Draft Genome Sequencing of the Pathogenic Fungus Cladosporium phlei ATCC 36193 Identifies Candidates of Novel Polyketide Synthase Genes Involved in Perylenequinone-Group Pigment Production

Cladosporium phlei (George) de Vries belongs to a group of hypomycetous fungi and causes purple eyespot disease, which is one of the most common foliar diseases in timothy (Phleum pratense). This disease is easily distinguishable by eye-shaped spots, with light grayish-fawn centers and purple margins on plant leaves. ¹ Phytopathogenic fungi, in general, produce toxins to infect the host plant, ² and the toxins play many roles, such as killing cells, infecting dead cells, or preventing the induction of a defense reaction. ³ Perylenequinones, which are a group of toxins secreted in an infection process, have chromophores that absorb light energy and produce active oxygen. The produced hydroxyl radicals (OH⁻), superoxide anions (O₂^–), hydrogen peroxide (H₂O₂), and singlet oxygen ( ¹ O₂) impair host cells. ⁴ Perylenequinones commonly have a 3,10-dihydroxy-4,9-perylenequinone chromophore and different side chains. ⁵ Since the compounds in the perylenequinone group have light-sensitive properties, they act as photosensitizers or photosensitizing agents. Because of the sensitivity to oxygen and laser light, the agents are used for photodynamic therapy (PDT). Until now, the United States Food and Drug Administration (USFDA) only approved Photofrin as a photosensitizer for the treatment of esophageal cancer and early lung cancer. In addition, the clinical efficacy has been confirmed for superficial early gastric cancer, early cervical cancer, and dendritic cells. Hypocrellin, a major component of Photofrin, is produced from Hypocrella bambusae. Hypocrellin is the most promising photoreceptor in the perylenequinone group. ⁶ However, H. bambusae only grows in limited areas, such as the northwest part of Yunnan Province in China and the southeast region of Tibet, and it is difficult to obtain mass production because of the growth conditions. Hypocrellin belongs to the 3,10-dihydroxy-4,9-perylenequinone group, together with phleichrome, cladochrome, cercosporin, and isocercosporin. ⁵ To overcome the limitation of hypocrellin production, we have focused on phleichrome, which can be used as a precursor of hypocrellin. Phleichrome is a deep red mycelial pigment, produced by C. phlei as a secondary metabolite. Perylenequinones, including phleichrome, are produced by the polyketide synthase (PKS) gene family of pigment-related genes. ⁷ However, the presence of the genes in C. phlei is not clear because of the lack of genome information. Thus, we assembled the draft genome of C. phlei and identified 9571 genes, including four new PKS genes.

The strain used in this study was ATCC 36193. The fungus was grown in V8 juice (20°C), and after 12 days, the mycelium was harvested by filtration and cryogenically pulverized. Genomic DNA was isolated from 1 g of pulverized tissue, according to a previously described protocol. ⁸ We constructed a sequencing library with 350 and 550 bp inserts with the Illumina Truseq nano DNA library preparation kit. The library was quantified using the Qubit dsDNA high-sensitivity (HS) assay and quantitative polymerase chain reaction (qPCR). The library was sequenced on the Illumina NextSeq500 system. The total amount of raw data was 10.1 Gbase.

To generate the best genome sequence, we assembled the genome by three different tools, Platanus (v.1.2.4), ⁹ SOAPdenovo2, ¹⁰ and CLCAssemblyCell (v4.2.0), with the default parameters. Scaffolding and gap closing for the contigs assembled by CLCAssemblyCell were performed using an in-house pipeline employing SSPACE ¹¹ and Gapfiller ¹² and considering homology regions in genomes of related species. The number of scaffolds produced by SOAPdenovo (6197) was smaller than that produced by Platanus (23 597), while the NG50 value of the Platanus results (25 429 bp) was, interestingly, larger than that of SOAPdenovo (16 255 bp). CLCAssemblyCell showed the best results, with the least number of scaffolds (5006) and the longest NG50 value (132 918 bp; Supplementary Table 1).

The annotation was performed using the Maker annotation pipeline, ¹³ resulting in the prediction of 9571 genes. To determine the completeness of the genome sequence and the annotation, BUSCO ¹⁴ analysis was performed with the BUSCO fungal data set including 1438 genes. As a result, 1412 complete single-copy BUSCOs, 4 duplicated BUSCOs, and 17 fragmented BUSCOs were found in the genome sequence, while only 5 BUSCOs were missing.

