Abstract
Background
Flammulina filiformis (previously known as Asian F. velutipes) is a
popular commercial edible mushroom. Many bioactive compounds with
medicinal effects, such as polysaccharides and sesquiterpenoids, have
been isolated and identified from F. filiformis, but their biosynthesis
and regulation at the molecular level remains unclear. In this study,
we sequenced the genome of the wild strain F. filiformis Liu355,
predicted its biosynthetic gene clusters (BGCs) and profiled the
expression of these genes in wild and cultivar strains and in different
developmental stages of the wild F. filiformis strain by a comparative
transcriptomic analysis.
Results
We found that the genome of the F. filiformis was 35.01 Mb in length
and harbored 10,396 gene models. Thirteen putative terpenoid gene
clusters were predicted and 12 sesquiterpene synthase genes belonging
to four different groups and two type I polyketide synthase gene
clusters were identified in the F. filiformis genome. The number of
genes related to terpenoid biosynthesis was higher in the wild strain
(119 genes) than in the cultivar strain (81 genes). Most terpenoid
biosynthesis genes were upregulated in the primordium and fruiting body
of the wild strain, while the polyketide synthase genes were generally
upregulated in the mycelium of the wild strain. Moreover, genes
encoding UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase,
which are involved in polysaccharide biosynthesis, had relatively high
transcript levels both in the mycelium and fruiting body of the wild F.
filiformis strain.
Conclusions
F. filiformis is enriched in a number of gene clusters involved in the
biosynthesis of polysaccharides and terpenoid bioactive compounds and
these genes usually display differential expression between wild and
cultivar strains, even in different developmental stages. This study
expands our knowledge of the biology of F. filiformis and provides
valuable data for elucidating the regulation of secondary metabolites
in this unique F. filiformis strain.
Keywords: Edible mushroom, Gene cluster, Gene expression,
Polysaccharides, Sesquiterpene, High-temperature-tolerance
Background
Flammulina filiformis, also known as enokitake, winter mushroom or
golden needling mushroom, is a species endemic to Asia and belongs to
the family Physalacriaceae, Agaricales [[49]1]. Previously, F.
filiformis from eastern Asia was regarded as Asian F. velutipes or F.
velutipes var. filiformis,but recently phylogenetic results based on
multi-gene markers and morphological comparisons demonstrated that “F.
velutipes” in eastern Asia is not identical to the European winter
mushroom F. velutipes and should be treated as a separate species,
namely F. filiformis, which includes all cultivated enokitake strains
in East Asia and those from South Korea and Japan with genome sequences
[[50]2]. Thus, we apply the name “F. filiformis” instead of the Asian
F. velutipes in our study.
F. filiformis is one of the most important and popular edible mushrooms
available commercially in China. It is widely cultivated and consumed
in Asian countries due to its high nutritional value and desirable
taste. It has been reported that China is currently the largest
producer of F. filiformis, with an annual production of 2.4 million
tons [[51]3]. F. filiformis also possesses tremendous pharmaceutical
value, and many bioactive constituents have been identified, such as
polysaccharides [[52]4–[53]6], flavonoids [[54]7], sesquiterpenes,
glycosides, proteins, and phenols [[55]8–[56]10]. These compounds have
been shown to exhibit antitumour, anticancer, anti-atherosclerotic
thrombosis inhibition, anti-aging and antioxidant effects [[57]11,
[58]12]. In addition, as a typical white-rot fungus, F. filiformis can
effectively degrade lignin and produce alcohol dehydrogenase, and thus
exhibiting potential for application in bioethanol production [[59]13].
In recent decades, research has mainly focused on the phylogenetic
taxonomy [[60]1, [61]14], genetic diversity [[62]15, [63]16],
nutritional and chemical constituents [[64]17–[65]19], pharmacological
bioactivity [[66]20, [67]21] and artificial cultivation of Flammulina
spp. [[68]22–[69]24]. Most studies have shown that F. filiformis
possesses relatively high carbohydrate, protein and amino acids
contents and low fat or lipid contents; thus, it generally was
recognized as a low energy delicacy [[70]25]. In addition, bioactive
polysaccharides (e.g., glucans and heteropolysaccharides),
immunomodulatory proteins (e.g., FIP-fve) and multiple bioactive
sesquiterpenes were also isolated and identified from the fermentation
broth, mycelia and fruiting bodies of F. filiformis [[71]26]. Tang et
al. [[72]12] reviewed the compounds derived from the F. filiformis and
their diverse biological activities. Increasing studies on the chemical
compounds and biological activities of this mushroom have supported
that F. filiformis should be exploited as a valuable resource for the
development of functional foods, nutraceuticals and even pharmaceutical
drugs [[73]27].
The development of genomic and transcriptomic sequencing technologies
has provided the powerful tools to understand the biology of edible
mushrooms, including the effective utilization of cultivation
substrates (lignocellulose) [[74]28, [75]29], the mechanism of fruiting
body formation and development and adaption to adverse environments,
such as high temperature environments or cold-stress conditions
[[76]30–[77]32]. For example, genome sequencing of the cultivars of F.
filiformis from Korea and Japan revealed their high capacity for
lignocellulose degradation [[78]28, [79]33]. Transcriptomic and
proteomic analyses of F. filiformis revealed key genes associated with
cold- and light-stress fruiting body morphogenesis [[80]34]. These
studies provided important information for the breeding and commercial
cultivation of F. filiformis.
Recent advances in genome sequencing have revealed that a large number
of putative biosynthetic gene clusters (BGCs) are hidden in fungal
genomes [[81]35, [82]36]. Genome mining efforts have also allowed us to
understand the silencing or activation of biosynthetic pathways in
microbes with the development of bioinformatics software, such as
antiSMASH, SMURF and PRISM [[83]37]. For instances, the genome-wide
investigation of 66 cosmopolitan strains of Aspergillus fumigatus
revealed 5 general types of variation in secondary metabolic gene
clusters [[84]38]. The identification of the tricyclic diterpene
antibiotic pleuromutilin gene clusters on the genome-scale increased
antibiotic production in Clitopilus passeckerianus [[85]39]; the
prediction of gene clusters involved in the biosynthesis of terperoid/
polyketide synthase (PKS) in the medicinal fungus Hericium erinaceus by
genome and transcriptome sequencing discovered a new family of
diterpene cyclases in fungi [[86]40, [87]41], and the identification of
the candidate cytochromes P450 gene cluster possibly related to
triterpenoid biosynthesis in the medicinal mushroom Ganoderma lucidum
by genome sequencing improved the production of effective medicinal
compounds [[88]42, [89]43].
However, as a popular edible mushroom that has a wide spectrum of
interesting biological activities, little is known about the synthesis
and regulation of bioactive secondary metabolites of F. filiformis. In
previous experiments, we collected the wild strain of F. filiformis
Liu355 from Longling, Yunnan and demonstrated that it could tolerate
relatively high temperatures during fruiting body formation (at
18 °C–22 °C) in the laboratory and that its temperature tolerance was
superior to that of the commercial strains of F. filiformis that
usually produce fruiting bodies at low temperatures ≤15 °C [[90]16].
