Sclerotium and mycelium of W. cocos (Figure 1) collected from Yingshan county, Hubei province, China (Shu et al., 2013) were kindly donated by Dr. Shaohua Shu at the Huazhong Agricultural University. W. cocos mycelium was grown on a cellophane membrane place on the surface of potato dextrose agar (PDA) medium at 28°C for 7 days. Both the mycelium and sclerotium were frozen in liquid nitrogen and stored at -80°C for total RNA extraction.

RNA-seq data from two samples of mycelium and sclerotium was from a previous study (Shu et al., 2013) and is accessible under accession number SRP018935 at NCBI¹. Genome data was retrieved from the JGI database².

Homologous sequence searches were performed with BLASTx against the S. cerevisiae database ³ and S. sclerotiorum database⁴ (≥e^-5) as described (Hegedus et al., 2016; Zhang et al., 2016).

To identify and classify the PKs, the protein sequences from W. cocos were searched against the Kinomer v.1.0 HMM library by the using of HMMSCAN program from the HMM software suite HMMer, and the cut off value was set to 20 as previously described (Miranda-Saavedra and Barton, 2007; Kosti et al., 2010; Wang et al., 2011; Zhang et al., 2016).

The RPKM method (Reads per kb per Million reads) was used to calculate RNA levels as previously described (Zhang et al., 2016; Mortazavi et al., 2008; Shu et al., 2013). To identify differentially expressed genes (DEGs) in mycelium and sclerotium, the statistical method of false discovery rate (FDR) was employed to correct the threshold of P-value in multiple tests. Those DEGs with a ratio ≥ 2 and an FDR ≤ 0.001 were chosen for this study. As previously described, the DEGs were analyzed with DEGSeq (Wang et al., 2010).

Total RNA of mycelium and sclerotium were extracted with TriZOL reagent (Invitrogen^TM, Carlsbad, CA, USA) and treated with RNase-free DNase I (Qiagen Inc, Duesseldorf, Germany) to remove residual DNA according to manufacturer’s protocols. The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems^TM, Foster, CA, USA) was used to generate the first strand cDNA according to the manufacturer’s instructions. Gene expression was analyzed by qRT-PCR using a Bio-Rad CFX96 Real Time System (Bio-Rad, America) and SYBR Premix Ex Taq II (TAKARA, Dalian, China), according to the manufacturer’s instructions. The PCR conditions were as follows: denaturation at 95°C for 3 min; 40 cycles of 95°C for 15 s, 55°C for 15 s and 72°C for 30 s; final step of 72°C for 10 min. The primers for qRT-PCR are listed in Table 1. The mRNA levels of the W. cocos alpha-tubulin gene (Shu et al., 2013; Zhang et al., 2016) were used to normalize the data for each qRT-PCR run. For each gene, qRT-PCR assays were repeated at least three times, with each repetition having three technical replicas.

Our search of the W. cocos genome sequence identified a total of 103 putative PKs, 87 of them with significant similarity (≥e^-5) to one or more of the 92 S. sclerotiorum and/or 115 S. cerevisiae PKs (Supplementary Table S1) (Hunter and Plowman, 1997; Hegedus et al., 2016). On the basis of conserved residues, 11 candidates belonged to the atypical PKs, 13 to the AGC Group, 10 to the CAMK Group, 2 candidates to the CK Group, 22 candidates to the CMGC Group, 11 to the STE Group, 10 to the TLK Group, and 24 to the Other Group (Supplementary Table S1). Compared with S. cerevisiae, W. cocos is predicted to have fewer PKs genes. It lacks orthologs of 30 S. sclerotiorum and/or S. cerevisiae PKs, including DBF2/4, ELM1, ALK1/2, PTK1/2, NPR1, HSL1, ISR1, YGR052W, YLR253W, YMR291W, HAL5, KKQ8, NNK1, PRR1/2, YPL150W, KCC4, MEK1, DUN1, KSP1, CAK1, SRB10, SSK2/SSK22, MPS1, PSK1/2, BUD32, TOS3, SAK1, SCY1, IKS1/2, and RAD53 (Supplementary Table S1). Moreover, W. cocos has two orthologs of CBK1 (Wolco1| 74908 and Wolco1| 70675), COQ8 (Wolco1| 138133 and Wolco1| 144299), PKP1 (Wolco1| 110818 and Wolco1| 92641), TEL1 (Wolco1| 108405 and Wolco1| 91919), VHS1 (Wolco1| 106038 and Wolco1| 155089), CDC28 (Wolco1| 91184 and Wolco1| 93694), SAT4 (Wolco1| 159823 and Wolco1| 134885), EVN7 (Wolco1| 137941 and Wolco1| 132412), ATG1 (Wolco1| 20796 and Wolco1| 109315), SPS1 (Wolco1| 95124 and Wolco1| 111584) and three orthologs of YAK1 (Wolco1| 64270, Wolco1| 97250 and Wolco1| 75698), SKY1 (Wolco1| 159141, Wolco1| 92467 and Wolco1| 95712), Pan3p (Wolco1| 164201, Wolco1| 138179 and Wolco1| 73826) (Supplementary Table S1). W. cocos also contains 16 putative PKs that have no distinct orthologs in S. cerevisiae and/or S. sclerotiorum.

