Abstract
Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean and other major food crops. Here we sequenced the genome of the sugar beet pathogen C. beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae. Although cercosporin biosynthesis has been thought to-date to rely on an eight gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein (CFP) previously shown to be involved with cercosporin auto-resistance, and four additional genes required for cercosporin biosynthesis including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Finally, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide new insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.
Significance Statement Species in the fungal genus Cercospora cause diseases in many important crops worldwide. Their success as pathogens is largely due to the secretion of cercosporin during infection. We report that the cercosporin toxin biosynthesis (CTB) cluster is ancient and was horizontally transferred to diverse fungal pathogens on an unprecedented scale. Since these analyses revealed genes adjacent to the established CTB cluster, we evaluated their role in C. beticola to show that four are necessary for cercosporin biosynthesis. Finally, we confirmed that the apple pathogen Colletotrichum fioriniae produces cercosporin, the first case outside the family Mycosphaerellaceae. Other Colletotrichum plant pathogens also harbor the CTB cluster, which points to a wider concern that this toxin may play in virulence and human health.
Cercospora are among the most speciose genera in all Fungi. First described in 1863 (1), the genus has sustained a long history, largely due to notoriety as the causal agent of leaf spot diseases in a wide range of plants including agriculturally important crops such as sugar beet, soybean, maize and rice that together account for hundreds of millions of dollars in lost revenue annually to growers worldwide. Although Cercospora spp. share several characteristics associated with pathogenicity, such as penetration through natural openings and extracellular growth during the biotrophic stage of infection, most rely on the production of the secondary metabolite (SM) cercosporin to facilitate infection (2, 3). Studies spanning nearly 60 years have made cercosporin a model perylenequinone (4), a class of SMs characterized by a core pentacyclic conjugated chromophore that gives rise to its photoactivity. When exposed to ambient light, cercosporin is a potent producer of reactive oxygen species in the presence of oxygen (5) with a quantum efficiency of >80% (6). This small molecule is lipophilic and can readily penetrate plant leaves leading to indiscriminate cellular damage within minutes of exposure (7). Indeed, cercosporin is nearly universally toxic to a wide array of organisms including bacteria, mammals, plants and most fungal species with the key exception of cercosporin-producing fungi, which exhibit cercosporin auto-resistance. To our knowledge, cercosporin is only known to be produced by Cercospora spp. with the single exception of the brassica pathogen Pseudocercosporella capsellae (8). However, Pseudocercosporella and Cercospora are phylogenetically closely-related, residing in a large clade within the Mycosphaerellaceae (9).
In contrast to the large body of information on cercosporin biology spanning several decades (10, 11), the cercosporin toxin biosynthesis (CTB) gene cluster was only recently resolved in C. nicotianae (12). The keystone enzyme for cercosporin biosynthesis, CTB1, bears all the hallmarks of an iterative, non-reducing polyketide synthase (NR-PKS) (13). Using CTB1 as a point of reference, the complete C. nicotianae CTB gene cluster was determined to consist of eight contiguous genes of which six are believed to be responsible for cercosporin assembly (CTB1, 2, 3, 5, 6, and 7) (12, 14). The zinc finger transcription factor CTB8 co-regulates expression of the cluster (12), while the major facilitator superfamily (MFS) transporter CTB4 exports the final metabolite (15). Downstream of the CTB cluster are two open reading frames (ORFs) encoding truncated transcription factors, while loci designated as ORF9 and ORF10 upstream of the CTB cluster are not regulated by light and are not hypothesized to encode proteins with metabolic functions (12). Consequently, the clustering of eight genes with demonstrated co-regulation by light that are flanked by ORFs with no apparent role in cercosporin biosynthesis has suggested that cercosporin production relies on the eight-gene CTB cluster (12). In this study, we used an evolutionary comparative genomics approach to show that the CTB gene cluster underwent multiple duplication events and was transferred horizontally across large taxonomic distances. Since these horizontal transfer events included genes adjacent to the canonical eight gene CTB cluster, we used reverse genetics to show that the CTB cluster includes additional genes in C. beticola, including one gene that was previously shown to be involved with cercosporin auto-resistance (16) and four previously unrecognized genes involved with biosynthesis. The CTB cluster was found in several Colletotrichum (Co.) species, and we confirmed that the apple pathogen Co. fioriniae can also produce cercosporin. As all earlier understanding of cercosporin biosynthesis has been unwittingly limited by a truncated set of genes in Cercospora spp., the full dimension of the gene cluster provides deeper insight into the evolution and dissemination of a fungal toxin critical to world-wide agriculture.
