Abstract
Trichothecenes are sesquiterpene toxins produced by diverse fungi, including some species of Trichoderma that are potential plant disease biocontrol agents. Trichoderma arundinaceum produces the trichothecene harzianum A (HA), which consists of the core trichothecene structure (12,13-epoxytrichothec-9-ene, EPT) with a linear polyketide-derived substituent (octa-2,4,6-trienedioyl) esterified to an oxygen at carbon atom 4. The genes required for biosynthesis of EPT and the eight-carbon polyketide precursor of the octa-2,4,6-trienedioyl substituent, as well as for esterification of the substituent to EPT have been described. However, genes required for conversion of the polyketide (octa-2,4,6-trienoic acid) to octa-2,4,6-trienedioyl-CoA, the immediate precursor of the substituent, have not been described. Here, we identified 91 cytochrome P450 monooxygenase genes in the genome sequence of T. arundinaceum, and provided evidence from gene deletion, complementation, cross-culture feeding, and chemical analyses that one of them (tri23) is required for conversion of octa-2,4,6-trienoic acid to octa-2,4,6-trienedioyl-CoA. The gene was detected in other HA-producing Trichoderma species, but not in species of other fungal genera that produce trichothecenes with an octa-2,4,6-trienoic acid-derived substituent. These findings indicate that tri23 is a trichothecene biosynthetic gene unique to Trichoderma species, which in turn suggests that modification of octa-2,4,6-trienoic acid during trichothecene biosynthesis has evolved independently in some fungi.
Similar content being viewed by others
References
Alexander NJ, McCormick SP, Hohn TM (1999) TRI12, a trichothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast. Mol Gen Genet 261:977–984
Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124:128–145. https://doi.org/10.1016/j.jbiotec.2006.01.026
Cardoza RE, Vizcaino JA, Hermosa MR, Monte E, Gutiérrez S (2006) A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation. J Microbiol 44:383–395
Cardoza RE, Malmierca MG, Hermosa MR, Alexander NJ, McCormick SP, Proctor RH, Tijerino AM, Rumbero A, Monte E, Gutiérrez S (2011) Identification of loci and functional characterization of trichothecene biosynthesis genes in filamentous fungi of the genus Trichoderma. Appl Environ Microbiol 77:4867–4877. https://doi.org/10.1128/AEM.00595-11
Cardoza RE, McCormick SP, Malmierca MG, Olivera ER, Alexander NJ, Monte E, Gutiérrez S (2015) Effects of trichothecene production on the plant defense response and fungal physiology: overexpression of the Trichoderma arundinaceum tri4 gene in T. harzianum. Appl Environ Microbiol 81:6355–6366. https://doi.org/10.1128/AEM.01626-15
Chadha S, Mehetre ST, Bansl R, Kuo A, Aerts A, Grigoriev IV, Druzhinina IS, Mukherjee PK (2018) Genome-wide analysis of cytochrome P450s of Trichoderma spp.: annotation and evolutionary relationships. Fungal Biol Biotechnol 5:12. https://doi.org/10.1186/s40694-018-0056-3
Chen W, Lee MK, Jefcoate C, Kim SC, Chen F, Yu JH (2014) Fungal cytochrome p450 monooxygenases: their distribution, structure, functions, family expansion, and evolutionary origin. Genome Biol Evol 6:1620–1634. https://doi.org/10.1093/gbe/evu132
Crooks GE, Hon G, Chandonia J-M, Brenner SE (2004) WebLogo: a sequence logo generator. Genome Res 14:1188–1190. https://doi.org/10.1101/gr.849004
Fan J, Urban M, Parker JE, Brewer HC, Kelly SL, Hammond-Kosack KE, Fraaije BA, Liu X, Cools HJ (2013) Characterization of the sterol 14α-demethylases of Fusarium graminearum identifies a novel genus-specific CYP51 function. New Phytol 198:821–835. https://doi.org/10.1111/nph.12193
Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species--opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56. https://doi.org/10.1038/nrmicro797
Helliwell CA, Poole A, Peacock J, Dennis ES (1999) Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis. Plant Physiol 119:507–510
Hermosa R, Viterbo A, Chet I, Monte E (2012) Plant-beneficial effects of Trichoderma and of its genes. Microbiology (Reading, Engl) 158:17–25. https://doi.org/10.1099/mic.0.052274-0
Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42:182–192. https://doi.org/10.1093/sysbio/42.2.182
Hofmann K, Stoffel W (1993) TMbase - a database of membrane spanning proteins segments. Biol Chem Hoppe Seyler 374:166
Hohn TM, Krishna R, Proctor RH (1999) Characterization of a transcriptional activator controlling trichothecene toxin biosynthesis. Fungal Genet Biol 26:224–235. https://doi.org/10.1006/fgbi.1999.1122
Käll L, Krogh A, Sonnhammer ELL (2004) A combined transmembrane topology and signal peptide prediction method. J Mol Biol 338:1027–1036. https://doi.org/10.1016/j.jmb.2004.03.016
