-wide approach revealed that His153Gly confers translational resistance to aglafoline on C. orbiculare. Consistent with the mRNA-selective action of the compound, we observed that a subset of mRNAs showed high aglafoline sensitivity in terms of translation efficiency in conidia (Figure 3C) and that the reduction was compound dose dependent (Figure 3D). Intriguingly, we observed that genes associated with the ribosome and its assembly were susceptible to rocaglate-mediated translational repression (Figure 3—figure supplement 3A). The reduction in translation efficiency mediated by aglafoline was attenuated by the His153Gly substitution (Figure 3E). This conclusion was also supported by global translation assessment (Figure 3F), which is based on cytosolic ribosome footprint alterations normalized to the mitochondrial footprints as internal spike-ins (Iwasaki et al., 2016). Consistent with earlier reports (Chen et al., 2021; Chu et al., 2020; Chu et al., 2019; Iwasaki et al., 2016; Iwasaki et al., 2019), the reduction in translation efficiency was associated with the presence of polypurine motifs in the 5′ UTR (Figure 3G). However, the His153Gly substitution compromised the polypurine-dependent translational repression. Although the mycelial stage of the fungus showed the similar trends in translational repression mediated by aglafoline (Figure 3—figure supplement 3B–F), the sensitive mRNAs were distinct from those in conidia (Figure 3—figure supplement 3G), suggesting differential impacts of rocaglates during the fungal life cycle.

Rocaglate-resistant fungi show an advantage in infection of plants with rocaglates

We were intrigued to test the role of Gly substitution in the parasitic property of fungi. Here, we used the infection process of C. orbiculare on cucumber leaves as a model system. The conidia of WT or His153Gly eIF4A-recombined strains were sprayed on Cucumis sativus (cucumber) cotyledons, and the biomass after inoculation with rocaglate was quantified (Figure 4A). Indeed, aglafoline reduced the biomass of the WT eIF4A-recombined strain on cucumber leaves, showing the antifungal effect of rocaglate (Figure 4B). In stark contrast, the His153Gly mutation in eIF4A affected fungal growth on cucumber leaves and resulted in rocaglate resistance in the fungi (Figure 4B). We note that the differential biomass of C. orbiculare could not be explained by the damage to cucumber leaves by aglafoline treatment as no morphological alteration of the leaves was observed under our conditions (Figure 4—figure supplement 1A). These results demonstrated that the Gly substitution found in Ophiocordyceps sp. BRM1 eIF4A provides the molecular basis of antirocaglate properties and allows the growth of the parasitic fungus in the presence of rocaglate (Figure 5).

Figure 4 with 1 supplement see all
Phenotypic comparison of the C. orbiculare eIF4AWT and eIF4AHis153Gly strains during infection in the presence of rocaglate.

(A) Workflow for monitoring the biomass of C. orbiculare eIF4AWT or eIF4AHis153Gly strains on cucumber leaves under treatment with aglafoline. (B) Comparison of in planta fungal biomass of C. orbiculare eIF4AWT or eIF4AHis153Gly strains with or without treatment with 1 µM aglafoline. Relative expression levels of the C. orbiculare 60 S ribosomal protein L5 gene (GenBank: Cob_v012718) normalized to that of a cucumber cyclophilin gene (GenBank: AY942800.1) were determined by RT–qPCR at 3 dpi (n = 8). The relative fungal biomasses of C. orbiculare were normalized to those of eIF4AWT without aglafoline. Significance was calculated by Student’s t-test (two-tailed). Three independent experiments showed similar results.

Figure 5 with 1 supplement see all
Model of the plant–fungus arms race evoked by rocaglates.

The ancestors of the Aglaia plants may have been subjected to fungal infection. To counteract this, Aglaia plants may have developed rocaglates to target the conserved translation factor eIF4A and to suppress in planta fungal growth. Simultaneously, Aglaia plants exhibit amino acid substitutions in the rocaglate binding pocket of eIF4As to prevent self-poisoning. Some fungi may impede rocaglate toxin by converting eIF4A to a rocaglate-insensitive form, enabling them to parasitize these plants.

Discussion

Since plants often produce antifungal secondary metabolites, a specific compound in the host plant may define the interaction between that plants and parasitic fungi (Pusztahelyi et al., 2015). The antifungal activity of rocaglates may protect Aglaia plants from phytopathogenic fungi (Figure 5, top and middle). Rocaglate may suppress protein synthesis from survival-essential genes such as translation machinery. To survive the presence of rocaglate, which targets the general translation initiation factor eIF4A, this plant adapts eIF4A through specific amino acid substitutions (Phe163Leu-Ile199Met: hereafter, we use the human position to specify amino acid residues) to evade the toxicity of the compounds (Iwasaki et al., 2019). This study showed that the parasitic fungus Ophiocordyceps sp. BRM1, which possibly originates from Ophiocordyceps spp. with an endophytic life stage, on Aglaia could also overcome this barrier by introducing an amino acid conversion (Phe163Gly) in eIF4A (Figure 5, bottom). Our results highlighted a tug-of-war between host plants and parasitic fungi through the production of translation inhibitory compounds and mutagenization in the target translation factor.

The molecular basis of secondary metabolite resistance in Ophiocordyceps sp. BRM1 is markedly distinct from the known strategies developed in other fungi. Avenacin from oats — an example of a plant-secreted antimicrobial substance (Morrissey and Osbourn, 1999) — is a triterpenoid that forms complexes with sterols in fungal cell membranes, causes a loss of membrane integrity, and thus exerts an antifungal effect (Armah et al., 1999; Osbourn et al., 1994). To counteract this compound and infect oats, the phytopathogenic fungus Gaeumannomyces graminis var. avenae (Gga) secretes avenaciase (Crombie et al., 1986; Osbourn et al., 1995), a β-glycosyl hydrolase that hydrolyzes terminal D-glucose in the sugar chain of avenacin. Indeed, avenacin degradation by this enzyme determines the host range of the fungus (Bowyer et al., 1995). In contrast to the detoxification strategy, Ophiocordyceps sp. BRM1 may cope with rocaglates through desensitization of the target protein eIF4A by an amino acid substitution (Figure 2), leaving the compound intact.

The different resistance mechanisms to toxic small molecules should be highly related to the compound targets. Since sterols targeted by avenacin are biosynthesized via complicated multiple steps with diverse enzymes, thus generating diverse sterol structures, the conversion of target sterols to evade avenacin requires many enzyme modifications and occurs only rarely. On the other hand, the target of rocaglates is an eIF4A protein (and a DDX3 protein, see below for details), and thus, evasion by a single amino acid mutation is relatively likely. These results exemplify the mechanistic diversity of attack and counterattack during plant–fungal pathogen interactions.

Although we observed that Gly163 in Ophiocordyceps sp. BRM1 eIF4A produced a substantial change in sensitivity to rocaglate, the resistance may not be as complete as that obtained by the substitution found in Aglaia Phe163Leu (Iwasaki et al., 2019). Additionally, the translation factor DDX3, which was recently found to be an alternative target of rocaglate (Chen et al., 2021), did not have amino acid substitutions in Ophiocordyceps sp. BRM1 (Figure 5—figure supplement 1A), whereas Aglaia DDX3s harbor a substitution at Gln360 (Chen et al., 2021). This may indicate that Ophiocordyceps sp. BRM1 is still in the process of evolving fitness for growth in Aglaia plants. Alternatively, the rocaglate-resistant amino acid conversions may involve a trade-off with the basal translation activity. Even with the inefficiency in translation, given that other fungi could not use the resources from the plant, this substitution may still be beneficial to fungi because of the lack of competition from other fungal species. These possibilities are not mutually exclusive.

