Six type-I PKS classes and highly conserved melanin and elsinochrome gene clusters found in diverse Elsinoë species

doi:10.21203/rs.3.rs-4788746/v1

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Six type-I PKS classes and highly conserved melanin and elsinochrome gene clusters found in diverse Elsinoë species

https://doi.org/10.21203/rs.3.rs-4788746/v1

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Elsinoë species are phytopathogenic fungi that cause serious scab diseases on economically important plants. The disease symptoms arise from the effects of a group of phytotoxins known as elsinochromes, produced via a type-I polyketide synthase (PKS) biosynthetic pathway. The elsinochrome gene cluster was first annotated in Elsinoë fawcettii where the main type-I PKS gene was characterized as EfPKS1. A later study showed that this gene and the associated cluster had not been correctly annotated, and that EfPKS1 was actually the anchor gene of the melanin biosynthetic pathway. A new type-I PKS gene EfETB1 associated with elsinochrome production was also identified. The aim of this study was to identify all type-I PKS genes in the genomes of seven Elsinoë species with the goal of independently verifying the PKS containing clusters for both melanin and elsinochrome production. A total of six type-I PKS classes were identified, although there was variation between the species in the number and type of classes present. Genes similar to the E. fawcettii EfPKS1 and EfETB1 type-I PKS genes were associated with melanin and elsinochrome production respectively in all species. The complete melanin and elsinochrome PKS containing clusters were subsequently annotated in all the species with high levels of synteny across Elsinoë species. This study provides a genus-level overview of type-I PKS distribution in Elsinoë species, including an additional line of support for the annotation of the melanin and elsinochrome PKS containing clusters in these important plant pathogens.

Perylenequinone

Phylogenetic analysis

Polyketide synthase

Secondary metabolites

Synteny analysis

Species of Elsinoë (Elsinoaceae, Myriangiales) are specialized phytopathogenic fungi that cause serious shoot and foliar diseases on many plant species, including agronomic crops and trees of economic importance [1]. Well-known species include E. fawcettii and E. australis causing citrus scab [2], E. perseae that causes avocado scab [3], E. ampelina the cause of grapevine spot anthracnose [4], and the recently described E. necatrix and E. masingae that result in a devastating Eucalyptus scab and shoot malformation disease in Indonesia [5] and South Africa [6]. Elsinoë infections are characterized by cork-like, cracked lesions known as scabs that can often lead to distortion and necrosis of the infected tissues [1, 5, 7]. These disease symptoms are initiated by a group of secondary metabolites known as elsinochromes [8, 9].

Elsinochromes are well-known secondary metabolites found in Elsinoë spp. that are crucial components of fungal virulence and lesion formation [9–11]. The red and orange pigmented elsinochromes are described as photosynthesizing agents as they produce reactive oxygen species in aerobic and light activated conditions [9]. These reactive oxygen species damage cell membranes and cause electrolyte leakage, which ultimately lead to tissue necrosis [9, 12]. Elsinochromes are structurally similar to perylenequinone phytotoxins synthesized by many other fungi, including aspergillin produced by Aspergillus niger [13], hypomycin produced by Hypomyces spp. [14], and cercosporin produced by Cercospora spp. [12].

Perylenequinone forms part of a class of secondary metabolites known as the polyketides [15]. Polyketides are among the most abundant secondary metabolites present in fungal cells [16]. They represent an important family of natural compounds that have a broad range of biological activities such as antifungal, antiparasitic, antibiotic, antitumor or immunosuppressant agents [17]. Polyketides include chemical compounds such as aflatoxins, naphthoquinones, anthraquinones, melanin, perylenequinones and others [18]. The chemical processes that are responsible for the structural and functional diversity of these compounds are executed by a group of multi-functional enzymes known as polyketide synthases (PKS) encoded by co-regulated genes [19].

