bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1
2
3
4 Transcriptional profiling elucidates the essential role of
5 glycogen synthase kinase 3 to fruiting body formation in
7
8
9 Kathy PoLam Chan1¶, JinHui Chang1¶, YiChun Xie1, Man Kit Cheung1,
10 Ka Lee Ma1, Hoi Shan Kwan1*
11
12
13 1 School of Life Sciences, The Chinese University of Hong Kong, Shatin, New
14 Territories, Hong Kong
15
16
17 *Corresponding author:
18 E-mail: [email protected]
19
20 ¶These authors contributed equally to this work.
21
1 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
22 Abstract
23 The functions of glycogen synthase kinase 3 (GSK3) have been well-studied in animal, plant
24 and yeast. However, information on its roles in basidiomycetous fungi is still limited. In this
25 study, we used the model mushroom Coprinopsis cinerea to study the characteristics of
26 GSK3 in fruiting body development. Application of a GSK3 inhibitor Lithium chloride (LiCl)
27 induced enhanced mycelial growth and inhibited fruiting body formation in C. cinerea. RNA-
28 Seq of LiCl-treated C. cinerea resulted in a total of 14128 unigenes. There were 1210
29 differentially expressed genes (DEGs) between the LiCl-treated samples and control samples
30 in the mycelium stage (first time point), whereas 1402 DEGs were detected at the stage when
31 the control samples formed hyphal knots and the treatment samples were still in mycelium
32 (second time point). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway
33 enrichment analysis of the DEGs revealed significant associations between the enhanced
34 mycelium growth in LiCl treated C. cinerea and metabolism pathways such as “biosynthesis
35 of secondary metabolite” and “biosynthesis of antibiotics”. In addition, DEGs involved in
36 cellular process pathways, including “cell cycle-yeast” and “meiosis-yeast”, were identified
37 in C. cinerea fruiting body formation suppressed by LiCl under favorable environmental
38 conditions. Our findings suggest that GSK3 activity is essential for fruiting body formation as
39 it affects the expression of fruiting body induction genes and genes in cellular processes.
40 Further functional studies of GSK3 in basidiomycetous fungi may help understand the
41 relationships between environmental signals and fruiting body development.
42
43
44
45
46
2 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
47 Introduction
48 Coprinopsis cinerea is a model organism for studying the development and growth regulation
49 in homobasidiomycetous fungi. It can complete its life cycle in only two weeks under
50 laboratory conditions. There are at least six developmental stages in the life cycle of C.
51 cinerea: mycelium, initial, stage 1 primordium, stage 2 primordium, young fruiting body, and
52 mature fruiting body with spore formation. Its gill and frequently the entire cap autolyzes to
53 have an inky black fluid at maturity [1]. Due to its rapid morphogenesis, C. cinerea is a
54 favorable model to be used in genetic studies of fruiting body development [2]. In particular,
55 strain #326 (AmutBmut pab1-1) is a homokaryotic strain that forms fruiting body without
56 mating, making it especially suitable for molecular studies of basidomycetous fungi under
57 different environmental conditions [3].
58
59 The development of C. cinerea depends on the sensing of environmental conditions,
60 including light, temperature, humidity, and nutrients [1,37]. After the mycelium reaches the
61 surface of horse dung, the habitat changes drastically, especially the day-night change of light,
62 temperature and humidity. Circadian rhythm, especially light, not only serves as a trigger for
63 adaptation to this changing environment, but also regulates the sexual development. The
64 transformation from mycelium to hyphal knot requires light, glucose depletion and lower
65 temperature [1]. Blue light is required to enter fruiting body initiation, primordial maturation
66 and karyogamy, which ensures spores to be eaten and spread by animals in the early morning
67 [1,4]. How environmental signals stimulate regulators of adaptation and reproduction has
68 attracted wide research interests. Kinases and transcription factors are among the most
69 intensively investigated [37,38].
70
3 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
71 Glycogen synthase kinase, GSK3, was initially identified as a serine/threonine protein kinase
72 of the CMGC family that phosphorylates and inactivates glycogen synthase [5]. Studies of
73 GSK3 in mammalian cells have indicated its involvement in many signaling pathways,
74 including cell proliferation and differentiation, transcription factor and microtubule dynamics.
75 As a result, GSK3 is now considered as a central regulator among signaling pathways [6].
76 Apart from in mammals, GSK3 also has a wide variety of essential cell signaling role in plant
77 and fungi. In plant, GSK3 integrates multiple hormonal signals, such as in hormone crosstalk,
78 to regulate plant stress tolerance and acts as an important component to suppress xylem in the
79 TDIF-TDR signaling pathway [7,8]. GSK3 also regulates the growth, conidiogenesis,
80 mycotoxin production, pathogenicity, and stress response mechanism in phytopathogenic
81 fungi, such as Fusarium graminearum and Magnaporthe oryzae [9,10], and is considered a
82 control regulator in cellular processes. Moreover, genes encoded as glycogen synthase kinase
83 in ascomycetous fungi, such as Saccharomyces cerevisiae and Neurospora crassa, regulate
84 protein-protein interaction, meiotic gene activation, and transcription factors of circadian
85 clock under environmental stress like nutrient, temperature and light [11,12,13]. Until now,
86 there is little knowledge on the role of GSK3 in mushroom-forming fungi. Lithium chloride
87 (LiCl), a mood stabilizer for mental illnesses treatment, has been used to study the
88 characteristics of glycogen synthase kinase in human and other animal models [14,15]. The
89 inhibition of GSK3 is the main action of LiCl from pharmacological and genetic studies [16].