Katoh K, Miswa K, Kuma K-I, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066
Kikuchi H, Miyagawa Y, Sahashi Y, Inatomi S, Haganuma A, Nakahata N, Oshima Y (2004) Novel spirocyclic trichothecanes, spirotenuipesine A and B, isolated from entomopathogenic fungus, Paecilomyces tenuipes. J Org Chem 69:352–356. https://doi.org/10.1021/jo035137x
Kistler HC, Broz K (2015) Cellular compartmentalization of secondary metabolism. Front Microbiol 6:68. https://doi.org/10.3389/fmicb.2015.00068
Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580. https://doi.org/10.1006/jmbi.2000.4315
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054
Lamb DC, Skaug T, Song H-L, Jackson CJ, Podust LM, Waterman MR, Kell DB, Kelly DE, Kelly SL (2002) The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2). J Biol Chem 277:24000–24005. https://doi.org/10.1074/jbc.M111109200
Lee HB, Kim Y, Jin HZ, Lee JJ, Kim C-J, Park JY, Jung HS (2005) A new Hypocrea strain producing harzianum A cytotoxic to tumour cell lines. Lett Appl Microbiol 40:497–503. https://doi.org/10.1111/j.1472-765X.2005.01719.x
Lindo L, McCormick SP, Cardoza RE, Brown DW, Kim H-S, Alexander NJ, Proctor RH, Gutiérrez S (2018) Effect of deletion of a trichothecene toxin regulatory gene on the secondary metabolism transcriptome of the saprotrophic fungus Trichoderma arundinaceum. Fungal Genet Biol 119:29–46. https://doi.org/10.1016/j.fgb.2018.08.002
Lindo L, McCormick SP, Cardoza RE, Busman M, Alexander NJ, Proctor RH, Gutiérrez S (2019a) Requirement of two acyltransferases for 4-O-acylation during biosynthesis of harzianum A, an antifungal trichothecene produced by Trichoderma arundinaceum. J Agric Food Chem 67:723–734. https://doi.org/10.1021/acs.jafc.8b05564
Lindo L, McCormick SP, Cardoza RE, Kim H-S, Brown DW, Alexander NJ, Proctor RH, Gutiérrez S (2019b) Role of Trichoderma arundinaceum tri10 in regulation of terpene biosynthetic genes and in control of metabolic flux. Fungal Genet Biol 122:31–46. https://doi.org/10.1016/j.fgb.2018.11.001
Malmierca MG, Cardoza RE, Alexander NJ, McCormick SP, Hermosa R, Monte E, Gutiérrez S (2012) Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes. Appl Environ Microbiol 78:4856–4868. https://doi.org/10.1128/AEM.00385-12
Malmierca MG, Cardoza RE, Alexander NJ, McCormick SP, Collado IG, Hermosa R, Monte E, Gutiérrez S (2013) Relevance of trichothecenes in fungal physiology: disruption of tri5 in Trichoderma arundinaceum. Fungal Genet Biol 53:22–33. https://doi.org/10.1016/j.fgb.2013.02.001
Malmierca MG, Barua J, McCormick SP, Izquierdo-Bueno I, Cardoza RE, Alexander NJ, Hermosa R, Collado IG, Monte E, Gutiérrez S (2015) Novel aspinolide production by Trichoderma arundinaceum with a potential role in Botrytis cinerea antagonistic activity and plant defence priming. Environ Microbiol 17:1103–1118. https://doi.org/10.1111/1462-2920.12514
McCormick SP, Stanley AM, Stover NA, Alexander NJ (2011) Trichothecenes: from simple to complex mycotoxins. Toxins 3:802–814. https://doi.org/10.3390/toxins3070802
Nakamura T, Yamada KD, Tomii K, Katoh K (2018) Parallelization of MAFFT for large-scale multiple sequence alignments. Bioinformatics 34:2490–2492. https://doi.org/10.1093/bioinformatics/bty121
Nasmith CG, Walkowiak S, Wang L, Leung WWY, Gong Y, Johnston A, Harris LJ, Guttman DS, Subramaniam R (2011) Tri6 is a global transcription regulator in the phytopathogen Fusarium graminearum. PLoS Pathog 7:e1002266. https://doi.org/10.1371/journal.ppat.1002266
Nelson DR (1999) Cytochrome P450 and the individuality of species. Arch Biochem Biophys 369:1–10. https://doi.org/10.1006/abbi.1999.1352
Nelson DR (2018) Cytochrome P450 diversity in the tree of life. Biochim Biophys Acta, Proteins Proteomics 1866:141–154. https://doi.org/10.1016/j.bbapap.2017.05.003
Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ (2014) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268–274. https://doi.org/10.1093/molbev/msu300
Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. https://doi.org/10.1038/nmeth.1701
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36
Pitt JI, Lange L, Lacey AE, Vuong D, Midgley DJ, Greenfield P, Bradbury MI, Lacey E, Busk PK, Pilgaard B, Chooi Y-H, Piggott AM (2017) Aspergillus hancockii sp. nov., a biosynthetically talented fungus endemic to southeastern Australian soils. PLoS ONE 12:e0170254. https://doi.org/10.1371/journal.pone.0170254
Poulos T, Johnson E (2005) Structures of cytochrome P450 enzymes. In: Cytochrome P450. Structure, mechanism and biochemistry. Kluwer Academic, New York, pp 88–114
