Kojic acid-mediated damage responses induce mycelial regeneration in the basidiomycete Hypsizygus marmoreus
Kojic acid-mediated damage responses induce mycelial regeneration in the basidiomycete Hypsizygus marmoreus
Jinjing Zhang 0 1 2
Hui Chen 0 1 2
Mingjie Chen 0 1 2
Hong Wang 0 1 2
Qian Wang 0 1 2
Xiaoxia Song 0 1 2
Haibo Hao 0 1 2
Zhiyong Feng 0 1 2
0 the National Natural Science Foundation of China (Grant No. 31601802 and 31401932), the Youth Talent Development Plan of Shanghai Municipal Agricultural System of China (Grant No. 20160111), the Project of Science and Technology
1 National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture and Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences , FengXian District, Shanghai, China, 2 Microbial Resources and Application Laboratory , School of Food Science and Engineering, Hefei University of Technology , Hefei , China , 3 College of Life Science, Nanjing Agricultural University , XuanWu District, Nanjing , China
2 Editor: Michael Freitag, Oregon State University , UNITED STATES
Mechanical damage can induce fruiting body production in fungi. In this study, the antioxidant kojic acid (KA) was found to enhance injured mycelial regeneration and increase fruiting body production in Hypsizygus marmoreus. KA reduced the level of reactive oxygen species (ROS), which are harmful to mycelia when excessively generated by mechanical damage. Moreover, KA increased catalase and superoxide dismutase activities and glutathione and ascorbic acid contents by up-regulating antioxidant gene expression. These results suggest that KA promotes mycelial regeneration in response to damage by activating a ªstress signalº and enhances the ability of H. marmoreus to resist oxidative damage by invoking the antioxidant system. In addition, KA increased the content of extracellular ATP, which serves as a ªstress signalº in response to injury, and modulated ROS signaling, decreasing NADPH oxidase gene expression and ROS levels in the mycelial-regeneration stage. KA treatment also up-regulated the MAPK, Ca2+ and oxylipin pathways, suggesting their involvement in the damage response. Furthermore, laccase and cellulase activities were stimulated by KA at different developmental stages. These results demonstrate that KA regulates gene expression and activates pathways for mycelial wound healing, regeneration of damaged mycelia and reproductive structure formation in the basidiomycete H. marmoreus.
Fruiting body production is induced in maturing mycelia upon exposure to adverse
environmental conditions, such as mechanical damage, which affect fungal growth, development, and
]. Although it is known that mechanical damage results in fruiting body
production in the damaged area in Schizophyllum commune [
], knowledge of how other
Commission of Shanghai Municipality
(No.13391912202) and the Shanghai Municipal
Agricultural Commission Development Program of
China (Grant No. 20130510). Additional funding
was provided by the earmarked fund for Shanghai
Modern edible fungi-industry Technology Research
System (201709). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
basidiomycetes respond to injury is limited. In ascomycetes, mechanical damage induces
sclerotia formation [
], and Fester and Hause [
] found that mechanical damage induces reactive
oxygen species (ROS) accumulation in the mycorrhizal fungus Glomus intraradices. In
addition, the mechanism by which injury induces the formation of conidia in the damaged area
was studied recently in Trichoderma atroviride [5±7], and HernaÂndez-Oñate et al. [
suggested that signaling molecules and pathways involved in the injury response of this fungus,
including injury, healing, and regeneration signals, share common features with injury
responses in plants and animals.
Indeed, animals and plants have developed mechanisms to rapidly and reliably respond to
mechanical damage [
]. Similarly, in fungi, extracellular ATP (eATP) serves as an ªinjury
signalº that triggers asexual development as an injury response [
]. ROS production also plays an
important role in inducing healing and regeneration in the damaged area [9±10], and there are
several important signaling pathways involved in the injury response, including ROS, MAPK,
and calcium signaling [
]. However, over-production of ROS also affects cellular components
through the oxidation of proteins, DNA, lipids, and other biomolecules, which might induce
cell damage or death [
]. To achieve normal growth, organisms have developed mechanisms
for regulating ROS levels [
]. For example, fungi can regulate their development by
maintaining ROS at a proper level [
], with important roles for antioxidant enzymes such as catalase
(CAT) and superoxide dismutase (SOD) in scavenging these harmful molecules [14±15].
