An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium

Accepted Manuscript An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium Frederik T. Hansen, Donald M. Gardiner, Erik Lysøe, Patricia Romans Fuertes, Bettina Tudzynski, Philipp Wiemann, Teis Esben Sondergaard, Henriette Giese, Ditlev E. Brodersen, Jens Laurids Sørensen PII: DOI: Reference:

S1087-1845(14)00219-9 http://dx.doi.org/10.1016/j.fgb.2014.12.004 YFGBI 2764

To appear in:

Fungal Genetics and Biology

Received Date: Accepted Date:

22 August 2014 17 December 2014

Please cite this article as: Hansen, F.T., Gardiner, D.M., Lysøe, E., Fuertes, P.R., Tudzynski, B., Wiemann, P., Sondergaard, T.E., Giese, H., Brodersen, D.E., Sørensen, J.L., An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium, Fungal Genetics and Biology (2014), doi: http://dx.doi.org/ 10.1016/j.fgb.2014.12.004

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An update to polyketide synthase and non-ribosomal synthetase genes and nomenclature in Fusarium Frederik T. Hansen1, Donald M. Gardiner2, Erik Lysøe3, Patricia Romans Fuertes1,4, Bettina Tudzynski5, Philipp Wiemann6, Teis Esben Sondergaard4, Henriette Giese4, Ditlev E. Brodersen1, Jens Laurids Sørensen4,* 1

NANORIPES Centre for Natural Non-Ribosomal Peptide Synthesis. Department of Molecular

Biology and Genetics, Aarhus University, Aarhus C, Denmark 2

Commonwealth Scientific and Industrial Research Organisation (CSIRO) Agriculture Flagship,

Queensland Bioscience Precinct, Brisbane, Queensland, Australia 3

Department of Plant Health and Plant Protection, Bioforsk–Norwegian Institute of Agricultural

and Environmental Research, 1430 Ås, Norway. 4

Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University,

Aalborg, Denmark. 5

Institut für Biologie und Biotechnologie der Pflanzen, Molecular Biology and Biotechnology of

Fungi, Westfälische Wilhelms-Universität Münster, Münster, Germany 6

Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI,

USA. * Corresponding author: Jens Laurids Sørensen, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. Tel. 0045 9940 8524; fax: 0045 9814 1808; e-mail: [email protected]

Abstract Members of the genus Fusarium produce a plethora of bioactive secondary metabolites, which can be harmful to humans and animals or have potential in drug development. In this study we have performed comparative analyses of polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) from ten different Fusarium species including F. graminearum (two strains), F. verticillioides, F. solani, F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti, and F. oxysporum (12 strains). This led to identification of 52 NRPS and 52 PKSs orthology groups, respectively, and although not all PKSs and NRPSs are assumed to be intact or functional, the analyses illustrate the huge secondary metabolite potential in Fusarium. In our analyses we identified a core collection of eight NRPSs (NRPS2-4, 6, 10-13) and two PKSs (PKS3 and PKS7) that are conserved in all strains analyzed in this study. The identified PKSs and NRPSs were named based on a previously developed classification system (www.FusariumNRPSPKS.dk). We suggest this system be used when PKSs and NRPSs have to be classified in future sequenced Fusarium strains. This system will facilitate identification of orthologous and non-orthologous NRPSs and PKSs from newly sequenced Fusarium genomes and will aid the scientific community by providing a common nomenclature for these two groups of genes/enzymes.

Keywords: Secondary metabolites, PKS, polyketide synthases, NPRS, non-ribosomal peptide synthetases

1. Introduction As genome sequencing has become a cost efficient way of providing high quality data we expect rapid increases in available genome sequences in the near future. These genomes will include some of the most economically important plant and human fungal pathogens (Summerell et al., 2010) belonging to the genus Fusarium. In recent years the genome sequences of F. fujikuroi, F. graminearum, F. oxysporum, F. pseudograminearum, F. solani, F. verticillioides and F. avenaceum (Coleman et al., 2009; Cuomo et al., 2007; Gardiner et al., 2012; Lysøe et al., 2014; Ma et al., 2010; Wiemann et al., 2013) has become publically available and the genome sequence of F. acuminatum has been announced together with a strain from the F. equiseti complex (Moolhuijzen et al., 2013), and an additional F. graminearum strain (Gardiner et al., 2014). Furthermore, the F. culmorum genome has been released in GenBank and large genome sequencing projects such as “The 1000 Fungal Genomes” initiated by the Joint Genome Institute (JGI, CA, USA) will provide additional genome sequences from Fusarium species (Spatafora et al., 2013). Members of the genus Fusarium have the ability to produce a vast array of bioactive secondary metabolites of which the two most abundant groups are polyketides and non-ribosomal peptides (Figure 1). A number of these have been linked to biosynthetic genes responsible for their production. The polyketide biosynthetic pathways are initiated by large multidomain polyketide synthases (PKSs), which contain the three basal core domains: β-ketosynthase (KS), acyltransferase (AT) and acyl-carrier protein (ACP) (McDaniel et al., 1994) and additional variable tailoring domains to ensure chemical diversity (Meier and Burkart, 2009). The non-ribosomal peptides are synthesized by multi-modular synthetases (NRPSs) where each module contains a specific order of catalytic domains. The adenylation domain (A) recognizes the specific amino acid substrate, which

is then transferred by the peptide acyl carrier domain (T or PCP) to the condensation domain (C) where the formation of the peptide bond takes place (Strieker et al., 2010). The existence of a PKS and NRPS gene does not directly imply the existence of a secondary metabolite. The products of the majority of PKSs and NRPSs are unknown, which is illustrated in F. graminearum where the products of 8/15 PKSs and 3/19 NRPSs are known (Hansen et al., 2012b; Jørgensen et al., 2014; Sørensen et al., 2014b). The products of the remaining gene clusters are currently unknown either because the genes can be nonfunctional or silent under the conditions tested or the products can be unstable, undetectable or produced in low quantities. We have previously published a guide of PKSs and NRPSs in F. graminearum, F. oxysporum, F. solani, and F. verticillioides, where we gave an overview of the distribution of these secondary metabolite genes (Hansen et al., 2012b). The variation of PKSs between different Fusarium species can be due to gene duplication, acquisition or loss events as well as nucleotide mutations conferring loss-of-function (Brown et al., 2012a). Furthermore, we have proposed a numeration system for PKS and NRPS genes which can be used in the annotation of new genes uncovered in future sequencing projects (Hansen et al., 2012b). The system was developed to reduce confusion as different PKSs or NRPSs could have the same number (Coleman et al., 2009; Cuomo et al., 2007; Kroken et al., 2003; Ma et al., 2010). The system has been widely accepted in genome announcements (Gardiner et al., 2012; Wiemann et al., 2013) and phylogenetic studies of secondary metabolite gene cluster (O'Donnell et al., 2013). In this review we update the list by including new data of PKSs and NRPSs from F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum. F. culmorum and F. equiseti as well as 11 additional F. oxysporum strains and one F. graminearum strain. Furthermore we provide an update on recent advances in linking PKSs and NRPSs to their

responsible products in Fusarium and announce the launch of a website where overview is constantly updated (www.FusariumNRPSPKS.dk).

2. Materials and methods The genome sequences of the following strains were used in the comparative analyses: F. graminearum (PH-1 and CS3005), F. culmorum (CS7071), F. pseudograminearum (CS3096), F. equiseti (CS3069), F. avenaceum (Fa05001), F. acuminatum (CS5907), F. fujikuroi (IMI58289), F. verticillioides (7600), F. oxysporum (Fo5176), F. oxysporum f. sp. raphani (NRRL 54005), F. oxysporum f. sp. vasinfectum (NRRL 25433), F. oxysporum (FOSC 3-a), F. oxysporum f. sp. lycopersici (4287), F. oxysporum f. sp. melonis (NRRL 26406), F. oxysporum f. sp. cubense tropical race 4 (NRRL 54006), F. oxysporum f. sp. radicis-lycopersici (NRRL 26381), F. oxysporum (Fo47), F. oxysporum f. sp. lycopersici (MN25), F. oxysporum f. sp. pisi (HDV247), F. oxysporum f. sp. conglutinans race 2 (NRRL 54008).

