Transcriptomic Profiling Identifies Candidate Genes Involved in Polyporus Umbellatus Sclerotial Formation Affected by Oxalic Acid

doi:10.21203/rs.3.rs-115257/v1

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Transcriptomic Profiling Identifies Candidate Genes Involved in Polyporus Umbellatus Sclerotial Formation Affected by Oxalic Acid

https://doi.org/10.21203/rs.3.rs-115257/v1

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published 29 Aug, 2021

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Polyporus umbellatus is a precious medicinal fungus. The whole transcriptome of P. umbellatus exposed to different concentrations of oxalic acid was performed and analyzed using RNA-seq based on sequencing technology. Reactive oxygen species (ROS) detection of P. umbellatus mycelia was visually conducted and using non-invasive micro-test technology (NMT), the net Ca²⁺ and H₂O₂ fluxes of P. umbellatus were measured. Totally, 22,523 unigenes were generated by De novo assembly of reads and there are 1223 differentially expressed genes (DEGs) between the control group and the high oxalic acid complemented (PD_S) group and 459 DEGs between the control group and the low concentration oxalic acid additive (PU_SM) group. The transcriptomic analysis indicated DEGs encoding enzymes related to oxidative stress, energy metabolism and so on. Compared to that of the control group, the biomass of the sclerotia in the PU_SM group increased 66%, however, no sclerotia formed in the PD_S group. The low concentration of oxalic acid could increase Ca²⁺ and H₂O₂ influx, while the high concentration of oxalic acid presented slight H₂O₂ efflux. There is a great significant positive correlation between the net Ca²⁺ and H₂O₂ fluxes. Different concentrations of exogenous oxalic acid affected P. umbellatus sclerotial formation in different ways.

Biotechnology and Bioengineering

General Microbiology

Polyporus umbellatus

transcriptomic sequencing

oxalic acid

non-invasive micro-test technology

Ca2+

H2O2

In fungi and plants, as one of the most simplest dicarboxylic acids, oxalic acid plays important roles all through the life of the creatures¹. In plant pathogenic fungus of Sclerotina sclerotiorum, oxalic acid played double roles during the pathogenic interactions with the host plant^2,3, thus, oxalic acid was shown to induce ROS and it can also inhibit ROS as well.

Polyporus umbellatus (Pers.) Fr., a valuable medicinal fungus, the sclerotium of which has been used as diuretic and antidote for more than two thousand of years in China. Steroids and polysaccharides are two principal groups of chemicals in P. umbellatus sclerotia⁴. Among these bioactive components, sterone compounds exhibited nephroprotective activty on renal intestitial fibrosis against aristolochic acid or adenine induced nephrotoxicity in rats⁵. In the previous study in our laboratory⁶, a HPLC method was performed for simultaneous quantitative determination of five sterone compounds including polyporusterone A and polyporusterone B which were isolated from the wild sclerotia of P. umbellatus. HPLC fingerprint was used to compare the wild sclerotia, the mycelia and scletotia under artificial conditions. The results showed that the cultured sclerotia possessed the similar spectrum with the wild sclerotia, while the mycelia only shared chromatographic peak of a few sterone compounds with the wild sclerotia, thus, it is necesssary to conduct experiments to induce sclerotia directly from hyphae effectively. In the previous study, it has been elucidated that P. umbellatus sclerotial formation is in close relationship with oxidative stress state and we have found that oxalic acid served as an antioxidant during P. umbellatus sclerotial formation⁷. However, whether oxalic acid affecting P. umbellatus sclerotial formation in a “low promotion and high suppression” way deserves further study. Based on the previous study, we are trying to find whether exogenous oxalic acid especially the minimum and the most suitable concentration to promote and the concentration of the oxalic acid to just completely inhibit P. umbellatus sclerotial formation, thus different concentrations of oxalic acid on sclerotial biomass was further studied in detail in the present study. According to the preveious study, it was demonstrated that calcium antagonists could inhibit P. umbellatus sclerotial formaiton⁸. Calcium ion and ROS are always stimulated by each other and function coordinately during signal transduction⁹. Due to stress, it was induced Ca²⁺influx and activate ROS production¹⁰. However, during P. umbellatus sclerotial formation exposed to different concentrations of oxalic acid, detailed molecular informaiton about calcium signalling pathway and the real-time dynamic changes as well as the correlationship between ROS and calcium ion is not clear.

RNA sequencing (RNA-seq), also named sequence-based assays of transcriptomes has been widely used these years and has become a relatively accurate tool for instantaneous gene expression analyses¹¹. To better understand the molecular process of P. umbellatus sclerotial formation under different concentrations of oxalic acid conditions, we perfomed RNA-seq transcriptomic analysis. In this study, DEGs between mycelia of P. umbellatus subjected to exogenous oxalic acid were described overall.

As an emerging detection, NMT has been widely used in many fields of research, such as recording H⁺, Ca²⁺, NO₃^- and H₂O₂ fluxes in plant systems¹². In order to confirm whether different concentrations of oxalic acid can affect intracellular ROS in P. umbellatus mycelia, fluorescence staining assay was conducted in this study. Furthermore, the real-time Ca²⁺ and H₂O₂fluxes in P. umbellatus mycelia by treatment of different concentrations of oxalic acid were detected by NMT. Thus, the aims of this study on the one hand was to identify the DEGs involved in the oxidative stress and calcium signal transduction as well as the profiles of their expression patterns based on high-throughput data. On the other hand, the correlationship of the fluxes of ROS and calcium ion was also analyzed by Pearson correlation analysis. The study will provide certain genetic evidence for the involvement of oxalic acid during P. umbellatus sclerotial formation.

