Abstract
TheTri4 gene ofFusarium sporotrichioides was isolated from a cloned DNA fragment carrying theTri5 gene by complementation of aTri4 − mutant. The nucleotide sequence ofTri4 was determined and the locations of three introns were identified. Analysis ofTri4 mRNA levels revealed that transcription reached maximum levels coincidently with the onset of trichothecene biosynthesis, and then declined 20-fold over the next 8 h. Disruption ofTri4 resulted in the loss of production of both trichothecenes and apotrichodiol and the accumulation of the unoxygenated pathway intermediate trichodiene. Transformants lacking a functionalTri4 gene were able to convert isotrichotriol, an early pathway intermediate, to T-2 toxin suggesting that most pathway enzymes are present inTri4 − mutants. These data suggest that the enzyme encoded byTri4 catalyzes the first oxygenation step in the trichothecene pathway and participates in apotrichodiol biosynthesis.Tri4 encodes a protein of 520 residues (M r =59 056) that shows significant homology with members of the superfamily of cytochromes P450. It appears most similar to the CYP3A subfamily (24.6% amino acid identity). Because it contains less than 40% positional identity with other cytochromes P450, theTri4 gene has been placed in a new cytochrome P450 gene family designatedCY P58.
Similar content being viewed by others
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Attar RM, Grotewold E, Taccioli GE, Aisemberg GO, Torres HN, Judewicz ND (1989) A cycloheximide-inducible gene ofNeurospora crassa belongs to the cytochrome P450 superfamily. Nucleic Acids Res 17:7535–7536
Beremand MN (1987) Isolation and characterization of mutants blocked in T-2 toxin biosynthesis. Appl Environ Microbiol 53:1855–1859
Cane DE (1990) Enzymatic formation of sesquiterpenes. Chem Rev 90:1089–1103
Colvin EW, Cameron S (1986) Synthesis and biological evaluation of a trichotheceneepi-epoxide 3a,4b,15-triacetoxy-12,13-epiepoxytrichothec-9-ene. J Chem Soc, Chem Commun 1642–1643
Desjardins AE, Plattner RD, VanMiddlesworth F (1986) Trichothecene biosynthesis inFusarium sporotrichioides: origin of the oxygen atoms of T-2 toxin. Appl Environ Microbiol 51:493–497
Desjardins AE, Plattner RD, Beremand MN (1987) Ancymidol blocks trichothecene biosynthesis and leads to accumulation of trichodiene inFusarium sporotrichioides andGibberella pulicaris. Appl Environ Microbiol 53:1860–1865
Desjardins AE, Spencer GF, Plattner RD, Beremand MN (1989) Furanocoumarin phytoalexins, trichothecene toxins and infection ofPastinaca sativa byFusarium sporotrichioides. Phytopathology 79:170–175
Desjardins AE, Hohn TM, McCormick SP (1992) The effect of gene disruption of trichodiene synthase (Tox5) on the virulence ofGibberella pulicaris. Mol Plant-Microbe Interact 5:214–222
Desjardins AE, Hohn TM, McCormick SP (1992) Trichothecene biosynthesis inFusarium species: chemistry, genetics, and significance. Microbiol Rev 57:595–604
Dumas JB, Edwards M, Delort J, Mallet J (1991) Oligodeoxyribonucleotide ligation to single stranded cDNAs: a new tool for cloning 5′ ends of mRNAs and for constructing cDNA libraries byin vitro amplification. Nucleic Acids Res 19:5227–5232
Funk C, Croteau R (1994) Diterpenoid resin acid biosynthesis in conifers: characterization of two cytochrome P450-dependent monooxygenases and an aldehyde dehydrogenase involved in abietic acid biosynthesis. Arch Biochem Biophys 308:258–266
Gledhill L, Hesketh AR, Bycroft BW, Dewick PM, Gilbert J (1991) Biosynthesis of trichothecene mycotoxins: cell-free epoxidation of a trichodiene derivative. FEMS Microbiol Lett 81:241–246
Gonzalez FJ, Nebert DW (1990) Evolution of the P450 gene superfamily: animal-plant ‘warfare’, molecular drive and human genetic differences in drug oxidation. Trends Genet 6:182–186
Gotoh O (1992) Substrate recognition sites in cytochrome P450 family 2(CYP2) proeins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem 267:83–90
