Kinase Hog1 and Adr1 Opposingly Regulate Haploid Cell Morphology by Controlling Vacuole Size in Sporisorium scitamineum

Morphogenesis is a strictly regulated efficient system in eukaryotes for adapting to environmental changes. However, the morphogenesis regulatory mechanism in smut fungi is not clear. This study reports a relationship between MAP kinase Hog1 and cAMP-dependent protein kinase A catalytic subunit (Adr1) for the morphological regulation in the sugarcane pathogen Sporisorium scitamineum. The results demonstrated that MAP kinase Hog1 and cAMP/PKA signaling pathways are essential for the morphological development of S. scitamineum. Interestingly, MAP kinase Hog1 and cAMP/PKA signaling pathways’ defective mutants exhibit an opposite morphological phenotype. The morphology of cAMP/PKA defective mutants is recovered by deleting the SsHOG1 gene. However, MAP kinase Hog1 and cAMP-dependent protein kinase catalytic subunit Adr1 do not interfere with each other. Further investigations showed that kinase Hog1 and Adr1 antagonistically regulates the vacuolar size, which contributes to the cell size and determines the cellular elongation rates. Kinase Hog1 and Adr1 also antagonistically balanced the cell wall integrity and permeability. Taken together, kinase Hog1- and Adr1-based opposing morphogenesis regulation of S. scitamineum by controlling the vacuolar size and cell wall permeability is established during the study.


Introduction
Several dimorphous fungi (animal and plant pathogens) respond to environmental conditions by changing budding growth and morphologies [1][2][3]. Morphogenetic regulation has been extensively studied including cAMP/PKA (G-protein, adenylyl cyclase, and protein kinase A) and mitogen-activated protein kinase (MAPK) pathways [4]. MAPK and cAMP/PKA signaling pathways regulate physiological processes, such as stress response, morphogenesis, polarized cell growth, mating, and virulence [5][6][7][8]. Saccharomyces cerevisiae cell size generally increased in response to cAMP addition [9]. MAP kinase Hog1 also played a key role in cellular responses to external stimuli and regulated morphogenesis and stress responses [10][11][12][13]. S. cerevisiae hog1∆ mutant cells have exhibited larger shape, abnormal bud-like extensions, and complex structure under hypertonic stress [12]. In Cryptococcus neoformans, mutants lacking the adenylyl cyclase Cac1 could not form titan cells, which reveals cAMP/PKA pathway involvement in the titan cells' formation [6,14,15]. The regulation of melanin and capsule production by the opposite phenotype of Hog1 and cAMP/PKA pathways in C. neoformans serotype A has been reported [16]. Disruption of the cAMP/PKA genes resulted in a constitutive pseudohypha phenotype in Ustilago maydis [1,17,18]. Sporisorium scitamineum was a basidiomycetous bipolar fungus of sugarcane

Strains and Growth Conditions
The wild-type MAT-1 of S. scitamineum was isolated and identified from teliospores of sugarcane smut by Yan [41]. Details of strains are shown in Table 1. The haploid sporidia was allowed to grow in Yeast Extraction-Peptone-Sucrose medium (YePS; pH 6.5) at 28 • C, 200 rpm for 24 h. For cell wall perturbing agent assessment, MAT-1, ss1adr1∆, ss1hog1∆, and ss1hog1∆adr1∆ haploid sporidia were allowed to grow till O.D. 600 = 1.0 (a spectrophotometer was used to measure the absorbance of haploid sporidia at 600 nm), and then 1.5 µL of sample was inoculated on YePSA medium with or without 1.0 mg/mL Congo Red (Sigma-Aldrich (Shanghai) Trading Co.Ltd, Shanghai, China) or 0.05 mg/mL Calcofluor White (Sigma-Aldrich (Shanghai) Trading Co.Ltd, Shanghai, China).

Strains Construction
Deletion of mutants was constructed with polyethylene glycol-mediated protoplast transformation methods as previously described [43]. The target gene was replaced by the Zeocin R (ZEO R ) resistance cassette. Transformants were selected with 100 g/mL Zeocin (Invitrogen, Carlsbad, CA, USA) and were identified by PCR using genomic DNA of MAT-1, ss1hog1∆gpa3∆, ss1hog1∆uac1∆, and ss1hog1∆adr1∆ as a template and the following primers: The primer sequence is shown in Table S1. In this study, transformants were constructed in the ss1hog1∆ background.