Materials and methods

RNA-Seq and de novo transcriptome assembly of Ophiocordyceps sp. BRM1

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Fungi on the stem of A. odorata (grown in Berkeley, CA) were harvested and subjected to RNA extraction with hot phenol. After further chloroform extraction, RNA was subjected to rRNA depletion by a Ribo-Zero Gold rRNA Removal Kit (Yeast) (Illumina). The RNA-Seq library was generated by a TruSeq Stranded mRNA Kit (Illumina) and sequenced by HiSeq4000 (Illumina) with a paired-end 100 bp option. Notably, reads from rRNA genes (i.e., internal transcribed spacer [ITS]) remained after rRNA depletion and were used for phylogenetic analysis.

Transcriptome assembly and functional annotation were performed as described previously (Iwasaki et al., 2019) using Trinity (Grabherr et al., 2011) and Trinotate (Haas et al., 2013). The eIF4A and DDX3 homologous sequences were aligned with MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) and depicted by ESPript 3.0 (Robert and Gouet, 2014; http://espript.ibcp.fr/ESPript/ESPript/). eIF4A and DDX3 homologous sequences of model species were obtained from UniProt. For Ophiocordyceps species, Tolypocladium species, and C. orbiculare, the ORF databases were obtained from EnsemblFungi (https://fungi.ensembl.org/index.html) or the Ohm laboratory (http://fungalgenomics.science.uu.nl; de Bekker et al., 2017). To survey the eIF4A and DDX3 homologs, the closest homologs of all the proteins in each species were searched with BLASTp (Camacho et al., 2009; https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/).

Phylogenetic analysis

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To identify the genus of the Aglaia-infecting fungus, closely related species were predicted. The de novo-assembled transcriptome sequence of the Aglaia-infecting fungus was searched by BLASTn (Camacho et al., 2009; https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) using the C. aotearoa ICMP 18537 ITS sequence (GenBank accession: NR_120136) (Schoch et al., 2014) as a query. Using the best hit sequence as a query, a BLASTn search was performed against the NCBI nucleotide collection (nr/nt) (Supplementary file 2).

A multilocus phylogenetic analysis of the Aglaia-infecting fungus with Ophiocordyceps species was performed. A total of 68 isolates were used for phylogenetic analysis, including an Aglaia-infecting fungus, 63 previously classified Ophiocordyceps strains consisting of 52 species, and 4 Tolypocladium species that were expected to serve as outgroups (Supplementary file 3). DNA sequences of ITS, SSU, LSU, TEF1α, and RPB1 were used as previously reported for the classification of Ophiocordyceps species (Xiao et al., 2017). Additional genomic sequences of Ophiocordyceps species identified by BLASTn were added to the analysis (Supplementary file 3). A phylogenetic tree was calculated as previously described (Gan et al., 2017; Xiao et al., 2017). Each locus (ITS, LSU, SSU, RPB1, and TEF1α) of the 68 isolates (Ban et al., 2015; Castlebury et al., 2004; Chen et al., 2013; de Bekker et al., 2017; Hu et al., 2013; Kepler et al., 2012; Liu et al., 2002; Luangsa-Ard et al., 2011; Luangsa-Ard et al., 2010; Quandt et al., 2018; Quandt et al., 2014; Sanjuan et al., 2015; Schoch et al., 2012; Spatafora et al., 2007; Sung et al., 2007; Wen et al., 2013; Will et al., 2020; Xiao et al., 2017) was aligned using MAFFT v7.480 (Katoh and Standley, 2013) and trimmed by trimAl v1.4.rev15 (Capella-Gutiérrez et al., 2009) with an automated setting. The processed sequences obtained from every 68 isolates were concatenated by catfasta2phyml v1.1.0 (Nylander, 2018; https://github.com/nylander/catfasta2phyml) to generate sequences comprising 3910 nucleotide positions, including gaps (gene boundaries ITS, 1–463; LSU, 464–1363; SSU, 1364–2248; RPB1, 2249–2922; TEF1α, 2923–3910). The best model for nucleotide substitutions under the BIC criterion was determined by ModelTest-NG v.0.1.6 (Darriba et al., 2020; https://github.com/ddarriba/modeltest) as follows: ITS, TIM3ef + G4; LSU, TIM1 + I + G4; SSU, TPM3 + I + G4; RPB1, TIM1 + I + G4; and TEF1α, TrN + I + G4. Then, the maximum likelihood phylogeny was estimated based on concatenated sequences by RAxML-NG v.0.9.0 (Kozlov et al., 2019; https://github.com/amkozlov/raxml-ng) using the ModelTest-NG specified best models for each partition with 1000 bootstrap replicates. The best-scoring maximum likelihood trees with bootstrap support values were visualized in iTOL v6 (Letunic and Bork, 2021; https://itol.embl.de/). Given sufficient separation from other known Ophiocordyceps, the fungus was named Ophiocordyceps sp. BRM1.

Compounds

RocA (Sigma-Aldrich) and aglafoline (MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO) and used for this study.

Plasmid construction

pColdI-H. sapiens eIF4A1 WT, Phe163Gly, and Phe163His

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pColdI-H. sapiens eIF4A1 WT has been reported previously (Iwasaki et al., 2019). Phe163Gly and Phe163His substitutions were induced by site-directed mutagenesis.

pColdI-O. sinensis eIF4A WT and His154Gly

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DNA fragments containing the O. sinensis eIF4A gene were synthesized by Integrated DNA Technologies (IDT) and inserted into pColdI (TaKaRa) downstream of the His tag with In-Fusion HD (TaKaRa). The His154Gly substitution was induced by site-directed mutagenesis.

pColdI-Ophiocordyceps sp. BRM1 eIF4A iso4 WT, Gly172His, and Gly172Phe

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The cDNA library of Ophiocordyceps sp. BRM1 was reverse-transcribed with ProtoScript II Reverse Transcriptase (New England Biolabs) and Random Primer (nonadeoxyribonucleotide mix: pd(N)9) (TaKaRa) from the total RNA of Ophiocordyceps sp. BRM1 (see details in the section 'RNA-Seq and de novo transcriptome assembly for Ophiocordyceps sp. BRM1'). Using the cDNA as a template, DNA fragments containing eIF4A iso4 were PCR-amplified and inserted into pColdI (TaKaRa) downstream of the His tag with In-Fusion HD (TaKaRa). The Gly172His and Gly172Phe substitutions were induced by site-directed mutagenesis.

pENTR4-C. orbiculare eIF4A WT and His153Gly

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To replace the eIF4A (GenBank: Cob_v000942) sequence in the C. orbiculare genome with synthesized C. orbiculare eIF4A WT or His153Gly, donor DNAs for homology-directed repair were constructed. DNA fragments, including 2 kb genome sequences upstream and downstream of C. orbiculare eIF4A (as homology arms), the C. orbiculare eIF4A genome sequence, and the neomycin phosphotransferase II (NPTII) expression cassette, were fused into the pENTR4 plasmid (Thermo Fisher Scientific) by HiFi DNA assembly (New England Biolabs). These fragments were PCR-amplified using C. orbiculare genomic DNA, which was isolated from the mycelium, or pII99 plasmid (Namiki et al., 2001). The His153Gly substitution was induced by site-directed mutagenesis. sgRNA-targeted sequences in homology arm sequences were deleted by site-directed deletion to prevent cleavage by CRISPR-Cas9.

Recombinant protein purification

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His-tagged recombinant proteins were purified as described previously (Chen et al., 2021). BL21 Star (DE3) (Thermo Fisher Scientific) cells were transformed with pColdI plasmids (see 'Plasmid construction' section). After the induction of protein expression by isopropyl-β-D-thiogalactopyranoside (IPTG) at 15°C overnight, cells were collected by centrifugation and flash-frozen in liquid nitrogen. Subsequently, the thawed cells were lysed by sonication.