P. Fungal growth conditions and exogenous oxalic acid treatments in cultivation experiments

umbellatus was isolated from Guxian, Shanxi Province of China and incubated on solid wheat bran medium at 25 °C in the dark. After inoculation, the petri dishes were placed in the dark at 25 °C. The modified maltose medium contains maltose 20 g·L⁻¹, peptone 4 g·L⁻¹, K₂HPO₄∙3H₂O 1 g·L⁻¹, KH₂PO₄ 0.5 g·L⁻¹, MgSO₄∙7H₂O 0.5 g·L⁻¹, CaCl₂ 0.005 g·L⁻¹, vitamin B1 0.05 g·L⁻¹ and agar 12 g·L⁻¹. Before inoculation, different concentrations of the exogenous sterile oxalic acid were added to the sterilized growth medium through a 0.22-μm Millipore filter (Merck, Germany). The final concentrations of oxalic acid in the maltose medium were 0 mg ml⁻¹, 0.005 mg ml⁻¹, 0.01 mg ml⁻¹, 0.05 mg ml⁻¹, 0.10 mg ml⁻¹ and 1.10 mg ml⁻¹. The group without oxalic acid (0 mg ml⁻¹) was treated as the control group in the experiments. After cultivation for 30 days, the colony morphology of sclerotia and mycelia were observed. The experiment was repeated three times, with 30 replicates in each group.

Fungal Growth Conditions and Treatments for Transcriptome Sequencing

The mycelia cultivated for 30 days in the optimal concentration of exogenous oxalic acid (PU_SM group) that promote sclerotial formation of P. umbellatus and the minimum exogenous oxalic acid that thoroughly inhibit sclerotial development (PD_S group) and the control group without oxalic acid named as PU_S (CK) were performed transcriptome sequencing. Each sample of the mycelia from different groups was collected respectively and immediately frozen in liquid nitrogen for total RNA extraction.

Transcriptome Sequencing

Transcriptome sequencing and analysis was performed by Beijing Novogene Co. Ltd (Headquarters). In order to obtain the transcriptome of P. umbellatus mycelia subjected to different concentrations of exogenous oxalic acid, the total RNA was isolated from the frozen tissues of the mycelial samples of the three groups of P. umbellatus (PU_SM, CK and PD_S), with three biological replicates in each group, using the RNA plant mini kit with column DNase digestion (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Then, RNA concentration was detected and the RNA integrity and purity were assessed. Then, 3 μg RNA per sample was used as input material for the RNA preparations. Finally, nine samples with RNA integrity number (RIN) values above 8 were used for libraries construction. Sequencing libraries were generated and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified and the fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer. First strand cDNA and the second strand cDNA synthesis were in succession synthesized. To prepare for hybridization, NEBNext Adaptor with hairpin loop structure was ligated. Subsequently, the library fragments were purified to select cDNA fragments of preferential 150~200 bp in length. After that, 3 μl USER Enzyme (NEB, USA) was added at 37 °C for 15 min, followed by 5 min at 95 °C prior to PCR. Then PCR was conducted with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, the PCR products were purified (AMPure XP system) and the library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). Then the library preparations were sequenced using Illumina Hiseq 2500 platform and paired-end reads of 125 bp were produced¹¹.

Sequence Annotation

Raw data of fastq format were firstly processed through in-house Perl scripts. During the process, clean data were obtained by removing reads containing ploy-N, adapter and low quality reads from raw data. Meanwhile, Q20, Q30, GC-content and sequence duplication level were also calculated. The DEGs analysis was based on the clean data with high quality. All the reads were then assembled using Trinity 50. Gene function was annotated based on the following databases: the non-redundant protein (NR) database, the nonredundant nucleotide (NT) database, Protein family (Pfam), Swiss-Prot (a manually annotated and reviewed protein sequence database), Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG) and Gene Ontology (GO), using BLASTX with a significance threshold of E-value < 10^-5.

Differential expression analysis of transcripts

The nine samples of the total RNA extracted from P. umbellatus mycelial tissue samples were used to perform three independent sequencing libraries. To better obtain the mechanism of P. umbellatus sclerotial formation affected by different concentrations of oxalic acid, it is important to identify the DEGs among the three groups. Then, to determine the functional annotations of the genes, the clean tags in each cDNA library were mapped to the all-unigenes data from the transcriptome sequencing using the short reads alignment program²⁹. Data from the two biological replicates of each group were merged, and the transcript abundance of each gene was determined by the reads per kilobase of exon model per million mapped read (RPKM) values.

To identify genes responding to oxalic acid and sclerotial formation-related DEGs, the log2 ratio of gene transcript abundance among the sequencing libraries for the mycelial tissue of the CK group and the oxalic acid- complemented groups were calculated. Differential expression analysis of the three groups was conducted using the DESeq R package (1.10.1). Based on the negative binomial distribution model, the statistical routines for determining differential expression in digital gene expression data were provided by DESeq³⁰. The statistical significance of the differential expression level for each gene was determined by evaluating the probability. For controlling the false discovery rate, the adjusted p values were used according to the Benjamini and Hochberg’s approach. The false discovery rate (FDR) was used to determine the threshold of the adjusted p-value. A gene with an adjusted p-value < 0.05 and the absolute value of log2 ratio > 1 was termed as a DEG.