90 LiCl inactivates the phosphorylation of GSK3 to its downstream targets. In this study, we
91 used high-throughput RNA sequencing (RNA-Seq) to reveal the transcriptional response of C.
92 cinerea on fruiting body formation and development with GSK3 inhibited by the addition of
93 LiCl. The transcriptional profile revealed could elucidate the role of GSK3 activity in fruiting
94 body development.
95
4 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
96 Material and methods
97 Strain and cultivation conditions
98 C. cinerea strain #326 (A43mut B43mut pab1-1), a homokaryotic fruiting strain, was grown
99 on yeast extract-malt extract-glucose (YMG) medium solidified with 1.5% (w/v) agar [17] at
100 37oC for 5-6 days until reaching the edge of plates. In the treatment experiments, a piece of
101 mycelium 5 mm diameter punch from the stock plates was inoculated on plates spread with
102 different concentrations (0.25 µM, 0.5 µM, 1 µM, 3 µM, 5 µM) of LiCl (Sigma-Aldrich)
103 after filter sterilization and covered with cellophane. Autoclaved water was added in the
104 controls. Mycelium in the treatment was incubated at 37oC in total darkness for 4 days and
105 then harvested for RNA extraction and transcriptome sequencing. Another set of RNA
106 extraction was carried out from the control plates after the formation of hyphal knots. After
107 the mycelium reached the edge of plates, the plates were incubated at 28oC with the 12/12 h
108 light/dark cycle for fruiting body development. Each treatment was in triplicate under the
109 same experiment conditions.
110
111 RNA isolation, library preparation and sequencing
112 For transcriptome sequencing, samples in two time points of C. cinerea were harvested, first
113 one in the mycelium stage were harvested after 4 days of incubation in total darkness, before
114 reaching the edge of plates, whereas second one in the hyphal knot stage were collected after
115 3-4 days of incubation under the 12/12 h light/dark cycle after the mycelium reached the edge
116 of plates. Samples collected were frozen in liquid nitrogen and stored at -80oC before RNA
117 extraction by using the RNeasy Plant Mini Kit (Qiagen). RNA was first treated with the
118 TURBO DNA-free kit according to the manufacturer’s instructions. Quality and quantity of
119 the extracted RNA were assessed on 1.5% agarose gels and an Agilent 2100 bioanalyzer with
5 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
120 the Agilent RNA 6000 Nano Kit. RNA with a RNA integrity number (RIN) greater than 8.0
121 was used for transcriptome library construction. RNA sequencing libraries were constructed
122 by using the TruSeq RNA Sample Prep Kit v2 (Illumina) according to the manufacturer’s
123 instructions. Qualified libraries were amplified on cBot to generate clusters on the flowcell
124 (TruSeq PE Cluster Kit V3–cBot–HS, Illumina). The amplified flowcell was then sequenced
125 paired-end on the HiSeq 2000 System (TruSeq SBS KIT-HS V3, Illumina). The entire
126 process was commissioned and performed by the Beijing Genomics Institute (BGI). The
127 RNA-seq row data was submitted to Gene Expression Omnibus (GEO) and its accession
128 number will be provided later.
129
130 Reads alignment and transcript assembly
131 Fastp was used to filter out low quality and unclean reads [18]. Hisat2 was used to align the
132 high quality and clear reads against the reference genome from JGI
133 (https://genome.jgi.doe.gov/programs/fungi/index.jsf) [3,19]. All transcripts were defined as
134 unigenes and annotated using the Gene Ontology (GO) database for defining their roles in
135 biological processes, molecular functions and cellular components. The unigenes were also
136 annotated using the EuKaryotic Orthologous Groups (KOG) database to define their roles in
137 metabolism, cellular processes and signaling, information storage and processing.
138
139 Differential expression analysis and gene functional annotation
140 After mapping to the reference genome, the gene expression levels of each sample were
141 calculated by Cufflinks [20]. Differentially expressed genes (DEGs) between the treatment
142 and control samples were then calculated using the Cuffdiff function in Cufflinks [21]. Only
143 transcripts with a p-value <0.05 and fold change >1 or <-1 were considered statistically
6 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
144 significant and used in GO and Kyoto Encyclopedia of Genes and Genome (KEGG)
145 enrichment analyses.
146
147 GO and KEGG enrichment analyses
148 GO and KEGG enrichment of DEGs were performed using the R package clusterProfiler [22].
149 The enrichGo function in clusterProfiler was used to identify the hypergeometric distribution
150 of DEGs based on the org.Cci.sgd.db Bioconductor annotation package [22], whereas the
151 enrichKEGG function was used to calculate the statistical enrichment of DEGs for KEGG
152 pathways. The threshold of p-value was set at 0.05 and the p.adjust method of Benjamini &
153 Hochberg (BH) was used to control the false discovery rate [39].
154
155 cDNA synthesis and qRT-PCR validation
156 Quantitative real time PCR (qRT-PCR) of selected genes from enriched KEGG pathways
157 was used to validate the RNA-Seq data. Approximately 500 ng of total RNA was used to
158 synthesize cDNA using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad) according to
159 the manufacturer’s instructions. SYBR green–based qRT-PCR was then performed using
160 SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on Applied Biosystems 7500.
161 Beta-tubulin was used as an endogenous control for normalization. Detail information of the
162 primers used is in S1 Table.
