University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Plant Pathology Plant Pathology Department 2007 Association of ergot alkaloids with conidiation in Aspergillus fumigatus Christine M. Coyle West Virginia University Shawn C. Kenaley West Virginia University William R. Rittenour University of Nebraska-Lincoln Daniel G. Panaccione West Virginia University, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/plantpathpapers Part of the Other Plant Sciences Commons, Plant Biology Commons, and the Plant Pathology Commons Coyle, Christine M.; Kenaley, Shawn C.; Rittenour, William R.; and Panaccione, Daniel G., "Association of ergot alkaloids with conidiation in Aspergillus fumigatus" (2007). Papers in Plant Pathology. 355. http://digitalcommons.unl.edu/plantpathpapers/355 This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Mycologia, 99(6), 2007, pp. 804–811. # 2007 by The Mycological Society of America, Lawrence, KS 66044-8897 Association of ergot alkaloids with conidiation in Aspergillus fumigatus Christine M. Coyle they have activities against mammals, insects, nema- Shawn C. Kenaley todes and bacteria (Schwarz and Eich 1983, Clay and William R. Rittenour1 Cheplick 1989, Ball et al 1997, Clay and Schardl 2002, Daniel G. Panaccione2 Panaccione 2005, Panaccione et al 2006). Their Division of Plant & Soil Sciences, West Virginia effects on humans are thought to be mediated by University, P.O. Box 6108, Morgantown, West their agonistic and/or antagonistic interactions with Virginia 26506-6108 monoamine neurotransmitter receptors (Pertz 1996, Gro¨ger and Floss 1998, Pertz and Eich 1999). Aspergillus fumigatus Fres., a common saprophyte Abstract: Ergot alkaloids are mycotoxins that affect and opportunistic pathogen, produces several ergot the nervous and reproductive systems of exposed alkaloids, including festuclavine and fumigaclavines individuals through interactions with monoamine A, B and C (Spilsbury and Wilkinson 1961, Cole et al receptors. They have been studied more widely in 1977). We recently reported a high performance ergot fungi and grass endophytes but also are found liquid chromatography (HPLC) procedure for iden- in Aspergillus fumigatus, an opportunistic human tification and quantification of these alkaloids and pathogen that reproduces and disseminates exclu- demonstrated them to be present in or on conidia of sively through conidia. The ergot alkaloids festucla- A. fumigatus in quantities that collectively total vine and fumigaclavines A, B and C are present in or approximately 1% of the dry mass of the conidium on conidia of A. fumigatus. Cultures of the fungus (Panaccione and Coyle 2005). Fumigaclavine C is the that are free of conidia are difficult to obtain, end product of the A. fumigatus pathway, with obscuring comparisons of conidia versus vegetative festuclavine, fumigaclavine B and fumigaclavine A hyphae as sources of the ergot alkaloids. To create (in that sequence) acting as the final three inter- conidiation-deficient strains of A. fumigatus we mediates in its biosynthesis (Panaccione 2005, manipulated the bristle A gene (brlA), which controls Schardl et al 2006, Unso¨ld and Li 2006). Differences vesicle formation or budding growth necessary for in abundance and activity of the enzymes responsible conidiation in Aspergillus spp. Disruption of brlA in A. for converting one intermediate to the next pre- fumigatus, via homologous recombination, resulted sumably account for the accumulation of festuclavine in a nonconidiating mutant that produced bristle-like and fumigaclavine A to relatively high concentrations structures instead of conidiophores and conidia. whereas fumigaclavine B is typically present in much Moreover the disrupted strain failed to produce ergot lower concentrations (Panaccione and Coyle 2005, alkaloids as verified by HPLC analyses. Complemen- Panaccione 2005). tation with a wild-type allele restored conidiation and A. fumigatus in vitro sporulates prolifically, and ergot alkaloid production. These results suggest that cultures free of conidia are difficult to obtain. Our ergot alkaloids are not produced within the vegetative unpublished observations of ergot alkaloid yields mycelium of the fungus and are associated directly from cultures of A. fumigatus on agar-based media with conidiation. (primarily conidia and limited vegetative hyphae) Key words: aspergillosis, bristle A gene, clavines, versus submerged, broth-based media (consisting mycotoxins mainly of hyphae and fewer conidia) suggested an apparent association between conidiation and ergot INTRODUCTION alkaloids. Research on Aspergillus spp. has associated second- Ergot