To construct a phylogenetic tree, we downloaded protein sequence files of available 15 species in Capnodiale order from GenBank and determined 71 proteins of single-copy genes that one species only has one ortholog gene by orthoMCL. ¹⁵ The tree was constructed using proml in the Phylip package ¹⁶ and the phangorn package ¹⁷ in the R environment, with the concatenated single-copy protein sequences multiple-aligned by MAFFT. Aureobasidium pullulans, belonging to the order Dothideales, was used as the outgroup. In the tree, C. phlei was included in a Cladosporiaceae family cluster with Rachicladosporium antarcticum (Figure 1), and the results corresponded to the taxonomic information.

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Figure 1.

Phylogenetic position of Cladosporium phlei based on 71 single-copy genes. Aureobasidium pullulans in Dothideales is used as the outgroup.

The candidates for PKS were selected based on the BLASTP similarity for protein sequences larger than 500 amino acids, with a 1e–10 e-value cutoff against a custom database containing 14 188 known PKS proteins. The PKS genes were determined by a National Center for Biotechnology Information (NCBI) CDD ¹⁸ domain search for the candidates. By the NCBI CDD domain search, eight PKSs were determined from 58 candidates. Among them, CpPKS1, CpPKS2, CpPKS3, and CpPKS4 have been known from previous studies. ⁷ However, four candidates, namely, CpPKS5, CpPKS6, CpPKS7, and CpPKS8, were new in C. phlei. Previous studies have shown that CpPKS1 is a non-reducing (NR)-PKS, which contains a duplicated acyl carrier protein (ACP) and thioesterase (TE) domain and is involved in pigment production. CpPKS2, a partially reducing (PR)-PKS, was found to be involved in the synthesis of methylsalicylic acids (MSAs) such as patulin. CpPKS3 and CpPKS4 are highly reducing (HR)-PKSs and contain domains such as dehydratase (DH), ketoreductase (KR), or enoylreductase (ER) domains. ⁷ Similarly, CpPKS5 had a NR-PKS domain similar to that of CpPKS1; CpPKS7 was similar to CpPKS2; CpPKS6 and CpPKS8 had HR-PKS domains similar to those of CpPKS3 and CpPKS4.

Here, we report the first draft genome of C. phlei, isolated from timothy, and four new PKS genes, which are potentially involved in phleichrome metabolism. Despite its importance, only four genomes, C. cladosporioides (https://www.ncbi.nlm.nih.gov/assembly/GCA_001931875.2), C. sphaerospermum UM843, ¹⁹ Rachicladosporium sp. CCFEE 5018, and R. antarcticum CCFEE5527, ²⁰ in Cladosporiaceae family were previously available (Table 1). We believe that the availability of the genome sequence, as well as the PKS gene information, provides a valuable basis for Cladosporium studies.

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Table 1.

Genome assembly results of five Cladosporium phlei–related genomes.

Species	Phlei	Clado	UM843	5018	Antar
Genome status	Draft	Draft	Draft	Draft	Draft
Assembly size (bp)	31.8 M	47.5 M	26.8 M	44.7 M	47.4 M
GC content	52.95%	42.9%	54.3%	54.9%	54.9%
Genome coverage	317×	100×	98.0×	175×	41.0×
No. of scaffolds	5006	843	155	232	267
N50	132 918	186 555	969 659	1 358 697	896 820
N90	25 411	20 301	200 226	194 991	162 724

PHLEI, CLADO, UM843, 5018, and ANTAR in the species row denote Cladosporium phlei, Cladosporium cladosporioides, Cladosporium sphaerospermum UM843, Rachicladosporium sp. CCFEE 5018, and Rachicladosporium antarcticum, respectively.

The draft genome sequence was deposited at NCBI (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/). The accession number is QZFA00000000.

Supplemental Material

Supplementary_Material – Supplemental material for Draft Genome Sequencing of the Pathogenic Fungus Cladosporium phlei ATCC 36193 Identifies Candidates of Novel Polyketide Synthase Genes Involved in Perylenequinone-Group Pigment Production

Supplemental material, Supplementary_Material for Draft Genome Sequencing of the Pathogenic Fungus Cladosporium phlei ATCC 36193 Identifies Candidates of Novel Polyketide Synthase Genes Involved in Perylenequinone-Group Pigment Production by Yu-Na Kang, Kum-Kang So, Do-Wan Kim, Dae-Hyuk Kim and Tae-Ho Lee in Evolutionary Bioinformatics