The strain of S. schenckii used, ATCC10268, was maintained at the Research Center for Pathogenic Fungi, Liaoning University, China. To obtain a mycelial culture, the ATCC10268 isolate was inoculated onto Sabouraud dextrose agar (SDA) solid medium (10 g/l tryptone, 40 g/l glucose) and incubated at 25°C. The mycelial colonies subsequently obtained were inoculated in liquid Sabouraud medium and cultured with shaking at 100 rpm at 25°C for 48 h. To induce the switch of S. schenckii from the mycelial phase to the 48-h induced yeast phase, mycelial culture was enriched and transferred to brain-heart infusion (BHI) liquid medium (HyClone; GE Healthcare Life Sciences) (18–23), which was incubated at 37°C and shaken at 100 rpm for 48 h.

S. schenckii cultures grown in SDA at 25°C or in BHI at 37°C for 48 h were collected, and the cell suspensions were fixed at 4°C by addition of an equal volume of fixing solution (0.2 M Na₂HPO₄, 0.2 M NaH₂PO₄) for 2–4 h. Cells were transferred to a centrifuge tube and spun at 12,000 × g for 15 min at 4°C to obtain a cell pellet, which was embedded in 1% agarose and then washed in 0.1 M PBS three times for 15 min each. Post-fixation staining was carried out with 1% OsO₄ in 0.1 M PBS (pH 7.4) for 2 h at room temperature, followed by removal of OsO₄, rinsing in 0.1 M PBS (pH 7.4) three times, and dehydration. Following infiltration and embedding the cell pellet, ultrathin sections (60–80 nm) were cut with an ultramicrotome. Sections were stained with uranyl acetate in pure ethanol for 15 min at 4°C rinsed with distilled water, stained with lead citrate for 15 min at 4°C, and rinsed with distilled water. The sections were allowed to air-dry overnight and were then observed using TEM (24). The thicknesses of the inner and outer layers of the mycelial cell wall were measured using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

S. schenckii cultured in SDA at 25°C or in BHI at 37°C for 48 h were smeared onto slides and stained with fungal fluorescence dyes (Lifetime bio) that bind specifically to chitin and cellulose for 15 min at 4°C. The slides were visualized using a Nikon 2000 fluorescent microscope (Nikon Instruments, Inc.) with a ×20 objective. For each slide, 10 microscopic fields were examined and the fields with median fluorescent brightness selected for comparation. Images were processed with NIS-Elements (version 4.10; Nikon Instruments, Inc.) imaging software.

S. schenckii in mycelial and 48-h induced yeast forms were collected for RNA extraction. Total RNA was extracted using RNAiso™ plus (Takara Bio, Inc.) and treated with RNase-free DNase I (Fermentas; Thermo Fisher Scientific, Inc.) to remove residual DNA. The quantity of RNA was determined using NanoDrop ND-1000 (Thermo Fisher Scientific, Inc.) and 1.2% agarose gels. The integrity of the total RNA was assessed using an Agilent 2200 Tape Station (Agilent Technologies, Inc.), and each sample had an RNA integrity number >7.5.

cDNA libraries were constructed using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina (Illumina, Inc.) according to the manufacturer's protocols. After the total RNA was extracted, the mRNA was purified using poly-T oligo-attached magnetic beads and fragmented into small pieces in fragmentation buffer from the kit. Using mRNA as a template, first-strand cDNA was synthesized using random primers and reverse transcriptase. Second-strand cDNA was synthesized using RNase H and DNA polymerase I (Takara Bio, Inc.) (25). Subsequently, the cDNA was purified, and subjected to end-repair, poly(A) addition, sequencing, adapter connecting and fragment size selection. Finally, the purified cDNA was PCR amplified and sequenced on the Illumina HiSeq™ Xten platform (Guangzhou RiboBio Co., Ltd.).

After analyzing the base composition and quality value, the sequencing data were filtered according to the raw data analysis results to remove the adaptor sequences, contaminated parts and low-quality reads to obtain clean reads. Next, clean reads were de novo assembled using Trinity (version no. v2013-02-25; Trinity Software, Inc.). Trinity software consists of three independent software modules, Inchworm, Chrysalis and Butterfly, which are used in turn to process large-scale RNA-Seq read data and obtain unigene sequences.

