A fragmented aflatoxin-like gene cluster in the forest pathogen Dothistroma septosporum Shuguang Zhang a,b, Arne Schwelm a,b, Hongping Jin a, Lesley J. Collins c, Rosie E. Bradshaw a,b,* a Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand b National Centre for Advanced Bio-Protection Technologies, Massey University, Private Bag 11222, Palmerston North, New Zealand c Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Private Bag 11222, Palmerston North, New Zealand Received 13 February 2007 accepted 11 June 2007 Available online 29 June 2007 Abstract The polyketide toxin dothistromin is very similar in structure to the aflatoxin precursor, versicolorin B. Dothistromin is made by a pine needle pathogen, Dothistroma septosporum, both in culture and in planta. Orthologs of aflatoxin biosynthetic genes have been iden- tified that are required for dothistromin biosynthesis in D. septosporum. In contrast to the situation in aflatoxin-producing fungi where 25 aflatoxin biosynthetic and regulatory genes are tightly clustered in one region of the genome, the dothistromin gene cluster is fragmented. Three mini-clusters of dothistromin genes have been identified, each located on a 1.3-Mb chromosome and each grouped with non-dothi- stromin genes. There are no obvious patterns of repeated sequences or transposon relics to suggest recent recombination events. Most dothistromin genes within the mini-clusters are co-regulated, suggesting that coordinate control of gene expression is achieved despite this unusual arrangement of secondary metabolite biosynthetic genes. �� 2007 Elsevier Inc. All rights reserved. Keywords: Dothistromin Red-band needle blight Mycotoxin Gene expression Gene regulation Secondary metabolite 1. Introduction Dothistroma septosporum is an ascomycete pathogen in the Order Dothideales. It causes red-band needle blight in a wide range of pine species, a disease that leads to needle death, premature defoliation and, in severe cases, tree death (reviewed in Bradshaw, 2004). The disease has been prevalent since the 1960s in pines planted as exotics in plantation forests, particularly in Southern hemisphere countries such as New Zealand where disease control has been achieved by fungicide sprays. During the last few years the incidence of red-band needle blight has increased dramatically in the Northern hemisphere. An epidemic in British Columbia, Canada, has caused exten- sive mortality of pines in their native ranges and the dis- ease incidence has been correlated with climate change (Woods et al., 2005). Dothistromin has been isolated from diseased needles and is thought to be responsible for the characteristic red-brown coloration commonly associated with the dis- ease (Shain and Franich, 1981). Although the role of the toxin in disease is not yet known, it does have a broad tox- icity to many types of organisms (Stoessl et al., 1990) and may confer a competitive advantage to D. septosporum against other microorganisms. As well as being produced in planta, the toxin is also produced and secreted by the fungus in culture, where some isolates produce more toxin than others (Bradshaw et al., 2000). Cultured isolates, how- ever, tend to be morphologically unstable, so it is not known whether isolates that are high toxin-producers in culture are also high-producers on pine needles. 1087-1845/$ - see front matter �� 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2007.06.005 * Corresponding author. Address: Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. Fax: +64 6 350 5688. E-mail address: R.E.Bradshaw@massey.ac.nz (R.E. Bradshaw). www.elsevier.com/locate/yfgbi Available online at www.sciencedirect.com Fungal Genetics and Biology 44 (2007) 1342���1354

One of the interesting aspects of Dothistroma biology is the close similarity of dothistromin to the aflatoxin precur- sor, versicolorin B. Aflatoxin (AF) and its close relative ste- rigmatocystin (ST) are produced predominantly by species within the Orders Eurotiales and Sordariales (Barnes et al., 1994 Klich et al., 2000). Within the genus Aspergillus, AF is made by the well-known section Flavi species such as A. parasiticus and A. flavus, as well as by non-section Flavi species such as A. ochraceoroseus (Cary and Ehrlich, 2006). The latter group largely originates from coastal regions of the tropics where they are associated with woody plants. The synthesis of AF requires around 25 genes, encoding enzymes and some regulatory proteins, clustered together in a 70-kb region of the genome (Yu et al., 2004). Charac- terisation of the ST cluster in A. nidulans showed that most of the ST genes are orthologs of AF genes, but that they are arranged in a different order within the cluster (Keller and Hohn, 1997). Analysis of AF/ST gene clusters from a range of section and non-section Flavi species suggested that the