Thus, the wild strain is a potential and an important material for
future breeding or engineering of new F. filiformis strains because
increasing the temperature tolerance can save a substantial amount of
energy. Most interestingly, the chemical composition of the wild strain
was different from that of other commercially cultivated strains of F.
filiformis, harboring more unique chemical compounds. A total of 13 new
sesquiterpenes with noreudesmane, spiroaxane, cadinane, and cuparane
skeletons were isolated and identified from the wild strain Liu355
[[91]9]. Fungi in Basidiomycota can produce diverse bioactive
sesquiterpenes but the knowledge about sesquiterpene synthases (STSs)
in these fungi are unclear. The identification of sesquiterpene
synthases from Coprinus cinereus and Omphalotus olearius provided
useful guidance for the subsequent development of in silico approaches
for the directed discovery of new sesquiterpene synthases and their
associated biosynthetic genes [[92]44].
Thus, the aims of our study are to explore the genetic features of this
interesting wild strain of F. filiformis on a genomic scale, to predict
the genes or gene clusters involved in the biosynthesis of
polysaccharide or secondary metabolites and to profile the expression
differences in these candidate genes during the development of F.
filiformis. In addition, the genes related to its
high-temperature-tolerance are also discussed. This research will
facilitate our understanding of the biology of the wild strain, provide
useful datasets for molecular breeding, improving compound production
and improve the production of novel compounds by heterologous pathway
and metabolic engineering in the future.
Results
General features of the F. filiformis genome
Prior to our study, three genomes classified as F. filiformis were
available in public databases: the relatively complete genome of strain
KACC42780 from Korea, a draft genome of TR19 from Japan and L11 from
China (previously named as Asian F.velutipes). In this study, we
sequenced the genome of a wild strain of F. filiformis by small
fragment library construction and performed a comparative genomic
analysis of secondary metabolite gene clusters. The assembled genome of
wild F. filiformis was 35.01 Mbp with approximately 118-fold genome
coverage. A total of 10,396 gene models were predicted, with an average
sequence length of 1445 bp. The genome size and the number of predicted
protein-encoding genes were very similar to the public published genome
of F. filiformis (Table [93]1). Functional annotation of the predicted
genes showed that more than half the predicted genes were annotated in
the NCBI Non-Redundant Protein Sequence Database (NR) (6383 genes) and
5794, 2582, 1972 and 837 genes were annotated in the databases Gene
ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters
of Orthologous Groups (COG) and SwissProt, respectively. In addition,
the wild F. filiformis genome contained 107 cytochrome P450 family
genes and 674 genes encoding secretory proteins.
Table 1.
Genomic features of four strains of Flammulina filiformis (=Asian F.
velutipes)
Strain voucher Liu355 L11 TR19 KACC42780
Accession number PRJNA531555 PRJNA191865 PRJNA191921 PRJDB4587
strain original Wild, Yunnan, China Clutivar, Fujian, China Cultivar,
Japan Cultivar, Korea
Genome size (Mb) 35.01 34.33 34.79 35.64
Genome Coverage 118× 132× 37.2×
No. of Scaffolds 2040 1858 5130 11
No. of Contigs 2060 28 590 6 405 500
Genes number 10 396 11 526 10 096 11 038
Gene total length (bp) 15 027 318(42.92%) 17 020 883 (49.58%) 14 905
273 (42.84%) 15 924 075 (44.68%)
Gene average length 1 445 1 477 1 476 1 443
G+C content(%) 52.31 52.46 52.35 52.31
P450 107 144 - -
CAZy 270 315 - 392
Secretory Protein 674 - - -
Transposon pre number 204 215 245 285
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Comparative genome analysis of four strains of F. filiformis showed
that the F. filiformis can be described by a pan-genome consisting of a
core genome (4074 genes) shared by four strains (on average 23.5% of
each genome) and a dispensable genome (13,219 genes) (Fig. [95]1a). A
total of 3104 orthologous genes were annotated in the KEGG database,
2722 genes were annotated in the GO database and 1055 genes were
specific to the wild strain Liu355.
Fig. 1.
[96]Fig. 1
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Samples information and venn diagram showing the numbers of orthologue
genes or differentially expressed genes. a The numbers of orthologue
genes between four strains of F. filiformis. L11(China) in red, TR19
(Japan) in purple, KACC42780 (Korea) in yellow and Liu355 (China) in
green. b The samples of wild and cultivar strains of F. filiformis.
Up-line: cultivar strain; down-line: wild strain, from left to right:
Dikaryotic mycelium (DK); Primordium (PD) and Fruiting bodies (FB). c
The numbers of differentially expressed genes (DEGs) in various
comparative groups of F. filiformis. Fruiting body of the wild strain
(FB) in blue, Primordium of the wild strain in red, Monokaryotic
mycelium of the wild strain (MK) in green, Dikaryotic mycelium of the
wild strain (DK) in yellow and Dikaryotic mycelium of the cultivar
strain of F. filiformis in brown. d Venn diagram showing the numbers of
DEGs at adjacent development stage of F. filiformis. Blue color
represented the number of DEGs of fruiting body (FB) versus primordium
(PD) and red color represented primordium (PD) versus dikaryotic
mycelium (DK) of the wild F. filiformis strain. Abbreviation: MK:
monokaryontic mycelium; DK: dikaryontic mycelium; PD: primordium; FB:
fruiting body
Functional characteristics of the predicted genes of F. filiformis
Functional annotation in KEGG database showed that the abundance of the
predicted genes of F. filiformis involved in translation (253 genes)
was the highest, followed by carbohydrate metabolism with 243 genes.
Twenty-one genes were involved in terpenoid and polyketide biosynthesis
(Additional file [98]1: Fig. S1).
Transcriptomic analysis and gene expression
We studied the gene expression differences across different
developmental stages, namely the monokaryotic (MK), dikaryotic mycelium
(DK), primordium (PD) and fruiting body (FB) stage of the wild strain
F. filiformis Liu355. Moreover, the DK of the cultivar strain of F.
filiformis (CGMCC 5.642) was also subjected to transcriptome sequencing
(Fig. [99]1b). Three biological replicates were designed for each
sample. The average clean data for each sample was 8.07–9.32 G. We
mapped the clean reads to the genome of F. filiformis Liu 355 using
HISAT software and obtained a relatively high total mapping rate
(92.63%). In addition, the expression variation between samples was the
smallest between the DK and FB stages (the average value of R^2 = 0.85)
and was the greatest between the wild strain’s MK and cultivar DK
stages of the wild F. filiformis strain (Additional file [100]2: Fig.
S2).