The type-I polyketide synthase (PKS) gene thought to be responsible for elsinochrome production was first characterized in E. fawcettii [10]. The authors cloned a type-I PKS gene that they named EfPKS1 and assigned this gene as the primary PKS responsible for elsinochrome production. Their findings were substantiated by a phylogenetic analysis, revealing that this gene was similar to other polyketide synthase (PKS) genes known to be involved in pigment production [20]. Targeted disruption of EfPKS1 produced mutants that showed lower levels of virulence and were unable to produce detectable levels of elsinochrome. In a subsequent study, several other genes in the vicinity of EfPKS1 were shown to form part of the elsinochrome cluster [20].

A study by Ebert et al. [18] refuted the findings of Chung and Liao [20], rather linking the EfPKS1 gene to melanin biosynthesis in E. fawcettii. The authors also showed that EfPKS1 was most closely related to type-I PKS genes responsible for melanin production in other fungi (Ebert et al 2019). This was experimentally substantiated by a gene knock-out that showed the ΔEfPKS1 mutant produced pale fungal colonies with lower levels of melanin, but left elsinochrome production unchanged. Using a draft genome sequence, Ebert et al. [18] escribed an amended gene cluster responsible for elsinochrome production, identifying EfETB1 as the core type-I PKS gene in this cluster. A mutant culture in which EfETB1 was disrupted lost the ability to produce elsinochrome, although melanin production was not altered [18]. These findings supported the role of EfPKS1 and the associated gene cluster in the melanin biosynthesis pathway, while the EfETB1 cluster was likely involved in elsinochrome production [18].

The studies of Chung and Liao [20] and Ebert et al. [18] rovided experimental and phylogenetic support for their findings, although these studies produced contrasting results. Both studies included only a single Elsinoë species for the bioinformatic analysis, but the recent availability of genome sequences for several economically important Elsinoë species [18, 21–24] provide the opportunity to further evaluate the pathways involved in melanin and elsinochrome production in these fungi.

A recent study by Cequeña and Sumabat-Dacones [25] provided an in-silico analysis of putative effectors and secondary metabolite synthetic clusters present in six Elsinoë genomes sequenced prior to 2021. Although the study provided insight into the potential secondary metabolite clusters present in different Elsinoë species, including that of melanin and elsinochrome biosynthesis, it did not address the missanotation of the elsinochrome cluster by Chung and Liao [20] as reported by Ebertet al. [18]. Furthermore, the structure of the large elsinochrome cluster was only annotated based on the output of a single annotation pipeline, with no further validation.

In the present study, a phylogenetics-based approach was used to independently identify and compare the type-I PKS genes present in the genomes of seven Elsinoë species, including the recently sequenced genome of E. necatrix [23]. For comparative purposes, the type-I PKS genes in two closely related Dothideomycetes, Myriangium duriaei and Aureobasidium pullulans, were also analysed. The results were used to identify the type-I PKS containing clusters thought to be involved in melanin and perylenequinone production across all species included in the study and to compare these with those from the previous studies by Chung and Liao [20] and Ebert et al. [18].

Identification and characterization of type-I PKS genes

Available whole genome sequences for seven Elsinoë (E. ampelina, E. arachidis, E. australis, E. batatas, E. fawcettii, E. murrayae and E. necatrix) and two related species, Myriangium duriaei (Myriangiaceae, Myriangiales) and Aureobasidium pullulans (Saccotheciaceae, Dothideales), were retrieved from the public assembly database at NCBI [26] (Table 1). These genomes were annotated using GeneMark-ES, with parameters optimised for fungi [27], and imported into Geneious Prime 2021.0.3 [28] to generate a genomic database. This database was mined for type-I PKS genes by predicting secondary metabolite (SM) clusters using the fungal version of antiSMASH [29]. For each of the type-I PKS SM clusters predicted by antiSMASH, the main type-I PKS gene was mapped back to the respective genome sequence using the genomic position provided by antiSMASH (Table 2). The respective genes were translated to proteins in Geneious Prime and submitted to the BLASTp server [30] at NCBI to confirm their identity.