Kojic acid [5-hydroxy-2-(hydroxymethy1)-y-pyrone] (KA), a fungal secondary metabolite,
is produced by many species of Aspergillus and Penicillium [
]. KA is now used in a variety of
applications, including as an antibiotic [
], an additive to prevent the browning of food
materials, and an antioxidant [
]. Recent studies have focused on its antibiotic activity, which may
help an organism resist oxidative stress via ROS scavenging [
]. In fungi, KA was shown to
increase cellular resistance to oxidative stress and to reduce damage [
]. In Aspergillus
parasiticus and Aspergillus flavus, increases in KA levels regulate mycelial growth and conidial
development by mediating the oxidative state [
]. KA can also induce repair of damaged tissues
In Hypsizygus marmoreus, fruiting body initiation in sawdust medium requires mechanical
damage induced by scraping mature mycelia, though the mechanism regulating this injury
response remains unknown. In this study, we found that adding the antioxidant KA to
scraping-damaged H. marmoreus mycelia induced mycelial healing and regeneration and
stimulated primordium initiation. Furthermore, KA regulated ªinjury signalsº, such as eATP and
ROS, and increased antioxidant activity. KA also up-regulated expression of genes involved in
certain signaling pathways associated with the mechanical damage response.
Materials and methods
Strain and culture conditions
H. marmoreus samples were obtained from the China General Microbiological Culture
Collection Center (Beijing) (no. CGMCC5.01974). After culturing on PDA (potato dextrose agar;
BD company, USA) medium for two weeks, the strain was transferred to PDB (PD broth; BD
company, USA) and cultured for another week. The liquid culture was inoculated onto
sawdust medium (sterilized at 121ÊC and 0.1 MPa for 2.5 h) in a 1100-mL bottle and cultured in
the dark at 25ÊC. After growth for 80 d, the surface mycelia were mechanically damaged using
a scraping machine, and the damaged mycelia were supplied with 15 mL tap water (CK) or 10
mM KA. The bottles were placed under reproduction conditions reported previously [
the 22nd day, when the fruiting bodies had matured, the production and number of fruiting
bodies were calculated.
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KA detection assays
Samples at the mycelial regeneration stage (H-M), pigmentation stage (H-V), and primordium
stage (H-P) were collected from the CK and KA groups, and extracellular KA concentrations
were assessed. Mycelia cultured on sawdust medium for 20, 60 and 80 d were also collected for
extracellular KA concentration determination. KA levels were evaluated using the colorimetric
]. KA forms a chelated compound with ferric ions, subsequently generating a red
color, as measured at 495 nm. The substrates from different developmental stages were
collected, 25 g of which was prepared in 50 mL of ddH2O in 250-mL flasks. The mixture was
shaken at 200 rpm for 30 min at 25ÊC followed by filtration and centrifugation at 6000×g for
20 min. A 1-mL aliquot of the liquid supernatant was mixed with 4 mL of ddH2O. The reaction
for KA detection contained 2 mL diluted liquid supernatant, 3 mL 1% (w/v) aqueous FeCl3
and 5 mL ddH2O.
RNA-Seq sample preparation, sequencing and analysis
After scraping mycelia, the damaged mycelia were treated with 15 mL 10 mM KA or 15 mL
tap water as a control. After approximately 2 days of regeneration (H-M), regenerated mycelia
were collected with the sawdust medium. On approximately the 6th day, the white mycelium
turned brown (H-V); the primordium appeared on approximately the 10th day (H-P). The
brown mycelia with little sawdust medium and primordia were then collected. To obtain an
overview of the effects of KA on gene expression profiles of H. marmoreus during the three
developmental stages, cDNA samples were prepared from the H-M, H-V and H-P stages of
the CK and KA groups and frozen at -80ÊC for RNA extraction.
Total RNA was extracted with TRIzol (Takara), reverse-transcribed to cDNA, and
sequenced as described previously [
]. Two independent repetitions for each sample were
sequenced separately using the Illumina HiSeqTM 2000 sequencing platform. All data were
analyzed as described previously [
]. Reads of 2×100 bp were obtained, and clean reads were
used to perform RNA de novo assembly with Trinity [
]. Genes were predicted and annotated
using local BLASTX programs against the National Center for Biotechnology Information
(NCBI) nr/nt, SwissProt, and STRING databases [
]. Differentially expressed genes (DEGs)
between the two samples were analyzed using RSEM [
] and edgeR [
], and the expression
level of each unigene was assessed according to fragments per kilobase of exon per million
reads mapped (FPKM) [
]. The raw data for the three developmental stages of the CK and
KA treatments have been submitted separately to NCBI under the accession number
SRA454268 and the bioproject number PRJNA339890.
Antioxidant enzyme detection assays
To examine changes in antioxidant enzyme activities, mycelia of the CK and KA groups were
collected at the three developmental stages (H-M, H-V and H-P); 1.0 g was ground in liquid
nitrogen, 9.0 mL normal saline was added, and the sample was centrifuged at 2500×g for 10
min. The supernatant (50 μL) was used to detect CAT and SOD activities. CAT activity was
detected according to the instructions of the CAT assay kit (Visible light) (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China). One unit of CAT was defined as the amount of
enzyme that decomposed 1 μmol of H2O2, as monitored at 405 nm. SOD activity was detected
as described by the Total SOD assay kit (hydroxylamine method) (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China). One unit of SOD activity was defined as the amount of
SOD that inhibited 50% of hydroxylamine oxidation per gram of tissue in 1 mL of solution, as
monitored at 550 nm.