2.1 PKS and NRPS sequences PKS and NRPS sequences for F. graminearum, F. oxysporum, F. solani and F. verticillioides were obtained in our previous study from the “Fusarium comparative genome database” Broad/MIT (http://www.broadinstitute.org/annotation/genome/fusarium_group/MultiHome.html)

and

Joint

Genome Institute database (JGI, http://genome.jgi-psf.org/Necha2/Necha2.home.html) (Hansen et al., 2012b). NRPS and PKS sequences of F. fujikuroi and F. pseudograminearum are now available in GenBank (Gardiner et al., 2012; Wiemann et al., 2013). Genome sequences of F. graminearum (CS3005), F. acuminatum, F. culmorum and F. equiseti were available from previous studies

(Gardiner et al., 2014; Moolhuijzen et al., 2013) and the protein sequences were predicted using FGENESH (Salamov and Solovyev, 2000) and Augustus (Keller et al., 2011). PKS and NRPS genes were obtained from the unpublished genome sequence of F. avenaceum isolate Fa05001 (ARS culture collection: NRRL 54939, Bioforsk collection: 202103, DTU collection: IBT 41708), isolated from barley in Finland (Kokkonen et al., 2010). Gene prediction was performed with Augustus v2.5.5 (Stanke et al., 2008), using default settings and F. graminearum as training set.

2.2 Comparative analyses of genome sequenced Fusarium strains. To illustrate the phylogenetic relationship of the genome sequenced Fusarium strains we constructed a phylogram based on the genomic DNA sequences of DNA-directed RNA polymerase II subunit (RPB2). The sequences were aligned with by multiple alignment using fast fourier transform (MAFFT) using T-REX web server (Boc et al., 2012). The alignments were analyzed with MetaPIGA v2.0 (Helaers and Milinkovitch, 2010) using maximum likelihood using 100 bootstraps and visualized with EvolView (http://evolgenius.info/evolview) (Zhang et al., 2012). A binary data set based presence/absence of PKSs and NRPSs was constructed and used to generate a cladogram with Dollo and Polymorphism Parsimony (dollop, PHYLIP) (Felsenstein, 1989) with 100 bootstrap replications. The resulting tree was visualized with EvolView.

2.3 Distribution of PKS and NRPSs in Fusarium

A diverse set of A and KS domains from available sequences were used to identify PKS and NRPS genes in the sequenced Fusarium strains. The sequences were aligned with MAFFT analyzed with MetaPIGA v2.0 (Helaers and Milinkovitch, 2010) using maximum likelihood using 100 bootstraps and visualized with EvolView. Conserved active sites and domains were identified using the NCBI Conserved Domain Database (Marchler-Bauer et al., 2011), InterPro (Hunter et al., 2012), PKS/NRPS Analysis Web-site (Bachmann and Ravel, 2009) and the ASMPKS web interfaces (Tae et al., 2007).

2.4 Analysis of the equisetin gene cluster In the comparative analyses we identified a proposed equisetin synthase (PKS18) in F. verticillioides, F. fujikuroi and F. oxysporum. The genomic regions surrounding PKS18 was aligned against the equisetin gene cluster of F. heterosporum (Kakule et al., 2013) using the ShuffleLAGAN global chaining algorithm (Brudno et al., 2003) using the default settings and visualized with the mVISTA browser (http://genome.lbl.gov/vista/).

3. Results and discussion 3.1 Comparison of genome sequenced Fusarium strains To examine the relationship of the 22 genome sequenced Fusarium strains we performed a phylogenetic analysis using the DNA sequence of the entire DNA-directed RNA polymerase II second largest subunit (RPB2) (Figure 2A). In the phylogenetic tree we placed F. solani as a outgroup and observed an organization in agreement with previous studies (O'Donnell et al., 2013)

placing F. oxysporum, F. verticillioides and F. fujikuroi in one clade, F. acuminatum and F. avenaceum in a second clade and F. pseudograminearum, F. graminearum and F. culmorum in a third, whereas and F. equiseti did not group together with other strains. To examine how distribution of PKSs and NRPSs affects grouping of the strains we generated a cladogram based on a binary matrix (presence/absence of PKSs and NRPSs) (Figure 2B). With this approach we saw a similar grouping of the strains as in the phylogenetic analyses. This indicates that the distribution of the PKSs and NRPSs reflects the evolutionary divergence between isolates where distantly related taxa show limited overlap in distribution, closely related ones show large overlap in distribution

3.2 Comparison of polyketide synthases PKSs were identified in the sequenced Fusarium strains through BlastP analyses using a diverse panel of PKSs. The KS domains were extracted and used in phylogenetic analyses in order to identify PKS orthology groups (Supplementary Figure 1). We identified 52 different PKS genes of which PKS3 and PKS7 and were present in all strains. PKS8 was also highly conserved among the Fusarium strains although part of KS domain was missing in F. culmorum and the entire gene was absent in F. oxysporum f. sp. melonis. In this update we have included a newly sequenced F. graminearum strain CS3005, which shares 15 PKSs with the originally sequenced F. graminearum strain (PH-1) based on our analyses. These includes the PKSs for biosynthesis of fusarubins (PKS3), zearalenone (PKS4+13), fusarins (PKS10), aurofusarin (PKS12) and the recently linked fusaristatin (PKS6+NRPS7) (Sørensen et al., 2014b). F. graminearum (CS3005) contains an additional unique PKS (PKS52), which is located in a putative cluster consisting of five genes (FG05_30421-FG05_30425). Homologs of the cluster are present in several Colletotrichum strains suggesting that F. graminearum CS3005 could have

obtained the gene cluster through a horizontal gene transfer event. The close relationship of F. culmorum and F. graminearum was visible in the PKS analyses as we identified 13 PKSs in F. culmorum, which all had a F. graminearum orthologue. PKS2 and PKS9 were however not identified in F. culmorum. We identified 14 putative PKSs in F. pseudograminearum, of which 13 had a F. graminearum orthologue and one (PKS40) was unique to F. pseudograminearum (Figure 3). PKS40 has recently been shown to provide the polyketide part of the polyketide non-ribosomal peptide (NRPS32) fused compounds W493 A and B (Sørensen et al., 2014b). The genome also contained the fusarielin synthase PKS9 (=FSL1) (Sørensen et al., 2012a) although three representative of the species were identified as non-producers of fusarielins (Sørensen et al., 2013). However, the respective cluster may be silenced under the experimental conditions tested or the F. pseudograminearum strains may produce other unidentified fusarielins, which were not detected by the targeted LC-MS/MS method. The PKSs and NRPSs in F. fujikuroi have previously been identified and numbered by Wiemann et al. (2013) using another system (Supplementary file 1), but here we have renumbered the genes to follow our system. The genome of F. fujikuroi contains 17 PKSs of which 13 have orthologue genes in F. verticillioides or F. oxysporum including the bikaverin synthase (PKS16 = BIK1) (Wiemann et al., 2009) as well as a hybrid PKS-NRPS (PKS18), which is also present in F. equiseti (Figure 3). PKS18 is a homologue of eqxS, which is responsible for production of equisetin in F. heterosporum (Kakule et al., 2013). This compound is a common metabolite produced by F. equiseti (Hestbjerg et al., 2002), but has not been reported from F. oxysporum, F. verticillioides and F. fujikuroi. In the biosynthetic pathway EqxS is proposed to release its product, trichosetin, by assistance of an enoylreductase (EqxC) (Kakule et al., 2013). Trichosetin is then methylated by the Nmethyltransferase EqxD, resulting in equisetin (Kakule et al., 2013) (Figure 4A). Analyses of the

