Transcriptome Analysis Revealed Highly Expressed Genes Encoding Secondary Metabolite Pathways and Small Cysteine-Rich Proteins in the Sclerotium of Lignosus rhinocerotis

Abstract

Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications.

Fig 1. Randomness assessment and gene coverage statistics of L. rhinocerotis.

(A) Distribution statistics of reads mapped to reference gene. The total number of reads aligned to reference genes was counted in the ratio of reads location in reference genes to the length of reference genes. (B) Distribution of genes’ coverage. The percentage of a gene covered by reads equals to ratio of the number of bases in a gene covered by unique mapping reads to number of total bases in that gene.

Fig 2. Alternative splicing events and genes in L. rhinocerotis.

Abbreviations: ES, exon skipping; IR, intron retention; A5SS, alternative 5' splice site; A3SS, alternative 3' splice site.

Fig 3. Gene expression distribution of L. rhinocerotis transcriptome.

Genes were grouped into eight log-scaled bins according to their expressions. Abbreviation: RPKM, reads per kilobase per million reads.

Fig 4. KEGG pathways classification of L. rhinocerotis transcriptome.

Fig 5. COG and GO annotations of L. rhinocerotis transcriptome.

(A) Histogram presentation of COG classification. (B) GO classification. Histogram was generated using web histogram tool WEGO at http://wego.genomics.org.cn/cgi-bin/wego/index.pl.

Fig 6. Alignment of putative cerato-platanins from L. rhinocerotis to cerato-platanin (CP) domain-containing protein sequences.

The interval of CP domains are shaded in grey. Residues in red are consensus (100%). Four cysteine residues within the CP domain (highlighted in yellow) are S—S-bonded as indicated by the green brackets. The CP domain-containing proteins were from Trametes versicolor (XP_008036158.1), T. cinnabarina (CDO71857.1), Dichomitus squalens (XP_007364464.1), Pleurotus ostreatus (KDQ34040.1), Grifola frondosa (BAN04650.1), Fistulina hepatica (KIY50660.1), Heterobasidion annosum (CPL2, AKA43766.1), Moniliophthora roreri (XP_007853249.1), Crinipellis campanella (AGL40518.1), Taiwanofungus camphoratus (Aca1, Q6J935), Colletotrichum gloeosporioides (EQB47844.1), Phaeosphaeria nodorum (SnodProt1, O74238.1), Aspergillus fumigatus (AspF13, KEY75437.1), and Ceratocystis platani (CP, P81702.1).

Fig 7. Phylogenetic analysis between putative cerato-platanins from L. rhinocerotis and related proteins.

Maximum-likelihood tree with the highest log likelihood (-2004.2139) is shown. WAG model based on the Bayesian information criterion (BIC) was used as the amino acid substitution model.

Fig 8. Sequence alignment of hydrophobin domain region within the putative hydrophobin-encoding genes of L. rhinocerotis and other fungi.

Residues in red are highly consensus (90%). Blue coloured fonts indicate low consensus residues (50% less than the first value) and residues in black are neutral. Conserved cysteine residues are highlighted in yellow. Green bracket indicates the position of conserved disulphide bonding between two cysteine residues. The hydrophobin domain-containing proteins were from Phlebiopsis gigantean (KIP08216.1), Ceriporiopsis subvermispora (EMD36640.1), Trametes cinnabarina (CDO70112.1), Trametes versicolor (XP_008040946.1), Dichomitus squalens (XP_007363423.1), Hydnomerulius pinastri (KIJ67293.1), Coniophora puteana (XP_007762953.1), Schizophyllum commune (SC6, XP_003038271.1), Coprinopsis cinerea (XP_001831661.1), Tricholoma terreum (AAL05426.1), Agrocybe aegerita (Pri2p, Q9Y8F0), Agaricus bisporus (ABH1, P49072.1), and Aspergillus fumigatus (RODA, P41746.2).

Fig 9. Phylogenetic analysis between putative hydrophobins from L. rhinocerotis and related proteins.

Maximum-likelihood tree with the highest log likelihood (-690.1136) is shown. CpREV model based on the Bayesian information criterion (BIC) was used as the amino acid substitution model.

Fig 10. Sequence alignment of lectin-encoding genes’ sugar binding site to top listed sequences from public databases.

Residues in red are consensus (100%). Hash marks (#) above the aligned sequences show the location of the conserved feature residues.