Validation of DEGs expression using Real-Time Quantitative PCR (qRT-PCR)

All the primers were designed using Primer 3.0 software, assessed by AmplifX1.5.0and synthesized by Genewiz Company (China). The 18S rRNA gene (EU442272) of P. umbellatus was used as a reference control¹¹. All the primers detected were PCR-amplified in the cDNA of the samples of the CK, PU_SM and PD_S groups respectively. All the randomly selected 9 genes from the RNA-seq data exhibited>2 folds higher or down-regulated<0.5-fold lower expression in the P. umbellatus in response to oxalic acid. Using qRT-PCR method, these DEGs were conducted to validate the expression patterns. The method was performed using the SYBR® Premix ExTaqTM (TaKaRa, Dalian, China) on the Roche Light Cycler 480Ⅱ. The reaction was conducted as follows: denaturated at 95 °C for 30 s for the first step, followed by 40 cycles of amplification at 95 °C for 5 s and 60 °C for 34 s. Each detected gene was repeated three times in independent runs. Gene expression was evaluated by the 2^−ΔΔCt method.

ROS detection in P. umbellatus Mycelial Cells

ROS measurement was conducted using a GMS10010.7 kit protocol. P. umbellatus mycelia of the PU_SM, PD_S and the control groups were respectively picked off using scotch tape. The fluorescence staining was prepared by mixing 5-(and-6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) with the dilution at the volume ratio of 1:20 on ice to avoid quenching of the fluorescent dye in the dark. Subsequently, 20 μl of the prepared staining fluid was dropped onto the glass slide and then the samples adhered to scotch tapes were placed down to closely contact the staining fluid on the slide. The samples were then incubated at 28°C for 1h in the dark. Finally, using an AxioCan HRc Zeiss microscopy, the labelled ROS in the mycelial cells from each sample was visualized with the excitation wavelength of 490nm and an emission wavelength of 530nm, according to the previous study³¹.

Real-time Ca²⁺ and H₂O₂fluxes measurement of P. umbellatus mycelia affected by different concentrations of oxalic acid using NMT

Ca²⁺and H₂O₂ was measured on the surface of P. umbellatus mycelia in different groups after cultivation for 30 days as mentioned above using NMT System (NMT100 Series, Younger USA LLC, Amherst, MA, USA). Ca²⁺-sensitive and ROS-sensitive microsensors were purchased from NMT Service Center, Xuyue Beijing Sci. & Tech Co. Ltd. The experimental voltage is +600 mV. The primary position (M1) of Ca²⁺ or ROS-sensitive microsensor was placed 30 μm from P. umbellatus mycelial surface, and the further away position (M2) is 60 μm (Sample detection images were shown in Supplement Figure 1).

Statistical Analysis

The data were analyzed with double-factor Pearson correlation analysis and all statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, IL, USA). Data were presented as means±SD from at least three independent experiments. P values<0.05 were considered a significant difference.

Colony morphology of P. umbellatus under exogenous different concentrations of oxalic acid

From the concentrations of the exogenous oxalic acid below 1mg mL^-1, it was found that the oxalic acid with 0.05mg·mL^-1served as the most suitable in P. umbellatus sclerotial formation (Fig. 1B), compared to that of the control group (Fig. 1A), P. umbellatus biomass in this group increased significantly (P<0.05). In addition, the minimum concentration of oxalic acid that completely inhibit P. umbellatus sclerotial development was 1.10 mg·mL^-1 (Fig. 1C).

Transcriptome sequencing and de novo Assembly and sequence annotation

To obtain the P. umbellatus mycelial transcriptome expression profile treated by oxalic acid, two libraries with different concentrations of oxalic acid and one library without oxalic acid as control were respectively constructed. Illumina sequencing data of P. umbellatus could be obtained in the NCBI BioProject and the Sequence Read Archive (SRA) database with the ID of PRJNA669949. Totally 284190976 raw reads were generated (Table 1). There were 276265498 clean reads left after removing adaptor sequences, ambiguous nucleotides and low-quality sequences. Assembly of clean reads brought about 22,523 unigenes in the range of 201~19,256 bp with a N50 length of 2834 bp (Fig. 2).

After the repeated and the short-length sequences were deleted, 22,523 non-redundant unigenes were subjected to similarity analysis according to the 7 public databases (Table 2). Among them, 12,864 (57.11%) unigenes had the highest matches in the NT database, followed by 11,236 (49.88%) unigenes in the SwissProt database. Specifically, 10,914, 10,645, 10,571, 6934 and 4063 unigenes had functional annotations in the other five databases (NR, GO, PFAM, KOG and KO), respectively accounting for 48.45%, 47.26%, 46.93%, 30.78% and 18.03%, as shown in Table 2.

According to GO, 10,645 unigenes were classified into three major functional ontologies, including biological process, cellular component and molecular function. For biological process, ‘cellular process’ and ‘metabolic process’ comprise huge proportions. For cellular component, ‘cell’ and ‘cell part’ account for considerable parts. In addition, ‘binding’ and ‘catalytic activity’ had great percentages in the molecular function part (Fig. 3).

Identification of DEGs in the mycelia of P. umbellatus treated with different concentrations of oxalic acid

Due to the analysis of the DEGs, RPKM values were calculated and the Pearson’s correlation coefficients (R²) were high between the biological replicates in the same group ranging from 0.902 to 1. The R² of the samples between the different groups ranged from 0.705 to 1.816 (Fig. 4). The DEGs between the CK and PU_SM, the CK and PD_S were analyzed and the DEGs with higher expression levels in PU_SM and PD_S of both groups were denoted as ‘up-regulated’, while those with lower expression levels in PU_SM and PD_S were ‘down-regulated’ (Fig. 5). There are 1223 DEGs between the control group without oxalic acid and the high oxalic acid group and among them, 679 were up-regulated and 544 were down-regulated. There are 459 DEGs between the control group and the low oxalic acid group and among them, 228 were up-regulated and 231 were down-regulated (Supplement Figure 2).