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Laccase and cellulase activity detection
Laccase activity was detected at the three developmental stages (H-M, H-V and H-P) in
samples treated or not with KA. Samples collected from the three developmental stages were
pretreated using a method developed for Pleurotus ostreatus [
]. After treatment, the filtrate was
recovered and centrifuged at 4ÊC at 6000×g for 20 min. laccase activity was detected according
to a previously described method [
]. The change in absorbance at 420 nm was recorded for
3 min with ABTS (2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) as the substrate.
One unit activity was defined as the amount of enzyme that oxidized 1 μmol of ABTS/min.
The same samples were also used to detect extracellular cellulase activity by the 3,
5-dinitrosalicylic acid method, which assesses the reducing sugar concentration as a result of by
cellulase catalysis. The liquid supernatant was used for detecting cellulase activity, as described by
the cellulase assay kit manual (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)
and monitored at 550 nm. One unit of cellulase activity was defined as the amount of gram of
mycelial protein that produced 1 μg of glucose per minute.
ROS detection assays
To evaluate changes in ROS, injured mycelia of the CK and KA groups were collected at 15,
30, and 60 min after damage and immediately frozen at -80ÊC. The samples were ground with
liquid nitrogen. Inhibition of superoxide anion (O2-) activity was performed as described by
the Inhibition and produced O2- assay kit (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China). In this reaction system, the amount of O2- inhibited by 1 g of mycelial protein at
37ÊC for 40 min was related to the amount of O2- inhibited by 1 mg of Vc, as monitored at 550
nm, and this value was consider as one unit of O2--inhibiting activity. Hydrogen peroxide
(H2O2) detection was performed as described by the H2O2 assay kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China), as monitored at 405 nm; hydroxy free radical (-OH)
detection was performed as described by the -OH assay kit (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China), as monitored at 550 nm. The O2--inhibiting activity and H2O2 and
-OH concentrations were calculated using the mycelial protein concentration.
Quantitation of reduced glutathione and ascorbic acid
Mycelia of the CK and KA groups the three stages (H-M, H-V and H-P) of collected at the
three developmental stages (H-M, H-V and H-P) were also to detect reduced glutathione
(GSH) and ascorbic acid (AsA). The mycelia were ground in liquid nitrogen using a
mortar and pestle, suspended in 6% (w/v) m-phosphoric acid containing 1 mM
ethylenediaminetetraacetic acid (EDTA) and centrifuged at 12,000×g for 10 min. Total GSH was
detected using a described method [
]. GSH contains sulfhydryls (±SH), which oxidize
DTNB (5', 5'-dithiobis-20-nitrobenzoic acid) to NTB (50-dithiobis-20-nitrobenoic acid),
generating a yellow color in an aqueous solution. The GSH level was detected at 412 nm
using a spectrophotometer.
In addition, crushed CK and KA mycelia were homogenized in 5% (w/v) m-phosphoric
acid on ice. The homogenate was centrifuged at 12,000×g for 15 min. Total AsA was
determined by a described method [
] based on the reduction of Fe3+ to Fe2+ with ascorbic acid
in acid solution followed by the formation of a red chelate between Fe2+ and 2,20-bipyridyl.
Total AsA was determined in a reaction mixture consisting of 0.2 mL supernatant, 0.5 mL
150 mM potassium phosphate buffer (pH 7.4) containing 5 mM EDTA, and 0.1 mL 10 mM
dithiothreitol (DTT) to reduce DHA to AsA. AsA levels were detected at 525 nm using a
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Extracellular ATP (eATP) detection assays
To assess eATP changes, CK and KA injured mycelia with sawdust medium were collected at
15, 30, and 60 min after damage, and the samples were weighed to exactly 1.0 g. The 1.0-g
substrate mixtures were added to 9.0 mL boiling water in a 50-mL centrifuge tube and boiled for
10 min. The mixture was then filtered, and the filtered solution was centrifuged at 3000×g for
10 min. The liquid supernatant was used for eATP content detection, as described in the ATP
assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and monitored at 636
Total protein detection
The mycelial protein concentration used for calculations was detected at 595 nm as described
by the Total protein quantitative assay kit (Coomassie Brilliant Blue) (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China). The standard protein concentration used in this assay
kit was 0.563 g/L, and Coomassie brilliant blue was employed.
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) validation
Approximately 2 μg total RNA from the CK and KA groups at the three stages (H-M, H-V and
H-P) was reverse-transcribed by M-MLV reverse transcriptase (Takara) using oligo (dT) as
the primer. Genes of interest were subjected to qRT-PCR analysis. The primers and accession
numbers of these genes and the internal reference gene (18S ribosomal RNA) are listed in S1
Table. Experiments were carried out using a Realplex2 System (Eppendorf, Germany), and the
amplification conditions were as follows [
]: 95ÊC for 10 min, 40 cycles of 95ÊC for 30 s, 55ÊC
for 30 s, and 72ÊC for 30 s. Three independent biological replicates were performed for each
gene. Relative gene expression was analyzed using the 2-ΔΔCt method [
Data presentation and statistical analysis
Values are shown as the mean ± SD of three independent experiments with three replicates
each. Differences among treatments were analyzed by one-way analysis of variance (ANOVA)
combined with Duncan's multiple range test at a probability of P < 0.05.