The W. cocos AGC Group has 13 members. The PKs from the AGC Group are important in the regulation of signaling pathways in response to cell wall or membrane stress and limited nutrients. In S. cerevisiae, in response to limited nutrients and other stresses, the activity of YPK1/2, SCH9, RIM15, DBF2/20, TPK1/2 (PKA catalytic subunit) and PKC1 are directly or indirectly regulated by the atypical PKs TOR1 and TOR2 (the target of rapamycin; Jacinto and Lorberg, 2008; Turrà et al., 2014).

Like many other filamentous fungi, W. cocos contains two genes, Wolco1| 115209 and Wolco1| 107737, that encode the catalytic subunits of PKA, similar to S. cerevisiae TPK1 and TPK2, and S. sclerotiorum SS1G_03171 and SS1G_13577, respectively. In Fusarium graminearum, deletion of the PKA-encoding gene cpk1 causes significantly decreased vegetative growth, conidiation and deoxynivalenol production. Furthermore, the cpk1 mutant was also defective in ascospore maturation and releasing. In contrast, the mutant of another PKA-encoding gene, cpk2, had no detectable phenotypes (Hu et al., 2014). In S. cerevisiae, given elevated glucose levels, the activated PKA regulates SNF1 (Wolco1| 160811) activity, thereby regulating downstream signaling pathways (Barrett et al., 2012). In S. sclerotiorum, deletion of SS1G_03171 did not result in an altered phenotype (Jurick et al., 2004), indicating the functional redundancy of the PKA. In the present study, mRNA levels originating from W. cocos PKA Wolco1| 107737, which is homologous to S. cerevisiae TPK2 (NP_015121.1) and S. sclerotiorum SS1G_13577, increased at the sclerotia stage. However, the mRNA level of the other PKA, Wolco1| 115209, which is homologous to S. cerevisiae TPK1 (NP_012371.2) and S. sclerotiorum SS1G_03171, did not change (Supplementary Table S1), indicating that Wolco1| 107737, but not Wolco1| 115209, may play a major role in the sclerotial development of W. cocos. The aurora kinase IPL1 (NP_015115.1) of S. cerevisiae and Fg06959 of F. graminearum are essential; their deletion is lethal (Wang et al., 2011). However, in W. cocos, there was no difference in mRNA levels for this kinase between the mycelium and sclerotia stages (Supplementary Table S1), indicating that W. cocos IPL1 may not be involved in sclerotial formation.

Wolfiporia cocos Wolco1| 71589 and Wolco1| 24527 encode PKs orthologous to S. cerevisiae SCH9 and RIM15, respectively. SCH9 is structurally related to PKA and works with PKA to negatively control RIM15 activity, thereby regulating the response to nutrient starvation or stress in yeast (Roosen et al., 2005). In F. graminearum, Fgsch9 (Fg00472) is involved in both deoxynivalenol (DON) production and growth, and Fgrim15 (Fg01312) plays important roles in DON production and conidiation (Wang et al., 2011). In the current study, W. cocos SCH9 (Wolco1| 71589) demonstrated higher mRNA levels in the sclerotial than the mycelial stage. In contrast, mRNA levels for W. cocos RIM15 (Wolco1| 24527) decreased at the sclerotial stage (Supplementary Table S1).

Several AGC Group PKs, including YPK1/YPK2, PKC1 and SCH9, are phosphorylated and activated by PHK1/PHK2 (Roelants et al., 2004). In S. cerevisiae, YPK1/YPK2 are involved in cell-wall integrity (Roelants et al., 2002), and YPK1 also phosphorylates and down regulates Fpk1 kinase activity (Roelants et al., 2010). In F. graminearum, deletion of Fgfpk1 (Fg04382) results in a reduced growth rate and increased sensitivity to osmotic and oxidative stress (Wang et al., 2011). PKC1 (PKC) targets BCK1 and regulates cell-wall integrity SLT2 MAPK (Reinoso-Martín et al., 2003; Turrà et al., 2014), and PKC1 is an essential gene in both F. graminearum and yeast. CBK1 is essential in wild-type S. cerevisiae strains and is involved in the regulation of polarized growth and cell-wall integrity by regulating the activity of SDP1 (Kurischko et al., 2005; Kuravi et al., 2011). In F. graminearum, the growth rate of CBK1 (Fg01188) mutants decreased by more than 90% and mutant strain formed compact colonies (Wang et al., 2011) (Table 2). In W. cocos, there are two PKs; one of them (Wolco1| 70675) is highly similar to the yeast CBK1 (NP_014238.3, E-value: 0.00E), indicating their potential roles in growth and cell-wall integrity (Table 2).

