Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement

Kozák, László; Szilágyi, Zoltán; Vágó, Barbara; Kakuk, Annamária; Tóth, László; Molnár, István; Pócsi, István

doi:10.1007/s00253-018-8807-x

Applied genetics and molecular biotechnology
Published: 19 February 2018

Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement

László Kozák^1,2,
Zoltán Szilágyi²,
Barbara Vágó²,
Annamária Kakuk²,
László Tóth²,
István Molnár³ &
István Pócsi¹

Applied Microbiology and Biotechnology volume 102, pages 3255–3266 (2018)Cite this article

825 Accesses
16 Citations
Metrics details

Abstract

The hypocrealean fungus Claviceps paspali is a parasite of wild grasses. This fungus is widely utilized in the pharmaceutical industry for the manufacture of ergot alkaloids, but also produces tremorgenic and neurotoxic indole-diterpene (IDT) secondary metabolites such as paspalitrems A and B. IDTs cause significant losses in agriculture and represent health hazards that threaten food security. Conversely, IDTs may also be utilized as lead compounds for pharmaceutical drug discovery. Current protoplast-mediated transformation protocols of C. paspali are inadequate as they suffer from inefficiencies in protoplast regeneration, a low frequency of DNA integration, and a low mitotic stability of the nascent transformants. We adapted and optimized Agrobacterium tumefaciens-mediated transformation (ATMT) for C. paspali and validated this method with the straightforward creation of a mutant strain of this fungus featuring a targeted replacement of key genes in the putative IDT biosynthetic gene cluster. Complete abrogation of IDT production in isolates of the mutant strain proved the predicted involvement of the target genes in the biosynthesis of IDTs. The mutant isolates continued to produce ergot alkaloids undisturbed, indicating that equivalent mutants generated in industrial ergot producers may have a better safety profile as they are devoid of IDT-type mycotoxins. Meanwhile, ATMT optimized for Claviceps spp. may open the door for the facile genetic engineering of these industrially and ecologically important organisms.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