Nucleic Acid Related Manipulation
Genomic DNA of MAT-1, ss1hog1∆, ss1hog1∆gpa3∆, ss1hog1∆uac1∆, and ss1hog1∆adr1∆ strains were isolated by following the SDS-based DNA extraction method [44]. PCR amplification was performed using the locus-specific primers (listed in Table S1) to confirm the replacement of targeted genes with the ZEO R selection marker. For Southern blot analysis, Genomic DNA of MAT-1, ss1hog1∆, ss1hog1∆gpa3∆, ss1hog1∆uac1∆, and ss1hog1∆adr1∆, and pDAN vector were digested with the restriction enzyme HindIII and the ZEO R sequence serves as the probe. For total RNA extraction, the fresh haploid was cultured in YePSA medium at 28 • C for 3 days, and then total RNA was isolated with RNeasy Mini Kit (QIAGEN, Hilden, Germany) following the established protocol [41]. For cDNA synthesis, the total RNA was used to synthesize the cDNA by HiScript ® II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The cDNA was performed by RT-qPCR analysis with Fast SYBR™ Green Master Mix (ThermoFisher Scientific, Carlsbad, CA, USA). The SsGPA3-, SsUAC1-, SsADR1-, SsHOG1-, wall integrity-and permeability-associated genes' expression level was calculated with the -∆∆Ct method [45] and used cytoskeletal protein gene GAPDH as internal control. The experiment was conducted in triplicate and for three independent biological replicates.

SsHog1 and SsAdr1 Phosphorylation Assays
The fresh haploid sporidia was cultured on YEPSA for 24 h and then the total protein was extracted as previously described [21]. Phosphorylated SsHog1 or SsAdr1 was detected by Western blot analysis with the primary antibody Phospho-p38 MAPK (Cell Signaling Technology, Boston, MA, USA) or Phospho-PKA C (Cell Signaling Technology, Boston, MA, USA). Total levels of SsHog1 or SsAdr1 were detected by probing with an anti-Hog1 or anti-Adr1 antibody (Genecreate Biological Engineering Company, Wuhan, China). Blot signals were displayed with the enhanced chemiluminescence (BIO-RAD, Hercules, CA, USA) after binding of an Anti-Rabbit IgG-Peroxidase secondary antibody (Sigma, Louis, MO, USA).

Neutral Red Stained and Microscopy
The fresh haploid sporidia were allowed to grow in YePS medium at 28 • C, 200 rpm until O.D. 600 = 1.0, and then were treated with Neutral Red. An amount of 5 µL of the sample was mounted on the slide and observed under Leica DMI8 Inverted Fluorescence Microscope with Leica DFC450 camera (Leica, Vizsla, Germany), using Leica Application Suite (LAS) X software to take picture (Scale bar = 10 µm).

Flow Cytometry Analysis
MAT-1, ss1adr1∆, ss1hog1∆, and ss1hog1∆adr1∆ haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then were treated with FITC-Dextran 70,000 (1 µM) for 5 min. Then, the haploid sporidia were washed with 1 × PBS twice and re-suspended with 1 × PBS. Flow cytometry (Beckman Coulter, Inc. Pasadena, CA, USA) analysis was performed using the fluorescein isothiocyanate (FITC) filter. The sample was independently performed with three independent biological repeats, each of which contained two replications. Data analysis was performed using the FlowJo 10 software.