The His-tagged protein was purified by Ni-NTA agarose (QIAGEN). Eluted proteins from beads were then applied to the NGC chromatography system (Bio-Rad). Using a HiTrap Heparin HP column (1 ml, GE Healthcare), proteins were fractionated via an increased gradient of NaCl. The peak fractions were collected, buffer-exchanged with NAP-5 or PD-10 (GE Healthcare) into the storage buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 10% glycerol, and 1 mM dithiothreitol [DTT]), concentrated with a Vivaspin 6 centrifugal concentrator (10 kDa MWCO) (Sartorius), flash-frozen in liquid nitrogen, and stored at −80°C. Proteins in the SDS-PAGE gel were stained with EzStainAQua (ATTO).

Fluorescence polarization assay

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The fluorescence polarization assay was performed as previously described (Chen et al., 2021). The reaction was prepared with 0–25 μM recombinant protein, 10 nM FAM-labeled [AG]10 RNA, 1 mM AMP-PNP (Roche), 1 mM MgCl2, 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol, and 1% DMSO (as a solvent of RocA) with or without 50 μM RocA/aglafoline. After incubation at room temperature for 30 min, the mixture was transferred to a black 384-well microplate (Corning), and the fluorescence polarization was measured by an Infinite F-200 PRO (Tecan). Under ADP + Pi conditions, 1 mM ADP (Fujifilm Wako Chemicals) and 1 mM Na2HPO4 were used as substitutes for AMP-PNP. The data were fitted to the Hill equation to calculate Kd values and visualized by Igor Pro v8.01 (WaveMetrics). The affinity fold change was calculated as the fold reduction in the Kd of RocA compared to the Kd of DMSO.

Reporter mRNA preparation

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The DNA fragments PCR-amplified from psiCHECK2−7×AGAGAG motifs or psiCHECK2-CAA repeats (Iwasaki et al., 2016) were used as a template for in vitro transcription with a T7-Scribe Standard RNA IVT Kit (CELLSCRIPT). RNA was capped and poly(A) tailed with a ScriptCap m7G Capping System, a ScriptCap 2′-O-Methyltransferase Kit, and an A-Plus Poly(A) Polymerase Tailing Kit (CELLSCRIPT).

In vitro translation assay in reconstituted system

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The reconstitution system for human translation has been described previously (Iwasaki et al., 2019; Machida et al., 2018; Yokoyama et al., 2019). The in vitro translation reaction and luciferase assay were performed as previously described (Iwasaki et al., 2019) with some modifications. The final concentrations of mRNA and the eIF4A protein were 60 ng/µl and 2.16 µM, respectively. The translation mixture was incubated for 2.5 hr. The fluorescence signal was detected using the Renilla-Glo Luciferase Assay System (Promega) and measured in an EnVision 2104 plate reader (PerkinElmer).

In vitro translation in RRL with complex-preformed mRNAs

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Preformation of the eIF4A, RocA, and mRNA complex and subsequent in vitro translation in RRL were performed as previously described (Iwasaki et al., 2019; Iwasaki et al., 2016), with modifications. For preformation of the complex containing eIF4A, RocA, and reporter mRNA, 1.4 µM recombinant eIF4A, 90.9 nM reporter mRNA, and 9.1 µM RocA were incubated at 30°C for 5 min in preformation buffer (16.6 mM HEPES-NaOH pH 7.5, 55.3 mM KOAc, 2.8 mM Mg(OAc)2, 1.8 mM ATP, 0.6 mM DTT, and 0.2% DMSO). After supplementation of Mg(OAc)2 to 26.3 mM, 30 µl of the reaction was loaded into a MicroSpin G-25 column (Cytiva) equilibrated with equilibration buffer (30 mM HEPES-NaOH pH 7.5, 100 mM KOAc, 1 mM Mg(OAc)2, and 1 mM DTT), centrifuged at 700 × g for 1 min at 4°C to remove free RocA, and mixed with 2.5 μl of storage buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 10% glycerol, and 1 mM DTT). Then, 4 µl of complex-preformed mRNA was incubated with 50% RRL (Promega) in a 10 µl reaction volume for 1 hr at 30°C, according to the manufacturer’s instructions. The fluorescence signal was detected using the Renilla-Glo Luciferase Assay System (Promega) and measured with the GloMax Navigator System (Promega). In the control experiments, instead of recombinant eIF4A proteins, storage buffer was used. Moreover, recombinant eIF4A proteins were added to the G-25 flowthrough solution in place of the storage buffer.

Fungal transformation

C. orbiculare strain 104-T (NARO GeneBank ID: MAFF 240422), a causal agent of anthracnose disease in Cucurbitaceae plants, was used. The isolated strains in this study are also listed in Supplementary file 4.

Preparation of protoplasts

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C. orbiculare protoplasts were prepared as previously described (Kubo, 1991; Rodriguez and Yoder, 1987; Vollmer and Yanofsky, 1986) with modifications. A frozen glycerol stock of C. orbiculare was streaked on 3.9% (w/v) potato dextrose agar (PDA) medium (Nissui) in a 90 mm dish and incubated at 25°C in the dark for 3 days. Outer edges of a colony were transferred to 20 ml of 2.4% (w/v) potato dextrose broth (BD Biosciences) and incubated for 2 days at 25°C in the dark. The proliferated mycelium was collected using a 70 µm cell strainer (Corning) and incubated in 150 ml of potato-sucrose liquid medium supplemented with 0.2% yeast extract (BD Biosciences) at 25°C with shaking at 140 rpm. The mycelium was harvested, washed with sterile water, and resuspended in 20 ml of filter-sterilized (0.2 µm pore size, GE Healthcare) osmotic medium (1.2 M MgSO4 and 5 mM Na2HPO4) containing 10 mg/ml driselase from Basidiomycetes sp. (Sigma-Aldrich) and 10 mg/ml lysing enzyme from Trichoderma harzianum (Sigma-Aldrich) in a 50 ml tube (Falcon, Corning). The suspension was gently agitated in a rotary shaker at 60 rpm for 90 min at 30°C. Then, the suspension was underlaid with 20 ml of trapping buffer (0.6 M sorbitol, 50 mM Tris-HCl pH 8.0, and 50 mM CaCl2) and centrifuged at 760 × g for 5 min using a swinging-bucket rotor (Hitachi, T4SS31). Protoplasts isolated from the interface of the two layers were pelleted, washed twice using STC (1 M sorbitol, 50 mM Tris-HCl pH 8.0, and 50 mM CaCl2), resuspended in STC at 108–109 protoplasts/ml, added to a 25% volume of polyethylene glycol (PEG) solution (40% [w/w] PEG3350, 500 mM KCl, 40 mM Tris-HCl pH 8.0, and 50 mM CaCl2), and stored at −80°C until use.

gRNA preparation

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Template DNA fragments for sgRNA in vitro transcription were PCR-amplified using the primers listed in Supplementary file 5. Using the DNA fragments, sgRNAs (sgRNAUP-1, sgRNAUP-2, sgRNADW-1, and sgRNADW-2) were prepared with a CUGA7 gRNA Synthesis Kit (Nippon Gene) following the manufacturer’s protocol.

Transformation

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The transformation was performed as previously described (Foster et al., 2018; Kubo, 1991; Yelton et al., 1984) with modifications. The mixture of plasmid DNA (5 µg, pENTR4-C. orbiculare eIF4A WT or His153Gly), the four sgRNAs (250 ng each), and Cas9 nuclease protein NLS (15 µg, Nippon Gene) were added to 150 µl of C. orbiculare protoplasts, followed by the addition of 1 ml of STC and 150 µl of PEG solution. The resulting suspension was incubated for 20 min on ice, supplemented with 500 µl of PEG solution, and gently agitated by hand. The suspension was serially diluted with a second addition of 500 µl, a third addition of 1 ml, and fourth and fifth additions of 2 ml of PEG solution, with gentle agitation at every dilution step. After incubation for 10 min at room temperature, the PEG solution was removed by centrifugation. The protoplasts were resuspended in 1 ml of STC, diluted with 15 ml of regeneration medium (3.12% [w/v] PDA and 0.6 M glucose), and then spread onto a plate containing 40 ml of selection medium (3.9% [w/v] PDA and 0.6 M glucose) containing 200 µg/ml G418 (Fujifilm Wako Chemicals). The plate was incubated for 5 days at 25°C in the dark. The G418-resistant colonies were further seeded in fresh selection medium containing G418 and subjected to selection for an additional 5 d.