References

Amici AM, Scotti T, Spalla C, Tognoli L (1967) Heterokaryosis and alkaloid production in Claviceps purpurea. Appl Microbiol 15(3):611–615
CAS PubMed PubMed Central Google Scholar
Arcamone F, Bonino C, Chain EB, Ferretti A, Pennella P, Tonolo A, Vero L (1960) Production of lysergic acid derivatives by a strain of Claviceps paspali Stevens and Hall in submerged culture. Nature 187(4733):238–239. https://doi.org/10.1038/187238a0
CAS Article PubMed Google Scholar
Bennett JW, Klich M (2003) Mycotoxins. Clin Microbiol Rev 16(3):497–516. https://doi.org/10.1128/CMR.16.3.497-516.2003
CAS Article PubMed PubMed Central Google Scholar
Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214
CAS PubMed PubMed Central Google Scholar
Byrne KM, Smith SK, Ondeyka JG (2002) Biosynthesis of nodulisporic acid A: precursor studies. J Am Chem Soc 124(24):7055–7060. https://doi.org/10.1021/ja017183p
CAS Article PubMed Google Scholar
Cawdell-Smith AJ, Scrivener CJ, Bryden WL (2010) Staggers in horses grazing paspalum infected with Claviceps paspali. Aust Vet J 88(10):393–395. https://doi.org/10.1111/j.1751-0813.2010.00624.x
CAS Article PubMed Google Scholar
Chain EB, Bonino C, Tonolo A (1962) Process for the production of alkaloid derivatives of lysergic acid. US Patent Office 3,038,840
Cole RJ, Dorner JW, Lansden JA, Cox RH, Pape C, Cunfer B, Nicholson SS, Bedell DM (1977) Paspalum staggers: isolation and identification of tremorgenic metabolites from sclerotia of Claviceps paspali. J Agric Food Chem 25(5):1197–1201. https://doi.org/10.1021/jf60213a061
CAS Article PubMed Google Scholar
de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16(9):839–842. https://doi.org/10.1038/nbt0998-839
Article PubMed Google Scholar
di Menna ME, Finch SC, Popay AJ, Smith BL (2012) A review of the Neotyphodium lolii/Lolium perenne symbiosis and its associated effects on animal and plant health, with particular emphasis on ryegrass staggers. N Z Vet J 60(6):315–328. https://doi.org/10.1080/00480169.2012.697429
Article PubMed Google Scholar
Ehrlich KC, Mack BM (2014) Comparison of expression of secondary metabolite biosynthesis cluster genes in Aspergillus flavus, A. parasiticus, and A. oryzae. Toxins (Basel) 6(6):1916–1928. https://doi.org/10.3390/toxins6061916
CAS Article Google Scholar
Esser K, Tudzynski P (1978) Genetics of the ergot fungus Claviceps purpurea: I. Proof of a monoecious life cycle and segregation patterns for mycelial morphology and alkaloid production. Theor Appl Genet 53(4):145–149. https://doi.org/10.1007/BF00273574
CAS Article PubMed Google Scholar
Flieger M, Mehta P, Mehta A (2003) Biotechnological potential of ergot alkaloids. In: Arora DK (ed) Fungal biotechnology in agricultural, food, and environmental applications. Marcel Dekker, New York, pp 91–99
Google Scholar
Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25(2-3):179–188. https://doi.org/10.1016/0378-1119(83)90223-8
CAS Article PubMed Google Scholar
Haarmann T, Machado C, Lübbe Y, Correia T, Schardl CL, Panaccione DG, Tudzynski P (2005) The ergot alkaloid gene cluster in Claviceps purpurea: extension of the cluster sequence and intra species evolution. Phytochemistry 66(11):1312–1320. https://doi.org/10.1016/j.phytochem.2005.04.011
CAS Article PubMed Google Scholar
Hareven D, Koltin Y (1970) Nuclear distribution in the mycelium of Claviceps and the problem of strain selection. Appl Microbiol 19(6):1005–1006
CAS PubMed PubMed Central Google Scholar
Hulvová H, Galuszka P, Frébortová J, Frébort I (2013) Parasitic fungus Claviceps as a source for biotechnological production of ergot alkaloids. Biotechnol Adv 31(1):79–89. https://doi.org/10.1016/j.biotechadv.2012.01.005
Article PubMed Google Scholar
Imlach WL, Finch SC, Zhang Y, Dunlop J, Dalziel JE (2011) Mechanism of action of lolitrem B, a fungal endophyte derived toxin that inhibits BK large conductance Ca²⁺-activated K⁺ channels. Toxicon 57(5):686–694. https://doi.org/10.1016/j.toxicon.2011.01.013
CAS Article PubMed Google Scholar
Keller U, Tudzynski P (2002) Ergot Alkaloids. In: Osiewacz HD (ed) Industrial applications. The Mycota (a comprehensive treatise on fungi as experimental systems for basic and applied research), vol 10. Springer, Berlin, pp 157–181. https://doi.org/10.1007/978-3-662-10378-4_8
Google Scholar
Kishimoto S, Sato M, Tsunematsu Y, Watanabe K (2016) Evaluation of biosynthetic pathway and engineered biosynthesis of alkaloids. Molecules 21(8):e1078. https://doi.org/10.3390/molecules21081078
Article PubMed Google Scholar
Kunitake E, Tani S, Sumitani J, Kawaguchi T (2013) A novel transcriptional regulator, ClbR, controls the cellobiose- and cellulose-responsive induction of cellulase and xylanase genes regulated by two distinct signaling pathways in Aspergillus aculeatus. Appl Microbiol Biotechnol 97(5):2017–2028. https://doi.org/10.1007/s00253-012-4305-8
CAS Article PubMed Google Scholar