The W. cocos CAMK Group has 10 members, eight of which are similar to S. cerevisiae CAMK-like (CAMKL) kinases (Supplementary Table S1; Table 3). Wolco1| 160811 encodes sucrose non-fermenting (SNF1) kinase, and its homolog in S. sclerotiorum and S. cerevisiae are involved in carbon catabolite repression (Sanz, 2003; Vacher et al., 2003). In the plant pathogenic fungus Gibberella zeae, GzSNF1 is important for vegetative growth, sexual reproduction and pathogenesis (Lee et al., 2009). Wolco1| 19611 is most similar to S. cerevisiae KIN1, which regulates exocytosis (Elbert et al., 2005). The homolog of Wolco1| 153019 in F. graminearum Fgkin4 is involved in growth, septum formation, conidiation and sexual reproduction (Wang et al., 2011), and its homolog in S. cerevisiae KIN4 plays a key role in the regulation of the spindle position checkpoint of cells exiting mitosis (Caydasi et al., 2010). Kinase Chk1 functions in DNA damage checkpoint in eukaryotes (Sanchez et al., 1999). In F. graminearum, FgChk1 (Fg01506) is important for DNA damage repair but not necessary for pathogenesis deletion (Wang et al., 2011). Wolco1| 137455 encodes kinase similar to S. sclerotiorum SS1G_06203, and S. cerevisiae RCK2. RCK2 is targeted by HOG1 and links to the pheromone response and hyperosmotic stress via MAPK pathways (Nagiec and Dohlman, 2012).

The W. cocos CMGC Group has 22 members, including the homolog of MAPKs (Supplementary Table S1; Table 4). MAPK cascades and MAPK signaling pathways are known to be involved in many major cell processes in fungi (Turrà et al., 2014). We found three MAPKs in W. cocos. Wolco1| 95503 orthologous to the HOG1-style MAPK in S. sclerotiorum (SS1G_07590) and S. cerevisiae (NP_013214.1) with high similarity. In B. cinerea, the closely related phytopathogen of S. sclerotiorum, the HOG1 ortholog is involved in osmotic stress, oxidative stress, conidia formation and sclerotial development (Segmüller et al., 2007). Wolco1| 107181 is orthologous to the FUS3-style MAPK SMK1 (SS1G_11866) in S. sclerotiorum. Silencing of SMK1 in S. sclerotiorum results in impaired sclerotial formation (Chen et al., 2004). Wolco1| 92114 encodes a kinase similar to S. sclerotiorum SMK3 (SS1G_06203) and S. cerevisiae SLT2-style MAPK (NP_011895.1). Deletion of SMK3 in S. sclerotiorum inhibits the production of sclerotia (Bashi et al., 2010). Deletion of the B. cinerea bmp3 gene encoding a homolog of the yeast MAPK Slt2 also results in the loss of sclerotial formation (Rui and Hahn, 2007).

Besides MAPKs, other members of CMGC Group kinases also play important roles in many major cell processes. Wolco1| 91184 and Wolco1| 93694 are the orthologs of yeast CDC28 (NP_009718.3) that regulate cell division in eukaryotes (Malumbres, 2014). Wolco1| 99718 is the ortholog of yeast CTK1(NP_012783.1) that is involved in sexual reproduction in yeast. In F. graminearum, orthologs of CTK1 Fgctk1 are involved in growth, conidiation, sexual reproduction and infection (Wang et al., 2011). W. cocos Wolco1| 93172 encodes PKs orthologous to S. cerevisiae PHO85. Deletion of PHO85 is not lethal in S. cerevisiae, whereas its ortholog is essential in Ustilago maydis and Cryptococcus neoformans (Castillo-Lluva et al., 2007; Virtudazo et al., 2010). In yeast, PHO85 is involved in the regulation of cell division in response to nutrient levels and environmental stresses (Nishizawa, 2015). In Aspergillus nidulans, the homologs of PHO85 play an essential role in cell cycle control and morphogenesis (Dou et al., 2003).