163
164 Statistical analysis
165 Data are presented as mean ± SD of three replicates for each treatment. Statistical analysis
166 was carried out using one-way analysis of variance (ANOVA) with p-values < 0.05
167 considered significant.
7 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
168
169 Result
170 Effect of LiCl on mycelium growth
171 Different concentrations of LiCl (0.25 µM, 0.5 µM, 1 µM, 3 µM and 5 µM; water in control)
172 were spread on the surface of YMG plates, on which a block of mycelium from the stock
173 plates was inoculated. After 24 hours, no difference in the colony size was observed between
174 the plates (Fig. 1). However, after 48 hours, C. cinerea mycelium treated with 1 µM and 3
175 µM LiCl grew larger colonies than that in other concentrations of LiCl and control plates. 2-3
176 days later, the mycelium treated with 0.25 µM, 0.5 µM and 5 µM LiCl grew larger colonies
177 than that of the control. Consecutively, the mycelium treated with 1 µM and 3 µM LiCl
178 reached the edge of plates after 5 days of incubation. However, other plates required 6 days
179 to reach the edge of plates. This suggests that the mycelium in 1 µM and 3 µM LiCl plates
180 had higher growth rate than that in other concentrations of LiCl and control plates.
181
182 Fig.1. Mycelium radial growth rate of C. cinerea in LiCl treated samples and control.
183 Control: treated with water; the concentration of LiCl: 0.25 µM, 0.5 µM, 1 µM, 3 µM and 5
184 µM. Vertical bars represent standard deviation of the mean (n=3).
185
186 Effect of LiCl on fruiting body initiation
187 To induce fruiting body formation, plates were transferred to an incubator with 12/12 hrs
188 light/dark cycle at 28OC. After 3-4 days of incubation, initials were observed on the control
189 plates and hyphal knots on plates with 0.25 µM LiCl. By contrast, plates with other
190 concentrations of LiCl were still in the mycelium stage. After 5-6 days, young fruiting bodies
191 were observed on the control plates and primary primordium on the 0.25 µM LiCl plates (Fig.
8 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
192 2). Initials and hyphal knots were observed on the 0.5 µM and 1 µM LiCl plates, respectively.
193 The 3 µM and 5 µM LiCl plates were still in the mycelium stage. Moreover, the mycelial
194 growth in the 5 µM LiCl plates was slightly faster than that in the control plates and the
195 mycelium stopped growth by reaching to the edge of plates. After one week of incubation,
196 the fruiting bodies in the control plates and the plates treated with 0.25 µM, 0.5 µM and 1 µM
197 LiCl autolyzed consecutively whereas the 3 µM and 5 µM LiCl plates remained in the
198 mycelium stage with no fruiting body formation.
199
200 Fig 2. Effect of different concentrations of LiCl in C. cinerea fruiting body development.
201 Primordium was observed on the YMG plates treated with 0.25 µM LiCl after 5 days of
202 incubation. Initials and hyphal knots were observed on the YMG plates treated with 0.5 µM
203 and 1 µM LiCl, respectively. YMG plates treated with 3 µM and 5 µM LiCl remained in the
204 mycelium stage, and mycelium treated with 5 µM LiCl stopped reaching the edge of plates.
205 Young fruiting bodies were observed on the control YMG plates treated with water.
206
207 Sequencing of the C. cinerea transcriptome
208 To study the transcriptional response of C. cinerea to GSK3 inhibition, transcriptomes of C.
209 cinerea treated with water and 3 µM LiCl in the mycelium and hyphal knots stages were
210 sequenced on the Illumina HiSeq 2000 platform. RNA-Seq resulted in 2.6-3.8x107 reads and
211 3.9-5.8x109 bases. Subsequent quality filtering removed 0.02% of the reads that were deemed
212 unclear. About 94% of the reads were mapped to the reference genome from JGI, resulting in
213 91240 transcripts and 14128 unigenes. All unigenes were annotated to GO and KOG,
214 comprising 2150 functional GO terms and 25 KOG classes.
215
9 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
216 Transcriptional profiling of the effect of LiCl on mycelial growth
217 enhancement
218 Of the 14128 unigenes expressed in the mycelium treated with water (HM) and treated with 3
219 µM LiCl (LM1), 1210 were significantly differentially expressed (P<0.05, |log2 fold
220 change|>1), including 531 upregulated and 679 downregulated unigenes in LM1(S1 Dataset).
221 GO annotation showed that both the upregulated and downregulated DEGs had a similar
222 distribution in the molecular function (~70%), biological process (~23%) and cellular
223 component (7%) categories (S1 Fig.).