Screening by PCR

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Then, the genomic DNA isolated from each colony was subjected to PCR to ensure the desired transformation (see Figure 3—figure supplement 1A for the design). The primers used for the PCR screening are listed in Supplementary file 5. The selected transformed conidia were suspended in 25% glycerol and stored at −80°C until use.

Growth comparison of C. orbiculare strains

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A C. orbiculare strain was seeded into 500 µl of PDA containing 0.04% (v/v) DMSO in one well of a 12-well plate with a toothpick and incubated in the dark at 25°C for 5 days. Colony sizes were measured with a ruler.

C. orbiculare mitochondrial genome assembly

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Reads from three PacBio RSII cells of the C. orbiculare 104-T whole genome sequencing (Gan et al., 2019) were mapped onto C. orbiculare scaffolds that were identified as potential mitochondrial sequences by the NCBI Genomic contamination screen with minimap2 v2.17-r941 (Li, 2018) using the map-bp setting. Aligned fasta reads were then assembled using flye v2.8.1-b1676 (Kolmogorov et al., 2019) with default settings (min overlap = 5000 bp). The assembly (GenBank accession: MZ424187) possessed a 36,318 bp contig with 2023.72× coverage and showed the highest homology to the C. lindemuthianum completed mitochondrial genome (KF953885) according to nucmer (Delcher et al., 2003). These genome data were used for data processing for ribosome profiling.

Ribosome profiling and RNA-Seq

Cell culture; mycelia

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Glycerol stocks of C. orbiculare eIF4AWT#1 and eIF4AH153G#1 strains were streaked on PDA in 90 mm plastic Petri dishes and incubated for 3 days. A single colony of each strain was transferred onto PDA and incubated for 3 days. The outer edges of colonies were transferred to 90 mm plastic dishes filled with 20 ml of PDB using plastic straws and incubated for 4 days. Aglafoline (0.3 or 3 µM) or DMSO was added to dishes and incubated for 6 h.

Cell culture; conidia

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A single colony from the glycerol stocks was cultured by the same method used for mycelium preparation. The outer edges of colonies of each strain were transferred into six 300 ml flasks filled with 100 ml of PDA. Two milliliters of sterilized water was added to each flask, and the flasks were shaken well to ensure that the mycelial cells adhered to the entire surface of the PDA evenly. After 6 days of incubation in the dark, conidia generated on the surface of PDA were suspended in 20 ml of sterilized water. The conidial suspension was filtered through a 100 µm pore-size cell strainer and collected by centrifugation at 760 × g for 5 min at room temperature. Twenty milliliters of resuspended conidia at 0.5 OD600 (approximately 2.5 × 106 conidia/ml) was dispensed in 50 ml ProteoSave SS tubes (Sumitomo Bakelite) and then treated with aglafoline (0.3 or 3 µM) or DMSO for 6 h in the dark with shaking at 140 rpm.

Cell harvest

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Cells were filtered by an MF membrane (0.45 µm pore size, Millipore), immediately scraped from the filter, and soaked in liquid nitrogen for 30 s. Then, 600 µl of lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide, 100 µg/ml chloramphenicol, and 1% Triton X-100) or 400 µl of TRIzol reagent (Thermo Fisher Scientific) was added dropwise into a tube containing the cell pellet and liquid nitrogen to form ice grains for ribosome profiling or RNA-Seq, respectively. The samples were stored at −80°C to evaporate the liquid nitrogen.

Library preparation

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For ribosome profiling, the frozen cells and lysis buffer grains were milled by a Multi-beads Shocker (YASUI KIKAI) at 2800 rpm for 15 s for one cycle. The lysates were thawed on ice and centrifuged at 3000 × g and 4°C for 5 min. The supernatant was treated with 25 U/ml Turbo DNase (Thermo Fisher Scientific) for 10 min and then clarified by centrifugation at 20,000 × g and 4°C for 10 min. Then, the supernatant was used for downstream ribosome profiling library preparation as described previously (Mito et al., 2020). Briefly, the lysates containing 10 µg of total RNA were treated with 20 U RNase I (Lucigen) at 25°C for 45 min. After ribosomes were collected by a sucrose cushion, the RNAs were separated in 15% urea PAGE gels, and the RNA fragments ranging from 17 to 34 nt were excised. Subsequently, the RNAs were dephosphorylated and ligated to linkers. Following rRNA removal with a Ribo-Minus Eukaryotes Kit for RNA-Seq (Thermo Fisher Scientific), the RNA fragments were reverse-transcribed, circularized, and PCR-amplified.

For RNA-Seq, frozen cells with TRIzol grains were lysed in a Multi-beads Shocker instrument at 2800 rpm for 15 s and thawed on ice. Then, 0.5 µg of RNA extracted with a Direct-zol RNA Microprep Kit (Zymo Research) was used for library preparation. Poly(A) selection and cDNA synthesis were performed using an Illumina Stranded mRNA Prep, Ligation (Illumina), and subsequent steps were performed with a TruSeq Stranded Total RNA Library Prep Gold (Illumina).

The final DNA libraries for ribosome profiling and RNA-Seq were sequenced on a HiSeq X (Illumina) with a paired-end 150 bp option.

Data processing

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Sequence data were processed as previously described (McGlincy and Ingolia, 2017) with modifications. For ribosome profiling, using the Fastp v0.21.0 (Chen et al., 2018) tool, sequences of reads 1 were corrected by reads 2, and quality filtering and adapter sequence removal were performed on reads 1. The adapter-removed reads 1 were split by the barcode sequence. Reads mapped to rRNA and tRNA sequences of C. orbiculare, which were predicted by RNAmmer (Lagesen et al., 2007) (http://www.cbs.dtu.dk/services/RNAmmer/) and tRNA-scan SE (Chan et al., 2021) (http://lowelab.ucsc.edu/tRNAscan-SE/) in the genome of C. orbiculare 104T (Gan et al., 2019) (PRJNA171217), using STAR v2.7.0a (Dobin et al., 2013), were removed from analysis. For all predicted tRNAs, the CCA sequence was added to the 3′ end. The remaining reads were mapped to the C. orbiculare genome (Gan et al., 2019) by STAR v2.7.0a. The A-site offset of footprints was empirically estimated to be 15 for the 19–21 nt and 24–30 nt footprints. Footprints located on the first and last five codons of each ORF were omitted from the analysis. For RNA-Seq, both reads 1 and 2 were used for analysis, and an offset of 15 was used for all mRNA fragments.

The translation efficiency change induced by aglafoline was quantified by DESeq2 (Love et al., 2014). Significance was calculated by a likelihood ratio test in a generalized linear model.

For Gene Ontology (GO) analysis, IDs of sensitive mRNAs in C. orbiculare conidia were converted to IDs of Saccharomyces cerevisiae homologs predicted using BLASTp (https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/; Camacho et al., 2009) and the S288C reference from the Saccharomyces Genome Database (SGD). A functional annotation chart for this list was obtained from DAVID (https://david.ncifcrf.gov/home.jsp; Huang et al., 2009a; Huang et al., 2009b). GO terms with a false discovery rate (FDR) of <0.05 were considered.

For 5′ UTR assignment of C. orbiculare, published RNA-Seq data (GSE178879) (Zhang et al., 2021) were aligned to the C. orbiculare genome by STAR 2.7.0a and were then assembled into transcript isoforms by StringTie v2.2.1 (Kovaka et al., 2019). The extensions upstream of the annotated start codons were assigned as the 5′ UTRs. The 5′ UTRs of transcripts expressed in conidia and mycelia were obtained separately. For analysis of the polypurine sequence in Figure 3F and Figure 3—figure supplement 3E, we used the 5′ UTR with the highest coverage in StringTie when multiple 5′ UTR isoforms were assigned.