gene clusters showed that a homologue of EqxD is present in F. equiseti, but not in F. fujikuroi, F. verticillioides and F. oxysporum (Figure 4B), suggesting that the biosynthetic pathway is terminated at trichosetin in these three species. Indeed trichosetin has recently been identified in a F. oxysporum strain (Inokoshi et al., 2013) and we have also detected the compound in F. oxysporum f. sp. lycopersici (fol4287) by LC-MS/MS (unpubl. results). PKS39 (FFUJ_12239), which is one of two unique PKSs in F. fujikuroi, is located in a putative gene cluster together with five additional genes including a transcription factor. Overexpression of the transcription factor as well as PKS39 led to production of four novel compounds with the elemental composition of the two most abundant compounds being C12H18O4 and structural elucidation is still ongoing (Wiemann et al., 2013). F. fujikuroi, F. avenaceum and F. acuminatum are producers of moniliformin, which previously has been suggested to be a polyketide (ApSimon, 1994) although recent results have indicated this is most likely not the case (Wiemann et al., 2012). PKS36 and PKS37 are located 15kbp apart in F. fujikuroi and share homology to the two PKSs involved in asperfuranone biosynthesis in Aspergillus nidulans (Chiang et al., 2009; Wiemann et al., 2013). This gene cluster is also present in F. avenaceum, but asperfuranone has not been identified in any Fusarium species it therefore unknown whether the PKS36/PKS37 produces asperfuranone or related compounds. F. avenaceum had the largest number of PKSs in the study (24) of which two were unique to this species. The large PKS number is reflected in the metabolite profile of F. avenaceum, which generally contains more secondary metabolites than other Fusarium species The secondary metabolite profile of F. avenaceum includes fusarin C (PKS10) and aurofusarin (PKS12) as well as the potential polyketides chlamydosporol, 2-AOD-3-ol, chrysogine and antibiotic Y (Sørensen and Giese, 2013; Sørensen et al., 2009; Uhlig et al., 2005), which have not been linked to synthases. Recently PKS6 and NRPS7 have been linked to biosynthesis of fusaristatin A in F. avenaceum (Sørensen et al., 2014c) similarly as in F. graminearum (Sørensen et al., 2014b). In F. acuminatum

we identified 14 PKSs including those responsible for fusarubins (PKS3), fusaristatin (PKS6), fusarins (PKS10) and aurofusarin (PKS12) and one unique PKS (PKS46). A homologue of the fusarielin synthase (PKS9) is also present in F. acuminatum and preliminary experiments have shown that it produces fusarielin A (Sørensen, unpublished data), which has previously been isolated from F. tricinctum (Nenkep et al., 2010) and from an unidentified Fusarium strain (Kobayashi et al., 1995). The fewest number of identified PKS genes was observed in F. equiseti (11), of which three were unique. F. equiseti is the only species in this analysis which can produce fusarochromanone (Hestbjerg et al., 2002), which probably is a polyketide. It is therefore likely that one of these three PKSs is responsible for this compound. More than 150 host specific formae speciales have been described in the F. oxysporum complex (Baayen et al., 2000) and currently the genomes of 12 strains of different F. oxysporum formae speciales are publically available. In our analyses we identified a variable number of PKSs among the different F. oxysporum strains ranging from 10 to 14 (Figure 3, Supplementary table 1). Six PKSs were present in all 12 strains (PKS3, 7, 18, 20, 21 and 27), although the KS, AT, MET and KR domains are missing in F. oxysporum (Fo5176).

3.2 Comparison of non-ribosomal peptide synthetases NRPSs were identified through BlastP analyses using a selected panel of variable A domains. The A domains were extracted from each NRPS and used for phylogenetic analyses (Supplementary Figure 2). Six NRPS are present in all sequenced strains (NRPS2, 3, 6, 10, 11 and 12) indicating that they are involved in synthesis of metabolites which are important for the fungi. For example

NRPS2 and NRPS6 are responsible for production of the siderophores ferricrocin and fusarinine, respectively (Oide et al., 2006; Tobiasen et al., 2007). Furthermore, NRPS4 was identified in all strains except F. oxysporum (Fo5176) (Supplementary data) and although its product remains unknown it appears to be involved in surface hydrophobicity in F. graminearum (Hansen et al., 2012a). In F. pseudograminearum we identified 16 NRPS genes of which one (NRPS32) is uniquely found in this species (Figure 5). This six modular NRPS is present in a hypothetical gene cluster together with PKS40, which together are responsible for production of W493A and B (Sørensen et al., 2014b). These compounds consist of six amino acid and one polyketide moiety and have previously been isolated from an unidentified Fusarium species (Nihei et al., 1998) and are similar to the compound acuminatum (Carr et al., 1985). The only difference between W493 B and acuminatum is that isoleucine is present in W493 B while acuminatum contains leucine. In F. culmorum we identified 18 NRPSs of which 18 are present in F. graminearum with only the one modular NRPS17 missing. In Fusarium fujikuroi we identified one NRPS (NRPS31) that is not present in the other sequenced Fusarium species (Wiemann et al., 2013). This four module NRPS is a homologue of the apicidin synthetase (APS1), which has previously been identified in F. semitectum as part of a cluster containing 11 additional genes (Jin et al., 2010). The gene cluster was largely intact with only the putative reductase APS10 missing (Niehaus et al., 2014a; Wiemann et al., 2013). This species does not produce any of the previously described apicidin analogues, instead overexpression of the transcription factor located in the cluster led to production of a novel analogue, named apicidin F (Von Bargen et al., 2013). As was found for the PKSs, the largest number of NRPS was observed in F. avenaceum (24), which includes synthetases for the three siderophores and for enniatins. Eight NRPSs were unique to F.

avenaceum including five multimodular NRPSs. In F. acuminatum the enniatin synthetase (NRPS22) contains a disrupted condensation domain, which is also observed in F. verticillioides. The synthetase is functional in F. acuminatum as we discovered enniatin A, A1, B and B1 in F. acuminatum by LC-MS/MS (unpubl. data), whereas it most likely is a pseudogene in F. verticillioides because it lacks the condensation domain well as the ketoisovalerate reductase gene (kivR) required for beauvericin and enniatin biosynthesis (Xu et al., 2008). We identified 14 NRPSs in F. equiseti, of which one (NRPS33) is not present in the other strains. Furthermore we identified a 11 module NRPS (NRPS34) in F. equiseti, making it the largest synthetase observed in the study. A proposed ortholog of NRPS34 was identified in F. acuminatum where the initial module (TCCATC) is missing. A NRPS7 homolog was detected in F. acuminatum, which is present in a gene cluster together with PKS6. This gene cluster is also present in F. avenaceum, F. culmorum and F. graminearum, respectively, which are the only species in the current study that have the genetic potential (PKS6 + NRPS7) to produce fusaristatins. Two of the 12 F. oxysporum strains had identical distribution of NRPS genes (F. oxysporum f. sp. cubense (NRRL 54006) and F. oxysporum f. sp. pisi (HDV247)), whereas the remaining differed in number and distributions. Nine NRPSs are present in all F. oxysporum strains, including the two siderophore synthetases NRPS2 and NRPS6 and the enniatin/beauvericin synthetase NRPS22. NRPS1, which is required for synthesis of the external siderophore malonichrome (Sørensen et al., 2014a) is however not present in two of the strains (F. oxysporum f. sp. raphanin and F. oxysporum (Fo5176)) as also observed for F. fujikuroi. Wiemann et al (2012) speculated that the absence of NRPS1 in some species fungi which thrive is a reflection of the redundancy in iron uptake systems (Wiemann et al., 2012). However, in seven F. oxysporum strains we identified orthologue genes (NRPS39) of the ferrirhodin synthetase FNR1, which has previously been identified in F. sacchari