DEGs Related to oxidases and dehydrogenases

In comparison to that of the control (PU_S) group, c43_g1 and c159_g1 encoding cytochrome c oxidase in the control group decreased 158.80 and 189.27 folds respectively in the PD_S group; NADPH oxidase encoded by c3315_g1 and c8851_g1 in the PD_S group was down-regulated 1.54 and 2.03 folds. c7390_g2 encoding galactose oxidase in the control group increased 2.16 folds than that of the PD_S group. In addition, D-amino-acid oxidase encoded by c7941_g1, L-gulonolactone/D-arabinono-1,4-lactone oxidase and so on encoded by c7166_g1 decreased in the PD_S group. Compared to that of the control group, alcohol oxidase encoded by c8987_g1 and c8020_g1 increased by 7.31 and 2.78 folds in the PU_SM group, however, c8176_g4 encoding alcohol oxidase in the PD_S decreased 2.20 folds.

NADH dehydrogenase encoded by c1494_g1, c17518_g1 and c6419_g1 in the PD_S group decreased by 109.18, 114.90 and 2.06 folds respectively, in comparison to that in the control group. In addition, the expression of c524_g1, c1758_g1 and c2305_g1 encoding NADH dehydrogenase was up regulated in the control group, while they did not express in the PD_S group at all.

c7742_g1, c6369_g1 and c7145_g1 encoding alcohol dehydrogenases in the PD-S group increased respectively 2.33, 1.85 and 2.09 folds compared to that in the control group (Table 3, Supplement Table S2).

DEGs Associated with Reductases and glutathione S-transferase

Aldo/keto reductase encoded by c8217_g1 decreased 2.43 folds in the PU-SM group, compared to that of the control group. Meanwhile, in the PD_S group, c9378_g1 encoding aldo/keto reductase decreased 2.15 folds less than that of the control group. The same enzyme encoded by c9881_g2 in the control group expressed 3.27 folds more than that of the PU_SM group. In addition, c8932_g1 encoding quinone reductase and nitrite reductase encoded by c10001_g1 down-regulated in the PU_SM group, compared to that of the control group. c16064_g1 encoding cytochrome c reductase was up regulated in the PD_S group, while it did not express in the control group at all. Cytochrome-b5 reductase encoded by c4751_g1, ribonucleotide reductase alpha subunit and gamma-glutamyl phosphate reductase encoded by c4563_g1 and c5317_g1 all down-regulated expressed in the control group, in comparison to that of the PD_S group.

Compared to the control group, c9468_g4 and c7959_g1 encoding glutathione S-transferase in the PD_S group increased 19.30 and 1.93 folds respectively. The same enzyme encoded by c6055_g1 decreased 6.92 folds in the PU_SM group, in comparison to that of the control group (Supplement Table S3).

DEGs Related to Peroxidases and Catalase

Compared to that of the control group, c5235_g1 encoding heme peroxidase increased 1.85 folds in the PD_S group. Manganese peroxidase encoded by c6600_gl of the mycelia in the control group decreased by 2.46 times compared to the PD_S group, in addition, c6769_g1 encoding the same protein decreased by 6.15 folds in the PU_SM group, compared to that of the control group. In the control group, c6329_g1 encoding catalase decreased by 2.30 folds compared to the PD-S group. Glutathione peroxidase encoded by c4316_g1 was specifically expressed in the PD_S group but did not express in the PU_S group (Supplement Table S3).

DEGs related to energy metabolism

In comparison to the the control group, isocitrate lyase encoded by c7209_g1 increased by 34.67 folds in the PU_SM group. The expression of the same gene increased 56.85 folds in the low oxalic acid group, compared to the high oxalic acid group. Regard to the DEG (c6742_g2) encoding fatty acid hydroxylase, the gene in the mycelia exposed to the PU_SM group was up-regulated about 29.21 times that of the control group. Due to fatty acid synthase encoded by c3295_g1 and c4529_g1, it was down-regulated 1.74 and 1.62 folds in the PD_S group, in comparison to the control group. In addition, c9487_g1 encoding fatty acid desaturase (delta-12 desaturase) expressed in the PD-S group 3.41 folds less than that in the control group. c8540_g1 encoding NADP⁺-dependent malic enzyme (1.1.1.40) in the PD_S group was increased 2.84 folds than that of the control group.

In comparison to that of the control group, c4703_g1 encoding L-rhamnose 1-dehydrogenase was up-regulated 12.38 folds in the mycelia of the PU_SM group, while the same gene was down-regulated 12.80 folds in that of the PD_S group. c7929_g2 encoding L-threo-3-deoxy-hexylosonate aldolase was down-regulated 3.55 folds in the PU_SM group, compared to that of the control group. Rhamnogalacturonase encoded by c4388_g1 in the PD_S was down-regulated 3.36 folds less than that of the CK group, in addition, c4271_g1 encoding rhamnogalacturonan acetylesterase was up-regulated 6.43 folds (Table 4). Compared with the high oxalic acid group, HMG-CoA reductase encoded by c8652_g1 and C-4 methyl sterol oxidase encoded by c8791_g2 were both up-regulated in the control group.

DEGs related to Calcium signaling pathway

The expression of c8739_g2 encoding calmodulin in the PU_SM group increased 2.66 folds than that of the control group. In addition, c2043_g1 and c3257_g1 encoding calmodulin expressed in the control group, while it also did not express in that of the PD_S group. Calmodulin-binding motif encoded by c4558_g1 expressed in the PU_SM group while it did not express at all in the control group. c8133_g1 encoding calmodulin-binding motif in the control group increased 2.13 folds than that of the PD_S group. Furthermore, c9830_g2 which participated in calcium ion transport in the PU_SM group expressed 2.71 folds more than that of the control group. In the PD_S group, c3191_g1 and c6291_g1 took part in cellular calcium ion homeostasis and calcium ion transport was down-regulated 5.62 and 1.91 times in comparison to that of the control group (Table 5).