Effects of KA on mycelial regeneration and fruiting body production in H.
Before the reproductive growth of H. marmoreus, external mycelia were machine scraped, and
15 mL of CK or 10 mM KA was added. After the mechanical damage stimulus, the mycelia
were transferred to a reproductive culture room. In this study, we found that adding 10 mM
KA induces damaged mycelia to heal and regenerate quickly (Fig 1A), and mycelial
pigmentation and primordium formation were also stimulated when compared to CK, which was only
treated with tap water (Fig 1B and 1C). Brown mycelia in the CK group were observed when
the mycelia in the KA group were completely dark brown (Fig 1B). When primordia were
approximately 0.5 cm long in the KA group, those in the CK group were barely visible.
Moreover, fruiting bodies matured much earlier in the KA group than in the CK group (Fig 1C).
KA treatment also significantly increased the production and number of fruiting bodies in
H. marmoreus (Fig 1D). However, no significant differences were found when KA was added
at any other developmental stage (data not shown).
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Fig 1. Effects of kojic acid on fruiting body development in H. marmoreus. A: Mycelial regeneration (H-M); B: mycelial
pigmentation (H-V); C: primordium (H-P); D: the production and number of fruiting bodies; E: the content of kojic acid (KA) at
different developmental stages; F: the content of KA during the developmental stages of H. marmoreus. CK: control group;
FBN: fruiting body number, FBP: fruiting body production, 20±80 d: the time for mycelium growth in sawdust medium. The
data are presented as the means ± SD of three independent experiments. Bars with different letters or asterisk denote a
statistically significant difference compared with the control according to multiple comparisons (P < 0.05).
KA levels in the developmental stages of H. marmoreus
KA is a major secondary metabolite produced by fungi [
]. We detected KA in the substrate
medium of H. marmoreus and found significant differences in the concentration, along with
mycelial vegetative growth and fruiting body development (Fig 1F). In addition, KA
production was significantly increased at the three developmental stages (H-M, H-V and H-P) upon
KA addition (Fig 1E). These results were in accordance with reports showing that KA
production is triggered by KA supplementation [34±35].
KA alters the transcriptome of H. marmoreus
To detect the role of KA in mycelial regeneration and fruiting body production in H.
marmoreus, transcript profiling was performed at the stages of mycelial regeneration (H-M), mycelial
pigmentation (H-V) and primordium (H-P) in the CK and KA groups. In total, 24,529
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Fig 2. Number of differentially expressed unigenes in each comparison. KA_M_vs_CK_M: the number
of up- and down-expressed unigenes in the comparison KA_M to CK_M; KA_V_vs_CK_V: the number of
upand down-expressed unigenes in the comparison KA_V to CK_V; KA_P_vs_CK_P: the number of up- and
down-expressed unigenes in the comparison KA_P to CK_P; KA_M_vs_KA_V: the number of up- and
downexpressed unigenes in the comparison KA_M to KA_V; KA_P_vs_KA_V: the number of up- and
downexpressed unigenes in the comparison KA_P to KA_V; CK_M_vs_CK_V: the number of up- and
downexpressed unigenes in the comparison CK_M to CK_V; CK_V_vs_CK_P: the number of up- and
downexpressed unigenes in the comparison CK_V to CK_P.
unigenes were detected, as listed in S1 Dataset. Gene Ontology (GO) assignments were used to
classify the functions of the annotated unigenes related to other fungi. Among the annotated
unigenes, 20,659 were categorized into 41 functional groups (S1 Fig). Moreover, 5246
identified unigenes were assigned to 24 functional classes in clusters of orthologous groups of
proteins (COG) analysis (S2 Fig). KA altered the number of DEGs and the ratio of up-regulated to
down-regulated unigenes (Fig 2). Moreover, during the transitions from H-M to H-V and
from H-V to H-P, the number of up-regulated unigenes in KA treatment exceeded that of
down-regulated unigenes. In the CK group, fewer up-regulated unigenes than down-regulated
unigenes were observed in both transition phases. Although the numbers of up-regulated
unigenes were fewer in the KA-M transition compared with the CK-M transition and in the
KA-V transition compared with the CK-V transition, there were more up-regulated unigenes
in the KA-P transition than in the CK-P transition. Our findings suggest a predominantly
positive regulatory role for KA in H. marmoreus primordium initiation.