The global translation change (i.e., the ribosome footprint change without consideration of the RNA abundance) was quantified by DESeq2 (Love et al., 2014) and renormalized to the mitochondrial footprints (as an internal spike-in standard) (Iwasaki et al., 2016).

Fungal inoculation

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Fungal inoculation was performed as previously described (Hiruma and Saijo, 2016; Kumakura et al., 2019) with modifications. Cucumber cotyledons were used for C. orbiculare inoculation. Seeds of cucumber, Cucumis sativus Suyo strain (Sakata Seed Corp.), were planted on a mix of equal amounts of vermiculite (VS Kakou) and Supermix A (Sakata Seed Corp.). Cucumbers were grown at 24°C under a 10 hr light/14 hr dark cycle using biotrons (NK Systems). Cotyledons were detached from seedlings of cucumbers and inoculated with C. orbiculare at 13 days post-germination. C. orbiculare strains (eIF4AWT#1 and eIF4AH153G#1, Supplementary file 4) were cultured on 100 ml of 3.9% PDA in a 300 ml flask at 25°C for 6 days in the dark. Conidia that appeared on the surface of PDA were suspended in 20 ml of sterilized water, filtered through cell strainers (100 µm pore size, Corning), pelleted by centrifugation at 760 × g for 5 min, and resuspended in sterilized water. The concentration of conidia was measured with disposable hemacytometers (Funakoshi) and adjusted to 105 conidia/ml with or without aglafoline (1 µM). Both conidial suspensions contained DMSO at 0.005% (v/v). Conidial suspensions were sprayed onto detached cotyledons using a glass spray (Sansho) and an air compressor (NRK Japan). Inoculated leaves were placed in plastic trays and incubated at 100% humidity for 3.5 days under the same conditions used for plant growth. Using a 6 mm trepan (Kai Medical), 6 leaf discs (LDs) were cut from each leaf, and 48 LDs were collected per sample. Six LDs were placed in a 2 ml steel top tube (BMS) with Φ5-mm zirconia beads (Nikkato), and eight tubes were prepared for each sample (n = 8). Samples were frozen in liquid nitrogen, ground at 1500 rpm for 2 min using a Shakemaster NEO (BMS), and stored at −80°C until RNA extraction.