(Munawar et al., 2013). Ferrirhodin belong to the major group of ferrichrome siderophores together with malonichrome (NRPS1) and ferricrocin (NRPS2), which is also reflected by the similar domain structure (ATC-ATC-ATCTCTC). In F. oxysporum f. sp. raphani we identified a possible ortholog (NRPS43) of the Aspergillus fumigatus fumarylalanine synthetase SidE (AFUB_04490), which share 55% identity on amino acid level. SidE is a bimodular NRPS (ATC-ATC), which synthetize fumarylalanine from fumarate and L-alanine (Steinchen et al., 2013). This compound has also been identified in A.s indicus (Birch et al., 1968) and Penicillium resticulosum (Birkinshaw et al., 1942), but so far not in any Fusarium species. Furthermore three NRPS-KS hybrid genes (NRPS40, 41 and 49) displaying a rare C-A-T-KS domain architecture was also identified. These are deviating from the common PKS-NRPS hybrid architecture and are indeed lacking all other PKS domains except for their single KS domain. Hybrid NRPS-PKS genes are known from other organisms but mainly from bacteria (Lawrence et al., 2011). An exception is the NRPS-PKS ChNPS7;PKS24 gene from Cochliobolus heterostrophus which is most likely the result of a horizontal gene transfer from bacteria (Bushley and Turgeon, 2010). However this unique gene only contains the A-T domain of the NRPS module followed by a full set of PKS domains and is probably not comparable with these NRPS-KS hybrids which appear to have orthologous genes in other fungi such as Metarhizium, Beauveria and Grosmannia among others.

3.4 Update on PKSs and NRPSs linked to products in sequenced Fusarium

After our first quick guide to PKSs and NRPSs in Fusarium (Hansen et al., 2012b) several genes have been linked to their products. Growing F. fujikuroi under alkaline conditions with sodium nitrate as the sole nitrogen source resulted in production of fusarubins in the mycelium of liquid cultures with 8-O-methylfusarubin as the major product (Studt et al., 2012). The responsible gene was identified as a PKS3 homologue (Studt et al., 2012), which has previously been linked to perithecial pigmentation in F. graminearum (Gaffoor et al., 2005), F. solani (Graziani et al., 2004) and F. verticillioides (Proctor et al., 2007) without isolating the compounds. Heterologous expression of PKS3 from F. solani in Aspergillus oryzae resulted in production of 6-Odemethylfusarubinaldehyde (Awakawa et al., 2012), which was also identified as the primary PKS product in the fusarubin biosynthetic pathway in F. fujikuroi (Studt et al., 2012). Deletion of PKS3 in F. fujikuroi also resulted in discoloration of perithecia (Studt et al., 2012), but it is still unknown whether fusarubins occur as monomer, building blocks or as polymers in perithecia, because isolation of pigments from Fusarium perithecia has so far been unsuccessful. Another PKS was linked to its resulting metabolite when PKS21 orthologs (FUB1) from F. verticillioides and F. fujikuroi, respectively, was associated with fusaric acid (5-butylpicolinic acid) production by gene replacement approaches (Brown et al., 2012b; Niehaus et al., 2014b). The biosynthetic pathway of fusaric acid is predicted to be initiated by the formation of a fully reduced 6-carbon polyketide chain by the PKS21 from three acetate units. The putative fusaric acid gene cluster contains four additional genes (FUB2-5), of which FUB3 encodes a putative amino acid kinase and is likely to be involved in assimilating the nitrogen atom to form fusaric acid (Brown et al., 2012b). Many Fusarium species appear reddish due to the production of mycelial pigments such as bikaverin and aurofusarin produced by PKS4 and PKS12, respectively. The two PKSs have similar

architectures containing KS, AT and ACP domains in addition to an N-terminal Claisen-type cyclase domain (CLC), which form a claisen cyclization and release the chemical scaffold in the bikaverin and aurofusarin biosynthetic pathways, pre-bikaverin (Wiemann et al., 2009) and YWA1 (Frandsen et al., 2011), respectively. Deletion of the CLC domain in the PKS12 homolog wA in A. nidulans has previously been shown to result in formation of citreoisocoumarin instead of YWA1 (Fujii et al., 2001) and similarly, deletion of the CLC domain in PKS16 resulted in production of bikisocoumarin (Li et al., 2010). Growing F. graminearum and three other aurofusarin producers on a medium made from a waste product from the candy industry resulted in production of citreoisocoumarin through PKS12 (Sørensen et al., 2012b). Likewise the bikaverin biosynthetic pathway was redirected to another isocoumarin, bikisocoumarin, in F. oxysporum and F. proliferatum when they were grown on the medium suggesting an alternative release mechanism under these growth conditions (Sørensen et al., 2012b). The hybrid NRPS-PKS (PKS10 = fus1) responsible for production of fusarin C has previously been identified in F. verticillioides (= F. moniliforme), F. venenatum (Brown et al., 2012b; Song et al., 2004) and F. graminearum (Gaffoor et al., 2005). The biosynthetic pathway has now been resolved through deletion and overexpression of the individual genes of the gene cluster in F. fujikuroi (Niehaus et al., 2013). The fusarin gene cluster consists of nine genes, of which only four were shown to be required for the biosynthesis of fusarin C (Niehaus et al., 2013). PKS10 is predicted to release its product with an open ring structure followed by ring closure, oxidation and methylation by three tailoring enzymes (Fus2, 8 and 9, respectively) (Niehaus et al., 2013).

Future updates

We are expecting a rapid increase in available genome sequences from additional Fusarium species in the near future, which will provide new PKSs and NRPSs. In order to keep the numbering system updated we have launched a website where the list of PKSs and NRPSs and their products is constantly updated (www.FusariumNRPSPKS.dk). We invite and encourage the Fusarium community to use this interactive resource for numbering PKSs and NRPSs when new Fusarium strains are sequenced in the future.

Conclusion Our original guide to PKSs and NRPSs in Fusarium was based on F. graminearum, F. verticillioides, F. solani and F. oxysporum and we have now extended the list by adding genes from sequenced strains of F. culmorum, F. pseudograminearum, F. fujikuroi, F. acuminatum, F. avenaceum, F. equiseti as well as one additional F. graminearum strain and 11 F. oxysporum strains. Extension of the existing numbering system by addition of 19 NRPSs and 17 PKSs, refines our view on the current phylogenetic relation between these key enzymes in distinct Fusarium species and individual strains. In the future this system can be further expanded when additional Fusarium strains are sequenced. The website will ensure that the system is updated to prevent overlapping numbering and will simultaneously ease identification of novel/unique as well as orthologs of existing PKSs and NRPSs, thereby facilitating the characterization of new secondary metabolites. As the existence of a given PKS or NRPS does not imply existence of a specific secondary metabolite, there is a need to characterize the PKSs and NRPSs to determine whether they are functional and are involved in production of secondary metabolites.