Among the 10,396 gene models of F. filiformis, 9931 gene models were
expressed (FPKM > 5) across the four different tissues (MK, DK, PD and
FB) of the wild strain and the dikaryotic mycelium of a cultivar strain
of F. filiformis. A total of 6577 genes were commonly expressed in all
tissues. One hundred fifty-one genes were specifically expressed in the
cultivar strain, and 199, 152, 116, 46 genes were specifically
expressed in FB, MK, DK and PD of the wild strain of F. filiformis,
respectively (Fig. [101]1c). The tissue-specific and high expression
transcripts in F. filiformis Liu355 are listed in
Additional file [102]3: Table S1. Two genes encoding ornithine
decarboxylase (involved in polyamine synthesis) were highly expressed
in the mycelium of the cultivar strain (Nove l01369, Nove l01744), and
the genes encoding oxidoreductase also had the highest expression level
(gene 830, FPKM > 1000). The genes encoding agroclavine dehydrogenase,
acetylxylan esxterase, β-glucan synthesis-associated protein and
arabinogalactan endo-1,4-β-galactosidase protein were significantly
highly expressed in the FB of the wild F. filiformis strain, with a
more than 20–100-fold change compared to their expression in the
mycelium. Agroclavine dehydrogenase is involved in the biosynthesis of
the fungal ergot alkaloid ergovaline [[103]45] and-β-glucan
synthesis-associated protein is likely linked to the biosynthesis of
fungal cell wall polysaccharides. The high expression of these genes
indicates that they probably play an important role in fruiting body
development and compound enrichment.
A total of 5131 genes (51.67%) were up or downregulated in at least one
stage of transition, such as from mycelium to primordium (PD vs DK,
3889 genes) and from primordium to fruiting body (FB vs PD, 3308 genes)
(Fig. [104]1d). During primordial formation, 1780 genes are
upregulated, and most of the genes were annotated as oxidoreductase
activity (GO:0016491), hydrolase activity (GO:0004553) and carbohydrate
metabolism (GO:0005975). The downregulated genes were mainly enriched
in transmembrane transport (GO:0055085). During fruiting body
development, genes related to the fungal-type cell wall (GO:0009277)
and the structural constituent of the cell wall (GO:0005199) were
upregulated, reflecting the dramatic changes in cell wall structure
during the developmental process. In addition, GO term enrichment of
differentially expressed genes (DEGs) between the wild strain Liu355
and cultivar strain CGMCC 5.642 showed that most genes displayed a
similar expression profile, but peptide biosynthetic and metabolic
process (GO:0006518; GO:0043043), amide biosynthetic process (GO:
0043604) and ribonucleoprotein complex (GO: 1901566) were upregulated
in the cultivar strain of CGMCC 5.642.
KEGG enrichment analysis showed that DEGs involved in glutathione
metabolism were significantly enriched in DK of the wild strain Liu 355
compared to the cultivar strain (Fig. [105]2). Thirty-three DEGs,
including genes encoding glutathione S-transferase,
ribonucleoside-diphosphate reductase, 6-phosphogluconate dehydrogenase,
cytosolic non-specific dipeptidase, gamma-glutamyltranspeptidase, and
glutathione peroxidase, participated in this pathway. In addition,
during the primordial and fruiting body development stages, the MAPK
signaling pathway (45 DEGs) and starch and sucrose metabolism pathway
(26 DEGs) were significantly enriched. Tyrosine metabolism,
biosynthesis of secondary metabolites and glycosphingolipid
biosynthesis were also significantly enriched in the fruiting body
formation stage.
Fig. 2.
[106]Fig. 2
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KEGG pathway enrichment analysis of differentially expressed genes
(DEGs) during F. filiformis development. Left columns: pathway
enrichment at mycelium stage of wild strain Liu355 compared to cultivar
strain CGMCC 5.642; Middle columns: pathway enrichment at primordium
stage compared to mycelium stage of wild strain Liu355; Right columns:
pathway enrichment at fruiting body stage compared to primordium stage.
Abbreviation: MK: monokaryontic mycelium; DK: dikaryontic mycelium; PD:
primordium; FB: fruiting body
Genes involved in polysaccharide biosynthesis in F. filiformis
We identified a total of 80 genes related to polysaccharide (PS)
biosynthesis involved in glycolysis and gluconeogenesis in the KEGG
pathway analysis (KEGG map 00010) [[108]46] at the genomic level,
including glucose-6-phosphate isomerase (GPI),
fructose-1,6-biphosphatase (FBP), and mannose-6-phosphate isomerase
(MPI). Genes encoding Zinc-type alcohol dehydrogenase were upregulated
in both the mycelium of the wild strain compared to the cultivar strain
and in the fruiting body compared to the mycelium of the wild of F.
filiformis strain (Additional file [109]4: Fig. S3 and
Additional file [110]5: Table S2). The genes encoding glycerol
2-dehydrogenase (gene9557, gene2028), 7-bisphosphatase (gene 2929),
alcohol dehydrogenase (gene7891-D2, gene 9773-D2) and aryl-alcohol
dehydrogenase (gene 4871, gene 612) were upregulated in mycelium of the
wild strain. The expression level of the gene encoding
mannose-1-phosphate guanylyltransferase (GDP) (gene 11,132-D3) was the
highest in the mycelium of the wild strain, with a more than 200-fold
change compared to that in the mycelium of the cultivar strain. The
genes encoding glycerol 2-dehydrogenase (gene 894) and sugar
phosphatase (gene 11,052-D2) were upregulated in the fruiting body
stage of the wild strain.
To identify PS related genes, several predicted metabolic enzymes
related to PS biosynthesis in G. lucidum [[111]47] were also blasted by
homology searches in the F. filiformis genome. We identified 21
putative essential enzymes involved in PS biosynthesis in F.
filiformis, including GPI, MPI, UDP-glucose dehydrogenases (UGD),
UDP-glucose pyrophosphorylase (UGP), hexokinase, galactokinase and
transketolase (Table [112]2). Among them, genes encoding UGP, UGD and
fructose-bisphosphate aldolase (FDA) had relatively high transcript
levels in all samples analyzed (FPKM > 100).
Table 2.