Laws I, Mantle PG (1989) Experimental constraints in the study of the biosynthesis of indole alkaloids in fungi. J Gen Microbiol 135(10):2679–2692. https://doi.org/10.1099/00221287-135-10-2679
CAS Google Scholar
Liu C, Noike M, Minami A, Oikawa H, Dairi T (2014) A fungal prenyltransferase catalyzes the regular di-prenylation at positions 20 and 21 of paxilline. Biosci Biotechnol Biochem 78(3):448–454. https://doi.org/10.1080/09168451.2014.882759
CAS Article PubMed Google Scholar
McMillan LK, Carr RL, Young CA, Astin JW, Lowe RG, Parker EJ, Jameson GB, Finch SC, Miles CO, McManus OB, Schmalhofer WA, Garcia ML, Kaczorowski GJ, Goetz M, Tkacz JS, Scott B (2003) Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli, and effects of paxilline intermediates on mammalian maxi-K ion channels. Mol Gen Genomics 270(1):9–23. https://doi.org/10.1007/s00438-003-0887-2
CAS Article Google Scholar
Michielse CB, Hooykaas PJ, van den Hondel CA, Ram AF (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet 48(1):1–17. https://doi.org/10.1007/s00294-005-0578-0
CAS Article PubMed Google Scholar
Nicholson MJ, Koulman A, Monahan BJ, Pritchard BL, Payne GA, Scott B (2009) Identification of two aflatrem biosynthesis gene loci in Aspergillus flavus and metabolic engineering of Penicillium paxilli to elucidate their function. Appl Environ Microbiol 75(23):7469–7481. https://doi.org/10.1128/AEM.02146-08
CAS Article PubMed PubMed Central Google Scholar
Nicholson MJ, Eaton CJ, Stärkel C, Tapper BA, Cox MP, Scott B (2015) Molecular cloning and functional analysis of gene clusters for the biosynthesis of indole-diterpenes in Penicillium crustosum and P. janthinellum. Toxins (Basel) 7(8):2701–2722. https://doi.org/10.3390/toxins7082701
CAS Article Google Scholar
Păcurar DI, Thordal-Christensen H, Păcurar ML, Pamfil D, Botez C, Bellini C (2011) Agrobacterium tumefaciens: from crown gall tumors to genetic transformation. Physiol Mol Plant Pathol 76(2):76–81. https://doi.org/10.1016/j.pmpp.2011.06.004
Article Google Scholar
Panaccione DG, Schardl CL (2003) Molecular genetics of ergot alkaloid biosynthesis. In: White JF Jr, Bacon CW, Hywel-Jones NL, Spatafora JW (eds) The clavicipitalean fungi: evolutionary biology, chemistry, biocontrol, and cultural impacts. Marcel-Dekker, New York, pp 399–424. https://doi.org/10.1201/9780203912706.ch13
Google Scholar
Panaccione DG, Cipoletti JR, Sedlock AB, Blemings KP, Schardl CL, Machado C, Seidel GE (2006) Effects of ergot alkaloids on food preference and satiety in rabbits, as assessed with gene-knockout endophytes in perennial ryegrass (Lolium perenne). J Agric Food Chem 54(13):4582–4587. https://doi.org/10.1021/jf060626u
CAS Article PubMed Google Scholar
Panaccione DG, Beaulieu WT, Cook D (2014) Bioactive alkaloids in vertically transmitted fungal endophytes. Funct Ecol 28(2):299–314. https://doi.org/10.1111/1365-2435.12076
Article Google Scholar
Parker EJ, Scott DB (2004) Indole-diterpene biosynthesis in ascomycetous fungi. In: An Z (ed) Handbook of Industrial Mycology, Vol. 22. Marcel Dekker, New York, pp. 405–426
Ricicová A, Flieger M, Rehácek Z (1982) Quantitative changes of the alkaloid complex in a submerged culture of Claviceps paspali. Folia Microbiol (Praha) 27(6):433–445. https://doi.org/10.1007/BF02876456
Article Google Scholar
Saikia S, Parker EJ, Koulman A, Scott B (2006) Four gene products are required for the fungal synthesis of the indole-diterpene, paspaline. FEBS Lett 580(6):1625–1630. https://doi.org/10.1016/j.febslet.2006.02.008
CAS Article PubMed Google Scholar
Saikkonen K, Young CA, Helander M, Schardl CL (2016) Endophytic Epichloë species and their grass hosts: from evolution to applications. Plant Mol Biol 90(6):665–675. https://doi.org/10.1007/s11103-015-0399-6
CAS Article PubMed Google Scholar
Sallam AA, Ayoub NM, Foudah AI, Gissendanner CR, Meyer SA, El Sayed KA (2013) Indole diterpene alkaloids as novel inhibitors of the Wnt/β-catenin pathway in breast cancer cells. Eur J Med Chem 70:594–606. https://doi.org/10.1016/j.ejmech.2013.09.045
CAS Article PubMed Google Scholar
Schardl CL, Panaccione DG, Tudzynski P (2006) Ergot alkaloids—biology and molecular biology. Alkaloids Chem Biol 63:45–86. https://doi.org/10.1016/S1099-4831(06)63002-2
CAS Article PubMed Google Scholar
Schardl CL, Young CA, Hesse U, Amyotte SG, Andreeva K, Calie PJ, Fleetwood DJ, Haws DC, Moore N, Oeser B, Panaccione DG, Schweri KK, Voisey CR, Farman ML, Jaromczyk JW, Roe BA, O’Sullivan DM, Scott B, Tudzynski P, An Z, Arnaoudova EG, Bullock CT, Charlton ND, Chen L, Cox M, Dinkins RD, Florea S, Glenn AE, Gordon A, Güldener U, Harris DR, Hollin W, Jaromczyk J, Johnson RD, Khan AK, Leistner E, Leuchtmann A, Li C, Liu J, Liu J, Liu M, Mace W, Machado C, Nagabhyru P, Pan J, Schmid J, Sugawara K, Steiner U, Takach JE, Tanaka E, Webb JS, Wilson EV, Wiseman JL, Yoshida R, Zeng Z (2013) Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. PLoS Genet 9(2):e1003323. https://doi.org/10.1371/journal.pgen.1003323
CAS Article PubMed PubMed Central Google Scholar