Wolfiporia cocos has three orthologs of YAK1 (Wolco1| 64270, Wolco1| 97250 and Wolco1| 75698). The YAK1 members control cell proliferation, differentiation and homeostasis (Aranda et al., 2011). In F. graminearum, the yak1 mutant is more sensitive to H₂O₂ than the wild type strain (Wang et al., 2011). Wolco1| 64147 is an ortholog of yeast IME2 (NP_012429.1), which is essential for meiosis initiation (Honigberg, 2004). It also regulates spore formation in response to nutrient levels and cAMP (McDonald et al., 2009). W. cocos has three orthologs of SKY1 (Wolco1| 159141, Wolco1| 92467 and Wolco1| 95712). SKY1 is a serine-arginine PK that is involved in metabolic signaling, cell-cycle regulation and chromatin reorganization (Giannakouros et al., 2011). In F. graminearum, the sky1 mutant was reduced in hyphal branching and produced fewer aerial hyphae (Wang et al., 2011).

The W. cocos STE Group contains 11 members (Supplementary Table S1; Table 5). Wolco1| 166770, Wolco1| 43954 and Wolco1| 150169 are the orthologs of yeast STE20, STE11, and STE7, respectively. In the MAPK cascade, MAPKKs (STE7) phosphorylate MAPKs, MAPKKKs (STE11) phosphorylate MAPKKs (STE7) and STE20 is the upstream kinase (Gustin et al., 1998; Chen and Thorner, 2007). Deletion of STE7 and STE11 orthologs in B. cinerea results in reduced growth and virulence (Schamber et al., 2010). The BMP1 MAPK mutant of B. cinerea was unable to form sclerotia and exhibited decreased virulence (Doehlemann et al., 2006). Wolco1| 75024 is orthologous to S. cerevisiae MKK2 MAPKKs, which act in the upstream SLT2-style MAPK pathway and activate this signaling pathway. Wolco1| 144702 is orthologous to S. cerevisiae PBS2 MAPKKs, which act upstream of the HOG1-style MAPK pathway. Wolco1| 135363 is orthologous to S. cerevisiae BCK1, which acts upstream of the SLT2-style MAPK pathway (Turrà et al., 2014). Finally, the CLA4 orthologs in M. grisea and Claviceps purpurea are involved in pathogenicity, mycelial growth and conidial morphology (Li et al., 2004; Rolke and Tudzynski, 2008).

There are only two members of the W. cocos CK Group (Supplementary Table S1; Table 6). Wolco1| 22440 is orthologous to S. cerevisiae HRR25. HRR25 is a multifunctional kinase that is involved in autophagy and endocytosis (Tanaka et al., 2014; Peng et al., 2015). The other CK member, Wolco1| 152548, encodes a PK similar to the S. cerevisiae PKs YCK1/YCK2. YCK1/YCK2 are membrane-localized kinases that phosphorylate another membrane anchor protein OPY2 and active the HOG1 signaling pathway in response to high-glucose conditions (Yamamoto et al., 2010).

There are 10 members in the W. cocos TKL Group. However, their homologous PKs are not typically found in S. sclerotiorum and S. cerevisiae, indicating that the TKL Group may only exist in higher fungi and eukaryotes.

There are 24 PKs members in Other Group that do not cluster into the major groups described above (Supplementary Table S1; Table 7). Wolco1| 159823 and Wolco1| 134885 are orthologous to S. cerevisiae SAT4, which is involved in regulating permeases activity and salt tolerance (Mulet et al., 1999; Pérez-Valle et al., 2010). In F. graminearum, the Fgsat4 mutant was more sensitive to 0.7 M NaCl but more tolerant to 0.7 M KCl (Wang et al., 2011). HRK1 (Wolco1| 142396) contributes to regulating membrane ATPase activity, which is important for glucose uptake (Goossens et al., 2000). Wolco1| 19350 is orthologous to SKS1, which is involved in the response to glucose and hyphal development (Johnson et al., 2014). BUB1 (Wolco1| 144048) and ARK1 (Wolco1| 130299) are involved in the formation of the mitotic spindle pole, cytokinesis, chromosome orientation and separation (Leverson et al., 2002; Storchová et al., 2011). There are six members in this group; however, their homologous PKs are not typically found in S. sclerotiorum and S. cerevisiae.

Nine W. cocos PKs whose orthologs in other fungi are involved in sclerotial formation were chosen for a validation procedure of their RNA levels were determined via qRT-PCR. The results revealed that mRNAs from genes encoding TPK2 (Wolco1| 107737), HOG1 (Wolco1| 95503), FUS3 (Wolco1| 107181), SLT2 (Wolco1| 92114), STE11 (Wolco1| 43954) and STE7 (Wolco1| 150169) were present in higher amounts during the sclerotial stage; CLA4 (Wolco1| 121529), STE20 (Wolco1| 166770), and MKK2 (Wolco1| 75024) mRNAs were present in lower amounts (Figure 2). These results are consistent with the de novo transcriptome sequencing data (Supplementary Table S1), indicating that W. cocos PKs regulate sclerotial formation in both positive and negative ways.