Putative enzymes involved in PS biosynthsis of and their gene
expression in F.filiformis
EC No. Gene ID gene length Enzyme name FPKM mean
E-value FB Liu355 PD Liu355 MK Liu355 FPKM DK Liu355 Cultivar 5.642
5.3.1.9 gene3100 2559 Glucose-6-phosphate isomerase 0 66.43 69.62
104.75 95.85 80.55
2.7.1.1 gene8329 1551 Hexokinase 1E-124 71.13 75.91 100.65 90.18 72.05
2.7.1.1 gene6893 1515 Hexokinase 7E-81 54.55 133.98 63.93 124.08 132.22
5.3.1.8 gene3253 1215 Mannose-6-phosphate isomerase 1E-99 50.54 30.08
48.00 49.57 64.08
4.2.1.47 gene2044 1131 GDP-D-mannose dehydratase 77.37 69.54 139.04
106.22 122.26
2.7.7.9 gene3603 2301 UDP-glucose pyrophosphorylase 0 237.51 235.28
371.96 198.44 229.93
2.7.7.9 gene3631 4578 UDP-glucose pyrophosphorylase 4E-135 10.34 3.80
29.95 6.22 8.14
5.1.3.2 gene6737 1158 UDP-glucose 4-epimerase 3E-63 63.69 77.37 53.52
47.21 74.03
1.1.1.22 gene10364 1458 UDP-glucose dehydrogenase 9E-84 106.55 121.62
199.55 108.85 150.53
4.1.1.35 gene6505 1350 UDP-glucuronic acid decarboxylase 0 182.47
118.70 186.57 117.16 252.40
2.7.1.6 gene2127 1581 Galactokinase 3E-89 29.62 26.75 27.22 37.11 45.66
2.7.7.12 gene3782 1128 Galactose-1-phosphate uridyltransferase 6E-103
5.64 22.78 7.52 8.43 11.61
1.1.1.9 gene9850 864 D-xylose reductase 1E-106 211.66 169.46 83.98
150.70 180.47
1.1.1.14 gene10388 1218 Zinc-dependent alcohol dehydrogenase 4E-46
109.79 89.73 115.09 88.14 127.83
4.1.2.13 gene7057 1074 Fructose-bisphosphate aldolase 2E-160 359.67
334.01 354.86 298.99 304.30
3.1.3.11 gene9805 2235 Fructose-1,6-bisphosphatase 4E-117 73.51 112.62
64.35 124.76 93.38
2.7.1.17 gene52 1653 D-xylulose kinase 9E-126 12.52 15.67 2.25 8.38
5.15
2.2.1.1 gene5296 2049 Transketolase 0 234.88 171.98 187.99 189.28
138.91
2.2.1.1 gene10236 2109 Transketolase 6E-180 9.77 13.12 2.46 18.96 24.79
2.2.1.1 gene9220 2172 Transketolase 3E-172 3.54 3.86 4.42 4.41 0.41
2.7.1.11 gene4194 3438 6-phosphofructokinase 0 68.27 59.02 87.68 71.13
74.46
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FPKM value is mean of three biological replicates.
Abbreviations: MK monokaryotic mycelium, DK dikaryotic mycelium, FB
fruiting body, PD primordium
Predicted bioactive secondary metabolite gene clusters of F. filiformis
In total, 13 gene clusters related to terpenoid biosynthesis and two
gene clusters for polyketide biosynthesis were predicted in the wild
strain of F. filiformis (Fig. [114]3 and Additional file [115]6: Table
S3). The numbers of gene clusters involved in terpene, PKS and NRPS
biosynthesis were different in the wild strain Liu355 compared with the
other three cultivar strains (KACC42780, TR19 and L11 with genome
sequencing) and the gene number related to terpene synthesis was higher
in the wild strain Liu355 (119 genes) than in the cultivar strain L11
(81 genes) (Table [116]3). We performed sequences’ similarity
comparison of genes involved in predicted terpene and type I PKS gene
clusters among different strains of F. filiformis using blastall
v2.2.26 software and the result was provided in Additional file [117]7:
Table S4. This result showed that a high similarity of genes sequence
and gene cluster existence between different strains of F. filiformis
but several individual genes specific occurred only in wild strain
Liu355 compared to cultivar L11,TR19 and KACC42780 (e.g. gene cluster
in scaffold 548).
Fig. 3.
[118]Fig. 3
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Identification of the 13 putative gene clusters for terpene and two
polyketides gene clusters (PKS) in F. filiformis genome by antiSMASH
software. Genes with SwissProt functional annotation were marked in red
color
Table 3.
Putative genes and gene clusters related to secondary metabolitic
biosynthesis for the F. filiformis
Strains Liu 355 (wild strain) L11(cultivar) KACC42780(Korea)
TR19(Japan)
Clusters Clusters_number gene_ number Clusters_number gene_ number
Clusters_number gene_ number Clusters_number gene_ number
NRPS 3 22 2 20 2 59 2 17
Terpene 13 119 10 81 17 139 15 115
typeI_PKS 2 32 1 18 2 34 2 30
cf_putative 6 71 3 53 52 1286 39 696
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Putative genes for terpenoid biosynthesis in F. filiformis
A total of 119 genes of 13 terpenoid clusters were divided into 10
clades according to their expression levels in different developmental
stages of the wild strain or cultivar strains (Additional file [121]8:
Fig. S4). Most genes in clade II, including encoding 4,5-DOPA
dioxygenase extradiol-like protein (gene3103) and squalene synthase
(gene3428), which is involved in the biosynthesis of squalene, a
precursor of terpenoid compounds, were upregulated in the primordium
compared to the mycelium of wild strain Liu355. The genes in clades
VIII-X, including key enzymes involved in terpenoid biosynthesis, such
as protoilludene synthase, candidate peroxisomal acyl-coenzyme A
oxidase, L-amino acid amidase and cytochorme P450, were significantly
differentially expressed in the fruiting body compared with the
mycelium of the wild strain Liu355 and in the mycelium of the wild
strain Liu355 compared to the cultivar strain CGMCC5.642. For example,
the putative terpene synthase (gene 9115) was highly expressed and
significantly upregulated in the mycelium of the wild strain (1.4-fold
change) compared to the mycelium of the cultivar strain (CGMCC 5.642)
and in the fruiting bodies compared with the primordium in the wild
strain Liu355 (19.4-fold change). Two putative cytochrome P450 genes
(gene 9114 and gene 7212) were also significantly upregulated in the
dikaryotic mycelium compared to the monokaryotic mycelium of the wild
strain (7.61- and 7.50- fold change, respectively) and the cultivar
strain mycelium (2.1- and 1.7- fold change, respectively). This result
likely explained the greater diversity of bioactive compounds in the
wild strain than in the cultivar strain in a previous study.
Putative genes for sesquiterpenoid biosynthesis in F. filiformis
We performed a genome-scale homologous search with sesquiterpene
synthases of O. olearius, C. cinereus and H. erinaceus based on the
genomic data of the wild strain Liu355. Twelve homologous sequences
with considerable similarity (e-value < 10^− 5) to the known
biochemically characterized sesquiterpene synthases (STS) were
identified in the genome of the wild strain F. filiformis Liu355.
Twelve STS genes of the wild F. filiformis strain included five genes
encoding delta (6)-protoilludene synthase (gene1663-D2, gene9115,
gene2784 and gene9115-D2, gene6325-D2), two genes encoding trichodiene
synthase (gene1140, gene2254), two genes encoding alpha-muurolene
synthase (gene1358-D2, gene1358), and one gene encoding a hypothetical
protein (gene3100). The phylogenetic analysis showed that these genes
grouped into four clades (Additional file [122]9: Fig.S5). Five genes
from the wild strain Liu355 (gene1140, gene10498-D2, gene4450,
gene2785, gene2254) and two genes from the cultivar strain L11 (Fla10,
Fla11) of F. filiformis were clustered together with the cuprenene
synthases Cop6 and Omp 8-Omp10 in the clade 1. These genes are likely
responsible for the 1,6- or 1,7-cyclization of 3R-nerolidyl diphosphate
(NPP) (Cop6) or involved in the biosynthesis of α- and β-barbatene
(Omp9), compounds known to be produced by fungi and plants and carotane
sesquiterpenes (Omp10). Gene 1358-D2 (from the wild strain) and Fla6
(from the cultivar strain) in clade 2 grouped with Omp1 and Omp2 and
were speculated to catalyse a 1,10-cyclization of E, E-farnesyl
diphosphate (FPP) to yield cadinane, a precursor of sesquiterpenoids
with noreudesmane, spiroaxane, cadinane and seco-cuparane. The
expression of gene1358-D2 and gene1358 in mycelium of the wild F.