Results
Secondary metabolite cluster expansion in Cercospora beticola
C. beticola strain 09-40 was sequenced to 100-fold coverage and scaffolded with optical and genome maps, resulting in 96.5% of the 37.06 Mbp assembly being placed in 12 supercontigs of which 10 are assumed to be chromosomes. Despite their ubiquitous presence in nature and cropping systems, genome sequences of Cercospora spp. are not well-represented in public databases. Therefore, to aid comparative analysis within the Cercospora genus we also sequenced the genome of C. berteroae and reassembled the genome of C. canescens (17) (SI Appendix, Table S1). To identify gene clusters responsible for biosynthesis of aromatic polyketides in C. beticola, we mined the genome to identify all SM clusters (18) and compared these with predicted clusters in related Dothideomycete fungi. The C. beticola genome possesses a total of 63 predicted SM clusters of several classes (Table S2), representing a greatly expanded SM repertoire compared to closely related Dothideomycetes (SI Appendix, Table S3). To identify the C. beticola PKS cluster responsible for cercosporin biosynthesis, we compared the sequence of the C. nicotianae CTB cluster (12) with predicted PKS clusters of C. beticola. The C. beticola PKS CBET3_00833 (CbCTB1) and flanking genes (CBET3_00830 – CBET3_00837) were ~96% identical to C. nicotianae CTB1 – CTB8 and all genes were collinear, strongly suggesting this region houses the CTB cluster in C. beticola.
Repeated duplication and lateral transfer of the cercosporin biosynthetic cluster
To study the evolutionary relationships of C. beticola PKSs, we conducted large-scale phylogenomic analyses that included various previously characterized PKSs from selected species (19). Since resolving orthologous relationships among PKSs can predict the type of SM that will be synthesized, we first built a phylogenetic tree of the conserved core β-ketoacyl synthase (KS) domains of each PKS that resulted in separating PKS enzymes into four major groups (SI Appendix, Fig. S1A). Among the eight C. beticola NR-PKSs, phylogenetic analysis revealed significant similarity between CbCTB1, CBET3_10910-RA, and CBET3_11350-RA which cluster at the base of the cercosporin clade (SI Appendix, Fig. S1B). Interestingly, genes flanking CBET3_10910-RA, but not CBET3_11350-RA, were also strikingly similar to CbCTB cluster genes (Fig. 1). Consequently, we hypothesize that the CBET3_10910 SM cluster is the result of a CTB cluster duplication. Since duplicated SM gene clusters appeared to be relatively rare in fungi (20), we investigated the origin and specificity of the CTB cluster and the putative duplication by searching for CbCTB1 homologs against a selected set of 48 published Ascomycete proteomes (SI Appendix, Table S4) representing a diverse group of fungal orders. We identified CbCTB1 orthologs in Cercospora spp. C. berteroae and C. canescens and confirmed its presence in Cladosporium fulvum (19) and Parastagonospora nodorum (21). Surprisingly, seven additional orthologs were identified in Sordariomycete species Co. orbiculare, Co. gloeosporioides, Co. fioriniae, Co. graminicola, Co. higginsianum, and Magnaporthe oryzae as well as one in the Leotiomycete Sclerotinia sclerotiorum (SI Appendix, Fig. S2A), representing diverse taxa harboring CTB1. Analysis of sequence similarity showed that intra-species (CbCTB1 - CBET3_10910-RA) sequence identity (45%) was lower than the inter-species identity (e.g. CbCTB1 and C. fulvum CTB1 (Clafu1_196875) are 55% similar; SI Appendix, Table S5), suggesting that the CTB1 duplication event was ancient and occurred prior to Dothideomycete speciation.