Quantification of fungal biomass in planta

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The living fungal biomass in cucumber leaves at 3.5 days postinoculation (dpi) was measured by RT-qPCR. Relative expression levels of the C. orbiculare 60S ribosomal protein L5 gene (GenBank: Cob_v012718) (Gan et al., 2013) normalized to that of a cucumber cyclophilin gene (GenBank: AY942800.1) (Liang et al., 2018) were determined. Total RNA was extracted with the Maxwell RSC Plant RNA Kit (Promega) and Maxwell RSC 48 Instrument (Promega) with the removal of genomic DNA according to the manufacturer’s protocol. cDNA was synthesized from 500 to 1000 ng of total RNA per sample with a ReverTraAce qPCR RT Kit (TOYOBO) following the manufacturer’s instructions. All RT-qPCRs were performed with THUNDERBIRD Next SYBR qPCR Mix (TOYOBO) and an MX3000P Real-Time qPCR System (Stratagene). The primers used are listed in Supplementary file 5.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)eIF4A1NCBIGenBank:CCDS11113.1
Gene (Ophiocordyceps sinensis)eIF4AEnsemblFungi (https://fungi.ensembl.org/index.html)EnsemblFungi:OCS_04979
Gene (Ophiocordyceps sp. BRM1)eIF4A iso4This studyIwasaki lab
Gene (Colletotrichum orbiculare)eIF4AGenBankGenBank:Cob_v000942
Gene (C. orbiculare)60S ribosomal protein L5GenBankGenBank:Cob_v012718
Gene (Cucumis sativus)CyclophilinGenBankGenBank:AY942800.1
Strain, strain background (Aglaia odorata)Aglaia odorataThis paperGrown in Berkeley, CA; Ingolia lab
Strain, strain background (Ophiocordyceps sp. BRM1)Ophiocordyceps sp. BRM1This paperGrown in Berkeley, CA; Ingolia lab
Strain, strain background (Escherichia coli)BL21 Star (DE3)Thermo Fisher ScientificCat. #:C601003
Strain, strain background (C. orbiculare)104-TNARO GenBankMAFF 240422
Strain, strain background (C. orbiculare)eIF4AWT#1This paperCoNK1171Supplementary file 4; Shirasu lab
Strain, strain background (C. orbiculare)eIF4AWT#2This paperCoNK1172Supplementary file 4; Shirasu lab
Strain, strain background (C. orbiculare)eIF4AH153G#1This paperCoNK1181Supplementary file 4; Shirasu lab
Strain, strain background (C. orbiculare)eIF4AH153G#2This paperCoNK1182Supplementary file 4; Shirasu lab
Strain, strain background (C. sativus)Suyo strainSakata Seed Corp.
Recombinant DNA reagentpColdI (plasmid)TaKaRaCat. #:3361
Recombinant DNA reagentpColdI-H. sapiens eIF4A1 WT (plasmid)RIEN BRCRDB17299Iwasaki et al., 2019
Recombinant DNA reagentpColdI-H. sapiens eIF4A1 Phe163Gly (plasmid)This paperIwasaki lab
Recombinant DNA reagentpColdI-H. sapiens eIF4A1 Phe163His (plasmid)This paperIwasaki lab
Recombinant DNA reagentpColdI-O. sinensis eIF4A WT (plasmid)This paperIwasaki lab
Recombinant DNA reagentpColdI-O. sinensis eIF4A His154Gly (plasmid)This paperIwasaki lab
Recombinant DNA reagentpColdI-Ophiocordyceps sp. BRM1 eIF4A iso4 WT (plasmid)This paperIwasaki lab
Recombinant DNA reagentpColdI-Ophiocordyceps sp. BRM1 eIF4A iso4 Gly172His (plasmid)This paperIwasaki lab
Recombinant DNA reagentpColdI-Ophiocordyceps sp. BRM1 eIF4A iso4 Gly172Phe (plasmid)This paperIwasaki lab
Recombinant DNA reagentpENTR4 (plasmid)Thermo Fisher ScientificCat. #:A10465
Recombinant DNA reagentpENTR4-C. orbiculare eIF4A WT (plasmid)This paperShirasu lab
Recombinant DNA reagentpENTR4-C. orbiculare eIF4A His153Gly (plasmid)This paperShirasu lab
Recombinant DNA reagentpII99 (plasmid)Namiki et al., 2001
Recombinant DNA reagentpsiCHECK2−7×AGAGAG motifsIwasaki et al., 2016
Recombinant DNA reagentpsiCHECK2-CAA repeatsIwasaki et al., 2016
Sequence-based reagentRandom Primer (nonadeoxyribonucleotide mix: pd(N)9)TaKaRaCat. #:3802
Sequence-based reagentFAM-labeled [AG]10 RNAIwasaki et al., 2019
Sequence-based reagentPrimersThis paperSupplementary file 5; Shirasu lab
Peptide, recombinant proteinH. sapiens eIF4A1 WTThis paperIwasaki lab
Peptide, recombinant proteinH. sapiens eIF4A1 Phe163GlyThis paperIwasaki lab
Peptide, recombinant proteinH. sapiens eIF4A1 Phe163HisThis paperIwasaki lab
Peptide, recombinant proteinO. sinensis eIF4A WTThis paperIwasaki lab
Peptide, recombinant proteinO. sinensis eIF4A His154GlyThis paperIwasaki lab
Peptide, recombinant proteinOphiocordyceps sp. BRM1 eIF4A iso4 WTThis paperIwasaki lab
Peptide, recombinant proteinOphiocordyceps sp. BRM1 eIF4A iso4 Gly172HisThis paperIwasaki lab
Peptide, recombinant proteinOphiocordyceps sp. BRM1 eIF4A iso4 Gly172PheThis paperIwasaki lab
Peptide, recombinant proteinDriselase from Basidiomycetes sp.Sigma-AldrichCat. #:D9515
Peptide, recombinant proteinLysing enzyme from Trichoderma harzianumSigma-AldrichCat. #:L1412
Peptide, recombinant proteinCas9 nuclease protein NLSNippon GeneCat. #:316-08651
Peptide, recombinant proteinTurbo DNaseThermo Fisher ScientificCat. #:AM2238
Peptide, recombinant proteinRNase ILucigenCat. #:N6901K
Commercial assay or kitrRNA depletion by a Ribo-Zero Gold rRNA Removal Kit (Yeast)IlluminaCat. #:RZY1324
Commercial assay or kitTruSeq Stranded mRNA KitIlluminaCat. #:15027078
Commercial assay or kitIn-Fusion HDTaKaRaCat. #:639650
Commercial assay or kitProtoScript II Reverse TranscriptaseNew England BiolabsCat. #:M0368L
Commercial assay or kitHiFi DNA assemblyNew England BiolabsCat. #:E2621
Commercial assay or kitNi-NTA agaroseQIAGENCat. #:30230
Commercial assay or kitHiTrap Heparin HP column, 1 mlGE HealthcareCat. #:17040601
Commercial assay or kitNAP-5GE HealthcareCat. #:17085302
Commercial assay or kitPD-10GE HealthcareCat. #:17085101
Commercial assay or kitVivaspin 6 (10 kDa MWCO)SartoriusCat. #:VS0601
Commercial assay or kitEzStainAQuaATTOCat. #:2332370
Commercial assay or kitBlack 384-well microplateCorningCat. #:3820
Commercial assay or kitT7-Scribe Standard RNA IVT KitCELLSCRIPTCat. #:C-AS3107
Commercial assay or kitScriptCap m7G Capping SystemCELLSCRIPTCat. #:C-SCCE0625
Commercial assay or kitScriptCap 2′-O-Methyltransferase KitCELLSCRIPTCat. #:C-SCMT0625
Commercial assay or kitA-Plus Poly(A) Polymerase Tailing KitCELLSCRIPTCat. #:C-PAP5104H
Commercial assay or kitRenilla-Glo Luciferase Assay SystemPromegaCat. #:E2720
Commercial assay or kitMicroSpin G-25 columnCytivaCat. #:27532501
Commercial assay or kitRabbit Reticulocyte Lysate, Nuclease-TreatedPromegaCat. #:L4960
Commercial assay or kitPotato dextrose agar (PDA) mediumNissuiCat. #:05709
Commercial assay or kitPotato dextrose brothBD BiosciencesCat. #:254920
Commercial assay or kit70 µm cell strainerCorningCat. #:352350
Commercial assay or kitYeast extractBD BiosciencesCat. #:212750
Commercial assay or kitFilter (0.2 µm pore size)GE HealthcareCat. #:6900-2502
Commercial assay or kit50 ml tubeFalcon, CorningCat. #:352070
Commercial assay or kitCUGA7 gRNA Synthesis KitNippon GeneCat. #:314-08691
Commercial assay or kit50 ml ProteoSave SS tubeSumitomo BakeliteCat. #:631-28111
Commercial assay or kitMF membrane (0.45 µm pore size)MilliporeCat. #:HAWP04700
Commercial assay or kitTRIzol reagentThermo Fisher ScientificCat. #:15596018
Commercial assay or kitRibo-Minus Eukaryotes Kit for RNA-SeqThermo Fisher ScientificCat. #:A1083708
Commercial assay or kitDirect-zol RNA Microprep KitZymo ResearchCat. #:R2060
Commercial assay or kitIllumina Stranded mRNA Prep, LigationIlluminaCat. #:20040532
Commercial assay or kitTruSeq Stranded Total RNA Library Prep GoldIlluminaCat. #:20020598
Commercial assay or kitVermiculiteVS Kakou
Commercial assay or kitSupermix ASakata Seed Corp.Cat. #:72000083
Commercial assay or kitCell strainer (100 µm pore size)CorningCat. #:431752
Commercial assay or kitDisposable hemacytometersFunakoshiCat. #:521-10
Commercial assay or kitMaxwell RSC Plant RNA KitPromegaCat. #:AS1500
Commercial assay or kitReverTraAce qPCR RT KitTOYOBOCat. #:FSQ-101
Commercial assay or kitTHUNDERBIRD Next SYBR qPCR MixTOYOBOCat. #:QPX-201
Chemical compound, drugRocASigma-AldrichCat. #:SML0656
Chemical compound, drugAglafolineMedChemExpressCat. #:HY-19354
Chemical compound, drugADPFujifilm Wako ChemicalsCat. #:019-25091
Chemical compound, drugAMP-PNPRocheCat. #:10102547001
Chemical compound, drugG418Fujifilm Wako ChemicalsCat. #:078-05961
Software, algorithmTrinityhttps://github.com/Trinotate/Trinotate/wikiGrabherr et al., 2011
Software, algorithmTrinotatehttps://github.com/Trinotate/Trinotate/wikiHaas et al., 2013
Software, algorithmMUSCLEhttps://www.ebi.ac.uk/Tools/msa/muscle/
Software, algorithmESPript 3.0http://espript.ibcp.fr/ESPript/ESPript/Robert and Gouet, 2014
Software, algorithmBLASTphttps://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/Camacho et al., 2009
Software, algorithmBLASTnhttps://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/Camacho et al., 2009
Software, algorithmMAFFT v7.480https://mafft.cbrc.jp/alignment/software/Katoh and Standley, 2013
Software, algorithmtrimAl v1.4.rev15https://anaconda.org/bioconda/trimal/filesCapella-Gutiérrez et al., 2009
Software, algorithmcatfasta2phyml v1.1.0https://github.com/nylander/catfasta2phyml
Software, algorithmModelTest-NG v0.1.6https://github.com/ddarriba/modeltesDarriba et al., 2020
Software, algorithmRAxML-NG v0.9.0https://github.com/amkozlov/raxml-ngKozlov et al., 2019
Software, algorithmiTOL v6https://itol.embl.de/Letunic and Bork, 2021
Software, algorithmIgor Pro v8.01WaveMetrics: https://www.wavemetrics.com/products/igorpro
Software, algorithmminimap2 v2.17-r941https://anaconda.org/bioconda/minimap2/filesLi, 2018
Software, algorithmflye v2.8.1-b1676https://anaconda.org/bioconda/flye/files?page=2Kolmogorov et al., 2019
Software, algorithmnucmerhttps://mummer4.github.io/manual/manual.htmlDelcher et al., 2003
Software, algorithmFastp v0.21.0https://github.com/OpenGene/fastpChen et al., 2018
Software, algorithmRNAmmerhttp://www.cbs.dtu.dk/services/RNAmmer/Lagesen et al., 2007
Software, algorithmtRNA-scan SEhttp://lowelab.ucsc.edu/tRNAscan-SE/Chan et al., 2021
Software, algorithmSTAR v2.7.0ahttps://github.com/alexdobin/STARDobin et al., 2013
Software, algorithmDESeq2https://bioconductor.org/packages/release/bioc/html/DESeq2.htmlLove et al., 2014
Software, algorithmDAVIDhttps://david.ncifcrf.gov/home.jspHuang et al., 2009a; Huang et al., 2009b
Software, algorithmStringTie v2.2.1https://github.com/gpertea/stringtieKovaka et al., 2019
OtherNGC chromatography systemBio-RadHigh-performance liquid chromatography
OtherInfinite F-200 PROTecanPlate reader
OtherEnVision 2104 plate readerPerkinElmerPlate reader
OtherGloMax Navigator SystemPromegaCat. #: GM2010Microplate luminometer
OtherSwinging-bucket rotorHitachiCat. #:T4SS31Centrifuge rotor
OtherCentrifugeHitachiCat. #:CF16RXIICentrifuge
OtherMulti-beads ShockerYASUI KIKAICat. #:MB2200(S)Bead mill homogenizer
OtherBiotronNK SystemsCat. #:LPH-410S and NH350SBiotron
OtherGlass spraySanshoCat. #:81-1192Spray
OtherAir compressorNRK JapanCat. #:UP-5FAir compressor
Other6 mm trepanKai MedicalCat. #:BP-60FBiopsy Punch
Other2 ml steel top tubeBMSCat. #:MT020-01HSSample tube
OtherΦ5-mm zirconia beadsNikkatoCat. #:5-4060-13Zirconia beads
OtherShakemaster NEOBMSCat. #:BMS-mini16Bead mill homogenizer
OtherMaxwell RSC 48 InstrumentPromegaCat. #:AS8500Automated nucleic acid purification platform
OtherMX3000P Real-Time qPCR SystemStratageneCat. #:401511Real-time qPCR system