filiformis strain is significantly upregulated compared to cultivar
strain based on our transcriptomic data (1.3- and 2.7- fold change,
respectively) (Table [123]4). The genes from the wild strain
(gene6325-D2, gene1663-D2, gene9115-D2 and gene9115) and from the
cultivar strain of F. filiformis (Fla2, Fla4, Fla7 and Fla12) that
clustered with Omp6 and Omp7 in clade 3 may be capable of catalyzing a
1,11-cyclization of (E, E)-FPP leading to major groups of bioactive
sesquiterpenes in Basidiomycota [[124]44]. Gene1358 and Fla9 of clade 4
clustered with Cop4, Omp4, Omp5a and Omp5b and may synthesize major
compounds that require the 1, 10-cyclization of (3R)-nerolidyl
diphosphate (NPP).
Table 4.
Expression level of 12 genes encoding enzymes involved into
sesquiterpenoid biosynthesis in F. filiformis
Gene ID chromosome length wild_FB_FPKM wild_primordia_FPKM wild_MK_FPKM
wild_DK_FPKM clutivar_DK_FPKM log2ratio wild_DK/cultivar DK) log2ratio
wild_DK/MK log2ratio wild FB/DK log2ratio wild_FB/PD log2 ratio wild
PD/DK Description
gene2785 Scaffold121 909 0.24 0.15 0.1 0.25 0.16 0.39 0.97 -0.02 0.2
-0.21 Trichodiene synthase
gene 1358 Scaffold78 1005 4.52 32.1 2.15 5.16 1.57 1.47 0.82 -0.15
-3.21 3.06 Linoleate 10R-lipoxygenase
gene1358-D2 Scaffold262 1005 45.38 48.94 124.54 82.53 38.31 0.86 -1.03
-0.82 -0.50 -0.32 Alpha-muurolene synthase
gene3100 Scaffold10 2559 66.43 69.62 104.75 95.85 80.55 0.01 -0.56
-0.49 -0.49 0.00 Hypothetic protein
gene1663-D2 Scaffold157 1035 1.87 0.17 1.34 0.27 3.49 -3.90 -2.72 2.89
3.10 -0.20 Delta(6)-protoilludene synthase
gene9115 Scaffold2 1038 111.29 4.54 2.69 21.74 10.70 0.77 2.59 2.37
4.17 -1.80 Delta(6)-protoilludene synthase
gene1140 Scaffold110 891 5.16 4.34 2.22 5.34 4.36 0.07 0.84 -0.03 -0.21
0.18 Trichodiene synthase
gene4450 Scaffold150 936 101.68 38.30 6.24 29.57 22.48 0.15 1.81 1.76
0.92 0.83 -
gene2254 Scaffold201 996 0.82 0.26 0.09 0.21 171.85 -9.93 0.71 2.04
1.25 0.79 Trichodiene synthase
gene10498-D2 Scaffold62 864 0.14 0.06 0.14 0.02 0.01 0.71 -3.10 2.65
0.75 1.89 -
gene9115-D2 Scaffold49 1038 87.98 3.11 3.31 23.40 16.45 0.25 2.40 1.93
4.39 -2.46 Delta(6)-protoilludene synthase
gene6325-D2 Scaffold61 1017 43.08 3.28 0.89 42.16 11.23 1.66 5.14 0.06
3.27 -3.21 Delta(6)-protoilludene synthase
[125]Open in a new tab
FPKM value is the mean expression value of three biological replicates
black bont means genes significantly upregulated
Abbreviation: MK monokaryontic mycelium, DK Dikaryontic mycelium, FB
fruiting body, PD primordium
Putative genes for polyketide biosynthesis in F. filiformis
The diverse structures of polyketides are biosynthesized from
short-chain carboxylic acid units by polyketide synthases (PKSs), PKSs
have been classified into type I, type II and type III based on their
product profiles and catalytic domain architecture [[126]48]. By gene
cluster prediction using antiSMASH, we found 30 genes in two gene
clusters annotated as type I PKSs, and they were mainly located in a
single scaffold, 24 and 78, in the F. filiformis genome, respectively
(Fig. [127]4 and Additional file [128]6: Table S3). The two gene
clusters both included core genes encoding polyketide synthases
(gene8217 and gene1373). The genes located on scaffold 78, including
putative polyketide synthase (gene1373, gene1374) and benzoate
4-monooxygenase (gene 1372), were most upregulated in the wild strain
mycelium compared with the cultivar strain mycelium, indicating that
polypeptide compounds are probably abundant in the mycelia of this
mushroom and especially in the wild strain.
Fig. 4.
[129]Fig. 4
[130]Open in a new tab
Hierarchical clustering analysis of 28 putative heat-shock protein
encoding genes in F. filiformis genome between wild strain Liu355 and
cultivar strain CGMCC5.642 and among four development stages of wild
strain Liu355. Expression ratios were plotted in a heatmap on a log2
scale. The red and green colors indicate up- and down-regulation, black
represents no significant expression change and grey represents
missing. The abbreviation: MK, monokaryotic mycelium; DK, Dikaryotic
mycelium; PD, primordium; FB, Fruiting body
Cytochrome P450s in the F. filiformis genome
We identified 107 genes in the cytochrome P450 family, including nine
putative trichodiene oxygenases, 31 O-methylsterigmatocystin
oxidoreductases, five benzoate 4-monooxygenases, two linoleate
10R-lipoxygenases, two ent-kaurene oxidases, lanosterol 14-alpha and
flavonoid hydroxylases and other candidate cytochrome P450s. Of these,
102 genes had diverse expression profiles across different tissues of
F. filiformis. Twenty-six CYP450 genes were upregulated in the mycelium
of the wild strain compared to the cultivar strain and the cytochrome
P450 (gene 5820-D3) had the highest expression level, with more than a
500-fold change. Twenty-one CYP450 genes were upregulated in the
fruiting body stage compared to the mycelium stage, and genes encoding
benzoate 4-monooxygenases had the highest transcript level, with
15-fold change. In the primordim stage, the gene encoding
docosahexaenoic acid omega-hydroxylase was the highest differentially
expressed gene.
Heat-shock proteins correspond to temperature changes in F. filiformis
In our study, the wild strain of Liu355 could grow fruiting bodies at
18 °C–22 °C in the laboratory. The heat-shock protein (HSP) family is
known to be positively correlated with organism thermotolerance
[[131]49]. Twenty-eight genes annotated as HSPs were identified in the
wild F. filiformis genome (Fig. [132]4). Among them, six genes were
significantly upregulated in the wild strain Liu355 compared to the
cultivar strain, including encoding protein HSP12, HSPC4, HSP104, LHS1
and GRP78, respectively. HSP12 (gene5359), HSP7F (gene10428), HSP75
(gene4363), HSP71 (gene4424) and HSP60 (gene9277) are differentially
upregulated expression at the beginning of fruiting bodies formation
(PD stage) compared to vegetative growth (DK stage).