Scott B, Young CA, Saikia S, McMillan LK, Monahan BJ, Koulman A, Astin J, Eaton CJ, Bryant A, Wrenn RE, Finch SC, Tapper BA, Parker EJ, Jameson GB (2013) Deletion and gene expression analyses define the paxilline biosynthetic gene cluster in Penicillium paxilli. Toxins (Basel) 5(8):1422–1446. https://doi.org/10.3390/toxins5081422
CAS Article Google Scholar
Socic H, Gaberc-Porekar V, Pertot E, Puc A, Milicić S (1986) Developmental studies of Claviceps paspali seed cultures for the submerged production of lysergic acid derivatives. J Basic Microbiol 26(9):533–539. https://doi.org/10.1002/jobm.3620260906
CAS Article PubMed Google Scholar
Thom ER, Popay AJ, Waugh CD, Minne EMK (2014) Impact of novel endophytes in perennial ryegrass on herbage production and insect pests from pastures under dairy cow grazing in northern New Zealand. Grass Forage Sci 69(1):191–204. https://doi.org/10.1111/gfs.12040
CAS Article Google Scholar
Tudzynski P, Correia T, Keller U (2001) Biotechnology and genetics of ergot alkaloids. Appl Microbiol Biotechnol 57(5-6):593–605. https://doi.org/10.1007/s002530100801
CAS Article PubMed Google Scholar
Uhlig S, Botha CJ, Vrålstad T, Rolén E, Miles CO (2009) Indole-diterpenes and ergot alkaloids in Cynodon dactylon (Bermuda grass) infected with Claviceps cynodontis from an outbreak of tremors in cattle. J Agric Food Chem 57(23):11112–11119. https://doi.org/10.1021/jf902208w
CAS Article PubMed Google Scholar
Uhlig S, Egge-Jacobsen W, Vrålstad T, Miles CO (2014) Indole-diterpenoid profiles of Claviceps paspali and Claviceps purpurea from high-resolution Fourier transform Orbitrap mass spectrometry. Rapid Commun Mass Spectrom 28(14):1621–1634. https://doi.org/10.1002/rcm.6938
CAS Article PubMed Google Scholar
van Engelenburg F, Smit R, Goosen T, van den Broek H, Tudzynski P (1989) Transformation of Claviceps purpurea using a bleomycin resistance gene. Appl Microbiol Biotechnol 30(4):364–370. https://doi.org/10.1007/BF00296625
Article Google Scholar
Wiewióra B, Żurek G, Pańka D (2015) Is vertical transmission of Neotyphodium lolli in perennial ryegrass the only possible way to the spread of endophytes? PLoS One 10(2):e0117231. https://doi.org/10.1371/journal.pone.0117231
Article PubMed PubMed Central Google Scholar
Xu Y, Orozco R, Wijeratne KEM, Gunatilaka LAA, Stock SP, Molnár I (2008) Biosynthesis of the cyclooligomer depsipeptide beauvericin, a virulence factor of the entomopathogenic fungus Beauveria bassiana. Chem Biol 15(9):898–907. https://doi.org/10.1016/j.chembiol.2008.07.011
CAS Article PubMed Google Scholar
Xu Y, Orozco R, Wijeratne KEM, Espinosa-Artiles P, Gunatilaka LAA, Stock SP, Molnár I (2009) Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal virulence factor of Beauveria bassiana. Fungal Genet Biol 46(5):353–364. https://doi.org/10.1016/j.fgb.2009.03.001
CAS Article PubMed Google Scholar
Yamada M, Yawata K, Orino Y, Ueda S, Isogai Y, Taguchi G, Shimosaka M, Hashimoto S (2009) Agrobacterium tumefaciens-mediated transformation of antifungal-lipopeptide-producing fungus Coleophoma empetri F-11899. Curr Genet 55(6):623–630. https://doi.org/10.1007/s00294-009-0275-5
CAS Article PubMed Google Scholar
Young C, McMillan L, Telfer E, Scott B (2001) Molecular cloning and genetic analysis of an indole-diterpene gene cluster from Penicillium paxilli. Mol Microbiol 39(3):754–764. https://doi.org/10.1046/j.1365-2958.2001.02265.x
CAS Article PubMed Google Scholar
Young CA, Bryant MK, Christensen MJ, Tapper BA, Bryan GT, Scott B (2005) Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Mol Gen Genomics 274(1):13–29. https://doi.org/10.1007/s00438-005-1130-0
CAS Article Google Scholar
Young CA, Felitti S, Shields K, Spangenberg G, Johnson RD, Bryan GT, Saikia S, Scott B (2006) A complex gene cluster for indole-diterpene biosynthesis in the grass endophyte Neotyphodium lolii. Fungal Genet Biol 43(10):679–693. https://doi.org/10.1016/j.fgb.2006.04.004
CAS Article PubMed Google Scholar
Young C, Schardl CL, Panaccione DG, Florea S, Takach JE, Charlton ND, Moore N, Webb JS, Jaromczyk J (2015) Genetics, genomics and evolution of ergot alkaloid diversity. Toxins (Basel) 7(4):1273–1302. https://doi.org/10.3390/toxins7041273
CAS Article Google Scholar
Zhang A, Lu P, Dahl-Roshak AM, Paress PS, Kennedy S, Tkacz JS, An Z (2003) Efficient disruption of a polyketide synthase gene (pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis. Mol Gen Genomics 268(5):645–655. https://doi.org/10.1007/s00438-002-0780-4
CAS Google Scholar
Zhang S, Monahan BJ, Tkacz JS, Scott B (2004) Indole-diterpene gene cluster from Aspergillus flavus. Appl Environ Microbiol 70(11):6875–6883. https://doi.org/10.1128/AEM.70.11.6875-6883.2004
CAS Article PubMed PubMed Central Google Scholar
Zhong YH, Wang XL, Wang TH, Jiang Q (2007) Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis. Appl Microbiol Biotechnol 73(6):1348–1354. https://doi.org/10.1007/s00253-006-0603-3
CAS Article PubMed Google Scholar