Greenhalgh R, Fielder DA, Morrison LA, Charland J-P, Blackwell BA, Savard ME, ApSimon JW (1989) Secondary metabolites ofFusarium species: apotrichothecene derivatives. J Agric Food Chemistry 37:699–705
Hohn TM, Beremand MN (1989) Regulation of trichodine synthase inFusarium sporotrichioides andGibberella pulicaris (Fusarium sambucinum). Appl Environ Microbiol 55:1500–1503
Hohn TM, Desjardins AE (1992) Isolation and gene disruption of theTox5 gene encoding trichodine synthase inGibberella pulicaris. Mol Plant-Microbe Interact 5:249–256
Hohn TM, McCormick SP, Desjardins AE (1993) Evidence for a gene cluster involving trichothecene pathway biosynthetic genes inFusarium sporotrichioides. Curr Genet 24:291–295
Jarvis BB (1992) Macrocyclic trichothecenes from BrazilianBaccharis species: from microanalysis to large scale isolation. Phytochem Anal 3:241–249
Karp F, Harris JL, Croteau R (1987) Metabolism of monoterpenes: demonstration of the hydroxylation of (+)-sabinene to (+)-cis-sabinol by an enzyme preparation from sage (Salvia officinalis) leaves. Arch Biochem Biophys 256:179–193
Kizawa H, Tomura D, Oda M, Fukamizu A, Hoshino T, Gotoh O, Yasui T, Shoun H (1991) Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P450 cDNA fromFusarium oxysporum. J Biol Chem 266:10632–10637
Maloney AP, VanEtten HD (1994) A gene from the plant pathogenNectria haematococca that encodes the phytoalexin-detoxifying enzyme pisatin demethylase defines a new cytochrome P450 family. Mol Gen Genet 243:506–514
McLaughlin CS, Vaughn MH, Campbell JM, et al (1977) Inhibition of protein synthesis by trichothecenes. In: Rodricks JV, Hesseltine CW, Mehlman MA (eds) Mycotoxins in human and animal health. Pathotoxin Publishers, Park Forest pp 263–273
Nelson DR, Nebert DW (1993) The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol 12:1–51
Nelson DR, Strobel HW (1988) On the membrane topology of vertebrate cytochrome P450 proteins. J Biol Chem 263:6038–6050
Nelson DR, Strobel HW (1989) Secondary structure prediction of 52 membrane-bound cytochromes P450 shows a strong structural similarity to P450cam. Biochemistry 28:656–660
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448
Proctor RH, Hohn TM (1993) Aristolochene synthase. Isolation, characterization, and bacterial expression of a sesquiterpenoid biosynthetic gene (Aril) fromPenicillium roqueforti. J Biol Chem 268:4543–4548
Proctor RH, Hohn TM, McCormick SP, Desjardins AE (1995)Tri6 encodes an unusual zinc finger protein involved in the regulation of trichothecene biosynthesis inFusarium sporotrichioides. Appl Environ Microbiol 61:1923–1930, 1995
Rambosek JA, Leach J (1987) Recombinant DNA in the filamentous fungi: progress and prospects. Crit Rev Biotechnol 6:357–393
Turgeon BG, Garber RC, Yoder OC (1987) Development of a fungal transformation system based on selection of sequences with promoter activity. Mol Cell Biol 7:3297–3305
Ueno Y, Sawano M, Ishii K (1975) Production of trichothecene mycotoxins byFusarium species in liquid shake culture. Appl Microbiol 30:4–9
Van Gorcom RFM, Boschloo JG, Kuijvenhoven A, Lange J, Van Vark AJ, Bos CJ, Van Balken JAM, Pouwels PH, Van den Hondel CAMJJ (1990) Isolation and molecular characterization of the benzoate-para-hydroxylase gene (bphA) ofAspergillus niger: a member of a new gene family of the cytochrome P450 superfamily. Mol Gen Genet 223:192–197
Author information
Authors and Affiliations
Additional information
Communicated by C. A. M. J. J. Van den Hondel
The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.
Rights and permissions
About this article
Cite this article
Hohn, T.M., Desjardins, A.E. & McCormick, S.P. TheTri4 gene ofFusarium sporotrichioides encodes a cytochrome P450 monooxygenase involved in trichothecene biosynthesis. Molec. Gen. Genet. 248, 95–102 (1995). https://doi.org/10.1007/BF02456618
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02456618