224
225 The DEGs were also annotated into 667 KOG terms, including 261 upregulated and 406
226 downregulated KOG terms. Almost half of the upregulated DEGs were annotated to
227 metabolism, 22% to cellular processes and signaling, and 14% to information storage and
228 processing. Downregulated DEGs had similar number of genes (about 33%) annotated to
229 metabolism and cellular processes and signaling, with 9% annotated to information storage
230 and processing (Fig 3).
231
232 Fig. 3 KOG annotation of DEGs. The distribution of KOG annotations of (A) upregulated
233 and (B) downregulated DEGs in HM vs LM1, HM: C. cinerea mycelium treated with water,
234 LM1: C. cinerea mycelium treated with 3 µM LiCl; and (C) upregulated and (D)
235 downregulated DEGs in HHK vs LM2, HHK: hyphal knots of the water treated samples,
236 LM2: consistent mycelium of 3 µM LiCl-treated samples.
237
238 GO functional enrichment analysis resulted in two categories, biological process and
239 molecular function, with P-value ≤0.05 (Fig 4). Most of the DEGs between HM and LM1
240 were enriched in molecular function, including “cofactor binding”, “heme binding”,
10 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
241 “tetrapyrrole binding”, and “coenzyme binding”. Besides, six DEGs were enriched in
242 biological process, including “carbohydrate metabolic process”, “carbohydrate biosynthetic
243 process”, “tetrapyrrole biosynthetic process”, “cellular carbohydrate biosynthetic process”,
244 “tetrapyrrole metabolic process”, and “porphyrin-containing compound biosynthetic process”.
245 No DEGs were significantly enriched in the cellular component category, indicating that
246 cellular component has no significant effect on the transcriptional response of C. cinerea
247 mycelium to LiCl treatment.
248
249 Fig.4. GO functional enrichment analysis. The size of the dots indicates the number of
250 DEGs involved in the pathway and the color represents the P-value. HM: C. cinerea
251 mycelium treated with water; LM1: C. cinerea mycelium treated with 3 µM LiCl; HHK:
252 hyphal knots of the water treated samples; LM2: consistent mycelium of 3 µM LiCl-treated
253 samples.
254
255 KEGG pathway enrichment analysis resulted in five pathways statistically enriched in the
256 LiCl-treated samples with P-value ≤0.05, including “biosynthesis of secondary metabolites”,
257 “biosynthesis of antibiotics”, “starch and sucrose metabolism”, “glycerophospholipid
258 metabolism”, and “tyrosine metabolism” (Fig 5). This result indicates that the enhanced
259 mycelial growth of C. cinerea treated with 3 µM LiCl was mainly associated with
260 metabolism pathways, especially those in secondary metabolism.
261
262 Fig 5. KEGG pathway enrichment analysis. The size of the dots indicates the number of
263 DEGs involved in the pathway and the color represents the P-value. HM: C. cinerea
264 mycelium treated with water; LM1: C. cinerea mycelium treated with 3 µM LiCl; HHK:
11 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
265 hyphal knots of the water treated samples; LM2: consistent mycelium of 3 µM LiCl-treated
266 samples.
267
268 Transcriptional profiling of the effect of LiCl on fruiting body
269 initiation repression
270 A total of 1402 unigenes were significantly differentially expressed between hyphal knots of
271 the water treated samples (HHK) and consistent mycelium of LiCl-treated samples (LM2)
272 (P<0.05, |log2 fold change|>1), of which 664 were upregulated and 738 were downregulated
273 in LM2 (S2 Dataset). GO annotation showed that both the upregulated and downregulated
274 DEGs had a similar distribution in the molecular function (71-75%), biological process
275 (17~22%) and cellular component (7~8%) categories (S1 Fig.).
276
277 The DEGs were annotated into 769 KOG terms, 355 of which were upregulated and 414
278 were downregulated in LM2. Forty-one percent of the upregulated DEGs belonged to
279 metabolism, followed by 25% to cellular processes and signaling, and 13% to information
280 storage and processing (Fig 3). For the downregulated DEGs, 34% were involved in cellular
281 process and signaling, 28% in metabolism and 26% in information storage and processing.
282 The number of upregulated DEGs in LM2 annotated to KOG classes were less than that in
283 LM1, however, the number of downregulated DEGs in LM2 annotated to KOG classes were
284 greater than that in LM1 (Fig 6 &7).
285
286 Fig 6. KOG classification of upregulated DEGs in the LiCl-treated samples. A: Cellular
287 processes and signaling; B: Information storage and processing; C: Metabolism. HM: C.
288 cinerea mycelium treated with water; LM1: C. cinerea mycelium treated with 3 µM LiCl;
12 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
289 HHK: hyphal knots of the water treated samples; LM2: consistent mycelium of 3 µM LiCl-
290 treated samples.
291
292 Fig 7. KOG classification of downregulated DEGs in the LiCl-treated samples. Cellular
293 processes and signaling; B: Information storage and processing; C: Metabolism; HM: C.
294 cinerea mycelium treated with water; LM1: C. cinerea mycelium treated with 3 µM LiCl;
295 HHK: hyphal knots of the water treated samples; LM2: consistent mycelium of 3 µM LiCl-
296 treated samples.
