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Research Article | Molecular Biology and Physiology

Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment

Xiuqing Yang, Peng Feng, Ying Yin, Kathryn Bushley, Joseph W. Spatafora, Chengshu Wang
B. Gillian Turgeon, Editor
Xiuqing Yang
aKey Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
bUniversity of Chinese Academy of Sciences, Beijing, China
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Peng Feng
aKey Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
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Ying Yin
aKey Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
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Kathryn Bushley
cDepartment of Plant Biology, University of Minnesota, St. Paul, Minnesota, USA
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Joseph W. Spatafora
dDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, USA
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Chengshu Wang
aKey Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China
eSchool of Life Science and Technology, ShanghaiTech University, Shanghai, China
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B. Gillian Turgeon
Cornell University
Roles: Editor
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DOI: 10.1128/mBio.01211-18
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  • FIG 1
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    FIG 1

    Prediction and functional verification of the CSN biosynthetic gene cluster. (A) Schematic map of the biosynthetic gene cluster. The genes are named following the previously designated SimA gene for the core NRPS gene. (B) Annotation of the gene contents within the gene cluster. ID, identifier. (C) Loss-of-function verification of the contributions of different genes to CSN biosynthesis. HPLC analysis of CSN production by the WT and different null mutants of T. inflatum. The standards CsA, CsB, and CsC were included in parallel analysis.

  • FIG 2
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    FIG 2

    Verification of the genes involved in d-Ala conversion and Bmt biosynthesis. (A) HPLC analysis of CsA production by WT and different mutants with or without the addition of d-Ala. The inset shows the mass spectra detected for the CsA and ΔsimB samples. m/z, [M+H]+; ΔSimBΔ6009, ΔSimB ΔTINF06009 double mutant. (B) Quantification analysis of CsA production by WT and different mutants. The strains were grown in fructose CSN induction medium with or without the supplementation of d-Ala (at a final concentration of 20 mM) for 10 days. The mycelia were then harvested for CSN extraction. Values are means plus standard errors (SE) (error bars). DW, mycelium dry weight. (C) LC-MS analysis of the extracted ion chromatography (EIC) showing the production or nonproduction of Bmt by WT and mutant strains. m/z, [M+H]+. (D) Chemical structure of Bmt. (E) Supplementation of Bmt (at a final concentration of 85 μM) in the growth medium enabled the null mutants to produce CsA (peaks shown in blue).

  • FIG 3
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    FIG 3

    Functional verification of the pathway-specific transcription factor SimL. (A) RT-PCR analysis of gene expression. The WT, ΔSimL, and WT::SimL strains were grown in fructose CSN induction medium for 10 days, and the mycelia were harvested for RNA extraction and gene expression analysis. TINF00183 and TINF07874 are indicated as 183 and 7874, respectively. β-Tub, β-Tubulin. (B) In silico analysis of the putative binding motif by the bZIP-type TF SimL. (C) Comparative quantification of CsA production. The WT and WT::SimL strains were grown in fructose CSN induction medium for 10 days, and the mycelia were harvested for cyclosporine extraction. There were three replicates for each sample. Values are means plus SE.

  • FIG 4
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    FIG 4

    Proposed pathway for CsA biosynthesis. (A) Bmt biosynthesis by the PKS pathway. The PKS SimG domains include the following: β-ketoacyl synthase (KS), acyltransferase (AT), dehydrogenase (DH), methyltransferase (MT), enoylreductase (ER), ketoreductase (KR), acyl carrier protein (ACP), S-adenosylmethionine (SAM). The chemical structure of compounds b1 to b3 and Bmt are shown. (B) Schematic structure of NRPS SimA and the machinery of CsA biosynthesis. There are 11 modules of SimA, and each module contains the condensation (C), adenylation (A), thiolation (T), and/or N-methylation (NM) domains. The terminal C domain (CT) is implicated in cyclization of the peptidyl chains to form CsA and its analogs. The cyclophilin SimC and exporter SimD may jointly contribute to cell tolerance of CSNs. Abu, aminobutyric acid; Sar, sarcocine; Nva, norvaline; MeLeu, methylleucine; aa, amino acids.

  • FIG 5
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    FIG 5

    Antifungal effect of CsA production. (A) Fungal cocultivation tests. The WT and mutants of T. inflatum were inoculated on PDA plates in parallel for 3 days, and the strain of A. flavus was then inoculated between the two T. inflatum colonies for 4 days. (B) Schematic diagram showing how the colony edge distances between the WT T. inflatum and Aspergillus (D1) or between the T. inflatum mutant and Aspergillus (D2) were measured. (C) Comparison of the colony edge distances between strains. Values are means plus SE. Values that are significantly different (P < 0.001 by two-tailed t test) are indicated by a bar and three asterisks. (D) Comparison of the colony edge distances between WT and WT::SimL strains after different incubation times (7 or 14 days [d]). Values are means plus SE. ***, P < 0.001. (E) Representative phenotypes of a fungal pair after inoculation and 3 weeks of growth of T. inflatum.