Table 1

Genome sequences used in this study.
Species	Strain	Accession number	Reference
Elsinoë ampelina	YL-1	SMYM00000000	Li et al. [51]
Elsinoë arachidis	LNFT-H01	JAAPAX00000000	Jiao et al. [22]
Elsinoë australis	Hillstone_3	QGIG00000000	NCBI – BioProject PRJNA419078
Elsinoë batatas	CRI-CJ2	JAESVG00000000	Zhang et al. [52]
Elsinoë fawcettii	DAR-70024	SWCR00000000	Shanmugam et al. [53]
Elsinoë murrayae	CQ-2017a	NKHZ00000000	NCBI – BioProject PRJNA385043
Elsinoë necatrix	CMW 56129	JANZYI000000000	Pham et al. [24]
Myriangium duriaei	CBS 260.36	JAAEIR000000000	Haridas et al. [54]
Aureobasidium pullulans	EXF-150	AYEO00000000	Gostinčar et al. [55]

Table 2

Type-I PKS genes identified by antiSMASH, mapped back to the *Elsinoë* genome sequences. Predicted PKS class determined by phylogenetic comparison.
	Genome position
Species	Contig number	Nucleotide position	Gene number	Predicted PKS family
Elsinoë necatrix	tig00000013	784,721–791,348	3489	Melanin
	tig00000013	1,558,466-1,565,254	3763	Elsinochrome
	tig00000019	429,679-437037	5192	T-toxin 2
	tig00000027	8,672 − 15,015	5917	Macrolide lactones
	tig00000031	26,392 − 32,738	6760	Macrolide lactones
	tig00000032	2,289,787-2,297,447	7648	T-toxin 1
	tig00000032	18,922 − 26,080	6764	T-toxin 3
Elsinoë fawcettii	SWCR01000008.1	125,267 − 132,055	8388	Elsinochrome
	SWCR01000008.1	926,368–932,998	8689	Melanin
	SWCR01000009.1	1,090,834–1,096,384	7017	Anthraquinone
	SWCR01000004.1	1,086,308–1,095,671	2761	T-toxin 5
Elsinoë ampelina	SMYMO100002.1	2,252,206-2,258,822	3531	Melanin
	SMYMO100002.1	3,139,385-3,146,179	3828	Elsinochrome
	SMYMO100002.1	1,622,720-1,629,309	3315	Naphthoquinone
Elsinoë arachidis	JAAPAX010000003.1	3,536,380-3,543,171	4012	Elsinochrome
	JAAPAX010000003.1	2,540,881-2,547,495	3699	Melanin
	JAAPAX010000003.1	1,905,809-1,912,398	3479	Naphthoquinone
	JAAPAX010000002.1	38,409 − 45,945	1563	T-toxin 3
	JAAPAX010000006.1	2,932,477-2,940,121	7382	T-toxin 1
Elsinoë batatas	JAESVG020000002.1	2,613,668 − 562,672	2396	Elsinochrome
	JAESVG020000002.1	1,315,266-1,321,882	2108	Melanin
	JAESVG020000002.1	1,805,220-1,810,667	1899	Naphthoquinone
	JAESVG020000005.1	2,090,919-2,100,402	5267	T-toxin 4
	JAESVG020000010.1	3,540,544-3,548,613	9482	T-toxin 1
Elsinoë murrayae	NKHZ01000011.1	83,358 − 90,130	4959	Elsinochrome
	NKHZ01000011.1	738,183–744,800	5223	Melanin
	NKHZ01000086.1	323,643 − 331,347	5484	T-toxin 1
Elsinoë australis	SWCS01000013.1	191,675 − 185,030	3198	Melanin
	SWCS01000013.1	996,526-1,003,321	3516	Elsinochrome
	SWCS01000010.1	1,256,623-1,262,193	8180	Anthraquinone
Myriangium duriaei	ML996083.1	1,696,308-1,702,980	3223
	ML996081.1	3,101,374-3,108,826	1166
	ML996086.1	1,365,136-1,357,142	5558
	ML996088.1	874,394–866,632	6568
Aureobasidium pullulans	KL584978.1	128,457 − 136,022	3177
	KL584984.1	845,879 − 852,633	6316
	KL584985.1	363,453 − 370,232	6540
	KL584986.1	318,552 − 325,043	6838
	KL585017.1	4,394 − 13,826	10666