Acknowledgements The study was supported by grants from The Danish Research Council, Technology and Production (12-132415 and 10-100105) and by the Grains Research and Development Corporation, an Australian Government statutory authority. References ApSimon, J. W., The biosynthetic diversity of secondary metabolites. In: J. D. Miller, H. L. Trenholm, Eds.), Mycotoxins in grains: compounds other than aflatoxin. Eagan Press, St. Paul, MN, USA, 1994, pp. 3-18. Awakawa, T., Kaji, T., Wakimoto, T., Abe, I., 2012. A heptaketide naphthaldehyde produced by a polyketide synthase from Nectria haematococca. Bioorg. Med. Chem. Lett. 22, 4338-4340. Baayen, R. P., O'Donnell, K., Bonants, P. J. M., Cigelnik, E., Kroon, L. P. N. M., Roebroeck, E. J. A., Waalwijk, C., 2000. Gene genealogies and AFLP analyses in the Fusarium oxysporum complex identify monophyletic and nonmonophyletic formae speciales causing wilt and rot disease. Phytopathology. 90, 891-900. Bachmann, B. O., Ravel, J., Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data. In: D. A. Hopwood, (Ed.), Complex enzymes in microbial natural product biosynthesis. Methods in enzymology. Elsevier academic press inc, San Diego, CA, USA, 2009. Birch, A. J., Qureshi, A. A., Rickards, R. W., 1968. Metabolites of Aspergillus indicus: The structure and some aspects of the biosynthesis of dihydrocanadensolide. Aust. J. Chem. 21, 2775-&. Birkinshaw, J. H., Raistrick, H., Smith, G., 1942. Studies in the biochemistry of micro-organisms 71. Fumaryl-dl-alanine (Fumaromono-dl-alanide), a metabolic product of Penicillium resticulosum sp.nov. Biochem. J. 36, 829-835. Boc, A., Diallo, A. B., Makarenkov, V., 2012. T-REX: a web server for inferring, validating and visualizing phylogenetic trees and networks. Nucleic Acids Res. 40, W573-W579. Brown, D. W., Butchko, R. A. E., Baker, S. E., Proctor, R. H., 2012a. Phylogenomic and functional domain analysis of polyketide synthases in Fusarium. Fungal Biology. 116, 318-331. Brown, D. W., Butchko, R. A. E., Busman, M., Proctor, R. H., 2012b. Identification of gene clusters associated with fusaric acid, fusarin, and perithecial pigment production in Fusarium verticillioides. Fungal Genet. Biol. 49, 521-532. Brudno, M., Malde, S., Poliakov, A., Do, C. B., Couronne, O., Dubchak, I., Batzoglou, S., 2003. Glocal alignment: finding rearrangements during alignment. Bioinformatics. 19, i54-i62. Bushley, K. E., Turgeon, B. G., 2010. Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships. BMC Evol. Biol. 10. Carr, S. A., Block, E., Costello, C. E., Vesonder, R. F., Burmeister, H. R., 1985. Structure determination of a new cyclodepsipeptide antibiotic from Fusaria fungi. J. Org. Chem. 50, 2854-2858. Chiang, Y. M., Szewczyk, E., Davidson, A. D., Keller, N., Oakley, B. R., Wang, C. C. C., 2009. A gene cluster containing two fungal polyketide synthases encodes the biosynthetic pathway for a polyketide, asperfuranone, in Aspergillus nidulans. J. Am. Chem. Soc. 131, 2965-2970.

Coleman, J. J., et al., 2009. The genome of Nectria haematococca: Contribution of supernumerary chromosomes to gene expansion. PLoS Genet. 5. Cuomo, C. A., et al., 2007. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science. 317, 1400-1402. Felsenstein, J., 1989. PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics. 5, 164-166. Frandsen, R. J. N., Schutt, C., Lund, B. W., Staerk, D., Nielsen, J., Olsson, S., Giese, H., 2011. Two novel classes of enzymes are required for the biosynthesis of aurofusarin in Fusarium graminearum. J. Biol. Chem. 286, 10419-10428. Fujii, I., Watanabe, A., Sankawa, U., Ebizuka, Y., 2001. Identification of Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chem. Biol. 8, 189-197. Gaffoor, I., Brown, D. W., Plattner, R., Proctor, R. H., Qi, W. H., Trail, F., 2005. Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (Anamorph Fusarium graminearum). Eukaryot. Cell. 4, 1926-1933. Gardiner, D. M., et al., 2012. Comparative pathogenomics reveals horizontally acquired novel virulence genes in fungi infecting cereal hosts. PLoS Path. 8. Gardiner, D. M., Stiller, J., Kazan, K., 2014. Genome sequence of Fusarium graminearum isolate CS3005. Genome announcements. 2. Graziani, S., Vasnier, C., Daboussi, M. J., 2004. Novel polyketide synthase from Nectria haematococca. Appl. Environ. Microbiol. 70, 2984-2988. Hansen, F. T., Droce, A., Sørensen, J. L., Fojan, P., Giese, H., Sondergaard, T. E., 2012a. Overexpression of NRPS4 leads to increased surface hydrophobicity in Fusarium graminearum. Fungal Biology. 116, 855-862. Hansen, F. T., Sørensen, J. L., Giese, H., Sondergaard, T. E., Frandsen, R. J., 2012b. Quick guide to polyketide synthase and nonribosomal synthetase genes in Fusarium. Int. J. Food Microbiol. 155, 128-36. Helaers, R., Milinkovitch, M., 2010. MetaPIGA v2.0: maximum likelihood large phylogeny estimation using the metapopulation genetic algorithm and other stochastic heuristics. BMC Bioinformatics. 11, 379. Hestbjerg, H., Nielsen, K. F., Thrane, U., Elmholt, S., 2002. Production of trichothecenes and other secondary metabolites by Fusarium culmorum and Fusarium equiseti on common laboratory media and a soil organic matter agar: An ecological interpretation. J. Agric. Food. Chem. 50, 7593-7599. Hunter, S., et al., 2012. InterPro in 2011: new developments in the family and domain prediction database. Nucleic Acids Res. 40, D306-D312. Inokoshi, J., Shigeta, N., Fukuda, T., Uchida, R., Nonaka, K., Masuma, R., Tomoda, H., 2013. Epitrichosetin, a new undecaprenyl pyrophosphate synthase inhibitor, produced by Fusarium oxysporum FKI-4553. J. Antibiot. 66, 549-554. Jin, J. M., et al., 2010. Functional characterization and manipulation of the apicidin biosynthetic pathway in Fusarium semitectum. Mol. Microbiol. 76, 456-466. Jørgensen, S. H., et al., 2014. Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis Fungal Genet. Biol. 70, 24-31. Kakule, T. B., Sardar, D., Lin, Z. J., Schmidt, E. W., 2013. Two related pyrrolidinedione synthetase loci in Fusarium heterosporum ATCC 74349 produce divergent metabolites. ACS Chemical Biology. 8, 1549-1557. Keller, O., Kollmar, M., Stanke, M., Waack, S., 2011. A novel hybrid gene prediction method employing protein multiple sequence alignments. Bioinformatics. 27, 757-763.