We analyzed the metabolic and regulatory pathways related to the identified DEGs, and
heatmap analysis was performed based on the total FPKM values of all the DEGs in each
pathway (Fig 3 and S2 Dataset). Most of these pathways, including the MAPK signaling pathway,
glycerolipid metabolism, glycerophospholipid metabolism, peroxisomes, ascorbate and
aldarate metabolism, pyruvate metabolism, glutathione metabolism, nitrogen metabolism, starch
and sucrose metabolism, and the cell cycle, were up-regulated in the KA group. In contrast,
pathways of fatty acid elongation, lysine biosynthesis, fatty acid biosynthesis, and protein
export were up-regulated in the CK group.
The DEGs were assigned to 10 groups according to their transcript levels in the CK and KA
samples, and their kinetics over the three stages were analyzed (Fig 4 and S3 Dataset). The
majority of genes were classified into subclusters 4 and 5, with opposite transcript patterns. In
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Fig 3. KEGG annotation of differentially expressed genes (DEGs). A heatmap showing the annotated pathways of
DEGs in the six samples of H. marmoreus. Different colors represent different expression levels. Green represents
downregulated expression and red up-regulated expression. Each row represents a different pathway. The heatmap was
constructed based on the log10 values of the FPKM of all DEGs related to this pathway in the H-M, H-V and H-P
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Fig 4. Cluster analysis of the gene expression profiles in the three stages after kojic acid treatment. Cluster analysis was performed on 838 genes
detected in the mycelial regeneration, mycelial pigmentation, and primordium stages. The log2 value of the FPKM for each gene was used for the
hierarchical clustering analysis. These genes were classified into 10 regulatory patterns.
subclusters 1, 2, 3, 4, 5, and 10, the pattern of the transcript levels in the three developmental
stages was consistent. However, in subcluster 7, the transcript levels of genes at H-M in the CK
group were higher than those in the KA group, including DNA-binding protein HU,
ribosomal subunit interface protein, and DNA-directed RNA polymerase subunit alpha. In
subcluster 6, the transcript levels of genes at H-M in the KA group were higher than those in the
CK group, including laccase, NAD dependent epimerase/dehydratase family protein,
oxidoreductase, short chain dehydrogenase/reductase family, and hydrophobin. In subcluster 8, KA
altered gene transcript patterns, with the highest transcript level at H-V in the KA group and
at H-P in the CK group.
The effects of KA on gene expression at different developmental stages were analyzed by
co-expression analysis [
]. Heatmap analysis was performed based on the FPKM values of 27
DEGs involved in signal transduction, energy metabolism, cell cycle, and secondary
metabolism (Fig 5). The functional annotations for these unigenes are listed in S2 Table. As shown in
Fig 5, genes involved in carbon metabolism, such as unigenes encoding pyruvate kinase and
glyceraldehyde 3-phosphate dehydrogenase, were up-regulated in KA-M compared with
CK-M. Genes involved in signal transduction, encoding NADPH oxidase regulator NoxR,
CAMK/CAMKL/GIN4 protein kinase, CMGC/MAPK protein kinase, and alpha-ketoglutarate
catabolism dioxygenase, were up-regulated in the KA groups compared with the CK groups,
and genes encoding antioxidant enzymes, including catalase and superoxide dismutase, were
also up-regulated by KA treatment. In accordance with the gene expression results, the
activities of CAT and SOD were significantly higher in the KA group than in the CK group at the
three developmental stages, except that CAT activity showed no significant change at the H-P
stage (Table 1). The contents of the antioxidants GSH and AsA also increased significantly due
to KA treatment (Table 1). In addition, genes involved in KA metabolism such as Zn(2)-Cys
(6) binuclear cluster domain-containing protein (KA1) and C2H2-type Zn-finger protein
(KA2) were up-regulated by KA.
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Fig 5. DEGs between four developmental stages in H. marmoreus. Each column represents an experimental sample (e.g.,
CK-M, CK-V, CK-P, KA-M, KA-V and KA-P) and each row a gene. Expression differences are shown in different colors. Red
indicates high expression and green low expression.
The effects of KA on laccase gene expression were analyzed by co-expression analysis [
In total, 13 laccase genes were found in the transcriptome of H. marmoreus. In the H-M
transition, no significant differences in expression of most laccase genes due to KA treatment were
observed. However, in the H-P transition, expression of most laccase genes was enhanced in
SOD: superoxide dismutase; CAT: catalase; GSH: glutathione; AsA: ascorbic acid; H-M: mycelial regeneration; H-V: mycelial pigmentation; H-P:
primordium. KA: kojic acid. Data are presented as the means ± SD of three independent experiments. Values with different letters denote a statistically
significant difference compared with the control according to multiple comparisons (P < 0.05).