The ROS content in P. umbellatus mycelia affected by different concentrations of oxalic acid

In the control group without oxalic acid, the ROS content of P. umbellatus mycelia has accumulated to a certain extent after cultivation for 30 days as shown in Fig. 6A, while in the PU_SM group, ROS level was more than that of the control group (Fig. 6B), however, in the PD_S group, the ROS content was much lower than that of the control group (Fig. 6C).

Validation of the DEGs using qRT-PCR.

The 9 unigenes were randomly selected and examined at the transcriptional level by qRT-PCR, to validate the reliability of the RNA-Seq data (Fig. 7). All the unigenes were shown that the expression patterns were consistent with the RNA-Seq data, indicating the validity of the RNA-seq sequencing results.

Net Ca²⁺ and H₂O₂ fluxes during P. umbellatus growth affected by different concentrations of oxalic acid using NMT

In the PD_S group, the Ca²⁺ influx less were shown with the net fluxes ranging from -54.74~63.15 (pmol·cm^-2·s^-1) than that of the control group and the PU_SM group (Fig. 8A), but the H₂O₂ in the PD_S group represented efflux slightly with the net fluxes ranging from 4.05 to 9.77 pmol·cm^-2·s^-1 (Fig. 8B), in comparison, the Ca²⁺ performed influx more in the control group and much more in the PU_SM group (Fig. 8A), however, H₂O₂ influx a litter more in the control group and the PU_SM group than that of the PD_S group (Fig. 8B). The significant positive correlationship with the Pearson’s correlation coefficient (r=0.991, P<0.01) between the fluxes measurement of Ca²⁺ and H₂O₂ was performed by correlationship analysis using SPSS and the scatter plot was shown in Supplement Figure 3.

It was found that oxalic acid accumulation led to lowering ambient pH and favored Sclerotinia sclerotiorum sclerotial development¹³. Recently, it has also been reported that S. sclerotiorum sclerotial formation was inhibited by exogenous oxalic acid of 25mM and higher¹⁴. Thus, oxalic acid played dual roles in sclerotial differentiation. In the present study, low concentrations of exogenous oxalic acid (ranging from 0.05 mg ml^-1 to 0.1 mg ml^-1) promote but high concentrations of oxalic acid (1.10 mg ml^-1 and higher) completely inhibit P. umbellatus sclerotial formation (Fig. 1C). We presented detailed molecular information about effects of exogenous oxalic acid on P. umbellatus sclerotial formation and found that oxalic acid affected P. umbellatus sclerotial formation, with performance in the special “low promotion and high suppression” way.

It has been well documented that during S. sclerotiorum infection, initially oxalic acid inhibits ROS-mediated plant defense responses, but later increases ROS production in Nicotiana benthamiana followed by programmed cell death. Therefore, oxalic acid has dual opposing roles in Sclerotinia pathogenesis².

Oxalic acid (10mM) application suppressed H₂O₂ and O₂^- contents and increased ascorbic acid concentration in lotus root slices¹⁵. In addition, oxalate can lead to ROS production in renal epithelial cells¹⁶.

ROS are unstable and highly-reactive chemical species¹⁷. Since NADPH oxidase activation is a key enzymatic source of ROS formation¹⁸, and in eukaryotes, it is an important source of ROS including H₂O₂¹⁹. According to the high-throughput data, two DEGs encoding this enzyme was downregulated in the PD_S group, compared to that of the control group, indicating the weak oxidizability of the mycelia of the group complemented with high concentrations of oxalic acid. Other oxidases play important roles in maintaining oxidation-reduction balance in fungi, for example, alcohol oxidase, homooctameric flavoprotein, is capable of catalyzing the oxidation of methanol to formaldehyde and hydrogen peroxide. This protein is involved in the pathway methane degradation, which is part of energy metabolism of certain yeasts²⁰. In the present study, the DEGs of c8176_g4, c8987_g1 and c8020_g1 encoding alcohol oxidase in both of the control group and the PU_SM group contribute to the high oxidative stress in the mycelia of P. umbellatus, which will compel sclerotial formation. Galactose oxidase can catalyze D-galactose and O₂ to form D-galacto-hexodialdose and H₂O₂. In this study, c7390_g2 encoding this gene was highly expressed in the control group, indicating it contributed to produce ROS and will be more beneficial to sclerotial formation, compared to that of the PD_S group.

The functions of reductases and other ROS-scavenging enzymes are also of the essence. For instance, acetoin reductase encoded by c9034_g4 was relatively up regulated in the control group, but it was relatively down regulated in the PU_S group, indicating the relative reducibility of the mycelia in the control group, therefore, it was much easier for the mycelia to form sclerotia in the PU_SM group. Glutathione S-transferases (GSTs) have been reported to catalyze reactions involving the conjugation of glutathione to electrophilic compounds and to remove ROS^21,22. Based on the transcriptomic data, the expression of c9468_g4 and c7959_g1 encoding glutathione S-transferase in the PD_S group increased, meanwhile, the same enzyme encoded by c6055_g1 decreased in the PU_SM group, compared to the control group. This indicated that in comparison to that of the control group, the reductively of the P. umbellatus mycelia of the PD_S group was higher but the reductivity of the P. umbellatus mycelia of the PU_SM group was lower. As one of the antioxidant enzymes, catalase was upregulated in the mycelia of the PD_S group in comparison to that of the control group, which will help to maintain oxidative stress level in the latter group.