Kobayashi, H., Sunaga, R., Furihata, K., Morisaki, N., Iwasaki, S., 1995. Isolation and structures of an antifungal antibiotic, fusarielin A, and related compounds produced by a Fusarium sp. J. Antibiot. 48, 42-52. Kokkonen, M., Ojala, L., Parikka, P., Jestoi, M., 2010. Mycotoxin production of selected Fusarium species at different culture conditions. Int. J. Food Microbiol. 143, 17-25. Kroken, S., Glass, N. L., Taylor, J. W., Yoder, O. C., Turgeon, B. G., 2003. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc. Natl. Acad. Sci. U. S. A. 100, 15670-15675. Lawrence, D. P., Kroken, S., Pryor, B. M., Arnold, A. E., 2011. Interkingdom gene transfer of a hybrid NPS/PKS from bacteria to filamentous Ascomycota. PloS one. 6, e28231. Li, Y. R., Xu, W., Tang, Y., 2010. Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains. J. Biol. Chem. 285, 22762-22771. Lysøe, E., et al., 2014. The genome of the generalist plant pathogen Fusarium avenaceum is enriched with genes Involved in redox, signaling and secondary metabolism. PloS one. 9, e112703. Ma, L. J., et al., 2010. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature. 464, 367-373. Marchler-Bauer, A., et al., 2011. CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 39, D225-D229. McDaniel, R., Ebertkhosla, S., Hopwood, D. A., Khosla, C., 1994. Engineered biosynthesis of novel polyketides: actVII and actIV genes encode aromatase and cyclase enzymes, respectively. . J. Am. Chem. Soc. 116, 10855-10859. Meier, J. L., Burkart, M. D., 2009. The chemical biology of modular biosynthetic enzymes. Chemical Society Reviews. 38, 2012-2045. Moolhuijzen, P. M., Manners, J. M., Wilcox, S. A., Bellgard, M. I., Gardiner, D. M., 2013. Genome sequences of six wheat-infecting Fusarium species isolates. Genome announcements. 1. Munawar, A., Marshall, J. W., Cox, R. J., Bailey, A. M., Lazarus, C. M., 2013. Isolation and characterisation of a ferrirhodin synthetase gene from the sugarcane pathogen Fusarium sacchari. ChemBioChem. 14, 388-394. Nenkep, V., Yun, K., Zhang, D., Choi, H. D., Kang, J. S., Son, B. W., 2010. Induced production of bromomethylchlamydosporols A and B from the marine-derived fungus Fusarium tricinctum. J. Nat. Prod. 73, 2061-2063. Niehaus, E.-M., Janevska, S., von Bargen, K. W., Sieber, C. M. K., Harrer, H., Humpf, H.-U., Tudzynski, B., 2014a. Apicidin F: Characterization and genetic manipulation of a new secondary metabolite gene cluster in the rice pathogen Fusarium fujikuroi. PloS one. 9, e103336. Niehaus, E.-M., et al., 2013. Genetic manipulation of the Fusarium fujikuroi fusarin gene cluster yields insight into the complex regulation and fusarin biosynthetic pathway. Chem. Biol. 20, 1055-1066. Niehaus, E.-M., von Bargen, K. W., Espino, J. J., Pfannmueller, A., Humpf, H.-U., Tudzynski, B., 2014b. Characterization of the fusaric acid gene cluster in Fusarium fujikuroi. Appl. Microbiol. Biotechnol. 98, 1749-1762. Nihei, K., Itoh, H., Hashimoto, K., Miyairi, K., Okuno, T., 1998. Antifungal cyclodepsipeptides, W493 A and B, from Fusarium sp.: Isolation and structural determimation. Bioscience Biotechnology and Biochemistry. 62, 858-863.

O'Donnell, K., et al., 2013. Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fungal Genet. Biol. 52, 20-31. Oide, S., Moeder, W., Krasnoff, S., Gibson, D., Haas, H., Yoshioka, K., Turgeon, B. G., 2006. NPS6, encoding a nonribosomal peptide synthetase involved in siderophore-mediated iron metabolism, is a conserved virulence determinant of plant pathogenic ascomycetes. Plant Cell. 18, 2836-2853. Proctor, R. H., Butchko, R. A. E., Brown, D. W., Moretti, A., 2007. Functional characterization, sequence comparisons and distribution of a polyketide synthase gene required for perithecial pigmentation in some Fusarium species. Food Addit. Contam. 24, 1076-1087. Salamov, A. A., Solovyev, V. V., 2000. Ab initio gene finding in Drosophila genomic DNA. Genome Res. 10, 516-522. Song, Z. S., Cox, R. J., Lazarus, C. M., Simpson, T. J., 2004. Fusarin C biosynthesis in Fusarium moniliforme and Fusarium venenatum. ChemBioChem. 5, 1196-1203. Sørensen, J., Giese, H., 2013. Influence of carbohydrates on secondary metabolism in Fusarium avenaceum. Toxins. 5, 1655-1663. Sørensen, J. L., Akk, E., Thrane, U., Giese, H., Sondergaard, T. E., 2013. Production of fusarielins by Fusarium. Int. J. Food Microbiol. 160, 206-211. Sørensen, J. L., et al., 2012a. Production of novel fusarielins by ectopic activation of the polyketide synthase 9 cluster in Fusarium graminearum. Environ. Microbiol. 14, 1159-1170. Sørensen, J. L., et al., Fungal NRPS-dependent siderophores: from function to prediction. In: J.-F. Martín, et al., Eds.), Biosynthesis and Molecular Genetics of Fungal Secondary Metabolites. Springer, 2014a. Sørensen, J. L., Nielsen, K. F., Sondergaard, T. E., 2012b. Redirection of pigment biosynthesis to isocoumarins in Fusarium. Fungal Genet. Biol. 49, 413-418. Sørensen, J. L., Phipps, R. K., Nielsen, K. F., Frank, J., Schroers, H. J., Thrane, U., 2009. Analysis of Fusarium avenaceum metabolites produced during wet apple core rot. J. Agric. Food. Chem. 57, 1632-1639. Sørensen, J. L., et al., 2014b. Identification of the biosynthetic gene clusters for the lipopeptides fusaristatin A and W493 B in Fusarium graminearum and F. pseudograminearum. J. Nat. Prod. Submitted. Sørensen, L. Q., Lysøe, E., Larsen, J. E., Khorsand-Jamal, P., Nielsen, K. F., Frandsen, R. J. N., 2014c. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system. BMC Mol. Biol. 15, 15. Spatafora, J., Stajich, J., Grigoriev, I., 2013. 1000 Fungal Genomes project. Phytopathology. 103, 137-137. Stanke, M., Diekhans, M., Baertsch, R., Haussler, D., 2008. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics. 24, 637-644. Steinchen, W., Lackner, G., Yasmin, S., Schrettl, M., Dahse, H.-M., Haas, H., Hoffmeister, D., 2013. Bimodular peptide synthetase SidE produces fumarylalanine in the human pathogen Aspergillus fumigatus. Appl. Environ. Microbiol. 79, 6670-6676. Strieker, M., Tanovic, A., Marahiel, M. A., 2010. Nonribosomal peptide synthetases: structures and dynamics. Curr. Opin. Struct. Biol. 20, 234-240. Studt, L., Wiemann, P., Kleigrewe, K., Humpf, H.-U., Tudzynski, B., 2012. Biosynthesis of fusarubins accounts for pigmentation of Fusarium fujikuroi perithecia. Appl. Environ. Microbiol. 78, 4468-4480.

Summerell, B. A., Laurence, M. H., Liew, E. C. Y., Leslie, J. F., 2010. Biogeography and phylogeography of Fusarium: a review. Fungal Diversity. 44, 3-13. Tae, H., Kong, E. B., Park, K., 2007. ASMPKS: an analysis system for modular polyketide synthases. BMC Bioinformatics. 8. Tobiasen, C., Aahman, J., Ravnholt, K. S., Bjerrum, M. J., Grell, M. N., Giese, H., 2007. Nonribosomal peptide synthetase (NPS) genes in Fusarium graminearum, F. culmorum and F. pseudograminearium and identification of NPS2 as the producer of ferricrocin. Curr. Genet. 51, 43-58. Uhlig, S., Petersen, D., Flaoyen, A., Wilkins, A., 2005. 2-Amino-14,16-dimethyloctadecan-3-ol, a new sphingosine analogue toxin in the fungal genus Fusarium. Toxicon. 46, 513-522. Von Bargen, K. W., Niehaus, E. M., Bergander, K., Brun, R., Tudzynski, B., Humpfir, H. U., 2013. Structure elucidation and antimalarial activity of apicidin F: An apicidin-like compound produced by Fusarium fujikuroi. J. Nat. Prod. 76, 2136-2140. Wiemann, P., et al., 2012. The Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1 of Fusarium fujikuroi Controls Development, Secondary Metabolism and Pathogenicity. PloS one. 7, e37519. Wiemann, P., et al., 2013. Deciphering the cryptic genome: Genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Path. 9. Wiemann, P., Willmann, A., Straeten, M., Kleigrewe, K., Beyer, M., Humpf, H. U., Tudzynski, B., 2009. Biosynthesis of the red pigment bikaverin in Fusarium fujikuroi: genes, their function and regulation. Mol. Microbiol. 72, 931-946. Xu, Y. Q., Orozco, R., Wijeratne, E. M. K., Gunatilaka, A. A. L., Stock, S. P., Molnar, I., 2008. Biosynthesis of the Cyclooligomer Depsipeptide Beauvericin, a Virulence Factor of the Entomopathogenic Fungus Beauveria bassiana. Chem. Biol. 15, 898-907. Zhang, H., Gao, S., Lercher, M. J., Hu, S., Chen, W.-H., 2012. EvolView, an online tool for visualizing, annotating and managing phylogenetic trees. Nucleic Acids Res. 40, W569W572.