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a: The ratio of laccase genes at the mycelial knot stage treated with tap water (CK-M) or kojic acid (KA-M);
b: the ratio of laccase genes at the mycelial pigmentation stage treated with tap water (CK-V) or kojic acid
c: the ratio of laccase genes at the primordium stage treated with tap water (CK-P) or kojic acid (KA-P).
the KA group compared to that in the CK group (Table 2). Extracellular laccase activity was
also significantly reduced at H-M and H-V but increased by KA at H-P (Fig 6A), which was in
accord with the gene expression results. At the same time, we found significantly higher
cellulase activity in the KA group than in the CK group at the H-V and H-P transitions, with no
significant changes in the H-M transition (Fig 6B).
Signaling pathways involved in mechanical damage
In this transcriptome database, expression of certain genes involved in several important
signaling pathways, including Ca2+ signaling (S3 Fig), ROS signaling, MAPK signaling (S4 Fig),
and oxylipin signaling, was detected by qRT-PCR (Figs 3 and 7). In fungi, calcium can serve as
an important second messenger during growth, development, and cell differentiation. In this
study, genes encoding a calcium channel, a CAMK protein kinase, and a calcium/proton
exchanger, which are considered key components in calcium signaling in response to different
types of stress, were up-regulated in the KA groups (Fig 7).
Genes encoding CMGC/MAPK protein kinase, protein kinase C, CDC42 rho
GTPaseactivating protein, and Ras protein were also up-regulated by KA (Fig 7). MAPK signaling
not only regulates fungal growth and development but is also involved in signal
transduction induced by oxidative stress. In fungi, eATP serves as a ªstress signal" to activate the
MAPK pathway and induce the formation of asexual reproduction structures [
]. We found
that the content of eATP was significantly higher in scraped mycelia than in unscraped
mycelia, and eATP levels were significantly enhanced by KA treatment after scraping (Fig
ROS is another important ªstress signalº in the mechanical damage response, and ROS
production mainly depends on plasma membrane-bound NADPH oxidase (Nox) activity
[9±10]. Two genes, encoding an NADPH oxidase regulator NoxR and NADPH oxidase
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Fig 6. Effects of KA on laccase and cellulase activities at the three developmental stages of H.
marmoreus. After being scraped, damaged mycelia were treated with 15 mL of tap water (CK) or 10 mM KA.
Laccase and cellulase activities were detected at the H-M, H-V and H-P stages. The data are presented as
the means ± SD of three independent experiments. Bars with different letters denote a statistically significant
difference compared with the control according to multiple comparisons (P < 0.05).
NoxB, were regulated by KA treatment. We also detected -OH and O2- scavenging activity
(Fig 8B and 8C) and the content of H2O2 (Fig 8D). After scraping mycelia, KA significantly
reduced the content of H2O2, and scavenging of -OH and O2- was significantly promoted.
Genes encoding alpha-ketoglutarate catabolism dioxygenase, phospholipase B and
phospholipase D are all involved in the oxylipin biosynthetic pathway (Fig 7). Lipid metabolism has
been proven to play an important role in the response to mechanical damage in plants,
animals, and fungi, and we found that genes involved in oxylipin signaling were also
up-regulated by KA.
H. marmoreus is a very important mushroom species, primarily due to its medicinal and
organoleptic properties. Stress might be the most important effector for fruiting body
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Fig 7. qRT-PCR analysis of gene expression compared to RNA-Seq data for H. marmoreus. The Y-axis
represents the relative expression level in the three stages for the control and KA groups. The unit for the RNA-Seq
data is FPKM value. CK-M, CK-V and CK-P: control groups in the three developmental stages of mycelial knot,
mycelial pigmentation and primordium, respectively; KA-M, KA-V and KA-P: kojic acid groups in the three
developmental stages of mycelial knot, mycelial pigmentation and primordium, respectively. ras: Ras protein; pkc:
protein kinase C; cdc: CDC42 rho GTPase-activating protein; mapk: MAPK protein kinase; age: RhoA GTPase
effector DIA/Diaphanous; kcd: alpha-ketoglutarate catabolism dioxygenase; nrd: nitrate reductase; gpd:
glyceraldehyde-3-phosphate dehydrogenase; phod: phospholipase D; phob: phospholipase B; cac: calcium channel;
camk: CAMK protein kinase; cpe: calcium/proton exchanger; ka1: Zn(2)-Cys(6) binuclear cluster domain-containing
protein; ka2: C2H2-type Zn-finger protein; pyk: pyruvate kinase; cat: catalase; sod: superoxide dismutase; noxR:
NADPH oxidase regulator; noxB: NADPH oxidase B. The error bars represent the standard deviation of three
initiation in H. marmoreus [
], and in the H. marmoreus production process, mechanical
damage by scraping of mature mycelia is needed to induce primordium initiation. However,
over-injury of mycelia might delay fruiting body development in this species.
