Journal: bioRxiv

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

doi: 10.1101/2022.07.04.498659

Figure Lengend Snippet: (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 polypurine motifs (left) was translated in the reaction. (B) MA (M, log ratio; A, mean average) plot of ribosome footprint changes caused by 3 µM aglafoline treatment in C. orbiculare eIF4A WT conidia. Resistant and sensitive mRNAs (false discovery rate [FDR] < 0.05) are highlighted. Data were normalized to mitochondrial footprints, which were used as internal spike-ins . (C) Cumulative distribution of the ribosome footprint changes for aglafoline-sensitive mRNAs (defined in B) in C. orbiculare eIF4A WT conidia with 0.3 or 3 µM aglafoline treatment. (D) Gene Ontology (GO) term analysis for aglafoline-sensitive mRNAs (defined in B). GO terms associated with yeast homologs were analyzed by DAVID ( , ). (E and F) Cumulative distribution of the ribosome footprint changes for aglafoline-sensitive mRNAs (defined in B) by 3 µM (E) and 0.3 µM (F) aglafoline treatment in C. orbiculare eIF4A WT and eIF4A His153Gly conidia. (G) Box plot of ribosome footprint changes caused by 3 µM aglafoline treatment in conidia across mRNAs with or without [A/G] 6 motif in 5′ UTR. The p values in C, E, F, and G were calculated by the Mann–Whitney U test.

Article Snippet: RocA (Sigma–Aldrich) and aglafoline (MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO) and used for this study.

Techniques: In Vitro, Recombinant, MANN-WHITNEY

Journal: bioRxiv

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

doi: 10.1101/2022.07.04.498659

Figure Lengend Snippet: (A) Basal protein synthesis activity in an in vitro translation system with human factors. Recombinant proteins of H. sapiens eIF4A1 WT or Phe163Gly were added to the reaction. (B) Schematics of eIF4A recombination in C. orbiculare . NPTII , neomycin phosphotransferase II. (C) PCR-based screening of the recombined strains. The primer sets used for screening are depicted in B. (D) Fluorescence polarization assay for FAM-labeled RNA ([AG] 10 ) (10 nM). WT and mutated Ophiocordyceps sp. BRM1 eIF4A proteins from the indicated species were used. To measure ATP-independent RNA clamping induced by aglafoline (50 µM), ADP and Pi were included in the reaction. Data represent the mean and s.d. (n = 3). (E) The summary of K d in D under aglafoline is depicted. Data represent the mean and s.d.

Article Snippet: RocA (Sigma–Aldrich) and aglafoline (MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO) and used for this study.

Techniques: Activity Assay, In Vitro, Recombinant, Fluorescence, Labeling

Journal: bioRxiv

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

doi: 10.1101/2022.07.04.498659

Figure Lengend Snippet: (A) MA plot of ribosome footprint changes by 3 µM aglafoline treatment in C. orbiculare eIF4A WT mycelia. Resistant and sensitive mRNAs (false discovery rate [FDR] < 0.05) are highlighted. Data were normalized to mitochondrial footprints, which were used as internal spike-ins . (B) Cumulative distribution of the ribosome footprint changes for aglafoline-sensitive mRNAs (defined in A) in C. orbiculare eIF4A WT mycelia with 0.3 or 3 µM aglafoline treatment. (C and D) Cumulative distribution of the ribosome footprint changes for aglafoline-sensitive mRNAs (defined in A) by 3 µM (E) and 0.3 µM (F) aglafoline treatment in mycelia C. orbiculare eIF4A WT and eIF4A His153Gly . (E) Box plot of ribosome footprint changes caused by 3 µM aglafoline treatment in mycelia across mRNAs with or without [A/G] 6 motif in 5′ UTR. (F) Venn diagram of the overlap between aglafoline-sensitive mRNAs in conidia (defined in ) and mycelia (defined in ). (G) Cumulative distribution of the ribosome footprint changes by the cell states (conidia biased as negative and mycelia biased as positive) for aglafoline-sensitive mRNAs in conidia (defined in ) and mycelia (defined in ). The p values in B, C, D, E, and G were calculated by the Mann–Whitney U test.

Article Snippet: RocA (Sigma–Aldrich) and aglafoline (MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO) and used for this study.

Techniques: MANN-WHITNEY

Journal: bioRxiv

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

doi: 10.1101/2022.07.04.498659

Figure Lengend Snippet: (A) Workflow for monitoring the biomass of C. orbiculare eIF4A WT or eIF4A His153Gly strains on cucumber leaves under treatment with aglafoline. (B) Comparison of in planta fungal biomass of C. orbiculare eIF4A WT or eIF4A His153Gly strains with or without treatment with 1 µM aglafoline. Relative expression levels of the C. orbiculare 60S ribosomal protein L5 gene (GenBank: Cob_v000942) 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 eIF4A WT and eIF4A His153Gly with aglafoline were normalized to those of eIF4A WT and eIF4A His153Gly without aglafoline, respectively. Significance was calculated by Student’s t test (two-tailed). Three independent experiments showed similar results.

Article Snippet: RocA (Sigma–Aldrich) and aglafoline (MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO) and used for this study.

Techniques: Comparison, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

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

doi: 10.1101/2022.07.04.498659

Figure Lengend Snippet: (A) C. sativus leaves were sprayed with DMSO or aglafoline (1 µM) in water and incubated for 3 d using the same method as C. orbiculare inoculation. (B) Alignment of DDX3 protein sequences from higher eukaryotes and fungal species, including Ophiocordyceps sp. BRM1 DDX3s.

Article Snippet: RocA (Sigma–Aldrich) and aglafoline (MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO) and used for this study.

Techniques: Incubation