To develop a ‘phylogenetic roadmap’ that may explain CTB1 evolution, we used the process of ‘reconciliation’ that takes into account both species and gene histories (22). Although not conclusive, reconciliation considers the costs of evolutionary events (i.e. gene duplications, transfers, and/or losses) to explain the most parsimonious evolutionary route to the present scenario (23). Reconciliation of the species tree (SI Appendix, Fig. S3) with the CTB1 protein tree revealed that the predicted evolutionary history of CTB1 can be characterized by four duplications, three transfers and wide-spread loss to most species analyzed (SI Appendix, Fig. S4A), and further corroborates our hypothesis that the CTB1 duplication event (D1) occurred prior to Dothideomycete speciation. Reconciliation revealed an ancient transfer in which the lineage leading to S. sclerotiorum acquired the duplicated CTB1 from the last common ancestor of Cercospora spp. (T1; SI Appendix, Fig. S4A). Duplications 2-4 (D2-4) arose after lateral transfer (T2) of CTB1 into the last common ancestor of the Glomerellales. CTB1 was then transferred (T3) from a common ancestor in the Glomerellales to M. oryzae (SI Appendix, Fig. S4A).
We extended the search for CTB cluster protein orthologs by scanning the 48 proteomes for homologs of CbCTB2 (CBET3_00830) to CbCTB8 (CBET3_00837) followed by phylogenetic tree construction and subtree selection (SI Appendix, Fig. S2). This resulted in the identification of orthologs in the same set of species previously listed to contain CTB1, with the only exceptions in cases where CTB gene homologs were lost in a species. Although the loss of CTB6 and CTB7 orthologs limits reconciliation analysis of these gene families, reconciliation of the subtrees for CTB2, CTB3, CTB4, CTB5 and CTB8 (SI Appendix, Fig. S4) supported a similar scenario as proposed for CTB1, which is summarized as two duplications and two horizontal transfer events that explain the present-day CTB scenario (Fig. 2). However, a slightly less parsimonious explanation is a single transfer to an ancestral Glomerellales species, followed by wide-spread loss in most species in this lineage except for M. oryzae and the analyzed Colletotrichum spp. (Fig. 2; SI Appendix, Table S6).
Extension of the predicted cercosporin biosynthetic cluster based on microsynteny
To further examine the CTB clusters across all recipient species we generated pairwise alignments relative to the C. beticola CTB cluster and flanks. To our surprise, we observed a striking level of similarity outside of the known eight CTB genes on the 3’ end of the cluster (Fig. 3) in all CTB containing genomes. To investigate whether the amount of microsynteny observed for CTB cluster and these flanking genes can be reasonably expected when comparing Dothideomycete and Sordariomycete genomes, we assessed the genome-wide microsynteny between the genomes of C. beticola and Co. gloeosporioides and C. beticola and M. oryzae. This analysis identified the CTB cluster together with its flanking genes as having the highest level of microsynteny among all regions in the genome between C. beticola and Co. gloeosporioides, and showed that the observed CTB microsynteny between C. beticola and M. oryzae was significantly higher than the genome-wide average (Fig. 4). Likewise, CTB protein similarity between C. beticola and Colletotrichum spp., and to a lesser degree with M. oryzae, is higher compared to the genome-wide average (SI Appendix, Fig. S5, S6). Thus, we hypothesized that these flanking genes are likely part of the C. beticola CTB cluster, and consequently it is significantly larger than previously described (12). To test this, we first determined the relative expression of all eight established C. beticola CTB genes as well as a number of flanking genes (CBET3_00828 to CBET3_00848) under light (cercosporin-inducing) compared to dark (cercosporin-repressing) conditions, which showed that all candidate CTB genes on the 3’ flank were induced in the light except CBET3_00846 and CBET3_00848 (SI Appendix, Table S7). Functional annotation of these novel, induced genes revealed one non-conserved phenylalanine ammonia lyase (CBET3_00840), the cercosporin facilitator protein (CFP) (16) (CBET3_00841), a candidate α-ketoglutarate-dependent dioxygenase (CBET3_00842), an EthD-domain containing protein (CBET3_00843), a β-ig-h3 fasciclin (CBET3_00844), a laccase (CBET3_00845) and protein phosphatase 2A (CBET3_00847; SI Appendix, Table S7), which have functions associated with multi-domain enzymes or polyketide biosynthesis in fungi or bacteria (12, 24-29). Phylogenetic analyses of these flanking genes and reconciliation of their respective protein phylogenies (SI Appendix, Fig. S2) with the species tree suggest that all genes except CBET3_00840, CBET3_00846, and CBET3_00847 have undergone highly similar evolutionary trajectories as the established CTB cluster genes (Fig. 2, SI Appendix, Fig. S4) suggesting that the CTB cluster was transferred as a whole at least once, followed by species-specific evolutionary trajectories involving frequent gene loss (Fig. 2) as well as gene gain.