In this study, adding KA to scraping-damaged mycelia enhanced the mycelial regeneration
ability (Fig 1A) and significantly increased fruiting body production (Fig 1D). KA is an
antioxidant produced as a major secondary metabolite by a range of microorganisms [
]. We found
that genes encoding SOD and CAT were up-regulated to different levels by KA; the activities
of CAT and SOD were increased and the contents of GSH and AsA were significantly higher
in the KA than in the CK groups (Table 1). As shown in Fig 3, antioxidant pathways, such as
peroxisomes, ascorbate and aldarate metabolism, and glutathione metabolism, were
up-regulated in the KA-M and KA-V groups. Furthermore, the content of KA increased significantly
after adding KA (Fig 1E), which is in accordance with the results of a previous study [34±35].
Chang et al. [
] found that production of the secondary metabolite KA promoted the
response to oxidative stress in fungi. KA was also able to increase cell resistance to oxidative
stress and induce repair of damaged tissues [
]. These results suggest that KA might
increase antioxidative capacity of injured mycelia and enhance mycelial healing and
regeneration in H. marmoreus.
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Fig 8. Effects of KA on ATP and ROS contents at the three developmental stages in H. marmoreus. Mycelia were scraped (S)
or unscraped (N), and 15 mL of tap water (W) or KA was added. a: The content of ATP; b: the OH- scavenging activity; c: the
O2scavenging activity; d: the content of H2O2. The samples were collected at 5 min, 30 min, and 60 min after the different treatments.
5-W-S, 30-W-S, and 60-W-S indicate water added to scraped mycelia at 5, 30, and 60 min; 5-KA-S, 30-KA-S, and 60-KA-S indicate
KA added to scraped mycelia at 5, 30, and 60 min; 5-W-N, 30-W-N, and 60-W-N indicate water added to scraped unscraped mycelia
at 5, 30, and 60 min; 5-KA-N, 30-KA-N, and 60-KA-N indicate KA added to unscraped mycelia at 5, 30, and 60 min. The data are
presented as the means ± SD of three independent experiments. Bars with different letters denote a statistically significant difference
compared with the control according to multiple comparisons (P < 0.05).
Oxidative stress caused by mechanical damage may be an important factor inducing
fruiting body initiation in some fungi such as S. commune [
], S. rolfsii [
] and G. intraradices [
Some reports have demonstrated that damaged cells or tissue can release their constituents or
fragments into the extracellular milieu during cellular stress or damage [
6, 7, 9, 36
]. eATP has
been proven to be an important early damage signal in plants, animals and fungi [
7, 9, 36
Medina-Castellanos et al.  showed that exposure of T. atroviride mycelia to eATP induces
asexual reproduction structure formation. In our study, we found significantly increased eATP
levels in scraped mycelia compared to unscraped mycelia, and adding KA to damaged mycelia
further significantly increased the level of eATP (Fig 8A). These results suggest that KA might
trigger the damaged mycelium to release more eATP, which is required for the injury response
and for inducing asexual developmental in H. marmoreus.
ROS, which include H2O2, O2-, -OH and singlet oxygen (O2) [37±38], play important roles
in cell proliferation, signal transduction, cell differentiation, and injury response [
However, ROS also affect cellular components via oxidation of proteins, DNA, lipids, and other
molecules to induce cell damage or death [
]. In this study, the ROS signaling pathway was
down-regulated in the KA groups relative to CK groups. noxB was down-regulated by KA, and
the concentrations of O2-, -OH, and H2O2 in damaged mycelia were significantly higher in the
CK groups than in the KA groups, with significant increases in undamaged KA group mycelia
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compared to CK group mycelia (Fig 8). These results suggest that KA reduced oxidative harm
by decreasing ROS to stimulate damaged mycelial healing and regeneration in H. marmoreus.
The MAPK and Ca2+ signaling pathways also participate in the injury response in fungi [
In our study, genes encoding MAPK protein kinase, protein kinase C (PKC), CDC42 rho
GTPase-activating protein, Ras protein involved in the MAPK signaling pathway, a calcium
channel, CAMK protein kinase, and a calcium/proton exchanger involved in calcium signaling
were up-regulated by KA. In filamentous fungi, the MAPK pathway is involved in several
processes, such as asexual and sexual reproduction, general stress response, cell fusion, and spore
germination [40±41]. In Magnaporthe grisea, defective sexual and asexual development results
from MAP kinase mutations [
]. Juvvadi et al. [
] determined that PKC is important for the
correct localization of the dolipore septum at the septal pore. In basidiomycetes, the septal
pore cap, which is a membranous structure located at both sides of the dolipore septum, plays
a key role in plug formation after hyphal damage or stress [
]. In addition, calcium plays an
important role as a second messenger for wounding and participates in the injury response in
T. atroviride [
], and calcium signaling pathways also have important roles in primordium
initiation in H. marmoreus [
]. These results suggest that KA induces MAPK and calcium
pathways to stimulate the transition from mycelium to primordium in H. marmoreus.