  • FIG 6
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    FIG 6

    Insect bioassays. (A) Survival of insects after injection with the spores of the WT and different mutants. Control insects (CK) were injected with 0.05% Tween 20. (B) Comparison of the LT50 values for the WT and different mutants. Values are means plus SE. Values that are significantly different from the value for the WT by log rank tests are indicated by asterisks as follows: ****, P < 0.0001; **, P = 0.0087; *, P = 0.0182.

Supplemental Material

  • Figures
  • FIG S1

    Target gene deletion and PCR verification. (A) Schematic map for gene deletion. The 5′- and 3′-flanking regions of the target gene were amplified with primer pairs U1/L1 and U2/L2. The products were then cloned into the binary vector containing the drug resistance gene for transformation of the wild-type strain. Primers F (F stands for forward) and R (R stands for reverse) designed for each target gene are used for PCR verification. (B) PCR verification of the drug-resistant mutants with or without the wild-type (WT) strain as a reference. Ect, ectopic transformant resulting in two PCR bands where the smaller band in each panel is from the WT gene. The ΔTINF06009 (Δ06009) mutant was obtained in the ΔSimB background for double deletions of these two genes. Download FIG S1, TIF file, 0.83 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S2

    Bmt structure analysis. (A) 1D NMR analysis of Bmt in D2O. (B) 1H-1H correlation spectroscopy (COSY) analysis of Bmt in D2O. Download FIG S2, TIF file, 1.14 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S3

    2D NMR analysis of Bmt in D2O. HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear singular quantum correlation. Download FIG S3, TIF file, 1.17 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S4

    Isoabsorbance plots of the CsA standard, WT, and selected mutant strains. The data were generated from the HPLC diode array detector. Download FIG S4, TIF file, 1.91 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S1

    NMR data for identification of Bmt. Download Table S1, PDF file, 0.07 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S5

    Phylogenetic analysis of the adenylation and condensation domains retrieved from selected NRPSs. (A) Phylogenetic and substrate-specific signature analysis of SimA adenylation domains. Substrates: Nva, norvaline; Abu, aminobutyric acid; Sar, sarcocine. (B) Phylogenetic analysis of the condensation domains of SimA and selected NRPSs from different fungi. The NRPS BEAS from Beauveria bassiana is responsible for the biosynthesis of beauvericin. DtxS1 from Metarhizium robertsii is responsible for the biosynthesis of destruxins. EcdA from Emericella rugula is responsible for echinocandin biosynthesis, and TqaA from Penicillium aethiopicum is responsible for the biosynthesis of the indole alkaloid fumiquinazoline F. Download FIG S5, TIF file, 1.33 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S6

    Self-inhibition and transportation assay of cyclosporin production in T. inflatum. (A) Phenotyping of fungal growth on PDA with or without the addition of CsA. Spore suspensions (1 × 107 spores/ml) were individually inoculated (2 μl each) on PDA amended with or without CsA (at a final concentration of 150 μg/ml) and incubated for 14 days. (B) Comparison of colony diameters between WT and mutants after the growth shown in panel A. Values are means plus SE. Values that are significantly different are indicated by asterisks as follows: **, P < 0.01; *, P < 0.05. (C) Comparison of cellular accumulation of CsA between WT and ΔSimD. DW, mycelium dry weight. (D) Comparison of extracellular accumulation of CsA between WT and ΔSimD strains. Values are means plus SE. Values that are significantly different (P  < 0.05) are indicated by an asterisk. Download FIG S6, TIF file, 2.10 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S7

    Phylogenetic analysis of the cyclophilin proteins encoded by T. inflatum genes (shown in boldface type) and selected insect-pathogenic fungi. The cyclophilin genes characterized in Beauveria bassiana are also shown in boldface type. The sources of the proteins are indicated by prefixes as follows: CCM, Cordyceps militaris; OCS, Ophiocordyceps sinensis; MAA, Metarhizium robertsii; CCAD, Cordyceps cicadae; BBA or Bb, Beauveria bassiana. Download FIG S7, TIF file, 0.49 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • FIG S8

    Cocultivation test of T. inflatum with other insect-pathogenic fungi. The strains of other fungi were inoculated on PDA plates in the middle for 2 days, and the WT and ΔSimA strains of T. inflatum were then inoculated in parallel for 7 days. MRO, Metarhizium robertsii; CCA, Cordyceps cicadae; CMI, C. militaris; BBR, Beauveria brongniartii. Download FIG S8, TIF file, 1.47 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

  • TABLE S2

    Primers designed and used in this study. Download Table S2, PDF file, 0.06 MB.

    Copyright © 2018 Yang et al.

    This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment
Xiuqing Yang, Peng Feng, Ying Yin, Kathryn Bushley, Joseph W. Spatafora, Chengshu Wang
mBio Oct 2018, 9 (5) e01211-18; DOI: 10.1128/mBio.01211-18

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Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment
Xiuqing Yang, Peng Feng, Ying Yin, Kathryn Bushley, Joseph W. Spatafora, Chengshu Wang
mBio Oct 2018, 9 (5) e01211-18; DOI: 10.1128/mBio.01211-18
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KEYWORDS

cyclosporine
Tolypocladium inflatum
antifungal activity
biosynthetic pathway
virulence

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