Novel CTB genes are essential for cercosporin biosynthesis
To confirm individual gene contributions for cercosporin production, we generated single gene deletion mutants of all candidate genes from CBET3_00840 to CBET3_00846 and tested their ability to produce cercosporin. These assays showed that cercosporin production in ΔCBET3_00844 and ΔCBET3_00845 mutants was abolished, while ΔCBET3_00842 and ΔCBET3_00843 mutants accumulated only a red, cercosporin-like metabolite that migrated differently in potato dextrose agar (PDA) culture plates and thin layer chromatography (TLC) (SI Appendix, Fig. S7), and exhibited a different profile obtained via high-performance liquid chromatography (HPLC) compared to cercosporin (Fig. 5). Other mutants produced compounds with HPLC profiles like cercosporin (Fig. 5), suggesting these genes are not involved with cercosporin biosynthesis. Taken together, these results corroborate our hypothesis that the CTB cluster extends to at least CBET3_00845 at the 3’ side and includes four novel CTB biosynthetic genes as well as CbCFP. Consequently, we propose naming genes CBET3_00842, CBET3_00843, CBET3_00844 and CBET_00845 as CbCTB9 to CbCTB12, respectively (SI Appendix, Table S7).
Pre-cercosporin isolation and characterization
To characterize the red metabolite that accumulated in ΔCTB9 and ΔCTB10 mutants, an ethyl acetate extract of the collected mycelia was analyzed by reverse phase HPLC. At 280 nm, a single peak was observed in both mutant extracts with identical retention time and UV-Vis spectra (Fig. 5,6). This peak was compared to a reference sample of cercosporin produced by wild-type C. beticola. The retention time of this peak was shorter than that of cercosporin suggesting a more polar metabolite. Comparison of the UV-vis spectra (Fig. 6A-C) of the unknown compound and cercosporin revealed nearly identical chromophores, suggesting close structural relation. The exact mass of the metabolite from the mutants was determined (ΔCTB9: m/z = 537.1762, ΔCTB10: m/z = 537.1757, [M+H+]), consistent with the elemental composition C29H28O10. This mass is 2 Da greater than that of cercosporin (+2 hydrogens), which led to a proposed structure for pre-cercosporin (Fig. 6D). Alternative hydroquinones of cercosporin could be excluded simply on the basis of the UV-vis spectral information and chemical instability. The presence of a free phenol in pre-cercosporin in place of the unusual 7-membered methylenedioxy of cercosporin is consonant with the red shift of the long wavelength λmax and the shorter HPLC retention time.