NADPH could convert H₂O₂ into H₂O by glutathione peroxidase and this enzyme stands for antioxidant ability²³. In the present study, c4316_g1 encoding glutathione peroxidase was only expressed in the PD_S group but did not express in the PU_S group, which indicated that the mycelia in the PD_S group expressed stronger antioxidative activity than that in the mycelia of the control group. Glutathione transferase and glutathione peroxidase possesses the similar characteristics in P. umbellatus mycelia of different groups. It has been reported that thiol redox state (TRS)-related antioxidant enzymes glutathione reductase, glutathione-S-transferase and glucose 6-phosphate dehydrogenase peaked in the undifferentiated hyphae, indicated TRS-associated high oxidative stress was in close relationship with sclerotial formation in phytopathogenic fungi²⁴.

Unsaturated fatty acids is the intrinsic reason for lipids oxidation²⁵. That c3295_g1 and c4529_g1 encoding fatty acid synthase was down-regulated in the PD_S group compared to that of the control group, which indirectly reflects that lipid oxidation is lower in the PD_S group.This speculation has been verified in the previous study, which indicated that exogenous oxalic acid reduced the lipid peroxidation in a concentration-dependent manner⁷.

In this study, with CM-H₂DCFDA probe for visually detection, the ROS content in P. umbellatus mycelia of the PU_SM group was the most, followed by that of the control group. The fluorescence intensity of the ROS level in the PD_S group was the slightest. In addition, using NMT technique, in the PD_S group, H₂O₂ represented efflux slightly, while in the control and the PU_SM group, H₂O₂ influxes were demonstrated in this study (Fig. 8B), and it is conceivable that the intracellular H₂O₂ decreased in the mycelia of P. umbellatus in the PD_S group, in comparison to both of the other groups. The result is consistent with the ROS content in P. umbellatus mycelia affected by different concentrations of oxalic acid (Fig. 6). Thus, these results have further confirmed that the high oxidative stress has been formed in the control group and the PU_SM group and facilitates P. umbellatus sclerotial formation, while lower ROS content in the PD_S group cannot.

NADPH oxidases require different redox cofactors and are regulated either through regulatory subunits or by calcium ions, and calcium-dependent regulation has been verified via EF-hand motifs by experiments²⁶. NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family. When the activity of the Ca²⁺ and phosphorylation-dependent RBOHD is upregulated, ROS production is caused to increase during plant defense^18,27.

In the present study, real-time Ca²⁺fluxes measurement showed that Ca²⁺influxes in the PU_SM group was the most, followed by that of the control group, and Ca²⁺influxes in the PD_S group was the least among these three groups. The more Ca²⁺ uptake in the control group and the PU_SM group causes intracellular Ca²⁺ concentration to rise and facilitates Ca²⁺ spiking and interaction with other moleculars in calcium transduction signalling pathway²⁸. Therefore, the intracellular H₂O₂ and Ca²⁺ increased in the PU_SM group but decreased in the PD_S group, compared to that of the control group. In other words, in the control group and PU_SM groups that rendered mycelia to differentiate into sclerotia, the elevated ROS levels accompanied with increase in calcium ion levels. Meanwhile, the transcriptomic analysis showed that the DEGs about calmodulin, calmodulin-binding motif, calcium ion transport and so on were up-regulated in the control group and the PU_SM group, which was different from that of the PD_S group. This implied that DEGs related to calcium signal transduction and ROS cooperatively plays an important role in P. umbellatus sclerotial formation.

In conclusion, the low concentration of oxalic acid induces ROS production by H₂O₂ influx, on the other hand, the high concentration of oxalic acid inhibits P. umbellatus sclerotial differentiation and oxidative stress by H₂O₂ efflux. The significant positive correlationship was shown between the fluxes measurement of calcium ion and H₂O₂. Further investigation is required to determine the mechanism of the oxalic acid affecting oxidation-reduction during P. umbellatus sclerotial formation.

Acknowledgements: This research was financially supported by the CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-I2M-3-013), National Natural Sciences Foundation of China (NSFC81973425, 81773843, 81803666) and Peking Union Medical College Discipline Construction Project (Tsinghua 211-201920100901).

Author contributions

S.X.G. and X.M.C.: experimental design and manuscript review; Y.M.X.: manuscript writing, manuscript review and performed the experiments; B.L.: performed part of the experiments and manuscript review; X.Z. and L.S.Z.: data analysis and raw data uploading; T.S.L. and M.W.L.: manuscript review;. All authors have read and approved the manuscript.

Competing interests

The authors declare no competing interests.