To determine the putative function of the different PKS proteins, a phylogenetic approach was used. First, reference protein sequences for eight known PKS groups, i.e., melanin, perylenequinone, macrolide lactones, t-toxin, aflatoxin, azaphilones, anthraquinone, and naphthoquinone, were retrieved from the protein database on NCBI [30] (Table 3). Protein domains characteristic of PKS proteins were identified from all available PKS sequences using InterProScan [31]. The protein sequences were trimmed to retain only the KS and AT domains, and the resulting sequences were aligned using MAFFT v. 7 [32]. The veracity of the alignment was visually confirmed in MEGA v. 7 [33]. Maximum Likelihood (ML) analysis was conducted using RaxML v. 8.2.4 [34] on the CIPRES Science Gateway v. 3.3 [35] with a default GTR substitution matrix and 1,000 rapid bootstraps. Resulting phylogenetic tree was viewed and edited in MEGA v. 7 [33].

Table 3

Reference sequences for eight known PKS gene classes
PKS gene class	Pathogen	Protein accession number
Melanin	Colletotrichum lagenarium	BAA18956
	Nodulisporium sp.	AAD38786
	Sordaria macrospore	CAM35471
Perylenequinone	Cercospora nicotianae	AAT69682.1
	Cercospora zeina	ARU80380
	Cercospora beticola	XP023460065
	Mycosphaerella coffeicola	ADO14890
Anthraquinone	Aspergillus fumigatus	XP_746435.1
	Parastagonospora nodorum	EAT76667.2
	Aspergillus nidulans	XP_657754.1
	Aspergillus terreus	XP_001217072.1
T-toxin	Bipolaris maydis	N4WHA7
	Bipolaris maydis	AAB08104
	Bipolaris maydis	N4WHA3
Aflatoxin	Aspergillus flavis	AAS90093.1
	Dothistroma septosporum	EME39092.1
	Aspergillus nidulans	AAC49191.1
Naphthoquinones	Fusarium fujikuroi	CAB92399.1
	Fusarium graminearum	AAU10633.1
	Aspergillus nidulans	CAA46695.2
	Aspergillus niger	EHA28527.1
macrolide lactones	Hypomyces subiculosus	ACD39762.1
	Fusarium graminearum	ABB90282.1
Azaphilones	Monascus purpureus	BAD44749.1
	Penicillium brevicompactum	ADY00130.1
Supplemental Material

Annotation and synteny analysis

Cluster boundaries provided by the antiSMASH analysis were used to define the putative melanin and perylenequinone PKS containing clusters from the Elsinoë species and the two related taxa (M. duriaei and A. pullulans). These boundaries were then trimmed to align with those reported previously for E. fawcettii [18, 20]. Each of the genes within the newly defined cluster were translated to amino acid sequences using Geneious Prime and submitted to the BLASTp server at NCBI for confirmation of protein identity (Table S1).

The two gene clusters were extracted from the genomes of all the Elsinoë species and the two closely related taxa for a synteny analysis. Clinker 0.0.21 [36] was used to assess global alignment between the cluster regions, with the optimal display order determined by cluster similarity. An interactive visualisation of the synteny was also generated in Clinker using clustermap.js using default settings [36]. Genes were grouped based on cluster similarity as determined by Clinker and were labelled according to their assigned protein identity previously determined by BLASTp on NCBI (Fig. 3).