To firmly support the tentative structure of pre-cercosporin, the crude extract of ΔCTB9 was further purified by reverse-phase HPLC. To obtain sufficient material for 1H-NMR analysis, extractions were performed quickly and in the dark to prevent apparent polymerization of pre-cercosporin. The relative instability of pre-cercosporin compared to cercosporin suggests a possible role for the methylenedioxy bridge in overall stability. Immediately evident in the 1H-NMR spectrum was the absence of the methylenedioxy singlet at δ5.74 diagnostic of cercosporin, but the appearance of a new methoxyl signal at δ4.28 and a phenol at δ9.25. Consistent with the new asymmetry in pre-cercosporin, two strongly hydrogen-bonded peri-hydroxy groups could be seen far downfield at ca. 15 ppm and two aryl hydrogens were observed at δ6.92 and δ6.87. That these latter resonances are observed only in pairs, as are the two side chain methyl doublets at ca. 0.6 ppm, and the doubling of other signals imply that pre-cercosporin is formed as a single atropisomer having a helical configuration likely identical to that of cercosporin, although it is conceivable CTB9 or CTB10 sets the final stereochemistry.
Identification of cercosporin from Co. fioriniae
Since our phylogenomic analyses suggested that several Colletotrichum spp. harbored CTB clusters (Figs. 2, 3), we questioned whether any Colletotrichum spp. can produce cercosporin. To initially assess this, two Co. fioriniae strains (HC89 and HC91) isolated from apple were assayed for cercosporin production using the KOH assay (30). No cercosporin-like pigment was observed in the media under the same conditions that stimulate cercosporin production in C. beticola. Since epigenetic modifiers have been used to induce production of SMs in fungal species (31, 32), we questioned whether this strategy could be used to induce cercosporin production in Co. fioriniae. Media amended with the histone deacetylase inhibitor trichostatin A (31) induced production of a red cercosporin-like compound into the media. To characterize this red metabolite, mycelia from both Co. fioriniae strains were extracted with ethyl acetate. Reverse-phase HPLC analysis as before revealed a peak with a retention time and UV-vis spectrum consistent with cercosporin in both extracts (Fig. 7A, B). The presence of cercosporin was confirmed by UPLC-ESI-MS (Fig. 7C).
Discussion
Several hypotheses exist for the maintenance of SM biosynthetic genes as clusters. In one, unlinked SM pathway genes are at a greater risk for dissociation during meiotic recombination (33) or chromosomal rearrangements (34). Additionally, clustering may facilitate strict coordination of gene expression, which may be particularly important during the biosynthesis of SMs that have potentially toxic intermediates to ensure their efficient conversion to final end products (35). Horizontal transfer and maintenance of the ancient CTB cluster specifically among plant pathogens suggests that it was critical for disease development in diverse pathosystems, including rice blast caused by M. oryzae and various anthracnose diseases caused by Colletotrichum spp. on many different crops. The CTB clusters in Co. higginsianum and Co. graminicola were reported as one of the few SM clusters between these species that are microsyntenic (36). Moreover, O’Connell detected specific upregulation of the CTB cluster in Co. higginsianum during colonization of Arabidopsis (36). Indeed, nine of 14 Co. higginsianum CTB genes were among the top 100 most highly expressed genes in planta. Recent analysis of natural selection processes in Co. graminicola identified orthologs of CTB genes CTB1 and CFP as one of ~80 genes undergoing significant positive selection (37), further suggesting a role in pathogenicity. Interestingly, the CTB clusters of Colletotrichum spp. and M. oryzae contain additional genes (two short-chain dehydrogenases, an additional desaturase, a ferric-chelate reductase and an NmrA-like family protein) that have been reported (38) to act as negative transcriptional regulators.
The identification of cercosporin production in two isolates of Co. fioriniae has significant implications for the apple packing, storage, and processing industries. Bitter rot, caused by Colletotrichum spp., is one of the top pre- and postharvest pathogens of apple (39). This disease is a major problem for the apple industry as it limits fresh fruit in the field and during storage, and has a quiescent stage allowing decay to occur on seemingly high quality apples, only to come out of storage rotten (Jurick II, personal observation). Hence, contamination of processed apple products with cercosporin could be a significant health hazard. For example, other fungal-produced toxins (e.g. patulin, citrinin, penicillic acid) can contaminate processed apple products (40). Patulin, produced by Penicillium spp., is the most troubling as it is carcinogenic and consequently the United States and Europe have strict patulin limits in fruit juices and processed pome fruit products (40, 41). Future studies will focus on the role of cercosporin production during the Colletotrichum-apple fruit interactions in addition to assaying processed fruit products made from apples with bitter rot symptoms to determine levels of the toxin in fruit. Although only Co. fioriniae strains were analyzed for the ability to produce cercosporin, the identification of highly similar CTB clusters in other Colletotrichum species (Figs. 2, 4) suggest that cercosporin production is wide-spread in this genus.