297
298 GO functional enrichment analysis resulted in 11 statistically enriched GO terms from the
299 molecular function category, including “cofactor binding”, “iron ion binding”, “heme
300 binding”, and “structural constituent of cell wall”. Five enriched GO terms were from the
301 biological process category, namely “carbohydrate metabolic process”, “respiratory chain
302 complex III assembly”, “mitochondrial respiratory chain complex III assembly”, “cell cycle
303 checkpoint”, and “negative regulation of cell cycle”. Moreover, three enriched GO terms
304 were from the cellular component category, namely “cell wall”, “external encapsulating
305 structure” and “fungal-type cell wall”. Eight GO terms from the molecular function category
306 were enriched in both time points, namely “cofactor binding”, “heme binding”, “tetrapyrrole
307 binding”, “iron ion binding”, “coenzyme binding, “Flavin adenine dinucleotide binding”,
308 “monooxygenase activity”, and “oxidoreductase activity”, indicating their continuous
309 involvement in fruiting body initiation in C. cinerea (Fig 4.).
310
311 KEGG pathway enrichment analysis of DEGs between HHK and LM2 resulted in seven
312 statistically enriched pathways in the LiCl-treated samples (Fig 5). These included “steroid
313 biosynthesis” and “glutathione metabolism” from the metabolism pathways category and
13 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
314 “Cell cycle-yeast”, “DNA replication”, “Meiosis-yeast”, “Mismatch repair”, and
315 “Homologous recombination” from the cellular process category. All DEGs involved in the
316 cellular process pathways were downregulated (S5 Table), indicating that LiCl suppressed
317 the expression of these DEGs and the inactivation of GSK3 may affect their expression in
318 fruiting body induction.
319
320 Validation of the RNA-Seq results by qRT-PCR
321 Eight genes involved in the enrichment analysis at the mycelium and hyphal knots stages
322 were selected to validate the results of transcriptional profiling. The expression patterns of
323 genes revealed by qRT-PCR were consistent with those in the RNA-Seq analysis, indicating
324 that the results of transcriptional profiling analysis are reliable (S5 Table).
325
326 Discussion
327 To reveal the involvement of GSK3 in the fruiting body development of C. cinerea, we
328 treated C. cinerea mycelium with a GSK3 inhibitor, Lithium chloride (LiCl). The result
329 showed that mycelium treated with LiCl grew faster than that of the control. KEGG
330 enrichment analysis showed that the enriched DEGs in LM1 in metabolism pathways. Two
331 enzymes were upregulated and six were downregulated in the “starch and sucrose
332 metabolism pathway” (S2 Fig.). In the glycogen synthesis process, UDP-glucose is the
333 substrate of glycogen synthase [31]. The downregulated expression of glycogen synthase
334 detected here may then allow more UDP-glucose to be used in the synthesis of beta (1,3)-D-
335 glucan, a component of the cell wall, for mycelium growth [23]. Besides, the upregulation of
336 glucoamylase and downregulation of alpha-amylase allow breakdown of glycogen into
337 glucose for mycelium growth instead of dextrin. Moreover, the upregulation of beta-
14 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
338 glucosidase breaks down beta-D-glucosides or cellobiose into glucose, thereby allowing rapid
339 cell division and accumulation of cell mass [24]. Moreover, galectin regulates cell growth
340 and extracellular cell adhesion [25]. The expression of galectin in mycelium is low and it is
341 upregulated in nutrient depletion for hyphal aggregation in previous studies [26, 27]. In this
342 study, galectin-1 (CC1G_05003) and galectin-2 (CC1G_05005) (S1Dataset) were
343 upregulated in the LiCl-treated samples, it implies that the phosphorylation of GSK3
344 suppresses the expression of galectin in mycelium under excessive nutrient.
345
346 The time required for fruiting body formation increased when the concentration of LiCl
347 increased. Upon 3 µM LiCl, C. cinerea fruiting body formation was inhibited even under
348 favorable environmental conditions. When mycelium transits to hyphal knots, it required
349 expression of several genes for the construction of new membranes. In a previous
350 transcriptome study, genes involved in the phospholipid biosynthesis process were
351 upregulated in the fruiting body initiation of C. cinerea [3]. In this study, genes of the
352 phospholipid biosynthesis process, including phosphatidylserine decarboxylase
353 (CC1G_06249, CC1G_09238, CC1G_09240, and CC1G_09237), phosphatide
354 cytidylyltransferase (CC1G_06860), inositol-3-phosphate synthase (CC1G_06001), and a
355 hypothetical protein (CC1G_09235) (S6 Table) were downregulated in LM2 treated with 3
356 µM LiCl compared with the control samples that formed hyphal knots. Most of these genes
357 participate in glycerophospholipid metabolism. Therefore, we suggest that GSK3 activity
358 affects glycerophospholipid metabolism for the transition from mycelium to hyphal knots.
359 Besides, fruiting body induction requires some important chromosome remodeling genes
360 [2,28,29]. Those genes, however, were downregulated in this study (S6 Table), indicating
361 that the activity of GSK3 might associate with the expression of chromosome remodeling
362 genes for cell division, which results in hyphal knots formation [30].