After assembly, the unigene sequences were annotated into the NCBI non-redundant (NR; http://www.ncbi.nlm.nih.gov/), Swiss-Prot (http://www.expasy.ch/spot/), Clusters of eukaryotic Orthologous Group (KOG; http://www.ncbi.nlm.nih.gov/cog/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg) databases using the BLASTALL package (release 2.2.28) from NCBI with an E-value ≤10⁻⁵. Based on NR annotation, GO functional annotation and further functional classification were performed using Blast2GO (http://www.blast2go.com/b2ghome).

Bowtie2 (version no. 2.2.5) was used to map the clean reads back to the unigenes, and the unigene mapping rate was counted. The unigene expressed values and transcript levels were calculated by the fragments per kilobase of transcript per million mapped reads (FPKM) method using the mapping results (26). EdgeR (version no. 3.10.0) was used to calculate the expression difference, and multiple hypothesis testing was performed to correct the P-value of the difference test. The threshold P-value was determined by controlling the false discovery rate (FDR). When the FDR value of the difference test was obtained, the differential expression multiples of the gene in different samples were calculated according to the amount of gene expression, using |log₂FC| ≥1, and FDR <0.05 as the threshold for screening differential genes (27).

The hypergeometric distribution test was used for GO classification and KEGG analysis of the DEGs to understand the functional properties of the genes and regulatory pathways involved. The obtained GO categories with P<0.05 and pathways with q-value ≤0.05 were defined as significantly enriched GO classifications and KEGG pathways (28).

One-way analysis of variance was performed using SPSS 17.0 (SPSS, Inc.). Differences were tested with Duncan's test, unless otherwise specified.

The structural organization of hyphal and conidial cell walls of S. schenckii in the mycelial and 48-h induced yeast forms was studied using high-pressure freezing TEM (HPF-TEM). Fewer hyphal fragments and larger conidia were observed when comparing the character of 48-h induced yeast cells with that of mycelial cells scanned with HPF-TEM. The cell wall of S. schenckii was observed as having a double-layered structure, with a low electron-density inner layer and a high electron-density outer layer, decorated with thin fibrils. The inner layer thicknesses of the S. schenckii mycelial cell wall was ~102 nm, thinner than the 151 nm in 48-h induced yeast cells, which decreased the total thickness of the mycelial cell wall. Meanwhile, compared with the 48-h induced yeast form, the mycelial cell walls showed a thicker outer layer densely covered with fine fibrils (Fig. 1).

To confirm the difference in cell wall structure between the mycelial and 48-h induced yeast forms, the cells were stained with Calcofluor White, targeting the chitin in the cell wall of S. schenckii. The fluorescence pattern observed in 48-h induced yeast cells, compared with that in mycelial cells, had stronger chitin and cellulose labeling patterns on the 48-h induced yeast cell surface (Fig. 2).

Following sequencing two samples of the mycelial and 48-h induced yeast phases using the Illumina platform, the mycelial phase samples were sequenced to obtain 27,465,858 reads and 4.12 G data were obtained, in which the Q20 and Q30 base proportions were 95.70 and 90.91%, respectively. The GC content proportion was 54.84% and the N ratio in sequencing was 0.33%. Sequencing the 48-h induced yeast samples gave 23,746,248 reads and 3.56 G of data, with Q20 and Q30 base ratios of 97.66 and 94.69%, respectively, a GC content ratio of 46.80%, and N ratio in sequencing of 0.18%. Subsequently, quality filtration of the raw data was performed. Mycelial phase samples gave 24,904,510 clean reads, and 48-h induced yeast phase samples gave 22,814,406 clean reads (Table I).

Trinity was used to assemble high-quality sequences in the samples. Following assembly, 31,779 unigene sequences were obtained. The total number of sequences generated was 72.2 Mbp, and the N50 and N90 lengths were 4,155 and 993 bp, respectively. The Max-length and Min-length of the unigenes were 29,705 and 301 bp, respectively, and the GC content was 52.98% (Table II). The transcriptomic data supporting the results are available at NCBI under GEO accession number GSE133322.