The microsynteny outside of the established CTB cluster prompted us to test whether the flanking genes in C. beticola are also required for cercosporin biosynthesis. Notably, we observed that these flanking genes, similar to the established CTB genes, were up-regulated under cercospor-ininducing conditions. Furthermore, targeted gene replacement of CbCTB9, CbCTB10, CbCTB11 and CbCTB12 completely abolished cercosporin biosynthesis, while replacement of CbCTB9 and CbCTB10 also resulted in the accumulation of a new, red metabolite, pre-cercosporin. We thus conclude that the CTB cluster is significantly larger than previously described (12).
The isolation and characterization of a new intermediate in the cercosporin biosynthetic pathway, pre-cercosporin, strongly suggests that formation of the unique 7-membered methylenedioxy bridge in the final product is the result of a two-step process requiring three genes. First, one of two precursor aryl methoxyl groups is oxidatively removed (possibly by CTB7, a flavin-dependent oxidoreductase), followed by oxidative ring closure by CTB9, an apparent α-ketoglutarate-dependent dioxygenase, in collaboration with CTB10. The precise role of CTB10, a putative dehydratase, in ring closure is unclear, but it could serve to facilitate closure of the unfavorable 7-membered methylenedioxy ring. In contrast, a single cytochrome P450 is known to convert two aryl ortho-methoxyl groups into the relatively more common 5-membered methylenedioxy group in alkaloid biosynthesis (42). A tentative cercosporin biosynthesis scheme was recently proposed (14) without knowledge of the expanded CTB cluster. However, in light of the identification of pre-cercosporin and the potential functions of the other newly discovered CTB genes, the previously proposed biosynthetic pathway (14) will have to be revised. While these investigations will be reported in due course, we suspect the newly discovered laccase (CTB12) acts early in the pathway to dimerize the product of CTB3 (14) to the first perylenequinone intermediate, which would have precedent in synthetic chemistry (43).
Despite sustained research on cercosporin for several decades, there are significant knowledge gaps in cercosporin biosynthesis. Our data shed new light on cercosporin biology that will have significant impact on cercosporin research specifically and perylenequinone research in general. The finding that at least one species in the important plant pathogenic genus Colletotrichum can produce cercosporin has significant implications for disease management. Moreover, since Co. fioriniae may secrete cercosporin into apple food products that may be directly consumed by humans, the toxic effects of cercosporin on human health may need to be considered.
Materials and Methods
For further information, see SI Appendix Materials and Methods.
Cercospora and Colletotrichum spp. genome sequencing
Genomic DNA of C. beticola strain 09-40 was isolated using the CTAB method from mycelia scraped from the surface of V8 juice agar Petri plates (44). Library preparation of three genomic libraries with increasing insert size (500 bp, 5 Kbp and 10 Kbp) and subsequent paired-end (PE) and mate-pair (MP) genome sequencing was performed by BGI Americas Corporation (BGI) using the Illumina platform. For detailed information on C. beticola genome assembly, see SI Appendix Materials and Methods.
Cercospora berteroae strain CBS 538.71 was obtained from Centraal Bureau voor Schimmelcultures (CBS) and cultivated on Petri plates containing potato dextrose agar (PDA; Difco). High-quality DNA was extracted using the CTAB method (45). Library preparation (500 bp) and subsequent paired-end (PE) genome sequencing was performed by BGI via the Illumina platform. A total of 31 million high-quality filtered sequence reads with an average length of 100 bp were generated. A draft genome assembly was constructed using SOAPdenovo (version 2.04), applying default parameters and K-mer length 51.