Data availability

The results of ribosome profiling and RNA-Seq (GEO: GSE200060) for C. orbiculare and RNA-Seq for Ophiocordyceps sp. BRM1 (SRA: PRJNA821935) obtained in this study have been deposited in the National Center for Biotechnology Information (NCBI) database. The C. orbiculare mitochondrial genome assembly generated in this study was deposited under accession number MZ424187. The scripts for deep sequencing data analysis were deposited in Zenodo (DOI: 10.5281/zenodo.7477706). Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Shintaro Iwasaki (shintaro.iwasaki@riken.jp).

The following data sets were generated
    1. Chen M
    2. Kumakura N
    3. Muller R
    4. Shichino Y
    5. Nishimoto M
    6. Mito M
    7. Gan P
    8. Ingolia NT
    9. Shirasu K
    10. Ito T
    11. Iwasaki S
    (2022) NCBI Gene Expression Omnibus
    ID GSE200060. A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants.
    1. Shichino Y
    2. Iwasaki S
    (2023) Zenodo
    Custom scripts for "A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants".
    https://doi.org/10.5281/zenodo.7477706
The following previously published data sets were used
    1. Gan P
    (2021) NCBI Nucleotide
    ID MZ424187.1. Colletotrichum orbiculare MAFF 240422 mitochondrion, complete genome.
    1. Gan P
    (2021) NCBI Gene Expression Omnibus
    ID GSE178879. RNAseq of Colletotrichum orbiculare on Nicotiana benthamiana.

References

    1. Oerke EC
    (2006) Crop losses to pests
    The Journal of Agricultural Science 144:31–43.
    https://doi.org/10.1017/S0021859605005708

Decision letter

  1. Antonis Rokas
    Reviewing Editor; Vanderbilt University, United States
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Biology Tübingen, Germany
  3. Jonathan H Schatz
    Reviewer; University of Miami Miller School of Medicine, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Jonathan H Schatz (Reviewer #1).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Please include evidence that demonstrates the inability of Ophiocordyceps BRM1 to grow in the presence of rocaglate with a change to H153. Since H153 should restore gain-of-function to rocA, it should not be necessary to engineer the Ophiocordyceps BRM1 genome, if any way to express the H153 variant in the organism is achievable. If this can't be achieved, then H153 and G153 together in the same cell-free reporter reaction should result in dominant-negative repression by H153 due to the gain-of-function rocaglate mechanism. This alternative experiment should be very doable as the authors have already purified the protein (Figure 2) and reported a cell-free translation assay (Figure 3A). Even though this translation assay was a reconstituted system with human factors, Ophiocordyceps BRM1 eIF4A bound to RocA should only require a polypurine motif in the reporter 5' UTR to be a dominant negative inhibitor of the scanning ribosome. The authors should include the negative control reporter mRNA (they have previously published this and referred to it as "CAA repeat") along with the 7X AGAGAG reporter mRNA.

2) For Figure 3 and the ribosome profiling data, can the authors confirm the footprint changes are not due to changes in mRNA levels? Determining TE or some similar metric (ribo-seq/rna-seq) would be able to decipher which changes are at the translation or mRNA level. The 6 hr treatments are proper but secondary transcriptional and mRNA decay effects could be accumulating by this point.

3) The manuscript ends with data showing that the gene-edited C. orbiculare strain produces less biomass after 3 days with Aglafoline treatment. Can the authors confirm that this edited strain does not have a growth defect that could alternatively explain the results?

https://doi.org/10.7554/eLife.81302.sa1

Author response

Essential revisions:

1) Please include evidence that demonstrates the inability of Ophiocordyceps BRM1 to grow in the presence of rocaglate with a change to H153. Since H153 should restore gain-of-function to rocA, it should not be necessary to engineer the Ophiocordyceps BRM1 genome, if any way to express the H153 variant in the organism is achievable. If this can't be achieved, then H153 and G153 together in the same cell-free reporter reaction should result in dominant-negative repression by H153 due to the gain-of-function rocaglate mechanism. This alternative experiment should be very doable as the authors have already purified the protein (Figure 2) and reported a cell-free translation assay (Figure 3A). Even though this translation assay was a reconstituted system with human factors, Ophiocordyceps BRM1 eIF4A bound to RocA should only require a polypurine motif in the reporter 5' UTR to be a dominant negative inhibitor of the scanning ribosome.

To test the dominant-negative mode of translational repression in vitro by Ophiocordyceps sp. BRM1 eIF4A and its Gly-to-His mutant, we first tried to use the corresponding recombinant proteins in the human reconstitution system, as the reviewer suggested. However, Ophiocordyceps sp. BRM1 eIF4A could not substitute for human eIF4A in active translation initiation (data not shown), unfortunately.

Thus, we instead employed a translation assay with a preformed RocA-eIF4A-mRNA complex (Iwasaki et al. Nature 2016; Iwasaki et al. Mol Cell 2019). In this experiment, we preincubated recombinant Ophiocordyceps sp. BRM1 eIF4A or its Gly172His mutant with a reporter mRNA possessing polypurine motifs in the presence or absence of RocA. If RocA can target the eIF4A protein, eIF4A should be stably clamped on the polypurine tract, providing steric hindrance to scanning ribosomes and thus repressing protein synthesis in rabbit reticulocyte lysate (RRL).

To perform this assay, we first characterized the Gly172His mutant of Ophiocordyceps sp. BRM1 eIF4A by a fluorescence polarization assay. Consistent with the effects of His residues in the context of human and O. sinensis eIF4As, the BRM1 eIF4A Gly172His mutant had a higher affinity for polypurine RNA ([AG]10) in the presence of RocA and ADP + Pi than did the Gly residue in the wild-type (see Figure 2C, Figure 2 — figure supplement 2G-H, and Table 1).

Then, we used recombinant WT Gly172 and Gly172His mutant proteins for complex preformation and subsequent in vitro translation in RRL. The complex of RocA and the Gly172His mutant on the reporter mRNA supplied steric hindrance for translation initiation (see Figure 3B). On the other hand, WT Gly172 did not have such a function.

These data clearly indicated that the His substitution at Gly172 in Ophiocordyceps sp. BRM1 eIF4A results in a dominant-negative gain-of-function for RocA-mediated translational repression.

The authors should include the negative control reporter mRNA (they have previously published this and referred to it as "CAA repeat") along with the 7X AGAGAG reporter mRNA.

Regarding Figure 3A, we used the negative control reporter mRNA possessing CAA repeats in the in vitro translation system with purified human factors (see Figure 3A). Consistent with the mode of function of RocA, we did not observe translational repression of this reporter mRNA whether the WT or mutant eIF4A protein was included in the reaction. Notably, we reperformed the experiments with the 7×AGAGAG reporter for side-by-side comparison to the CAA repeat reporter.