Download references

Funding

This work was supported by the European Union and the European Social Fund through the project EFOP-3.6.1-16-2016-00022 (to IP) and by the National Institutes of Health project NIGMS R01GM114418-01A1 (to IM).

Author information

Authors and Affiliations

Department of Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary
László Kozák & István Pócsi
Teva Pharmaceutical Works Ltd., Debrecen, Hungary
László Kozák, Zoltán Szilágyi, Barbara Vágó, Annamária Kakuk & László Tóth
Natural Products Center, School of Natural Resources and the Environment, University of Arizona, Tucson, USA
István Molnár

Authors

László Kozák
View author publications
You can also search for this author in PubMed Google Scholar
Zoltán Szilágyi
View author publications
You can also search for this author in PubMed Google Scholar
Barbara Vágó
View author publications
You can also search for this author in PubMed Google Scholar
Annamária Kakuk
View author publications
You can also search for this author in PubMed Google Scholar
László Tóth
View author publications
You can also search for this author in PubMed Google Scholar
István Molnár
View author publications
You can also search for this author in PubMed Google Scholar
István Pócsi
View author publications
You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to István Molnár or István Pócsi.

Ethics declarations

Conflict of interest

IM has a disclosed financial interest in, and LK, ZS, BV, AK, and LT are employees of Teva Pharmaceutical Works Ltd., Hungary. Responsibility for the design of the experiments, the evaluation of the results, the conclusions drawn, and the opinions expressed in this article are solely those of the authors and are not shared by Teva Pharmaceutical Works Ltd. IP declares no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the Authors.

Electronic supplementary material

ESM 1

(PDF 737 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kozák, L., Szilágyi, Z., Vágó, B. et al. Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium-mediated gene replacement. Appl Microbiol Biotechnol 102, 3255–3266 (2018). https://doi.org/10.1007/s00253-018-8807-x

Download citation

Received: 21 October 2017
Revised: 13 January 2018
Accepted: 19 January 2018
Published: 19 February 2018
Issue Date: April 2018
DOI: https://doi.org/10.1007/s00253-018-8807-x

Keywords

Claviceps paspali
Indole-diterpene
Paspaline
Paspalitrem
Ergot
Agrobacterium tumefaciens