15 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
363
364 KEGG pathway enrichment analysis showed that DEGs involved in “cell cycle-yeast”,
365 “meiosis-yeast”, “DNA replication”, “mismatch repair”, and “homologous recombination” in
366 the cellular process category were downregulated in LM2 compared to the controls, showing
367 that C. cinerea treated with LiCl remained in the mycelium stage as a result of the
368 downregulation of genes involved in cell cycle and meiosis. Phosphorylation of a yeast
369 GSK3 homolog has been found to promote vegetative growth in cell cycle and meiotic
370 development pathway [32,33]. In Fusarium. graminearum, the signal GSK3 homolog is
371 suggested to promote the transcription of numerous early meiosis-specific genes as the Δfgk3
372 mutant failed in sexual development processes [9,34]. In this study, we suggest that the
373 phosphorylation of GSK3 is essential for cellular processes during fruiting body formation as
374 the inhibition of GSK3 activity by LiCl in C. cinerea suppressed fruiting body formation.
375
376 KOG analysis showed a similar distribution of upregulated DEGs involved in four KOG
377 categories at both time points, indicating that gene upregulation does not play a significant
378 role in the inhibition of fruiting body initiation via GSK3 inactivation. On the other hand, the
379 increased contribution of downregulated genes in information storage and processing at the
380 second time point indicates that the inhibition of fruiting body initiation is via
381 downregulation of relevant genes. The contribution of DEGs involved in cellular processes
382 and signaling was similar between the two time points, indicating that the activity of GSK3
383 inhibited by LiCl constantly downregulated genes involved in cellular processes and
384 signaling despite changes in environmental conditions. At first, the mycelia were incubated in
385 nutrient-rich plates at a high temperature under total darkness. After the mycelia reached the
386 edge of plates, the plates were then transferred to a nutrient-reduced, low temperature
387 environment under light/dark cycle. Due to the environmental changes, genes involved in
16 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
388 cellular processes and signaling responded to initiate fruiting body formation. The inhibition
389 of GSK3 suppressed the expression of genes involved in cellular processes and signaling
390 even when the environmental conditions were changed to a favorable condition for fruiting
391 body initiation. Therefore, we suggest that the activity of GSK3 is required for fruiting body
392 initiation. However, how environmental factors such as light, temperature and nutrient
393 interact with GSK3 to stimulate the global gene expression changes for fruiting body
394 induction requires further investigation.
395
396 Fruiting body formation in basidiomycetes is influenced by individual or multiple
397 environmental factors, such as light, temperature and nutrients. In this study, C. cinerea was
398 first grown at 37oC under total darkness with rich nutrient and then at 28oC in a light/dark
399 cycle after the mycelium reached the edge of plates. Light, cold shock and nutrient
400 diminution trigger or promote fruiting as well as the expression of stress response genes [1,
401 27, 35, 36]. Several studies have found that the expression of stress response genes to
402 environmental conditions are upregulated during fruiting body formation [1,3,37]. However,
403 in this study, the expression of nutrient response genes such as PKA protein kinase
404 (CC1G_01089) were significantly deregulated and adenylate cyclase (CC1G_02340) were
405 slightly downregulated in LM2 treated with LiCl compared with the controls (S2 Dataset).
406 Both participant in cAMP-PKA pathway and involve in sensing the nutrient level during
407 development transition [37]. As GSK3 homologs in yeast and Neurospora crassa take a
408 response in nutrient signal and light signal transmission [11,12,13], we propose that the
409 activity of GSK3 may be an important channel between extracellular signals to respective
410 response gene expression related to fruiting body formation.
411
412
17 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
413 Conclusion
414 To the best of our knowledge, this study is the first transcriptome profiling of the role of
415 GSK3 in C. cinerea fruiting formation. We found that the inhibition of GSK3 activity by the
416 addition of LiCl at the mycelium stage enhanced the mycelial growth. This phenotype could
417 be explained by the enriched metabolism pathways, such as “biosynthesis of secondary
418 metabolite” and “biosynthesis of antibiotic”, in which DEGs were upregulated to release
419 glucose for energy. Moreover, DEGs involved in cellular process pathways, including “cell
420 cycle-yeast” and “meiosis-yeast”, were identified in the C. cinerea fruiting body formation
421 suppressed by LiCl under favorable environmental conditions. Our data suggest that the
422 activity of GSK3 is essential for fruiting body formation by affecting the expression of
423 fruiting body induction genes and genes in cellular processes. Further studies on the
424 phosphorylation of GSK3 with external environmental signals may facilitate the
425 understanding of how environmental factors induce fruiting body development.
426
427 Acknowledgement
428 We thank Dr. Hajimme Muraguchi for sharing C. cinerea strains used in this study.
429
430 Reference
431 1. Kues U. Life History and Developmental Processes in the Basidiomycete Coprinus
432 cinereus. Microbiol Mol Biol Rev. 2000;64(2):316–53.
433 2. Sakamoto Y. Influences of environmental factors on fruiting body induction,
434 development and maturation in mushroom-forming fungi. Fungal Biol Rev.
435 2018;32(4):236–48.
436 3. Muraguchi H, Umezawa K, Niikura M, Yoshida M, Kozaki T, Ishii K, et al. Strand-
18 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
437 specific RNA-seq analyses of fruiting body development in Coprinopsis cinerea. PLoS
438 One. 2015;10(10):1–23.
439 4. Lu BC. The control of meiosis progression in the fungus Coprinus cinereus by
440 light/dark cycles. Fungal Genet Biol. 2000;31(1):33–41.