Figure legends Figure 1. Structures of polyketides and nonribosomal peptides that have been linked to genes in Fusarium. Figure 2. Comparison of genome sequenced Fusarium strains. A: Phylogenetic tree obtained by maximum likelihood analyses of DNA sequences of DNA-directed RNA polymerase II subunit (RPB2). B: Cladogram obtained through parsimony analysis of a binary data set based presence/absence of PKSs and NRPSs. The numbers at the internal nodes indicate bootstrap support from 100 bootstrap replications (>0.65). Figure 3. Overview of the PKSs and their domain structures in F. graminearum (Fg; two strains), F. culmorum (Fc), F. pseudograminearum (Fp), F. equiseti (Fe), F. acuminatum (Fac), F. avenaceum (Fav), F. verticillioides (Fv), F. fujikuroi (Ff), F. oxysporum (Fo; 12 strains) and F. solani (Fs). Known products as well as alternative numbers, functional names and alternative numbers Wiemann et al. (2013) are also listed.

Figure 4. Conservation and biosynthesis of equisetin. A: Shuffle-lagan alignment and plot of the equisetin gene cluster in F. heterosporum against F. equiseti, F. verticillioides, F. fujikuroi and F. oxysporum. B: Biosynthesis of equisetin proposed by Kakule et al. (2013) where trichosetin is released from the PKS assisted by the enoylreductase (EqxC). Trichosetin is then methylated by EqxD in F. heterosporum and F. equiseti.

Figure 5. Overview of NRPSs and their domain structures in F. graminearum (Fg; two strains), F. culmorum (Fc), F. pseudograminearum (Fp), F. equiseti (Fe), F. acuminatum (Fac), F. avenaceum (Fav), F. verticillioides (Fv), F. fujikuroi (Ff), F. oxysporum (Fo; 12 strains) and F. solani (Fs). *Partial deletion of A domains. Known products as well as alternative numbers and functional names are also listed.

Supplementary figure 1. Phylogenetic tree of KS domains from the 52 PKS obtained through multiple sequence alignments of with the amino acid sequences. Phylogenetic trees were generated by maximum likelihood analyses using a bootstrapping of 100 replications. F. graminearum (Fg; representative of two strains), F. culmorum (Fc), F. pseudograminearum (Fp), F. equiseti (Fe), F. acuminatum (Fac), F. avenaceum (Fav), F. verticillioides (Fv), F. fujikuroi (Ff), F. oxysporum (Fo; representative of 12 strains) and F. solani (Fs).

Supplementary figure 2. Phylogenetic tree of the adenylation domains of the 52 NRPSs obtained through multiple sequence alignments of with the amino acid sequences. Phylogenetic trees were generated by maximum likelihood analyses using a bootstrapping of 100 replications. F. graminearum (Fg; representative of two strains), F. culmorum (Fc), F. pseudograminearum (Fp), F. equiseti (Fe), F. acuminatum (Fac), F. avenaceum (Fav), F. verticillioides (Fv), F. fujikuroi (Ff), F. oxysporum (Fo; representative of 12 strains) and F. solani (Fs).

Supplementary file 1. Accession numbers of PKSs and NRPSs used in the study and binary table.

O O

O

OH

O

OH

O

O

O

O

O

O O

OH

PKS products

O O

O

HO

OH

HO

O

O

O

O

O

8-O-methylfusarubin (PKS3)

Aurofusarin (PKS12)

Bikaverin (PKS16)

OH

OH O

OH O

OH

O HO

O

HO

HO

O OH

O

O

HO

OH

O HO

OH O

Orcinol (PKS14)

Zearalenone (PKS4+PKS13)

O

O

O Fusarielin H (PKS9)

O OH

Bikisocoumarin (PKS16)

NH2

HO O

O

N

OH

OH

NRPS-PKS products

O

O

O

O

O

O O

O

N

O

OH

Fumonisin B1 (PKS24)

O

N

O

HO HO

O HO

O

OH

O

HO

O

Citreoisocoumarin (PKS12)

Fusaric acid (PKS21)

O

OH

O

O

N H

Fusaridione (epxS) Equisetin (PKS18)

Fusarin C (PKS10)

OH

PKS + NRPS products

OH

O

N

N O

O

O

NH2 O

H N O HN

O O

O

N

O O

N H

O

HN HN

H N

O

N

O

O

Fusaristatin A (PKS6 + NRPS7)

O

O

W493 B (PKS40 + NRPS32)

O O

O

NRPS products

O

O

N O

O

N

N

O

O

O

N

O

O

O

N N

O

O

O

O

O

O

Enniatin B (NRPS22)

O

O

HO O

NH

N H

O H N

O

O

H2 N

O

O N N H

O

N

O N O OH N OH OH N

Ferricrocin (NRPS2)

OH

O

Beauvericin (NRPS22)

H N

N

H N

HN

N O

O

H N

O

O

O

N OH

OH

O O

HO

O

NH2

Apicidin F (NRPS31)

O

NH

N O

O O NH2 Fusarinine C (NRPS6)

O

H N

O

O H N

N H

O NH O

O N H N OH

N OH O

OH

N OH O

OH

O O

OH

Malonichrome (NRPS1)

O

A

B F. verticillioides (7600) F. fujikuroi (IMI58289) F. oxysporum (Fo5176) 0.93 F. oxysporum f. sp. conglutinans (NRRL 54008) F. oxysporum f. sp. vasinfectum (NRRL 25433) 1 1 F. oxysporum f. sp. pisi (HDV247) 0.91 F. oxysporum f. sp. raphani (NRRL 54005) F. oxysporum f. sp. cubense (NRRL 54006) F. oxysporum f. sp. lycopersici (MN25) 0.98 1 0.75 F. oxysporum f. sp. radicis-lycopersici (NRRL 26381) F. oxysporum (Fo47) F. oxysporum (FOSC 3-a) 0.9 F. oxysporum f. sp. melonis (NRRL 26406) 1 F. oxysporum f. sp. lycopersici (4287) F. graminearum (CS3005) 1 1 F. graminearum (PH1) 1 F. culmorum (CS7071) F. pseudograminearum (CS3096) F. equiseti (CS3069) F. avenaceum (Fa05001) 1 F. acuminatum (CS5907) F. solani (77-13-4)

0.95

1

1 1

1

1

1

0.92

1 1 1

1 1 1 1 1

F. oxysporum (Fo5176) F. verticillioides (7600) F. fujikuroi (IMI58289) F. oxysporum f. sp. raphani (NRRL 54005) F. oxysporum f. sp. vasinfectum (NRRL 25433) F. oxysporum (FOSC 3-a) F. oxysporum f. sp. lycopersici (4287) F. oxysporum f. sp. melonis (NRRL 26406) F. oxysporum f. sp. cubense (NRRL 54006) F. oxysporum f. sp. radicis-lycopersici (NRRL 26381) F. oxysporum (Fo47) F. oxysporum f. sp. lycopersici (MN25) F. oxysporum f. sp. pisi (HDV247) F. oxysporum f. sp. conglutinans (NRRL 54008) F. equiseti (CS3069) F. graminearum (CS3005) F. graminearum (PH1) F. culmorum (CS7071) F. pseudograminearum (CS3096) F. avenaceum (Fa05001) F. acuminatum (CS5907) F. solani (77-13-4)

