Effects of xanthotoxin treatment on trichothecene production in Fusarium sporotrichioides Nancy J. Alexander, Susan P. McCormick, and Judith A. Blackburn Abstract: There are 4 P450 oxygenases involved in the biosynthesis of T-2 toxin in Fusarium sporotrichioides. Exactly how these enzymes react to antimicrobial plant defense compounds is unknown. Xanthotoxin (8-methoxypsoralen) is a phototoxic furanocoumarin that acts as a P450 oxygenase inhibitor. The current study shows that the addition of concentra- tions of 1.0 mmol/L or less of xanthotoxin to liquid cultures of F. sporotrichioides NRRL3299 can effectively block T-2 toxin production and cause an increase in accumulation of trichodiene, the hydrocarbon precursor of trichothecenes. The addition of xanthotoxin to liquid cultures of a trichodiene-accumulating F. sporotrichioides Tri4 – mutant caused a 3- to 10-fold increase in trichodiene accumulation, suggesting that xanthotoxin not only blocks trichothecene oxygenation reac- tions, but may in some way also promote the synthesis of trichodiene. Feeding studies showed that 2 of the 4 P450 oxy- genases, TRI4 and TRI1, were more sensitive to xanthotoxin, while oxygenases TRI11 and TRI13 were unaffected. Quantitative reverse-transcriptase PCR indicated that several of the genes in the toxin biosynthetic pathway were upregu- lated by xanthotoxin, with Tri4 showing the highest increase in expression. These results indicate that while xanthotoxin inhibits specific P450 oxygenase activity, it also has an effect on gene expression. Key words: mycotoxin, qRT-PCR, P450, trichodiene, furanocoumarin. Re ´sume ´: Il existe quatre oxyge ´nases P450 implique ´es dans la biosynthe `se de la toxine T-2 de Fusarium sporotrichoides. On ne sait pas exactement comment ces enzymes re ´agissent aux compose ´s associe ´s a ` la de ´fense antimicrobienne des plan- tes. La xanthotoxine (8-me ´thylpsorale `ne) est un furanocoumarin phototoxique qui agit comme inhibiteur des oxyge ´nases P450. L’e ´tude pre ´sente a de ´montre ´ que l’ajout de 1.0 mM ou moins de xanthotoxine a ` des cultures liquides de F. sporotri- choides NRRL3299 pouvait effectivement bloquer la production de la toxine T-2 et causer une augmentation de l’accumu- lation de trichodie `ne, l’hydrate de carbone pre ´curseur des trichothe ´ce `nes. L’ajout de xanthotoxine a ` des cultures liquides du mutant F. sporotrichoides Tri4 – qui accumule le trichodie `ne a cause ´ une augmentation de 3 a ` 10 fois de l’accumulation du trichodie `ne, ce qui sugge `re que la xanthotoxine ne bloque pas seulement les re ´actions d’oxyge ´nation du trichothe ´ce `ne, mais favorise d’une quelconque fac ¸on la synthe `se de trichodie `ne. Des e ´tudes d’alimentation ont de ´montre ´ que 2 des 4 oxy- ge ´nases, TRI4 et TRI1, e ´taient plus sensibles a ` la xanthotoxine alors que les oxyge ´nases TRI11 et TRI13 n’e ´taient pas af- fecte ´es. Des analyses en PCR re ´verse quantitative (qRT-PCR) ont indique ´ que plusieurs des ge `nes de la voie de biosynthe `se de la toxine e ´taient re ´gle ´s positivement par la xanthotoxine, l’expression de Tri4 e ´tant la plus augmente ´e. Ces re ´sultats indiquent qu’alors que la xanthotoxine inhibe l’activite ´ d’oxyge ´nases spe ´cifiques, elle exerce aussi un effet sur l’expression ge ´nique. Mots-cle ´s : mycotoxine, qRT-PCR, P450, trichodie `ne, furanocoumarine. [Traduit par la Re ´daction] Introduction Trichothecenes are a group of toxic sesquiterpenoids with an olefinic bond and an epoxide group produced by several related genera of fungi (for a review see Desjardins 2006). They are potent inhibitors of eukaryotic protein synthesis, and play a role in a number of human and animal mycotoxi- coses (Ueno 1977) and the wheat head scab plant disease (Proctor et al. 1995a; Desjardins et al. 1996). Trichothecenes are derived from the alicyclic hydrocarbon trichodiene (Fig. 1), which is a cyclization product of farnesyl pyrophos- phate. The conversion of trichodiene to more complex tri- chothecenes, such as T-2 toxin (Fig. 1), requires a series of oxygenations, cyclizations, and esterifications (Fig. 1) (Des- jardins et al. 1993) The genes controlling the biosynthesis of 15-acetyldeoxy- nivalenol and T-2 toxin in Fusarium graminearum and Fusa- rium sporotrichioides, respectively, have been identified and mapped to 4 unlinked loci in the genome (for a review see Desjardins 2006). Although there are up to 8 oxygenation steps in the biosynthesis of these toxins, only 4 P450 genes are required (Hohn et al. 1995; McCormick et al. 2004, 2006a, 2006b; Tokai et al. 2007). Three of the P450 genes, Tri4 (Hohn et al. 1995), Tri11 (Alexander et al. 1998), and Tri13 (Lee et al. 2002; Brown et al. 2002), are located within a large 28 kb cluster. The fourth P450 gene, Tri1, is at a sep- arate locus (Peplow et al. 2003; Brown et al. 2003). Plants often respond to invasion by fungi by producing Received 7 May 2008. Accepted 14 May 2008. Published on the NRC Research Press Web site at cjm.nrc.ca on 18 December 2008. Reposted on the Web site with corrections on 5 January 2009. N.J. Alexander, 1 S.P. McCormick, and J.A. Blackburn. Mycotoxin Research Unit, National Center for Agricultural Utilization Research, ARS/USDA, 1815 N. University St, Peoria, IL 61604, USA. 1 Corresponding author (e-mail: Nancy.Alexander@ars.usda.gov). 1023 Can. J. Microbiol. 54: 1023–1031 (2008) doi:10.1139/W08-100 # 2008 NRC Canada