Discussion
Flammulina filiformis is one of most widely cultivated white rot fungi
in large commercial scale in China. It was reported that the first
cultivar strain of F. filiformis in China was domesticated from a wild
strain isolated from Fujian province in 1974 [[133]15]. To date, the
genomes of three cultivars of F. filiformis from Japan, Korea and China
were sequenced respectively. In this study, we sequenced the genome of
a wild strain of F. filiformis with abundant sesquiterpenoid compounds
and high-temperature tolerance, which collected from Yunnan Province
recently. The genome size (35.01 Mbp) and the numbers of putative genes
(10396) of the wild F. filiformis were similar to the previous public
genome of the cultivar F. filiformis (Table [134]1). Pan-genomic
analysis indicated that only 23.5% orthologus genes were shared among
the four strains of F. filiformis (Fig. [135]1a). The proportion
(23.5%) of core genes in the pan-genomic analysis of F. filiformis was
similar to that in the pan-genomic analysis of 23 Corallococcus spp.
[[136]50]. The number seems relative lower than actual number. A
possible explain was that these genomes were different sequencing depth
or different methods applied for genomic assemble and annotation.
Transcriptomic analysis showed 30 genes were specific expression in
mycelium of the cultivar CGMCC5.642, including genes encoding ornithine
decarboxylase (ODC), N-acetyltransferase and malate dehydrogenase; four
genes without functional annotation were specific expression in
dikaryotic mycelium of the wild strain F. filiformis (Additional file
[137]3:Table S1).ODC was the first and rate-limiting enzyme in the
synthesis of polyamines and it was also involved in methyl
jasmonate-regulated postharvest quality retention in button mushrooms
[[138]51]. Specific expression of these genes in cultivar strain of F.
filiformis was possible related to human domestic activity, while the
function of the genes specifically expressed in the wild strain of F.
filiformis will be further studied.
In addition, the genes involved in glutathione metabolism were
significantly enriched in DK of the wild strain Liu 355 compared to the
cultivar strain CGMCC 5.642. A study on the glutathione metabolism in
the filamentous fungus Aspergillus nidulans indicated that glutathione
itself and glutathione metabolic enzymes play crucial roles in the
germination of conidiospores and markedly contribute to the general
stress tolerance of the fungus [[139]52]. The high expression of genes
related to glutathione metabolism in the wild strain of F. filiformis
implied that the strain probably had strong environmental adaptation
and it would be a potential better breeding resource.
Polysaccharides (PSs) are important and bioactive components of F.
filiformis and other edible and medicinal mushrooms [[140]47]. GPI,
FBP, UGD and UGP are known important enzymes in the biosynthetic
pathway of PSs of edible mushrooms [[141]43, [142]47]. In our study,
genes encoding to GPI, FBP and MPI were predicted to involve in PS
biosynthesis of F. filiformis by KEGG enrichment analysis and by
homologous protein search with known enzymes involved in PS
biosynthesis of medicinal mushroom of G. lucidum. The gene encoding
mannose-1-phosphate guanylyltransferase (GDP) exhibited differential
expression in mycelium of the wild strain Liu 355 compared to cultivar
strain CGMCC5.642 with over 200-fold change, indicating the potential
abundance of PS compounds, and the content difference of PSs between
the wild and the cultivar strain of F. filiformis will be determined in
the next study.
Besides PSs, sesquiterpene compounds are the main bioactive secondary
metabolites in Flammulina. The chemistry investigation of six strains
(one wild strain and five other cultivar strains) of F. filiformis in a
previous report revealed that the wild strain Liu355 contained many new
sesquiterpenes with various skeletons, including cuparane-type and
sterpurane-type sesquiterpenes. Noreudesmane, spiroaxane, cadinane and
seco-cuparane sesquiterpenoids were first identified as new compounds
in the mycelium of the wild F. filiformis strain and 12 putative
sesquiterpene synthase genes (Fla1–12) were also predicted from the
genomic sequence of the cultivar strain of F. filiformis L11 [[143]9].
These more sesquiterpenes from the wild strain of F. filiformis were
mainly derived from 1,10-cyclization of FPP mechanisms. Thus, the
enzymes encoded by gene1358-D2 and gene1358, clustered with known
functional Omp1 and Omp2 (O. olearius) are probably responsible for the
production of sesquiterpene with specific skeleton of the F.
filiformis. The expression level of gene1358-D2 and gene1358 will be
verified in different strains of F. filiformis by quantitative
real-time PCR in the next step. In addition, the yield of these
sesquiterpene compounds and the reason for the wild strain possessing
more kinds of sesquiterpene compounds will be further explained in
ongoing research.
Comparative studies of filamentous fungal species have shown that
secondary metabolism gene clusters are often either highly divergent or
uniquely present in one or a handful of species [[144]38].
Investigation of genome-wide within-species variation of 66
cosmopolitan strains of a single species Aspergillus fumigatus revealed
that genes cluster exist location polymorphisms (a cluster was found to
differ in its genomic location across strains) and it affect the
function of gene cluster [[145]38]. In our study, we did a preliminary
analysis of gene cluster prediction within four different strains and
revealed the more than 95% genes in predicted terpene and type I PKS
clusters in wild strain Liu355 also existed in other three strain.
However, the location confirmation of these gene clusters and the
function need to be verified by experiments in the further study.
Sometimes, the number of predicted genes clusters related to secondary
metabolism in fungi based on genome sequencing data was also effected
on sequencing method (platform) and sequencing depth and sample size
(only four strains can available in our study), therefore, more strains
from different geographic regions will be collected for further
analysis.
A temperature downshift (cold stimulation) is considered to be one of
the most important and essential environmental factors for the fruiting
initiation and fruiting body formation of F. filiformis [[146]34]. In
our study, six genes annotated with heat shock protein family (HSP12,
HSPC4, HSP104, LHS1 and GRP78) displayed significantly differential
expression in the wild strain Liu355 compared to the cultivar strain.
It is known that HSP12 is part of a group of small HSPs that function
as chaperone proteins and are ubiquitously involved in nascent protein
folding by protecting proteins from misfolding and are partially
characterized as a stress response; the expression of HSP12 protein was
observed in response to cold stress [[147]53]. The expression of HSP104
and HSP70 is regulated by the Hsf (heat-shock factor) interaction,
which can be stimulated by heat stress in yeast [[148]49]. In addition,
HSP70 chaperone and two putative HSP were also found were upregulated
at only primordium or young fruiting body of cultivar F. filiformis
using the iTRAQ labeling technique [[149]54]. Our study also predicted
that genes encoding HSP12, HSP 71, HSP60 probable involved in formation
and differentiation of fruiting bodies of F. filiformis. However, the
exact molecular function of HSPs in the high-temperature tolerance of
wild F. filiformis and its adaptive mechanisms for relatively high
temperatures need further study.