Magnuson, J.K. & Lasure, L.L. Organic acid production by filamentous fungi. Advances in Fungal Biotechnology for Industry, Agriculture and Medicine. Kluwer Academic/Plenum Publishers. 307-340 (2004).
Williams, B., Kabbage, M., Kim, H.J., Britt, R. & Dickman, M.B. Tipping the Balance: Sclerotinia sclerotiorum Secreted Oxalic Acid Suppresses Host Defenses by Manipulating the Host Redox Environment. PLoS. Pathog. 7(6), e1002107 (2011).
Kabbage, M., Williams, B. & Dickman, M.B.. Cell Death Control: The Interplay of Apoptosis and Autophagy in the Pathogenicity of Sclerotinia sclerotiorum. PLoS. Pathog. 9(4), e1003287 (2013).
Zhao, Y,Y. Traditional use, phtochemistry, pharmacology, pharmacokinetics and quality control of Polyporus umbellatus (Pers.) Fries: A review. J. Ethnopharmacol. 149, 35-48 (2013).
Zhao,Y.Y., Shen, X., Cheng, X.L., Wei, F., Bai, X. & Lin, R.C. Urinary metabonomics study on the protective effects of ergosta-4,6,8(14),22-tetraen-3-one on chronic renal failure inratsusing UPLCQ-TOF/MS and a novel MSE data collection technique. Process. Biochem. 47, 1980–1987 (2012).
Zhou, W,W. Studies on the constituents of scleotia and fermented mycelia of Polyporus umbellatus and the quality analysis. Thesis of Doctor’s degree, Chinese Academy of Medicinal Sciences and Peking Union Meidical College. 113-124 (2008).
Xing, Y.M., Yin, W.Q., Liu, M.M., Wang, C.L. and Guo, S.X. Oxalic acid and sclerotial differentiation of Polyporus umbellatus. Sci. Rep. 5, 10759 (2015).
Liu, Y.Y. & Guo, S,X. Involvement of Ca²⁺ channel signaling in Sclerotial formation of Polyporus umbellatus. Mycopathologia. 169,139–150 (2010).
Demidchik, V. & Shabala S. Mechanisms of cytosolic calcium elevation in plants: the role of ion channels, calcium extrusion systems and NADPH oxidase-mediated ROS-Ca²⁺ Hub. Funct. Plant. Biol. 45, 9-27 (2018).
Jha. S.K., Sharma, M. & Pandey, G.K. Role of cyclic nucleotide gated channels in stress management in plants. Curr Genomics. 17, 315-329 (2016).
Liu, M.M., Xing, Y.M., Zhang, D.W. & Guo, S.X.. Transcriptome analysis of genes involved in defence response in Polyporus umbellatus with Armillaria mellea infection. Sci Rep. 5, 16075 (2015).
McLamore, E.S., Jaroch, D., Chatni, M.R. & Porterfield, D.M. Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems. Planta. 232, 1087–1099 (2010).
Rollins, J,A. & Dickman, M.B. pH signaling in Sclerotinia sclerotiorum: identification of a pacC/RIM1 homolog. Appl. Environ. Microbiol. 67,75-81 (2001).
Atallah, O. & Yassin, S. Aspergillus spp. eliminate Sclerotinia sclerotiorum by imbalancing the ambient oxalic acid concentration and parasitizing its sclerotia. Environ Microbiol. online ahead of print, doi: 10.1111/1462-2920.15213. (2020).
Ali, S., Khan, A.S., Anjum, M.A., Nawaz, A., Nawaz, A., Naz, S., Ejaz, S. & Hussain, S. Effect of postharvest oxalic acid application on enzymatic browning and quality of lotus (Nelumbo nucifera Gaertn.) root slices. Food. Chem. 312, 126051 (2020).
Liu, Y.D., Yu, S.L., Wang, R., Liu, J.N., Jin, Y.S. & Li, Y.F. An RH. Rosiglitazone Suppresses Calcium Oxalate Crystal Binding and Oxalate-Induced Oxidative Stress in Renal Epithelial Cells by Promoting PPAR-γ Activation and Subsequent Regulation of TGF-β1 and HGF Expression. Oxid. Med. Cell. Longev. published online doi: 10.1097/JU.0000000000000997.02 (2020).
Im. K.H., Baek, S.A., Choi, J. & Lee, T.S. Antioxidant, Anti-Melanogenic and Anti-Wrinkle Effects of Phellinus vaninii. Mycobiology. 47(4), 494–505 (2019).
Kadota, Y., Shirasu, K. & Zipfel, C. Regulation of the NADPH oxidase RBOHD during plant immunity. Plant. Cell. Physiol. 56, 1472–1480 (2015).
Xing, Y.M., Chen, J., Song, C., Liu, Y.Y., Guo, S.X. & Wang, C.L. Nox gene expression and Cytochemical localization of hydrogen peroxide in P. umbellatus sclerotial formation. Int. J. Mol. Sci. 14, 22967-22981 (2013).
Waterham, H.R., Russell, K.A., Vries, Y.D. & Cregg, J.M. Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris. J. Cell. Biol. 139 (6), 1419–1431 (1997).
Labrou, N.E., Papageorgiou, A.C., Pavli, O. & Flemetakis, E. Plant GSTome: structure and functional role in xenome network and plant stress response. Curr. Opin. Biotechnol. 32, 186–194 (2015).
Estévez, I.H. & Hernández, M.H. Plant Glutathione S-transferases: An overview. Plant. Gene. 23, 100233 (2020).
Islam, T., Ganesan, K. & Xu, B.J. New Insight into Mycochemical Profiles and Antioxidant Potential of Edible and Medicinal Mushrooms: A Review. Int. J. Med. Mushrooms. 21(3), 237-251 (2019).
Patsoukis, N. & Georgiou, C.D. Thiol redox state and related enzymes in sclerotium-forming filamentous phytopathogenic fungi. Mycol. Res. 112, 602-610 (2008).
Giannakopoulos, E. et al. Long-Term Preservation of Total Phenolic Content and Antioxidant Activity in Extra Virgin Olive Oil: A Physico-biochemical Approach. Free Radicals Antioxidants. 10(1), 4-9 (2020).
Kawahara, T. & Lambeth, J.D. Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes. BMC. Evol. Biol. 7, 178 (2007).
Di Palmaa, A.A. et al. Nitro-oleic acid triggers ROS production via NADPH oxidase activation in plants: A pharmacological approach. J. Plant. Physiol. 153128, 246–247 (2020).
Xing, Y.M., Zhao, M.M., Guo, L.C., Li, B., Chen. J. & Guo, S.X. Identification and expression of DoCCaMK during Sebacina sp. symbiosis of Dendrobium officinale. Sci. Rep.10, 9733 (2020).
Cui, Y.N. et al. Transcriptomic Profiling Identifies Candidate Genes Involved in the Salt Tolerance of the Xerophyte Pugionium cornutum. Genes. 10, 1039 (2019).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome. Biol. 11, R106 (2010).
Xing, Y.M. et al. Sclerotial formation of Polyporus umbellatus by Low Temperature Treatment under Artificial Conditions. Plos. One. 8(2), e56190 (2013).