ST-type cluster is ancestral to the AF-type. Cary and Ehr- lich (2006) proposed an ancestral basal ������mini-cluster������ of genes (pksA, hexA, hexB, and nor-1) required to form and stabilize the initial polyketide, possibly along with some regulatory genes (aflR, aflJ). These authors specu- lated that duplication and recruitment of genes encoding polyketide-modifying enzymes subsequently occurred, fol- lowed by purifying selection and some gene loss. Indeed there is considerable evidence for duplication of genes in metabolic gene clusters (Cary and Ehrlich, 2006 Chang and Yu, 2002) and for the occurrence of transposable ele- ments and/or repeated sequences that suggest a mechanism whereby movement of sets of genes may have occurred dur- ing the evolution of gene clusters (Tanaka et al., 2005 Young et al., 2006). The D. septosporum genome contains orthologs of AF genes located on a 1.3-Mb minichromosome. The dotA and pksA genes encode versicolorin reductase and polyke- tide synthase, respectively, which have 80% and 57% amino acid identity to their A. parasiticus orthologs, Ver-1 (AflM) and PksA (AflC). Targeted replacement of dotA and pksA confirmed they are both essential for dothistromin biosyn- thesis and they each have several other AF-like genes alongside (Bradshaw et al., 2002, 2006), suggesting they comprise two parts of a cluster of dothistromin biosyn- thetic genes. The aim of this work was to identify the remaining genes and to build up a complete picture of the dothistromin gene cluster, in order to provide further insight into the evolution of AF-like gene clusters. Due to the close structural similarity of dothistromin and versi- colorin B it was expected that a cluster of AF gene homo- logs would be present in the D. septosporum genome. Unexpectedly, the two known dothistromin gene cluster fragments are not parts of one larger cluster but are each surrounded by other (non-dothistromin) genes. Another AF-like dothistromin gene, vbsA, was characterized and similarly shown to be located in an ���island��� of non-dothi- stromin genes. The results suggest a highly fragmented group of dothistromin genes in contrast to the tight clusters of AF/ST genes reported to date. 2. Materials and methods 2.1. Strains and culture conditions Escherichia coli strains KW251 (Promega Corp., Madi- son, WI), and Top 10 (Invitrogen Corp., Carlsbad, CA), were grown on Luria���Bertani (LB) agar plates (Sambrook et al., 1989) supplemented, when necessary, with either ampicillin (100 lg/ml) or kanamycin (50 lg/ml). D. septo- sporum (strain NZE7) was obtained from Pinus radiata needles near Rotorua, New Zealand and its identity con- firmed by its ability to produce dothistromin and by its ribosomal ITS sequence (Barnes et al., 2004 Bradshaw et al., 2000). Cultures were routinely maintained on dothistroma medium (DM) (Bradshaw et al., 2000) at 22 ��C unless otherwise stated. Cultures for genomic DNA preparation, from wild-type or transformed D. septospo- rum, were started from an inoculum of macerated mycelia one 5-mm diameter plug of mycelium was macerated with a pestle and placed in a 125 ml Erlenmeyer flask containing 25 ml of DB (Dothistroma broth) (2.5% (w/v) oxoid malt extract, 2% (w/v) oxoid nutrient broth). Cultures were grown for 7 days with agitation at 160 rpm, and then myce- lia collected by filtration through Miracloth (Calbiochem Corporation, La Jolla, CA). 2.2. Molecular biology Genomic DNA was isolated from freeze-dried mycelia of D. septosporum using the method of Moller et al. (1992). Plasmid and cosmid DNA was extracted using a QIAprep�� spin miniprep kit (Qiagen, Hilden, Germany). PCR products amplified with Taq DNA polymerase (Invit- rogen) were routinely cloned into pGEM��-T Easy (Pro- mega, Madison, WI) and transformed into E. coli TOP10. For Southern/colony hybridization, digested geno- mic DNA or library clones were transferred to Hybond-N+ nylon membranes (Amersham BioSciences, Buckingham- shire, UK) by capillary transfer (Southern, 1975). Probes were labeled either using a DIG high prime DNA labeling system (Roche) or by PCR following the description from Roche. Hybridizations were carried out at 42 ��C in buffer containing 50% deionized formamide, 5�� SSC, 0.1% (w/ v) N-lauroylsarcosine, 0.02% (w/v) SDS, and 2% (w/v) Roche blocking reagent. Separation of D. septosporum chromosomes on a contour-clamped homogeneous electric field (CHEF) gel and Southern hybridization with [a-32P]dCTP-labeled vbsA PCR fragment (amplified using primers vbs1aF and vbs-4R Supplementary Table S1) were as described in Bradshaw et al. (2006). DNA sequencing was carried out using an ABI Prism BigDye�� Terminator cycle sequencing ready reaction kit and an ABI3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). S. Zhang et al. / Fungal Genetics and Biology 44 (2007) 1342���1354 1343