Statistical Analysis
Data were expressed as mean ± standard error (SE). Differences among different treatments were analyzed using Student's t-test. The histogram was generated using GraphPad Prism 5 software.

cAMP/PKA Kinase SsAdr1 Is a Key Factor in Controlling Haploid Cell Size of S. scitamineum
The impact of the cAMP/PKA signaling pathway on morphogenesis was analyzed by culturing SsADR1 and its upstream genes' (SsGPA3 and SsUAC1) deletion mutants (details of strains are shown in Table 1) on the YePSA medium. The results showed that the morphogenesis of the cAMP/PKA defective mutant colonies was changed to the glossy surface phenotype. This phenomenon could be partially restored through the exogenous addition of cAMP in ss1gpa3∆ and ss1uac1∆ mutants as compared to wild type ( Figure 1A). However, ss1adr1∆ mutants still exhibited a distinct glossy surface, even after cAMP treatment ( Figure 1A). The disruption of the SsPRF1 gene [42] encoding the pheromone response factor and located downstream of SsADR1 shared a similar normal morphology with the wild type ( Figure 1A). The area and length of the haploid sporidia were also measured, which revealed significantly smaller cell size in cAMP/PKA defective mutants (ss1gpa3∆, ss1uac1∆, ss1adr1∆) as compared to the wild-type and complementary strains ( Figure 1B-D). These results indicated that the cell size of cAMP/PKA defective mutants was diminished by reducing the cAMP signal and was partially restored by the exogenous addition of cAMP in ss1gpa3∆ and ss1uac1∆ mutants except in the ss1adr1∆ mutant ( Figure 1B-D). Overall, the results indicated the necessity of the cAMP/PKA pathway in morphogenesis whereas SsAdr1 played a key role in the S. scitamineum morphological regulation.  MAT-1, ss1gpa3∆, ss1uac1∆, ss1adr1∆, Ss1ADR1-COM, and ss1prf1∆ haploid sporidia were cultured under YePSA medium with or without exogenetic cAMP (5 mM). Images were taken 4 days after cultivation. Scale bar = 5.0 mm. (B) The microscopic morphology of wild-type MAT-1, ss1gpa3∆, ss1uac1∆, ss1adr1∆, Ss1ADR1-COM, and ss1prf1∆ strains. MAT-1, ss1gpa3∆, ss1uac1∆, ss1adr1∆, Ss1ADR1-COM, and ss1prf1∆ haploid sporidia were allowed to grow till O.D. 600 = 1.0, and treated with or without exogenetic cAMP (5 mM) under YePSA medium at 28 • C for 24 h. Images were taken by Leica DMI8 Inverted Fluorescence Microscope. Scale bar = 10 µm. (C) The cell areas of MAT-1, ss1gpa3∆, ss1uac1∆, ss1adr1∆, Ss1ADR1-COM, and ss1prf1∆ strains were measured. MAT-1, ss1gpa3∆, ss1uac1∆, ss1adr1∆, Ss1ADR1-COM, and ss1prf1∆ haploid sporidia were allowed to grow till O.D. 600 = 1.0 with or without exogenetic cAMP (5 mM), and then images were taken by Leica DMI8 Inverted Fluorescence Microscope. Adobe Photoshop software was used to calculate the pixels of each cell. One square inch (Sq. In.) is 72 by 72 pixels. Bar chart depicts statistical difference between the mean values ( p < 0.01). The sample was independently performed with three independent biological repeats, each of which contained twenty replications. (D) The cell lengths of MAT-1, ss1gpa3∆, ss1uac1∆, ss1adr1∆, Ss1ADR1-COM, and ss1prf1∆ strains were measured. MAT-1, ss1gpa3∆, ss1uac1∆, ss1adr1∆, Ss1ADR1-COM, and ss1prf1∆ haploid sporidia were allowed to grow till O.D. 600 = 1.0 with or without exogenetic cAMP (5 mM), and then images were taken by Leica DMI8 Inverted Fluorescence Microscope and the length of the cell was measured. The unit is µm. Bar chart depicts statistical difference between the mean values ( p < 0.01). The sample was independently performed with three independent biological repeats, each of which contained twenty replications.