Table 1. Summary of quality evaluation of sequencing output data

Sample	Raw reads	Clean reads	Clean bases	Error (%)	Q20 (%)	Q30 (%)	GC (%)
PU_S1^a	48862698	47515246	7.13G	0.02	96.46	91.35	55.30
PU_S2^a	55825490	54510292	8.18G	0.02	96.61	91.62	55.54
PU_S3^a	45034602	43841868	6.58G	0.02	96.06	90.70	55.78
PU_SM1^b	44804502	43382478	6.51G	0.02	96.47	91.51	55.95
PU_SM2^b	46263970	44731548	6.71G	0.02	96.13	90.85	55.45
PU_SM3^b	47793444	46365222	6.95G	0.02	96.46	91.44	55.51
PD_S1^c	43868088	42749076	6.41G	0.02	96.65	91.81	55.14
PD_S2^c	44566228	43376858	6.51G	0.02	96.58	91.66	55.13
PD_S3^c	47705028	46319972	6.95G	0.02	96.51	91.50	56.66
Total	424814050	412792560	61.93G

^aPU_S1, PU_S2 and PU_S3: the mycelia of P. umbellatus in the control group. ^bPU_SM1, PU_SM2 and PU_SM3: the mycelia of P. umbellatus in the low oxalic acid group. ^cPD_S1, PD_S1 and PD_S3: the mycelia of P. umbellatus in the high oxalic acid group.

Table 2. Gene function annotation analysis in 7 public databases

	Number of Unigenes	Percentage (%)
Annotated in NR	10914	48.45
Annotated in NT	12864	57.11
Annotated in KO	4063	18.03
Annotated in SwissProt	11236	49.88
Annotated in PFAM	10571	46.93
Annotated in GO	10645	47.26
Annotated in KOG	6934	30.78
Annotated in all Databases	2469	10.96
Annotated in at least one Database	18855	83.71
Total Unigenes	22523	100

Table 3. The DEGs of Oxidase and dehydrogenase (partly).

DEGs NO. Gene ID	Annotation of enzyme	Compared groups	adjusted p-value	Log2 Fold change	Up or down regulated
c3315_g1	NADPH oxidase	CK vs. PD_S	0.036757	0.62	up
c8851_g1	NADPH oxidase	CK vs. PD_S	0.000177	2.02	up
c43_g1	cytochrome c oxidase subunit 1	CK vs. PD_S	1.1009E-40	7.31	up
c159_g1	cytochrome c oxidase subunit 2	CK vs. PD_S	1.8551E-20	7.56	up
c13857_g1	cytochrome c oxidase subunit 3	CK vs. PD_S	1.25E-18	7.44	up
c8987_g1	Alcohol oxidase	CK vs. PU_SM	3.22E-10	2.87	down
c8176_g4	Alcohol oxidase	CK vs. PD_S	1.84E-05	1.14	up
c17518_g1	NADH dehydrogenase subunit 1	CK vs. PD_S	9.20E-13	6.82	up
c1494_g1	NADH dehydrogenase	CK vs. PD_S	2.84E-30	6.77	up

Table 4. DEGs associated with energy metabolism

Annotation of enzymes	DEGs NO.	Compared groups		adjusted p-value	Log2 Fold change	Up or down regulated
L-rhamnose 1-dehydrogenase	c4703_g1		CK vs PU_SM	0.00060777	3.63	down
L-rhamnose 1-dehydrogenase	c4703_g1		CK vs PD_S	5.43E-09	3.68	up
L-threo-3-deoxy-hexylosonate aldolase	c7929_g2		CK vs PU_SM	0.00054459	1.83	up
Rhamnogalacturonase	c4388_g1		CK vs PD_S	0.033252	1.75	up
rhamnogalacturonan acetylesterase	c4271_g		CK vs PD_S	7.50E-12	2.69	up
Isocitrate lyase	c7209_g1		CK vs PU_SM	1.54E-13	5.12	down
Fatty acid hydroxylase (cytochrome P450)	6742_g2		CK vs PU_SM	3.53E-24	4.87	down
Fatty acid synthase	c3295_g1		CK vs PD_S	0.0034424	0.80	up
Fatty acid synthase	c4529_g1		CK vs PD_S	0.04753	0.70	up
fatty acid desaturase	c9487_g1		CK vs PD_S	0.0019857	1.76	up

Table 5. DEGs related to Calcium signaling pathway

DEGs No.	Annotation of enzyme	Compared groups	Adjusted p-value	Log2 Fold change	Up or down regulated
c8739_g2	calmodulin	CK vs. PU_SM	0.023908	1.413	down
c2043_g1	calmodulin	CK vs. PD_S	0.0001615	Inf	up
c3257_g1	calmodulin	CK vs. PD_S	0.0001615	Inf	up
c4558_g1	calmodulin-binding motif	CK vs. PU_SM	0.00019948	-Inf	down
c8133_g1	calmodulin-binding motif	CK vs. PD_S	0.00031588	1.09	up
c9830_g2	calcium ion transport	CK vs. PU_SM	0.0090109	1.44	down
c3191_g1	cellular calcium ion homeostasis	CK vs. PD_S	3.0275E-26	2.49	up
c6291_g1	calcium ion transport	CK vs. PD_S	0.001211	0.93	up

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Transcriptomic Profiling Identifies Candidate Genes Involved in Polyporus Umbellatus Sclerotial Formation Affected by Oxalic Acid

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