Conclusions
In our study, genome and transcriptome sequencing and the assembly and
annotation of the high-temperature-tolerant wild F. filiformis strain
were carried out, and the gene clusters associated with
polysaccharides, terpenoid and polyketide biosynthesis were predicted.
Comparative genomic analysis with three other Asian cultivar strains of
F. filiformis revealed that the wild strain has a similar genome size
and relatively more putative gene numbers related to secondary
metabolite biosynthesis. Most genes related to terpenoid biosynthesis
were upregulated in the primordium and fruiting body of the wild
strain, while PKS genes were generally upregulated in the mycelium of
the wild strain; however, the specific regulatory pathways involved
such synthesis pathways remain unresolved in this study.
Six genes belonging to the HSP family, including HSP12, HSPC4, HSP104,
LHS1 and GRP78, were significantly upregulated in the wild strain
Liu355 compared to the cultivar strain and may be responsible for the
development of fruiting bodies at relatively high temperatures in the
high-temperature-tolerant wild F. filiformis strain. However, the
expression of these genes in other strains of F. filiformis, especially
in strains under low-temperature developmental conditions, requires
verification in future studies. Our study provides an important genetic
dataset for F. filiformis as a potential breeding material, and
provides a foundation for enhancing the understanding of the biology of
F. filiformis.
Materials and methods
Fungal strains and strain culture
The wild strain Liu355 used for genomic sequencing was kindly provided
by Prof. H. W. Liu (State Key Laboratory of Mycology, Institute of
Microbiology, Chinese Academy of Sciences) and was first isolated from
the fruiting body of F. filiformis collected from Longling, Yunnan
Province, southwestern China. The voucher specimens of the F.
filiformis fruiting bodies were deposited in the Cryptogamic Herbarium,
Kunming Institute of Botany, Chinese Academy of Sciences (HKAS85819).
DNA sequencing of the internal transcribed spacer (ITS) region of F.
filiformis (HKAS85819) was listed under GenBank accession number
[150]KP867925 [[151]9]. The haploid monokaryotic strain F. filiformis
Liu355 (deposited in Mycological Laboratory of the Institute of
Medicinal Plant Development, Chinese Academy of Medical Sciences) was
prepared by the protoplast mononuclear method and was grown on potato
dextrose agar (PDA) at room temperature for 2–3 weeks in the dark. The
fruiting bodies were obtained in sterile plastic bottles containing on
growth substrate (cotton seed hulls, 78%; wheat bran, 20%; KH[2]PO[4],
0.1%; MgSO[4], 0.1%; sucrose, 1%; and ground limestone, 1%; with a
moisture content of 70%) at 25 °C for 30 d, followed by cold
stimulation at 18 °C and 90% humidity until primordial development
occurred. Cultures were maintained at low temperature (18 °C and 75%
humidity) to allow full fruiting body development [[152]55]. In
addition, the genomic data of two cultivar strains from Korea
(KACC42780, Bioproject PRJNA191921) and Japan (TR19, Bioproject
PRJDB4587) were available from the NCBI public database, and the
genomic sequence of strain L11 (Bioproject PRJNA191865) was kindly
provided by the Mycological Research Center, College of Life Sciences,
Fujian Agriculture and Forestry University [[153]56]. The cultivar
dikaryotic strain (CGMCC 5.642) was obtained from the China General
Microbiological Culture Collection Center (Beijing, China,
[154]http://www.cgmcc.net/) and stored in our laboratory.
Genome and transcriptome sequencing and analysis
Total genomic DNA of F. filiformis was extracted from the mononuclear
mycelia in PDA medium using the Omega E.Z.N.A. fungal DNA midi kit
(Omega, USA) according to the manufacturer’s instructions. Total DNA
was evaluated by agarose gel electrophoresis and quantified by Qubit
2.0 Fluorometer (Thermo Scientific). Library construction and
sequencing was performed at the Beijing Novogene Bioinformatics
Technology Co. Ltd. (China). The quality and quantity of libraries were
checked using an Agilent 2100 Bioanalyzer. The F. filiformis strain was
sequenced using 350 bp paired-end reads on an Illumina HiSeq 4000
platform via the PE150 method. The quality of sequencing results for
fastq files was evaluated using software FastQC
([155]http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and
readfq.v10 ([156]https://github.com/lh3/readfq) also was used for
sequence quality control. The raw data was filtered by removing the
low-quality reads, including reads with N content higher than 10%,
reads whose base quality value is less than 20 with the ratio is higher
than 40%, duplication (exactly the same PE reads) and PE reads
containing sequencing adapters (15 bases aligned to the adapter
sequence). After that, the high-quality reads were mapped to the
reference genome sequence of F. filiformis L11 (Bioproject PRJNA191865)
using BWA v0.5.9-r16 software. Functional annotation of the predicted
genes was performed using BLAST against Gene Ontology (GO), Kyoto
Encyclopedia of Genes and Genomes (KEGG), SwissProt and NCBI
Non-Redundant Protein Sequence Database (NR) [[157]40].
The term pan-genome was first proposed by Tettelin et al. in 2005 and
it defines the entire genomic repertoire of a given phylogenetic clade
and encodes for all possible lifestyles carried out by its organisms
[[158]57, [159]58]. Pan-genome usually comprises the core-genome
(essential nucleotide sequences shared by all genomes in the cohort),
dispensable genome (nucleotide sequences shared by a subset of genomes
in the cohort) and strain-specific genes (nucleotide sequences existing
only within a particular genome in the cohort) [[160]59]. Pan-genome
analysis in our study was carried out using the standalone CD-HIT tool
to cluster orthologous proteins [[161]60].
For transcriptomic sequencing, total RNA was extracted using the
RNAeasy Plant Mini kit (Qiagen) according to the manufacturer’s
protocols. Five samples were prepared; the monokaryotic mycelium (MK),
dikaryotic mycelium (DK), primordium (PD) and fruiting bodies (FB) of
the wild strain Liu355 and the dikaryotic mycelium of the cultivar
strain CGMCC 5.462. Each sample had three biological replicates. All
samples were subjected to RNA-Seq on the Illumina HiSeq2000 platform
(Illumina, San Diego, CA, USA). Raw data (raw reads) of fastq format
were firstly processed through FastQC
([162]http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). In
this step, clean data (clean reads) were obtained by removing reads
containing adapter that were added for reverse transcription and
sequencing, low-quality bases (> 50% of the bases with a quality
score ≤ 5), reads containing ploy-N and low quality reads that
sequences containing too many unknown bases (> 5%). At the same time,
Q20, Q30 and GC content the clean data were calculated. All the
downstream analyses were based on the clean data with high quality.
After that, the RNA-seq reads were mapped to the F. filiformis genome
(Liu355) using TopHat v2.0.1253 [[163]61]. HTSeq v0.6.1 software was
used to count the read numbers mapped to each gene [[164]62]. The FPKM
value was used to calculate gene expression, and the upper-quartile
algorithm was used to correct the gene expression. Gene differential
expression analysis was performed using the DESeq R package (1.10.0)
using a corrected p-value [[165]63]. Genes with an adjusted P-value
< 0.05 were considered differentially expressed. Hierarchical
clustering of gene expression was conducted using Genesis 1.7.7
[[166]64].