2) For Figure 3 and the ribosome profiling data, can the authors confirm the footprint changes are not due to changes in mRNA levels? Determining TE or some similar metric (ribo-seq/rna-seq) would be able to decipher which changes are at the translation or mRNA level. The 6 hr treatments are proper but secondary transcriptional and mRNA decay effects could be accumulating by this point.

We truly thank the reviewers for noting this consideration and providing the opportunity to improve our data. We conducted RNA-Seq for the corresponding conditions, measured the translation efficiency across the transcriptome, and redefined the aglafoline-sensitive mRNAs (see Figure 3C).

Even with this translation efficiency calculation, we reproduced the aglafoline-mediated translational repression in a dose-dependent and polypurine-selective manner (see Figure 3D, 3G).

Importantly, the His153Gly substitution conferred aglafoline resistance in terms of the translation efficiency (see Figure 3E).

Thus, our reassessment of the translation efficiency verified the net translation changes induced by aglafoline over the secondary alterations in the mRNA abundance.

3) The manuscript ends with data showing that the gene-edited C. orbiculare strain produces less biomass after 3 days with Aglafoline treatment. Can the authors confirm that this edited strain does not have a growth defect that could alternatively explain the results?

According to the reviewers’ suggestion, we measured the colony size as an indicator of cell growth in the engineered C. orbiculare strains (see Figure 3 — figure supplement 1C, 1D) and could not detect any difference.

https://doi.org/10.7554/eLife.81302.sa2

Article and author information

Author details

  1. Mingming Chen

    1. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan
    2. RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Contributed equally with
    Naoyoshi Kumakura
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4453-1105
  2. Naoyoshi Kumakura

    Plant Immunity Research Group, RIKEN Center for Sustainable Resource Science, Yokohama, Japan
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Contributed equally with
    Mingming Chen
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0259-2444
  3. Hironori Saito

    1. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan
    2. RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan
    Contribution
    Formal analysis, Investigation, Visualization, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9623-4132
  4. Ryan Muller

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Contribution
    Resources, Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8868-7841
  5. Madoka Nishimoto

    Laboratory for Translation Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan
    Contribution
    Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0948-2504
  6. Mari Mito

    RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan
    Contribution
    Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2564-6273
  7. Pamela Gan

    Plant Immunity Research Group, RIKEN Center for Sustainable Resource Science, Yokohama, Japan
    Contribution
    Resources, Formal analysis, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
  8. Nicholas T Ingolia

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Contribution
    Resources, Supervision, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3395-1545
  9. Ken Shirasu

    1. Plant Immunity Research Group, RIKEN Center for Sustainable Resource Science, Yokohama, Japan
    2. Department of Biological Science, Graduate School of Science, The University of Tokyo, Tokyo, Japan
    Contribution
    Supervision, Funding acquisition, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0349-3870
  10. Takuhiro Ito

    Laboratory for Translation Structural Biology, RIKEN Center for Biosystems Dynamics Research, Yokohama, Japan
    Contribution
    Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3704-5205
  11. Yuichi Shichino

    RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan
    Contribution
    Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0093-1185
  12. Shintaro Iwasaki

    1. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan
    2. RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    shintaro.iwasaki@riken.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7724-3754

Funding

Japan Society for the Promotion of Science (JP19H02959)

  • Shintaro Iwasaki

Ministry of Education, Culture, Sports, Science and Technology (JP20H05784)

  • Shintaro Iwasaki

Japan Agency for Medical Research and Development (JP21gm1410001)

  • Takuhiro Ito

RIKEN (Biology of Intracellular Environments)

  • Takuhiro Ito

RIKEN (Integrated life science research to challenge super aging society)

  • Takuhiro Ito

Japan Society for the Promotion of Science (JP19H03172)

  • Takuhiro Ito

RIKEN (Dynamic Structural Biology)

  • Takuhiro Ito

Japan Society for the Promotion of Science (JP17H06172)

  • Ken Shirasu

Japan Society for the Promotion of Science (JP20K15500)

  • Naoyoshi Kumakura

Japan Society for the Promotion of Science (JP18K14440)

  • Naoyoshi Kumakura

Japan Science and Technology Agency (JPMJAX20B4)

  • Naoyoshi Kumakura

RIKEN (Special Postdoctoral Researchers)

  • Naoyoshi Kumakura
  • Yuichi Shichino

RIKEN (Incentive Research Projects)

  • Naoyoshi Kumakura
  • Yuichi Shichino

Japan Society for the Promotion of Science (JP21K15023)

  • Yuichi Shichino

Ministry of Education, Culture, Sports, Science and Technology (JP21H05734)

  • Yuichi Shichino

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank all the members of the Iwasaki laboratory for constructive discussions, technical help, and critical reading of the manuscript. We are also grateful to the Support Unit for Bio-Material Analysis, RIKEN CBS Research Resources Division for technical help, supercomputer HOKUSAI Sailing Ship in RIKEN for computation, and the Vincent J Coates Genomics Sequencing Laboratory (supported by National Institutes for Health Instrumentation Grant, S10 OD018174) at UC Berkeley for DNA sequencing. SI was supported by the Japan Society for the Promotion of Science (JSPS) (a Grant-in-Aid for Scientific Research [B], JP19H02959), the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (a Grant-in-Aid for Transformative Research Areas [B] 'Parametric Translation', JP20H05784), the Japan Agency for Medical Research and Development (AMED) (AMED-CREST, JP21gm1410001), and RIKEN ('Biology of Intracellular Environments' and 'Integrated life science research to challenge super aging society'). TI was supported by JSPS (a Grant-in-Aid for Scientific Research [B], JP19H03172), AMED (AMED-CREST, JP21gm1410001), and RIKEN ('Dynamic Structural Biology', 'Biology of Intracellular Environments', and 'Integrated life science research to challenge super aging society'). KS was supported by JSPS (a Grant-in-Aid for Scientific Research [S], JP17H06172). NK was supported by JSPS (a Grant-in-Aid for Young Scientists, JP20K15500 and JP18K14440), the Japan Science and Technology Agency (JST) (ACT-X, JPMJAX20B4), and RIKEN (Special Postdoctoral Researchers and Incentive Research Projects). YS was supported by JSPS (a Grant-in-Aid for Early-Career Scientists, JP21K15023), MEXT (a Grant-in-Aid for Transformative Research Areas [A] 'Multifaceted Proteins', JP21H05734), and RIKEN (Special Postdoctoral Researchers and Incentive Research Projects). MC was an International Program Associate of RIKEN. NK and YS were recipients of the Special Postdoctoral Researchers Program of RIKEN. HS was a recipient of a Junior Research Associate Program of RIKEN.

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Biology Tübingen, Germany

Reviewing Editor

  1. Antonis Rokas, Vanderbilt University, United States

Reviewer

  1. Jonathan H Schatz, University of Miami Miller School of Medicine, United States

Version history

  1. Received: June 22, 2022
  2. Preprint posted: July 4, 2022 (view preprint)
  3. Accepted: January 12, 2023
  4. Version of Record published: February 28, 2023 (version 1)

Copyright

© 2023, Chen, Kumakura et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Mingming Chen
  2. Naoyoshi Kumakura
  3. Hironori Saito
  4. Ryan Muller
  5. Madoka Nishimoto
  6. Mari Mito
  7. Pamela Gan
  8. Nicholas T Ingolia
  9. Ken Shirasu
  10. Takuhiro Ito
  11. Yuichi Shichino
  12. Shintaro Iwasaki
(2023)
A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants
eLife 12:e81302.
https://doi.org/10.7554/eLife.81302

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https://doi.org/10.7554/eLife.81302

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    Background:

    Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown.

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    This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR.

    Funding:

    This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.