441 5. EMBI N, RYLATT DB, COHEN P. Glycogen Synthase Kinase‐3 from Rabbit
442 Skeletal Muscle: Separation from Cyclic‐AMP‐Dependent Protein Kinase and
443 Phosphorylase Kinase. Eur J Biochem. 1980;107(2):519–27.
444 6. Frame S, Cohen P. GSK3 takes centre stage more than 20 years after its discovery.
445 Biochem J. 2001;359:1–16.
446 7. Kondo Y, Ito T, Nakagami H, Hirakawa Y, Saito M, Tamaki T, et al. Plant GSK3
447 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling. Nat
448 Commun. 2014;5:1–11.
449 8. Saidi Y, Hearn TJ, Coates JC. Function and evolution of “green” GSK3/Shaggy-like
450 kinases. Trends Plant Sci. 2012;17(1):39–46.
451 9. Qin J, Wang G, Jiang C, Xu JR, Wang C. Fgk3 glycogen synthase kinase is important
452 for development, pathogenesis, and stress responses in Fusarium graminearum. Sci
453 Rep. 2015;5:1–8.
454 10. Zhou T, Dagdas YF, Zhu X, Zheng S, Chen L, Cartwright Z, et al. The glycogen
455 synthase kinase MoGsk1, regulated by Mps1 MAP kinase, is required for fungal
456 development and pathogenicity in Magnaporthe oryzae. Sci Rep. 2017;7(1):945.
457 11. Xiao Y, Mitchell AP. Shared Roles of Yeast Glycogen Synthase Kinase 3 Family
458 Members in Nitrogen-Responsive Phosphorylation of Meiotic Regulator Ume6p. Mol
459 Cell Biol [Internet]. 2000;20(15):5447–53.
460 12. Andoh T, Hirata Y, Kikuchi A. Yeast Glycogen Synthase Kinase 3 Is Involved in
461 Protein Degradation in Cooperation with Bul1, Bul2, and Rsp5. Mol Cell Biol.
19 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
462 2000;20(18):6712–20.
463 13. Tataroǧlu Ö, Lauinger L, Sancar G, Jakob K, Brunner M, Diernfellner ACR. Glycogen
464 synthase kinase is a regulator of the circadian clock of Neurospora crassa. J Biol Chem.
465 2012;287(44):36936–43.
466 14. Li L, Song H, Zhong L, Yang R, Yang XQ, Jiang KL, et al. Lithium chloride promotes
467 apoptosis in human leukemia NB4 cells by inhibiting glycogen synthase kinase-3 beta.
468 Int J Med Sci. 2015;12(10):805–10.
469 15. Liu Z, Li R, Jiang C, Zhao S, Li W, Tang X. The neuroprotective effect of lithium
470 chloride on cognitive impairment through glycogen synthase kinase-3β inhibition in
471 intracerebral hemorrhage rats. Eur J Pharmacol. 2018;840(October):50–9.
472 16. Chiu CT, Chuang DM. Molecular actions and therapeutic potential of lithium in
473 preclinical and clinical studies of CNS disorders. Pharmacol Ther. 2010;128(2):281–
474 304.
475 17. Rao PS, Niederpruem DJ. Carbohydrate metabolism during morphogenesis of
476 Coprinus lagopus (sensu Buller). J Bacteriol. 1969;100(3):1222–8.
477 18. Chen S, Zhou Y, Chen Y, Gu J. Fastp: An ultra-fast all-in-one FASTQ preprocessor.
478 Bioinformatics. 2018;34(17):i884–90.
479 19. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory
480 requirements. Nat Methods. 2015 Mar 9;12:357.
481 20. Trapnell C, Pachter L, Salzberg SL, Williams B a, Pertea G, Mortazavi A, et al.
482 Transcript assembly and abundance estimation from RNA-Seq reveals thousands of
483 new transcripts and switching among isoforms. Nat Biotechnol. 2011;28(5):511–5.
484 21. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene
485 and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.
486 Nat Protoc. 2012;7(3):562–78.
20 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
487 22. Yu G, Wang L-G, Han Y, He Q-Y. clusterProfiler: an R Package for Comparing
488 Biological Themes Among Gene Clusters. Omi A J Integr Biol. 2012;16(5):284–7.
489 23. Douglas C. Fungal b ( 1,3 ) - D -glucan synthesis. Med Mycol. 2001;39:55–66.
490 24. Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, et al.
491 Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus
492 Trichoderma reesei. J Biol Chem. 2003;278(34):31988–97.
493 25. Walser PJ, Haebel PW, Künzler M, Sargent D, Kües U, Aebi M, et al. Structure and
494 functional analysis of the fungal galectin CGL2. Structure. 2004;12(4):689–702.
495 26. Bertossa RC, Kües U, Aebi M, Künzler M. Promoter analysis of cgl2, a galectin
496 encoding gene transcribed during fruiting body formation in Coprinopsis cinerea
497 (Coprinus cinereus). Fungal Genet Biol. 2004;41(12):1120–31.