Identification and characterization of type-I PKS genes

AntiSMASH analysis of the Elsinoë genomes and those of the two related fungi included for comparative purposes revealed seven type-I PKS clusters in E. necatrix, five in E. arachidis, E. batatas and A. pullulans, four in E. fawcettii and M. duriaei and three in E. ampelina, E. murrayae and E. australis (Table 2). Each type-I PKS cluster had a single main type-I PKS gene which was mapped back to the respective genomes and for which gene identity was confirmed by BLASTp analysis. A phylogenetic comparison of the highly conserved ketosynthase (KS) and acyltransferase (AT) protein domains from the predicted type-I PKS genes made it possible to assign a putative function to each type-I PKS gene based on a set of reference proteins [18, 22, 37] (Table 3). Eight distinct classes of PKSs were identified, with all members within a class noted to synthesize structurally similar secondary metabolites (Fig. 1). Based on the clustering of the identified PKS protein sequences and the reference protein sequences within the tree, the classes were categorized as perylenequinone, aflatoxin, naphthoquinones, melanin, anthraquinone, macrolide lactones, t-toxin and azaphilones [18, 22, 37].

Only the melanin and perylenequinone gene clusters were represented in all the Elsinoë species considered in this study. The Elsinoë PKS protein domains present in the melanin class were representative of the melanin biosynthesis pathway, while the proteins clustering in the perylenequinone class represented the elsinochrome producing PKS gene cluster previously identified by Ebert et al. [18]. Putative amino acid sequences from specific Elsinoë species were found to represent the classes of macrolide lactones, naphthoquinones, t-toxin, and anthraquinones. All Elsinoë species other than E. ampelina and E. australis had one or more putative PKS proteins present in the t-toxin class, with E. necatrix having three representatives. E. australis and E. fawcettii had one putative PKS protein each in the anthraquinone class, while E. ampelina, E. arachidis and E. batatas each had a single putative PKS protein in the naphthoquinone class. Elsinoë necatrix was the only species that had any representation (two PKS proteins) in the macrolide lactone class. In addition, none of the Elsinoë species had any representative putative protein sequence in the aflatoxin class. M. duriaei and A. pullulans included for comparative purposes had representative putative protein sequences only in three classes. Both A. pullulans and M. duriaei had a single PKS gene in the melanin class, while four A. pullulans and one M. duriaei PKS genes were present in the t-toxin group. Two PKS genes from the genome of M. duriaei were present in the azaphilones outgroup, with A. pullulans not having a representative in this group.

InterProScan analysis of the different Elsinoë putative PKS proteins identified eleven different domains (Fig. 2). All of the identified PKSs contained the three main domains i.e., β-ketosynthase (KS; Pfam accession number PF00109), acyl-transferase (AT; PF00698) and ACP (PP-binding; PF00550). The melanin, elsinochrome and macrolide lactones PKSs were very similar with all three containing the additional starter unit (SAT; PF16073), dehydratase (DH; PF14765) and thioesterase (TE; PF00975) domains. Similarly, the naphthoquinone PKSs also contained an additional SAT and TE domain but lacked a DH domain. The t-toxin PKSs were divided into five groups depending on the additional domains that were present. t-toxin 1 PKS was defined by the DH, methyl-transferase (MT; PF08242), trans-acting enoyl (ER; PF00107) and β-ketoreductase (KR; PF08659) domains. t-toxin 2 PKSs contained the DH, MT, and ER domains, while t-toxin 3 contained DH, ER and KR domains. t-toxin 4 PKS contained a DH, MT, KR and a nonribosomal peptide synthetase (NRPS; PF00668) domain, while t-toxin 5 PKS contained an acetyl-CoA synthetase (AMP; PF00501) domain in addition to the DH, MT, and KR domains.

Annotation and synteny analysis of the elsinochrome and melanin biosynthetic gene cluster.