Moreover, lipid metabolism has an important function in the injury responses of plants,
animals, and fungi [7, 45±46]. Hernandez-Onate et al. [
] proved that fungi produce oxylipins
in response to mechanical damage through a set of enzymes shared with the biosynthetic
pathways of plants and animals. In the present study, oxylipin signaling was stimulated by KA
treatment, and transcriptional analyses showed that genes encoding phospholipase B,
phospholipase D, and α-dioxygenase were up-regulated by KA in H. marmoreus (Fig 7). Regulation
of the oxylipid pathway after mechanical damage is particularly important for plants to initiate
defense reactions in a timely fashion [
], and activation of oxylipid signaling might also
induce immune responses in plants. In T. atroviride, genes associated with oxylipin
biosynthesis are up-regulated during the injury response, which is similar to activation of the fungal
immune response [
]. These results suggest that adding KA to damaged H. marmoreus mycelia
might rapidly activate the defense response after mechanical damage.
In addition, genes involved in carbon metabolism, such as pyruvate kinase and
glyceraldehyde 3-phosphate dehydrogenase, were activated by KA (Fig 7). Laccase activity was
significantly increased at the H-P stage by KA, and cellulase activity was significantly increased at the
H-V and H-P stages by KA (Fig 6). Laccase and cellulase play important roles in lignin and
cellulose degradation in fungi [48±49] and also in mycelial maturation and fruit-body growth in
H. marmoreus [49±51]. These results suggest that KA might activate primary metabolism to
increase fruiting body production in H. marmoreus.
Mechanical damage can stimulate fruiting body initiation in fungi. However, the molecular
mechanism invoked in response to mechanical damage is less well understood. In this work,
we found that adding KA to damaged mycelia induced mycelial regeneration. A schematic
model by which KA regulates the fruiting body of H. marmoreus is shown in Fig 9. It was
found that adding KA increased eATP levels to stimulate damaged mycelia to quickly respond
to the injury, acting as a ªstress signalº. The mycelial regeneration capacity was enhanced by
increasing SOD and CAT activities and the contents of GSH and AsA. KA also increased the
content of KA and resistance to oxidative injury in H. marmoreus. Genes involved in MAPK
(mapk, cdc42, ras, pkc), Ca2+ (camk, cac and cpe) and oxylipin (phob, phod and kcd) signaling
were activated by KA, and these pathways might play important roles in fruiting body
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Fig 9. Schematic representation of the injury response in H. marmoreus after kojic acid treatment. Adding KA causes mycelia to respond to injury
faster by increasing the content of eATP (Fig 8). The mycelial regeneration capacity is enhanced by increasing antioxidative enzyme activity (SOD and
CAT) and antioxidants (GSH and AsA) (Table 1) as well as the KA content (Fig 1). KA also activates signaling pathways including the MAPK pathway
(mapk), Ca2+ signaling pathway (cam, cac, cpe), and oxylipin signaling pathway (phoa, phob, phoc and phod) (Figs 5 and 7). In addition, pyk, gpd, lacc,
and cel were up-regulated by KA (Figs 6 and 7). The solid line indicates that the results were confirmed by transcriptomic analysis and experimental data;
the dotted line indicates that the results were only confirmed by transcriptomic analysis.
initiation in H. marmoreus. In addition, expression of pyk, gpd, cel and lacc was affected by KA
treatment at different developmental stages, suggesting that KA regulates primary metabolism
for fruiting body production.
S1 Fig. Gene Ontology classification of the H. marmoreus transcriptome.
S2 Fig. COG functional categories of H. marmoreus unigenes.
S3 Fig. Calcium signaling pathway identified by KEGG annotation (http://www.kegg.jp/
kegg-bin/show_pathway?ko04020). The red boxes indicate that the genes identified in the
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transcriptome of H. marmoreus are annotated in the metabolic pathways.
S4 Fig. MAPK signaling pathway identified by KEGG annotation (http://www.kegg.jp/
kegg-bin/show_pathway?ko04011). The red boxes indicate that the genes identified in the
transcriptome of H. marmoreus are annotated in the metabolic pathways.
S1 Dataset. All unigenes from the H. marmoreus transcrptome.
S2 Dataset. The KEGG annotations in the six samples of H. marmoreus.
S3 Dataset. The DEGs information in the 10 subclusters of H. marmoreus.
S1 Table. Primers used for quantitative real-time PCR.
S2 Table. The functional annotation for 27 unigenes from six DGE libraries.
We thank Zhao Jing for giving us technological guidance on cultivating mushroom.
Data curation: Haibo Hao.
Formal analysis: Jinjing Zhang, Mingjie Chen.
Funding acquisition: Jinjing Zhang, Hui Chen.
Investigation: Hong Wang, Zhiyong Feng.
Project administration: Hui Chen, Zhiyong Feng.
Resources: Jinjing Zhang, Qian Wang.
Software: Xiaoxia Song.
Writing ± original draft: Jinjing Zhang, Hui Chen.
Writing ± review & editing: Jinjing Zhang.
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