Colletotrichum fioriniae strains HC89 and HC91 were isolated previously from infected apples and cultivated on Petri plates containing potato dextrose agar (PDA; Difco). High-quality DNA was extracted using the CTAB method (45). Library preparation (500 bp) and subsequent paired-end (PE) genome sequencing was performed by BGI via the Illumina platform. Draft genome assemblies were constructed using SOAPdenovo (version 2.04), applying default parameters and K-mer length 51.
Protein function characterization
For functional characterization of the predicted protein sequences, hardware-accelerated BLASTp on a DeCypher machine (TimeLogic; Carlsbad, USA) was used to identify homologous proteins in the non-redundant (nr) protein database obtained at the NCBI. InterProScan (version 5.44; http://www.ebi.ac.uk/Tools/pfa/iprscan5/) was used to identify conserved protein domains. The results of both analyses were imported into Blast2GO (46) and used to generate single, uniquely functional annotations for each protein as well as a list of all associated gene ontology (GO) terms.
Secondary metabolite cluster identification, characterization and visualization
Putative SM clusters were identified in the genome sequence of C. beticola and that of related fungi using antiSMASH2 (18) (version 2.1.0; https://bitbucket.org/antismash/antismash2/). To generate antiSMASH2-required EMBL formatted genome files, the GFF3 gene features files in combination with the respective genome sequences were converted to the EMBL sequence format using the custom Perl script GFF3_2_EMBL.pl. Subsequently, antiSMASH2 was run with default parameters, allowing for the identification of PKS, NRPS SM, Hybrid PKS-NRPS, terpene cyclase (TC), siderophore and lantipeptide SM clusters. SM clusters that showed similarity to a mixture of these clusters or only a minimal set of homologous protein domains were depicted as “other.” In addition, DMAT, for dimethylallyl tryptophan synthase clusters were identified by screening the InterProScan results for Pfam domain PF11991.
Secondary metabolite phylogenetic analyses
For phylogenetic analyses of the type I polyketide enzymes, we used Mafft (version 7.187), applying global alignment (–globalpair) and a 1000 cycles of iterative refinement (–maxiterate 1000), to align full-length sequences as well as selected domains of all PKS enzymes that were identified by antiSMASH2 in the genome sequences of the six Dothideomycetes: C. beticola, D. septosporum, Z. tritici, L. maculans, P. tritici-repentis and P. nodorum, and one Eurotiomycete: Aspergillus nidulans. In addition, previously characterized polyketide synthases (SI Appendix, Table S8) were included for reference. Prior to phylogenetic tree reconstruction, the alignments were trimmed with TrimAl (47) (version 1.2). Maximum likelihood phylogenetic trees were determined with RaxML (version 8.1.3), applying rapid bootstrapping (-# 100) and automated protein model selection (-m PROTGAMMAAUTO). Final trees were prepared online using EvolView (48). Species tree topologies were built with Cvtree (49) webserver by uploading the predicted proteomes of 48 published Ascomycete fungi (SI Appendix, Table S4).
For phylogenetic tree reconciliation analyses of the protein and species trees, the protein trees were pre-processed with treefixDTL (50) (version 1.1.10) to minimize errors introduced during tree reconstruction. TreefixDTL can correct phylogenetic trees in the presence of horizontal gene transfer. Reconciliation analyses as well as rooting were conducted in NOTUNG (23) according to the instructions (version 2.8 beta).
Genome-wide gene cluster microsynteny and protein identity analysis
Genome-wide gene-by-gene cluster analyses were performed using the custom Perl script calClusterSimilarity.pl, and plotted using ggplot2 in R using synteny.R. As input, this pipeline takes the typical output of an orthoMCL analysis, reformatted by analyseOrthoMCL.pl. In short, it requires each proteinId to have an associated clusterId. Furthermore, it requires properly formatted GFF3 files for each genome that are used to associate location of protein-coding genes and their flanks. Finally, the number of flanking genes to be used can be chosen freely, but must be set ODD. For the analyses presented in Figure 4, a cluster size of 30 was set. Genome-wide protein-by-protein best-BLAST percent identities were derived from the similarities table prepared during orthoMCL analyses and subsequently plotted in R using pairwise_pident_boxplots.R.