0.1

RPB2

Binary PKS and NRPS

Gene

Fg

Fc

Fp

Fe

Fac

Fav

Fv

Ff

Fo

Fs

Product

Funct. names

PKS1 PKS2

8/12

PKS3

10/12

PKS4

Size (kbp)

Domains KS

AT

DH

PKS2+30

KS

AT

DH MET ER

SAT KS

AT

PT ACP ACP TE

ER

KR ACP KR ACP

Fusarubins

fsr1/PGL1

PKS3

Zearalenone

ZEB1

PKS22

KS

AT

DH

PKS5

KS

AT

DH MET ER

KR ACP CT

PKS23

KS

AT

DH MET ER

KR ACP

PKS5 Fusaristatins

PKS6

Alt. names

PKS21

ER

KR ACP

PKS7

12/12

PKS7

KS

AT

DH

PKS8

11/12

PKS13

KS

AT

DH MET ER

DH MET KR ACP

ER

KR ACP

PKS9

Fusarielins

FSL1

PKS24

KS

AT

PKS10

Fusarins

FUS1/FUSS/FusA

PKS10

KS

AT MET KR ACP

PKS25

KS

AT

PKS11

DH MET ER

KR ACP

C

A

PKS12

Aurofusarin

AUR1

PKS26

SAT KS

AT

PT ACP TE

PKS13

Zearalenone

ZEB2

PKS27

SAT KS

AT

PT ACP TE

PKS14

Orcinol

PKS28

SAT KS

AT

PT ACP TE

PKS29

SAT KS

AT

PT ACP MET

PKS4

SAT KS

AT

PT ACP TE

PKS39

KS

AT

DH

PKS1

KS

AT MET KR ACP

PKS8

KS

AT

PKS9

KS

AT MET KR ACP

PKS6

KS

AT

DH

PKS12

KS

AT

DH MET ER

KR ACP

PKS40

KS

AT

DH MET ER

KR ACP

PKS11

KS

AT

DH MET ER

KR ACP

PKS14

KS

AT

DH

DH MET ER

PKS15 PKS16

Bikaverin

10/12

PKS17

5/12

PKS18

12/12

PKS19

4/12

PKS20

12/12

PKS21

12/12

PKS22

3/12

BIK1

Depudecin Equisetin

Fusaric acid

Eqx1

FUB1

PKS23 Fumonisins

PKS24

Fum1

PKS25

ER

R

KR ACP

KR C

A

R

A

R

DH MET KR ACP

ER

ER

C

KR ACP

KR ACP

PKS15

KS

AT

PKS27

12/12

PKS20

KS

AT MET KR ACP

PKS28

12/12

PKS41

KS

AT

DH MET ER

KR ACP

PKS29

PKS31

KS

AT

DH MET ER

KR ACP CT

PKS30

PKS32

KS

AT

DH MET ER

KR ACP CT

PKS31

PKS33

KS

AT

DH MET ER

KR ACP

PKS32

PKS34

KS

AT

DH

PKS33

PKS35

KS

AT

DH MET ER

KR ACP

PKS34

PKS36

KS

AT

DH MET ER

KR ACP

PKS37

SAT KS

AT

PT ACP ACP TE

PKS36

PKS17

KS

AT

DH MET ER

PKS37

PKS18

KS

AT ACP MET TE

PKS16

KS

AT

DH MET ER

PKS19

KS

AT

DH

PKS26

Pigment

PKS35

pksN

PKS38 PKS39

Unknown SM

PKS40

W493

ER

ER

KR ACP C

A

R

KR ACP

KR ACP

KR ACP

KR ACP

KS

AT

DH MET ER

PKS41

KS

AT

PT MET

KR ACP

PKS42

KS

AT

DH MET ER

KR ACP

PKS43

KS

AT

DH MET ER

KR ACP

PKS44

KS

AT

DH MET ER

KR ACP

PKS45

KS

AT

DH MET KR ACP

PKS46

KS

AT

PT ACP ACP TE

PKS47

KS

AT

PT ACP TE

PKS48

KS

AT

DH MET ER

PKS49

KS

AT

DH MET KR ACP

C C

C

T

R

A

T

R

A

T

R

A

KR ACP CT

PKS50

2/12

KS

AT

DH MET KR ACP

PKS51

5/12

KS

AT

DH MET ER

KR ACP TE

KS

AT

DH MET ER

KR ACP

PKS52

1/2

Total

16

13

14

11

14

24

16

17

15

13

3

6

9

12 kbp

SAT

starter unit acyltransferase

DH

Dehydratase

KR

Ketoreductase

CT

Carnitine acyltransferase

KS

keto-synthase

ER

Enoylreductase

PT

Product template

C

Condensation

TE

Thio-esterase / claisen cyclase

AT

Acetyltransferase

Acyl carrier protein

A

Adenylation

R

Reductase

MET

Methyltransferase

ACP

T

Peptidyl carrier protein

) KS 18

x9

xH

eq

eq

xF

xG

eq

eq

xD

xR eq

eq

eq

eq

eq

x3

xC

(P xS

x1 eq

F. heterosporum F. equiseti vs F. heterosporum

100%

F. verticillioides vs F. heterosporum

100% 50

F. fujikuroi vs F. heterosporum

100% 50

F. oxysporum vs F. heterosporum

50

0

5

10

15

25

20

100% 50

30 kbp

eqxS (PKS18) KS AT

DH

MT KR ACP C

O

EqxC

A

O

O

T R OH

O

S

NH

HO

N H OH

O

Trichosetin

EqxD

OH

O N

HO O

Equisetin

Fg

Gene

Fc

Fp

Fe

Fac

Fav

Fv

Ff

Fo

Fs

Product

Funct. names

NRPS1

10/12

Malonichrome

NRPS2

12/12

Ferricrocin

SidC

NRPS3

12/12

NRPS4

12/12

Fusarinine

SidD

NRPS5

1/12

NRPS6

12/12

Size (kbp)

Domains

*

Fusaristatins

NRPS7 NRPS8 NRPS9

12/12

NRPS10

12/12

NRPS11

12/12

NRPS12

12/12

NRPS13

12/12

NRPS14 NRPS15 NRPS16 NRPS17 NRPS18 NRPS19 11/12

NRPS20 NRPS21

11/12

NRPS22

12/12

NRPS23

12/12

Enniatin/Beauverin BeaS/ESYN1

NRPS24 NRPS25 NRPS26 NRPS27

*

NRPS28 NRPS29 NRPS30 NRPS31

Apicidins

NRPS32

W493

APS1

NRPS33 NRPS34 NRPS35 2/12

NRPS36 NRPS37 NRPS38

*

NRPS39

7/12

NRPS40

2/12

NRPS41

2/12

NRPS42

4/12

NRPS43

1/12

NRPS44

1/12

Ferrirhodin

FNR1

Fumarylalanine

SidE

NRPS45 NRPS46 NRPS47 NRPS48 NRPS49 NRPS50 NRPS51

*

*

NRPS52 Total

19

Adenylation

18

16

13

19

Condensation

24

17

15

22

13

Peptidyl carrier protein

10

Epimerization

20

Reductase

30

40 kbp

Methyltransferase

Keto-synthase

Thio-esterase

Highlights • 52 different PKSs and 52 different NRPSs were identified in 22 Fusarium strains • PKS3, 7 and 8 were conserved in all sequenced • NRPS2-4, 6 and 10-13 were conserved in all sequenced strains • Fusarium avenaceum has the highest number of PKSs (24) and NRPSs (24)