Proctor RH, Desjardins AE, Plattner RD, Hohn TM (1999) A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A. Fungal Genet Biol 27:100–112. https://doi.org/10.1006/fgbi.1999.1141
Proctor RH, McCormick SP, Kim H-S, Cardoza RE, Stanley AM, Lindo L, Kelly A, Brown DW, Lee T, Vaughan MM, Alexander NJ, Busman M, Gutiérrez S (2018) Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi. PLoS Pathog 14:e1006946. https://doi.org/10.1371/journal.ppat.1006946
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117–124
Quidde T, Osbourn AE, Tudzynski P (1998) Detoxification of α-tomatine by Botrytis cinerea. Physiol Mol Plant Pathol 52:151–165
Ro DK, Arimura GI, Lau SYW, Piers E, Bohlmann J (2005) Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP702B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase. Proc Natl Acad Sci U S A 102:8060–8065. https://doi.org/10.1073/pnas.0500825102
Rubio MB, Hermosa R, Vicente R, Gómez-Acosta FA, Morcuende R, Monte E, Bettiol W (2017) The combination of Trichoderma harzianum and chemical fertilization leads to the deregulation of phytohormone networking, preventing the adaptive responses of tomato plants to salt stress. Front Plant Sci 8:294. https://doi.org/10.3389/fpls.2017.00294
Ryu SM, Lee HM, Song EG, Seo YH, Lee J, Guo Y, Kim BS, Kim J-J, Hong JS, Ryu KH, Lee D (2017) Antiviral activities of trichothecenes isolated from Trichoderma albolutescens against pepper mottle virus. J Agric Food Chem 65:4273–4279. https://doi.org/10.1021/acs.jafc.7b01028
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Schneider TD, Stephens RM (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 19:6097–6100
Semeiks J, Borek D, Otwinowski Z, Grishin NV (2014) Comparative genome sequencing reveals chemotype-specific gene clusters in the toxigenic black mold Stachybotrys. BMC Genomics 15:590. https://doi.org/10.1186/1471-2164-15-590
Shoresh M, Harman GE, Mastouri F (2010) Induced systemic resistance and plant responses to fungal biocontrol agents. Annu Rev Phytopathol 48:21–43. https://doi.org/10.1146/annurev-phyto-073009-114450
Sievers F, Higgins DG (2014) Clustal Omega, accurate alignment of very large numbers of sequences. Methods Mol Biol 1079:105–116. https://doi.org/10.1007/978-1-62703-646-7_6
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. https://doi.org/10.1038/msb.2011.75
Tamm C, Breitenstein W (1980) The biosynthesis of trichothecene mycotoxins. In: The biosynthesis of mycotoxins: a study in secondary metabolism. Elsevier, New York, pp 69–101
Tudzynski B (2005) Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology. Appl Microbiol Biotechnol 66:597–611. https://doi.org/10.1007/s00253-004-1805-1
Urlacher VB, Girhard M (2012) Cytochrome P450 monooxygenases: an update on perspectives for synthetic application. Trends Biotechnol 30:26–36. https://doi.org/10.1016/j.tibtech.2011.06.012
Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story. Genome Biol 1:3003.1–3003.9. https://doi.org/10.1186/gb-2000-1-6-reviews3003
Xiaoming L, Mu P, Wen J, Deng Y (2017) Carrier-mediated and energy-dependent uptake and efflux of deoxynivalenol in mammalian cells. Sci Rep 5:5889. https://doi.org/10.1038/s41598-017-06199-8
Acknowledgements
We thank Crystal Probyn, José Alvarez, Jennifer Teresi, Amy McGovern, and Christine Hodges for technical assistance.
Data Availability Statement
Sequences of the genomic regions containing tri18-tri17 and tri23 when present, from Trichoderma protrudens, T. turrialbense, T. albolutescens, and Hypocrea rodmanii, have been submitted to the GenBank database of the National Center for Biotechnology Information. The accession numbers for these sequences are as follows: MN136191 Hypocrea rodmanii, MN136192 T. albolutescens, MN136193 T. protrudens, and MN136194 T. turrialbense.
Genome sequences of T. arundinaceum and T. brevicompactum are available (Proctor et al. 2018).
Funding
This work was supported by the Spanish Ministry of Science, Innovation and Universities (MICINN-RTI2018-099600-B-100 to S.G.). Funding was also provided by the USDA-ARS National Program for Food Safety (NP108). L. Lindo was granted a fellowship by the University of León (Spain).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human or animals performed by any of the authors.
Disclaimer
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 12.3 mb)
Rights and permissions
About this article
Cite this article
Cardoza, R.E., McCormick, S.P., Lindo, L. et al. A cytochrome P450 monooxygenase gene required for biosynthesis of the trichothecene toxin harzianum A in Trichoderma. Appl Microbiol Biotechnol 103, 8087–8103 (2019). https://doi.org/10.1007/s00253-019-10047-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-019-10047-2