The melanin and perylenequinone type-I PKS genes were used to annotate the complete melanin and elsinochrome gene clusters in all seven Elsinoë species. After adjusting the cluster boundaries to match those previously described [18, 20], the melanin and elsinochrome clusters contained six and ten genes, respectively. BLASTp using the putative cluster proteins as queries confirmed that each gene matched to a homolog previously characterised in E. fawcettii (Table S1) [18, 20]. For the melanin biosynthetic cluster, this included the core biosynthetic PKS gene encoding a polyketide synthase (EfPKS1), as well as genes for transcription factor (TSF1), ESC reductase (RDT1), ESC prefoldin protein subunit 3 (PRF1), and ECT1 transporter (ECT1). For the elsinochrome biosynthetic cluster, the predicted genes included the core biosynthetic PKS gene encoding the protein polyketide synthase CTB1-like protein (CTB1) as well as homologs to the genes for O-methyltransferase (OMT1), FAD-binding domain-containing protein (FAD1), major facilitator superfamily domain-containing protein (MFS1), O-methyltransferase 2 (OMT2), fungal Zn(2)-Cys(6) binuclear cluster domain-containing protein (ZNC1), protein STB3 (STB3), FAD binding/ oxidoreductase (OXR1), and a fasciclin domain-containing protein (FAS1).

Only a melanin biosynthesis cluster was detected in the genomes of the closely related Dothideomycetes taxa, M. duriaei and A. pullulans. In M. duriaei, all five genes in the melanin cluster could be identified, while only four of these genes (no BLAST identity to the ECT1 protein sequence) were present in A. pullulans. No homologs to any of the genes from the elsinochrome biosynthetic cluster were detected in the genome sequences of either of the two fungi.

High levels of synteny were apparent within both the melanin and elsinochrome biosynthetic clusters across the different Elsinoë species (Fig. 3). For the melanin cluster, the five main genes were present in the order RDT1 - TSF1 - PKS1 - PRF1 - ECT1. The only exception was the ECT1 gene that was inverted and positioned upstream of RDT1 in E. australis and E. murrayae, resulting in a gene order of ECT1 - RDT1 - TSF1 - PKS1 - PRF1. Additional genes were also identified between the inverted ECT1 gene and the RDT1 gene in these two species. The core melanin cluster genes in M. duriaei and A. pullulans had the same order and orientation as the consensus for the Elsinoë species (RDT1 - TSF1 - PKS1 - PRF1 - ECT1), apart from the ECT1 gene that was absent in A. pullulans. Similarly, the structure of the elsinochrome cluster was highly conserved between the different Elsinoë species. In all the species, the genes were present in the order CTB1 – OMT1 – FAD1 – MFS1 – OMT2 – ZNC1 – STB3 – FAD2 – OXR1 – FAS1. A gene encoding for a hypothetical protein, lacking a conserved domain, was found between MFS1 and OMT2 in the genomes of E. batatas and E. murrayae.

In this study, an extensive analysis of the type-I PKS genes present in the genomes of seven different Elsinoë species was conducted. This analysis not only provided an inventory of the PKS secondary metabolite clusters present in these species, but also identified the complete melanin and elsinochrome PKS containingclusters across all species. A high diversity of type-I PKSs was found in these Elsinoë genomes, with representatives of the main type-I PKS genes grouping in six of the eight known PKS classes analysed. Among these, only gene sequences from two classes were present in all seven Elsinoë species. These two classes included the perylenequinone type-I PKS class, representing the elsinochrome biosynthetic cluster, and the melanin type-I PKS class. Further analysis of these two clusters showed strong similarity to the E. fawcettii elsinochrome and melanin biosynthetic clusters previously described by Ebert et al. [18]. This study provides a robust comparison of type-I PKSs between the different Elsinoë species, and presents a genus level comparison and analysis of both the melanin and elsinochrome gene clusters in these fungi.