For functional annotation analysis, BLAST was used to align the assembled unigenes with five public databases. Overall, 23,329 (73.41%) unigenes could be aligned in one or more databases: 21,079 (66.33%) unigenes were similar to proteins in the NR database and 17,398 (54.75%) were annotated in Swiss-Prot (Table III). For GO annotation, 10,542 unigenes were assigned to the ‘cellular component’ category and ‘integral membrane component’ (n=2,808), ‘nucleus’ (n=1,604) and ‘cytosol’ (n=1442) were other main subcategories. In addition, 11,834 unigenes were classified as ‘biological processes’; the three main categories were ‘metabolic process’ (n=1,373), ‘oxidation-reduction process’ (n=1,117) and ‘transmembrane transport’ (706). Furthermore, 12,149 unigenes were assigned to the ‘molecular function’ category and genes assigned to ‘ATP binding’ (n=1,588) and ‘metal ion binding’ (747) accounted for the vast majority of this category. In the KOG classification, 13,854 unigenes were categorized into 25 KOG functional groups; ‘General function prediction only’ was the largest group, followed by ‘Post-translational modification, protein turnover, chaperones’ and ‘Translation, ribosomal structure and biogenesis’. In the KEGG pathway analysis, 11,062 unigenes could be mapped to 342 metabolic pathways and the largest category was ‘Carbon metabolism’ (n=424).

The sequences of sample reads and unigenes were compared using bowtie2; this aligned 22,315,744 (89.00%) reads of the mycelial phase and 19,552,494 (85.00%) reads of the 48-h induced yeast phase to unigene sequences.

Next, DEGs were identified by comparing the FPKM values for each gene between the mycelial and 48-h induced yeast forms of S. schenckii, and DEGs between the two forms were identified. The results demonstrated that 12,217 genes were expressed differentially between the two forms, including 7,271 upregulated and 4,946 downregulated genes (Fig. 3).

To identify the major functional categories represented by DEGs, with all unigene genes as background genes, the P-value was calculated using hypergeometric distribution method, and P<0.05 was taken as the threshold to obtain significant heights relative to the background (29). Among DEGs between the mycelial and 48-h induced yeast forms, GOs associated with ATP binding, metal ion binding and zinc ion binding in the molecular function category; GOs associated with metabolic processes, oxidation reduction reactions and transmembrane transport in the biological process category; and particularly those associated with integral components of the membrane, nucleus, cytosol, cytoplasm and mitochondrion in the cellular component group were significantly changed (Fig. 4).

During the process of switching, the form and function of the cell change notably, and those changes must be supported by changes in cell components, including cytoplasmic proteins, nucleoproteins, regulatory proteins, toxic proteins and cell wall component proteins. This led to the observation that genes associated with the integral components of the membrane changed most significantly. These changes require large amounts of protein and energy for anabolism, as well as raw materials absorbed from the environment; therefore, genes associated with metabolic processes, oxidation-reduction processes, transmembrane transport, and the steps of transcription and translation, including ATP binding, metal ion binding, zinc ion binding and DNA binding, changed markedly.

To further elucidate the molecular interactions among DEGs, KEGG analysis was performed (30). Among DEGs between the mycelial and 48-h induced yeast forms of S. schenckii, pathways for oxidative phosphorylation, ribosome, the MAPK pathway, carbon metabolism, RNA transport, protein processing in endoplasmic reticulum, pyrimidine metabolism, purine metabolism, biosynthesis of amino acids, meiosis and the cell cycle were enriched (Fig. 5). During the process of switching, enormous changes must occur in the expression of the genes in these pathways, which are necessary for transcription, translation, biosynthesis and energy synthesis. Beyond those predictable changes, it was also found that numerous genes, including DRK1, Hog1, Skn7 and Ste11, which are involved in the two-component system heterotrimeric G protein, cAMP and Ras-Hog1 signaling pathways, were altered (Table IV; Fig. 6). These signal transduction pathways serve important roles during the initiation of the dimorphic switch.