MAP Kinase SsHog1 Is Required for Maintaining Haploid Cell Morphology of S. scitamineum
The hog1∆ mutant cells exhibited an irregular and larger cell morphology in S. cerevisiae under hypertonic stress [12]. However, the cell morphology of ss1hog1∆ mutants (with or without hypertonic stress) in S. scitamineum were not significantly different (unshown). Nevertheless, ss1hog1∆ mutant colonies appeared bigger with a folded surface in YePSA medium, compared with the wild-type and Ss1HOG1-COM strains (Figure 2A). Furthermore, the ss1hog1∆ mutant displayed a larger cell area and longer cell length, in comparison to the wild-type and Ss1HOG1-COM strains ( Figure 2B-D). In short, SsHog1 maintained a stable S. scitamineum morphology. MAT-1, ss1hog1∆, and Ss1HOG1-COM haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then images were taken by Leica DMI8 Inverted Fluorescence Microscope. Adobe Photoshop software was used to calculate the pixels of each cell. One square inch (Sq. In.) is 72 by 72 pixels. Bar chart depicts statistical difference between the mean values ( p < 0.05). NS denotes a not statistically significant difference (p < 0.05). The sample was independently performed with three independent biological repeats, each of which contained twenty replications. (D) The cell lengths of MAT-1, ss1hog1∆, and Ss1HOG1-COM strains were measured. The fresh haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then images were taken by Leica DMI8 Inverted Fluorescence Microscope and the length of the cell was measured. The unit is µm. Bar chart depicts statistical difference between the mean values ( p < 0.01). NS denotes a not statistically significant difference (p < 0.05). The sample was independently performed with three independent biological repeats, each of which contained twenty replications.

The Deletions of SsADR1 and SsHOG1 Do Not Affect Expression of Each Other in S. scitamineum
Based on the above-mentioned results, the potential molecular interaction between SsHog1 and the cAMP/PKA pathway was investigated by analyzing the mRNA transcription and protein phosphorylation levels in ss1hog1∆ and cAMP/PKA defective mutants. The results demonstrated that the SsGPA3, SsUAC1, and SsADR1 gene transcription levels were not significantly different than wild-type and ss1hog1∆ mutants ( Figure 3A). Moreover, PKA phosphorylation levels in ss1hog1∆ mutants were almost similar to wild-type MAT-1 ( Figure 3B). Similarly, the transcription level of the SsHOG1 gene ( Figure 3C) and the phosphorylation level of SsHog1 ( Figure 3D) was not significantly different than wild-type and cAMP/PKA defective mutants. Taken together, these results suggested the lack of interference between SsHog1 and SsAdr1, and they might split for regulating the morphology. levels of genes SsGPA3, SsUAC1, and SsADR1 in the cAMP/PKA pathway were detected in the MAT-1 and ss1hog1∆. NS denotes a not statistically significant difference (p < 0.05). The sample was independently performed with three independent biological repeats, each of which contained two replications. (B) The phosphorylated levels of PKA in MAT-1, ss1adr1∆, and ss1hog1∆ strains. Total protein was extracted from the fresh haploid sporidia under YePSA medium. The phosphorylated PKA was detected with the primary antibody Phospho-PKA C and the total level of SsAdr1 was determined with anti-Adr1 antibody by Western blot analysis. (C) Transcriptional profile of SsHOG1 was detected in MAT-1, ss1gpa3∆, ss1uac1∆, and ss1adr1∆. NS denotes a not statistically significant difference (p < 0.05). The sample was independently performed with three independent biological repeats, each of which contained two replications. (D) The phosphorylated levels of SsHog1 in MAT-1, ss1hog1∆, ss1gpa3∆, ss1uac1∆, and ss1adr1∆ strains. Total protein was extracted from the fresh haploid sporidia under YePSA medium. The phosphorylated Hog1 was detected with the primary antibody Phospho-p38 MAPK (Thr180/Tyr182) and the total level of SsHog1 was determined with anti-Hog1 antibody by Western blot analysis.

Deletion of SsHOG1 Recovers the Morphology of cAMP/PKA Defective Mutants
The causes of ss1hog1∆ and cAMP/PKA defective mutants' contradictory impacts on morphology. The deletion strains of the cAMP/PKA pathway under the background of ss1hog1∆ mutants were individually generated by homologous recombination, and named as ss1hog1∆gpa3∆, ss1hog1∆uac1∆, and ss1hog1∆adr1∆. Target-sequence disruption in mutants was confirmed through PCR amplification ( Figure S1A,B). Detection of doubledeletion mutants served as the Zeocin gene probe through Southern blotting ( Figure S1C). The transcriptional profile confirmed the disappearance of SsGPA3, SsUAC1, and SsADR1 genes' expressions in double-deletion mutants, thus indicating the destruction of the original gene ( Figure S1D).