A phylogenetic analysis of all the identified type-I PKS genes across the genomes of the seven Elsinoë species identified these as responsible for melanin, perylenequinone, anthraquinone, t-toxin, naphthoquinone and macrolide lactone production. Although functional studies would be required to elucidate the role of each of these clusters in the biology of these fungi, previous studies provide some clues. Quinones such as naphthoquinones or anthraquinones can act as antifungal, antibacterial, anti-inflammatory or anticancer agents by inhibiting respiration and DNA causing damage [38, 39]. Macrolide lactones have been associated with important biological activities such as cytotoxicity, and acting as antiviral, antibacterial, anti-plasmodial or anti-inflammatory agents [40]. These functions are generally governed by the ecological niche in which the species occur [41]. It would consequently be interesting to determine the function of these secondary metabolites in the different Elsinoë species.

The two type-I PKS clusters present in all seven Elsinoë species were identified as the melanin and perylenequinone (presented as elsinochrome in Elsinoë species) type-I PKS clusters. These findings are consistent with those of Cequeña and Sumabat-Dacones [25] where a PKS gene was identified for both melanin and elsinochrome production in the genomes of the six Elsinoë species. Elsinochrome is an important polyketide secondary metabolite produced exclusively by Elsinoë species [9, 11, 42], while melanin is a pigment present in a wide range of fungi [43]. Although both the elsinochrome and melanin PKS containing clusters were present in all seven Elsinoë genomes, only the latter was also present in the two non-Elsinoë taxa included for comparitive purposes. These findings provide independent support for the clusters assigned by Ebert et al. [18], and together with the functional analyses provided by those authors, identifies the elsinochrome gene cluster with confidence. It would be interesting in the future, to explore the findings of Liao and Chung [10] suggesting that deletion of the PKS type-I gene responsible for melanin production resulted in lower levels of pathogenicity and elsinochrome production.

In the current study, both the elsinchrome cluster and the melanin cluster were conserved in all seven Elsinoë spp. with high levels of synteny between the different species. SM clusters often evolve in specific fungal linages as a response to unique ecological requirements and they tend to remain as a cluster to ensure functionality [44]. The structure and conservation of the elucidated clusters in the present study provides evidence that SM clusters are maintained in closely related species by factors such as natural selection, epistatic selection, or selection for co-regulation through chromatin remodelling [16, 45–48] or horizontal gene transfer [44, 49, 50]. The maintenance of these SM clusters even indicates that it could play a role in the pathogenicity of these fungal pathogens.

Characterization of different PKS clusters in this study relied on robust genomic evidence and accurate identification of the PKS clusters from previous studies [18, 22, 37]. Our in-silico approach offers a glimpse into the potential roles of these polyketide biosynthesis clusters, although such an analysis has inherent limitations. Detailed functional studies on these predicted PKS clusters, such as the those by Chung and Liao [20] and Ebert et al. [18] combining gene expression data and analytical chemistry, will provide stronger evidence of our results. Nevertheless, this study still provides a comprehensive resource with extensive analysis of the type-I PKS genes for future investigation into the evolution of type-I PKS genes and the function of these clusters in the biology of an economically important, but relatively understudied group of fungi.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request. Annotations of the elsinochrome and melanin cluster of all seven Elsinoë species and two outgroup species used in this study (GenBank format) were deposited in Figshare (10.6084/m9.figshare.26355331).

Competing interests

The authors declare that they have no competing interests.

Funding

Funding for this study was provided by the National Research Foundation, South Africa and the University of Pretoria.

Authors' contributions

Leadership and funding for this study was provided by P.M.W., B.D.W. and M.J.W. with conceptulisation and design contributed by all authors. N.Q.P annotated the genomes and A.V.H. performed all bioinformatic analysis. All authors contributed to the interpretation of the data and A.V.H. prepared the initial manuscript and all authors contributed to its completion.

Acknowledgments

This study was initiated through the bilateral agreement between the Forestry and Agricultural Biotechnology Institute (FABI) and the April Group, RGE, Indonesia.

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Six type-I PKS classes and highly conserved melanin and elsinochrome gene clusters found in diverse Elsinoë species

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