498 27. Boulianne RP, Liu Y, Aebi M, Lu BC, Kues U. Fruiting body development in
499 Coprinus cinereus: Regulated expression of two galectins secreted by a non-classical
500 pathway. Microbiology. 2000;146(8):1841–53.
501 28. Nakazawa T, Ando Y, Hata T, Nakahori K. A mutation in the Cc.arp9 gene encoding a
502 putative actin-related protein causes defects in fruiting initiation and asexual
503 development in the agaricomycete Coprinopsis cinerea. Curr Genet. 2016;62(3):565–
504 74.
505 29. Ando Y, Nakazawa T, Oka K, Nakahori K, Kamada T. Cc.snf5, a gene encoding a
506 putative component of the SWI/SNF chromatin remodeling complex, is essential for
507 sexual development in the agaricomycete Coprinopsis cinerea. Fungal Genet Biol.
508 2013;50(1):82–9.
509 30. Muraguchi H, Abe K, Nakagawa M, Nakamura K, Yanagi SO. Identification and
510 characterisation of structural maintenance of chromosome 1 (smc1) mutants of
511 Coprinopsis cinerea. Mol Genet Genomics. 2008;280(3):223–32.
21 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
512 31. De Paula R, Azzariti de Pinho C, Terenzi H, Bertolini M. Molecular and biochemical
513 characterization of the Neurospora crassa glycogen synthase encoded by the gsn
514 cDNA. Mol Genet Genomics. 2002;267(2):241–53.
515 32. Rubin-Bejerano I, Sagee S, Friedman O, Pnueli L, Kassir Y. The in vivo activity of
516 Ime1, the key transcriptional activator of meiosis-specific genes in Saccharomyces
517 cerevisiae, is inhibited by the cyclic AMP/protein kinase A signal pathway through the
518 glycogen synthase kinase 3-beta homolog Rim11. Mol Cell Biol. 2004;24(16):6967–
519 79.
520 33. Wang C, Zhang S, Hou R, Zhao Z, Zheng Q, Xu Q, et al. Functional analysis of the
521 kinome of the wheat scab fungus Fusarium graminearum. PLoS Pathog. 2011;7(12).
522 34. Turrà D, Segorbe D, Di Pietro A. Protein Kinases in Plant-Pathogenic Fungi:
523 Conserved Regulators of Infection. Annu Rev Phytopathol. 2014;52(1):267–88.
524 35. Tsusué YM. EXPERIMENTAL CONTROL OF FRUIT‐BODY FORMATION IN
525 COPRINUS MACRORHIZUS. Dev Growth Differ. 1969;11(1):164–78.
526 36. Stamets, P. Growing gourmet and medicinal mushrooms. 3rd ed. Ten Speed Press;
527 2000.
528 37. Cheng CK, Au CH, Wilke SK, Stajich JE, Zolan ME, Pukkila PJ, et al. 5’-Serial
529 Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting
530 body development in Coprinopsis cinerea. BMC Genomics. 2013;14(1).
531 38. Stajich JE, Wilke SK, Ahren D, Au CH, Birren BW, Borodovsky M, et al. Insights
532 into evolution of multicellular fungi from the assembled chromosomes of the
533 mushroom Coprinopsis cinerea (Coprinus cinereus). Proc Natl Acad Sci.
534 2010;107(26):11889–94.
535 39. Mao X, Cai T, Olyarchuk JG, Wei L. Automated genome annotation and pathway
536 identification using the KEGG Orthology (KO) as a controlled vocabulary.
22 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
537 Bioinformatics. 2005;21(19):3787–93.
538
539 Supporting information
540 S1 Dataset. Differentially expressed genes in LM1 against HM, HM: C. cinerea
541 mycelium treated with water; LM1: C. cinerea mycelium treated with 3 µM LiCl.
542
543 S2 Dataset. Differentially expressed genes in LM2 against HHK, HHK: hyphal knots of
544 the water treated samples; LM2: consistent mycelium of 3 µM LiCl-treated
545
546 S1 Table. Primers designed for validation of gene expression profile in C. cinerea
547 transcriptome data.
548
549 S2 Table. Unigenes used for validation of gene expression profile in C. cinerea
550 transcriptome data.
551
552 S3 Table. KEGG enrichment between different samples (p-value ≤ 0.05).
553
554 S4 Table. GO enrichment between different samples (p-value ≤ 0.05).
555
556 S5 Table. DEGs involved in KEGG enriched pathways between HHK and LM2.
557
558 S6 Table The downregulated expression gene of fruiting body induction based on RNA-
559 seq analysis.
560
23 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
561 S1 Fig. Go annotation of DEGs. The distribution of GO annotations of (A) upregulated and
562 (B) downregulated DEGs in HM vs LM1, and (C) upregulated and (D) downregulated DEGs
563 in HHK vs LM2.
564
565 S2 Fig. One of enriched KEGG pathways in HM vs LM1 transcriptome analysis.
566 Starch and sucrose metabolism pathway is one of the enriched metabolism pathways in
567 samples treated with 3 µM LiCl. Red boxes indicate upregulated DEGs and green boxes
568 indicate downregulated DEGs in HM vs LM1. Gray boxes indicate non-DEGs.
569
24 bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/492397; this version posted December 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.