Gene expression analysis
To investigate the expression of cercosporin cluster genes, C. beticola was grown in a 250 mL Erlenmeyer flask containing 100 mL potato dextrose broth (PDB; Difco) either in the light or dark, to promote and repress cercosporin production, respectively. Total RNA was isolated using TRIzol (ThermoFisher) following the manufacturer’s instructions followed by an on-column Dnase treatment (Qiagen). Total RNA was used for cDNA synthesis using an oligo-(dT) primer and the SuperScript II reverse transcriptase kit (Invitrogen) following manufacturer’s instructions. The resulting cDNA was used as a template for quantitative polymerase chain reaction (qPCR). Selected genes were queried for expression using the Power SYBR Green PCR Master Mix (Applied Biosytems) using a PTC-2000 thermal cycler (MJ Research) outfitted with a Chromo4 Real-Time PCR Detector (Bio-Rad) and MJ Opticon Monitor analysis software version 3.1 (Bio-Rad). Primers for gene expression analysis are listed in SI Appendix, Table S9).
Transformation and disruption of target genes
Split-marker PCR constructs for targeted gene replacement were prepared as described (44) using genomic DNA of 10-73-4 and 09-40 wild-type C. beticola and pDAN as PCR templates. Selected mutants were complemented using pFBT005, which encodes resistance to nourseothricin and allowed us to clone our gene of interest between the ToxA promoter and TrpC terminator using PacI and NotI (Promega) restriction sites. For detailed information, see SI Appendix Materials and Methods.
4,6,9-trihydroxy-1,12-bis(2-hydroxypropyl)-2,7,11-trimethoxyperylene-3,10-dione (pre-ercosporin)
Data obtained at 5 °C on a Bruker AVANCE spectrometer. 1H NMR (400 MHz, CDCl3, δ): 15.24 (s, 1H), 14.93 (s, 1H), 9.25 (s, 1H), 6.92 (s, 1H), 6.87 (s, 1H), 4.28 (s, 3H), 4.19, (s, 3H), 4.18 (s, 3H), 3.57 – 3.51 (m, 2H), 3.42 – 3.36 (sym 5-line overlapping signal, 2H), 2.86 – 2.74 (m, 2H), 0.64 (d, J = 6.1 Hz, 3H), 0.60 (d, J = 6.1 Hz, 3H). UPLC-ESI-HRMS: calculated for C29H29O10 [M+H+]: 537.1761, found [M+H+]: 537.1762.
Colletotrichum spp. cercosporin assay
To determine whether Colletotrichum species were able to produce cercosporin, two monoconidial isolates C. fioriniae (HC89 and HC91) were grown on 9 cm Petri plates containing 15 mL of PDA as described above to replicate conditions that were conducive for cercosporin production in vitro. Seven day old cultures of each isolate were grown in a temperature controlled incubator at 25 °C with natural light. A pinkish to dark red color was visible in the media for all isolates except HC75 which had a yellow-colored pigment. Using a #2 cork borer, three plugs were removed from each isolate from the edge, middle and center of each colony and placed in small screw cap glass vials. Three plugs were also removed from an uncolonized PDA plate and included as a negative control. Cercosporin (Sigma-Aldrich) was dissolved in acetone to 100 mM and used as a positive control. 5N KOH was added to each vial to cover the surface of the plugs and incubated on a shaking incubator at room temperature for 4 hours. Supernatants were examined for cercosporin spectrophotometrically. To induce cercosporin production, we followed the procedures described by Shwab et al. (31) except 10.0 µM trichostatin A (TSA; Sigma) was used. Cercosporin production by C. fioriniae HC89 and HC91 was confirmed by HPLC and UPLC-ESI-MS analysis as described above for pre-cercosporin.
Acknowledgements
We thank W. Underwood and T. L. Friesen (USDA – ARS) for review of the manuscript, A. G. Newman for helpful discussions and a reference sample of cercosporin and N. Metz (USDA – ARS) for excellent technical assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.