KEGG enrichment analysis of differentially expressed genes (DEGs)
We used KOBAS v2.0 software to test the statistical enrichment of
differentially expressed genes (DEGs) in KEGG pathways
([167]https://www.kegg.jp/kegg/). Based on the hypergeometric
distribution, we predicted the enriched pathway of DEGs with all the
annotation genes. The formula is below: N represented the all gene
number with pathway annotation, n is the DEGs number of N, M refers the
all gene number annotated in a specific pathway and m is the DEGs
number annotated in a specific pathway. The pathway was defined as
significant enrichment (Padj≤0.05).
[MATH: p=1−∑i=0
m−1MiN−Mn
−iNn :MATH]
Prediction of gene clusters involved in biosynthesis of secondary metabolites
The biosynthetic gene clusters were predicted using antiSMASH 3.0
software [[168]65]. AntiSMASH currently offers a broad collection of
tools and databases for automated genome mining and comparative
genomics for a wide variety of different classes of secondary
metabolites [[169]66]. In addition, Sequence homology searches method
(BlastP) was also used to identify genes related to terpenoid
biosynthesis. The sesquiterpene synthases were identified based on
multiple sequence alignments and phylogenetic analyses developed by the
Schmidt-Dannert group [[170]44]. It has been reported that a more
divergent cytochrome P450 oxidase could be involved in secondary
biosynthesis [[171]67]. Therefore, we searched the genome of F.
filiformis for proteins with a P450 conserved domain using the NCBI CDD
tool and BLASTp [[172]40] and also by homology BLAST in the Fungal
Cytochrome P450 Database ([173]p450.riceblast.snu.ac.kr/index.php?
a = view) to obtain the annotated gene for cytochrome P450. In
addition, CAZymes were identified using the Carbohydrate Active enZymes
(CAZy) database [[174]68]. We performed DIAMOND search against the CAZy
pre-annotated CAZyme sequence database and combined with corresponding
gene functional annotation to get the annotation result. TransposonPSI
software was used as transposon gene prediction and this software uses
PSI-BLAST to detect distant homology between genomic sequences and a TE
library bundled with the program [[175]69]. Secretory proteins was
predicted with Signal P4.1 server
([176]http://www.cbs.dtu.dk/services/signalP/) searching in all
encoding amino acid sequence of F. filiformis genome.
Supplementary information
[177]12864_2020_7108_MOESM1_ESM.jpg^ (1.3MB, jpg)
Additional file 1: Fig.S1. A KEGG functional annotation of the
predicted genes of F. filiformis. The highest number of genes related
to metabolism process and carbohydrate metabolism except for genetic
information processing.
[178]12864_2020_7108_MOESM2_ESM.jpg^ (1.3MB, jpg)
Additional file 2: Fig.S2. Relationships among five transcriptomes
samples of F. filiformis. Pairwise correlation of normalized FPKMs
between RNA samples. The Pearson correlation coefficient ranges from no
correlation (white) to perfect correlation (dark blue). Each sample has
three biological replicates. The abbreviation: MK, monokaryotic
mycelium; DK, Dikaryotic mycelium; PD, primordium; FB, Fruiting body.
[179]12864_2020_7108_MOESM3_ESM.xlsx^ (52.7KB, xlsx)
Additional file 3: Table S1. Tissue-specific expression transcript in
four different tissues of F. filiformis. (XLSX 52 kb)
[180]12864_2020_7108_MOESM4_ESM.jpg^ (1.2MB, jpg)
Additional file 4: Fig. S3. KEGG mapping (map 00010) of
glycolysis/gluconeogenesis pathway [[181]46] identified in F.
filiformis and the putative gene expression level on different tissue
of F. filiformis. Red stars indicate the hits of differentially
expressed genes in this map. The expression level of mapped genes (EC
5.4.2.2, EC 5.3.1.9, EC 5.3.1.1, EC 4.2.1.11, EC 1.2.4.1, EC 1.1.1.1,
EC1.1.1.2) in different tissues was displayed in map. Abbreviation: F1,
dikaryotic mycelium of cultivar strain CGMCC 5.642; F2, dikaryotic
mycelium of wild strain Liu355; F3, fruiting body of wild strain
Liu355; F4, primordium of wild strain Liu355; F5, monokaryotic mycelium
of wild strain Liu355. The red and green colors indicate up-and
down-regulation; black represents no significant expression change.
Detail information of about the gene can be found in Additional file
[182]5 (Note: obtained appropriate copyright permission to use the map
from KEGG).
[183]12864_2020_7108_MOESM5_ESM.xlsx^ (60.5KB, xlsx)
Additional file 5: Table S2. The expression of genes involved in
glycolysis and gluconeogenesis pathway in F. filiformis genome.
[184]12864_2020_7108_MOESM6_ESM.xlsx^ (49.1KB, xlsx)
Additional file 6: Table S3. Predicted biosynthetic gene clusters
involved in terpene, PKS, NRPS and siderophore in F. filiformis using
antiSMASH tool.
[185]12864_2020_7108_MOESM7_ESM.xlsx^ (57KB, xlsx)
Additional file 7: Table S4. A comparative analysis of distribution of
putative genes and gene clusters (terpene and type I PKS) related to
secondary metabolitic biosynthesis in different strains of the F.
filiformis. A blast search was performed using the software blastall
v2.2.26 and thethreshold parameter: identity > 40% coverage > 40%. “1”:
implied the gene distribution in other strain and “0” means the gene
probabaly is not distribution in other strain.
[186]12864_2020_7108_MOESM8_ESM.jpg^ (1.2MB, jpg)
Additional file 8: Fig. S4. Hierarchical clustering analysis of 119
putative genes related to terpenoid biosynthesis in F. filiformis
genome. Expression ratios were plotted in a heatmap on a log2 scale.
The red and green colors indicate up- and down-regulation, black
represents no significant expression change and grey represents missing
data. The abbreviation: MK, monokaryotic mycelium; DK, Dikaryotic
mycelium; PD, primordium; FB, Fruiting body. Detail information of
about the gene annotation can be found in Additional file [187]6.
[188]12864_2020_7108_MOESM9_ESM.jpg^ (439.7KB, jpg)
Additional file 9: Fig. S5. Neighbor-Joining phylogram of putative
sesquiterpene synthases (STS) of F. filiformis were constructed based
homologous protein sequences. The number along branch represent the
bootstrap value above 50%. The gene encoding sesquiterpene synthases
with red dot were identified in this study. Detail information of the
sequences used in phylogram can be found in reference [[189]40,
[190]44]. Labeled in “Cop” from the fungus Coprinopsis cinereus; “Omp”
from fungus Omphalotus olearius and “Sh” from fungus Stereum hirsutum.
(JPG 439 kb)
Acknowledgments