alkaloids are a complex family of indole-derived ary metabolism and sporulation (Calvo et al 2002, Bok secondary metabolites produced by several fungi and Keller 2004). For example sporulation and (Flieger et al 1997, Gro¨ger and Floss 1998, Panac- sterigmatocystin production have been shown to be cione 2005, Schardl et al 2006). The ecological roles regulated by a FadA Ga protein-dependent signaling of ergot alkaloids are poorly understood; however pathway in Aspergillus nidulans Eidam (Winter) (Hicks et al 1997). More specifically sterigmatocystin Accepted for publication 15 August 2007. production and conidiation both are regulated 1 Present address: Department of Plant Pathology, 406 Plant Sciences Hall, University of Nebraska at Lincoln, Lincoln, NE negatively by a cAMP-dependent protein kinase 68588-0722. catalytic subunit (pkaA) (Shimizu and Keller 2001). 2 Corresponding author. E-mail: [email protected] Conidiation has been studied extensively in A. 804 COYLE ET AL:ERGOT ALKALOIDS AND CONIDIATION 805 nidulans. Conidiophore development is contingent of 1 min at 94 C, 1 min at 57 C and 1 min at 72 C, with on differentiation of the fungus from polarized apical a final extension step of 72 C for 7 min. A 4.6 kb disruption extension to budding growth (Cole 1986). One gene construct, pBRLA1, was generated by ligating the PCR in particular, brlA, has been determined to control product into the T/A overlap vector pCR2.1 (Invitrogen, conidiophore development and encodes a nucleic Carlsbad, California). The construct was linearized at the unique NdeI site located within the brlA fragment before acid binding protein (BrlA) (Boylan et al 1987, transformation of the fungus. Protoplasts were prepared Adams et al 1988, Adams et al 1998). BrlA is an early and transformed according to the protocol described by protein in a signaling cascade that controls the Murray et al (1992) and with modifications noted by Coyle expression of many genes, including some that and Panaccione (2005). Protoplasts were cotransformed encode for other regulatory proteins by binding to with the linearized disruption construct, pBRLA1, and brlA response elements (BRE) within the promoter a hygromycin resistance plasmid, pMOcosX (Orbach regions of these genes (Chang and Timberlake 1992). 1994), linearized at a unique NotIsite(Coyleand In A. nidulans transcription of brlA may initiate at Panaccione 2005). Nonconidiating transformants were different sites resulting in two overlapping transcrip- identified by their altered colony morphology and color tion units, brlAa and brlAb (Prade and Timberlake and were confirmed by light microscopy. Nonconidiating 1993). The products of these two forms are in- mutants were purified to nuclear homogeneity through repetitive subculturing from hyphal tips (because nuclear terchangeable functionally, translated in the same purification via single conidium isolation was not possible). reading frame and differ only in that the product of Homologous recombination between pBRLA1 and the brlAb has an additional 23 amino acids at the amino native brlA locus within the transformants was verified by terminus (Prade and Timberlake 1993). In this paper three PCR assays similar to the PCR reaction described we report mutational analyses of the brlA gene of A. above but with different primers and annealing tempera- fumigatus to produce nonconidiating mutants for the tures. The 59 border of the recombination event was purpose of examining whether ergot alkaloids are confirmed by PCR (annealing temperature at 57 C) primed produced during vegetative growth or conidiation. with oligonucleotides UF (59-TGTAAAACGACGGCCAGT- GAAT-39, which anneals to vector sequences near the universal primer annealing site in pCR2.1) and brlAFscrn MATERIALS AND METHODS (59-CTCCAACGAATGTCCGTCTATG-39, complementary to brlA sequences near the 59-end of the brlA gene and Fungi and culture conditions.—Aspergillus fumigatus isolate flanking the intended site of integration) (FIG. 1a). The FGSC A1141 (Panaccione and Coyle 2005) and derivatives juxtaposition of sequences near the 39 border of the were cultured routinely on potato-dextrose agar (PDA) integration event was verified by PCR (annealing temp prepared from dehydrated potato flakes (Pillsbury, Minnea- 55 C) primed from oligonucleotides UR (59-AGCTATGAC- polis, Minnesota) (20 g/L), D-glucose (20 g/L) and agar CATGATTACGCCA-39, complementary to vector sequences (15 g/L). Cultures intended for preparation of protoplasts near the reverse primer annealing site of pCR2.1), and or DNA were grown overnight in 15 mL of malt-extract brlARscrn (59-TAGTGACAAGCTCTGCTTGGA