The SsHog1 and SsAdr1 Collaborate for Haploid Cell Morphology by Controlling Vacuole Size in S. scitamineum
The vacuole size is known to correlate with cell size in plants and vacuolar expansion inhibition could restrict cell growth [34,36,46]. Therefore, the vacuoles were observed as orange-red by Neutral Red staining in S. scitamineum ( Figure 5A). As expected, the vacuoles of the ss1adr1∆ mutants were diminished with a significant reduction in vacuole-to-cell size scaling ratio, compared with the wild-type and Ss1ADR1-COM strains ( Figure 5A,B). Contrarily, the larger vacuoles and the vacuole-to-cell size scaling ratio were present in ss1hog1∆ mutant cells in comparison to the wild-type and Ss1HOG1-COM strains. Interestingly, the size of the vacuole and the ratio of vacuole-to-cell scaling in ss1hog1∆adr1∆ mutant cells did not exhibit significantly changes as compared to the wild type ( Figure 5A,B). Taken together, SsHog1 and SsAdr1 exhibited a correlation with vacuole size, which might regulate the cell morphology by influencing the vacuole size.

SsHog1 and SsAdr1 Regulate Cell Wall Permeability to Maintain Cell Size
The regulatory network of SsHog1 and SsAdr1 involved in morphological regulation was further explored by performing transcriptome analysis with the ss1adr1∆, ss1hog1∆, and wild-type sporidia grown in the YePSA medium. Differentially expressed genes (DEGs) were identified in the ss1adr1∆ and ss1hog1∆ mutants, compared to the wild-type MAT-1 strain. Gene ontology (GO) enrichment of the DEGs is represented in Figure S2. The "transmembrane transport" (GO:0055085) and "transport activity" (GO:0005215) were found to be more significant descriptors in the ss1hog1∆ mutant. Interestingly, "transport activity" was also a highly significant descriptor in the ss1adr1∆ mutant, suggesting an important role of MAP kinase Hog1 and cAMP-dependent protein kinase catalytic subunit Adr1in transport activity.
We next wondered whether differential expression of "transport activity" genes affected cell wall integrity and permeability. The wall integrity-and permeability-associated genes, including CDR99456.1 (encoding an Sge1 drug-resistance protein), CDS01502.1 (encoding a Pho8 alkaline phosphatase), CDU25217.1 (encoding a probable purine permease), CDS00122.1 (encoding a glucan synthase subunit), CDR88142.1 (encoding glycosyl hydrolases), CDU25158.1 (encoding glycosyl hydrolases), and CDU24651.1 (encoding a chitinase), were subjected to quantitative real-time PCR (RT-qPCR) analysis. The results showed that transcriptional expression of the CDR99456.1, CDS00122.1, and CDU24651.1 genes were significantly (p < 0.05) up-regulated in the ss1adr1∆ mutant. Contrarily, gene expressions in the ss1hog1∆adr1∆ mutant were not significantly different from the wild type ( Figure 6A). Moreover, the CDR99456.1 and CDS00122.1 genes were reduced in the ss1hog1∆ mutant ( Figure 6A). The transcription level of CDS01502.1, CDU25217.1, CDR88142.1, and CDU25158.1 genes were significantly (p < 0.05) increased in the ss1hog1∆ mutant and slightly downregulated or remained undifferentiated in the ss1adr1∆ mutant as compared to the wild type ( Figure 6A). The transcription levels of these genes decreased in the ss1hog1∆adr1∆ mutant than ss1hog1∆ mutant ( Figure 6A). Previously, we have reported the necessity of MAP kinase Hog1 for the cell wall integrity in S. scitamineum [27]. Therefore, MAT-1, ss1adr1∆, ss1hog1∆, and ss1hog1∆adr1∆ strains were cultured on the YEPS medium containing the cell wall perturbing agent Congo Red (CR) or Calcofluor White (CFW). The results showed that the glossy surface phenotype in the ss1adr1∆ mutant could be fully restored by the exogenous addition of Congo Red or Calcofluor White, which returned the ss1hog1∆ mutant colony morphology to the level of the wild type ( Figure 6B). To compare the change of permeability between these mutants and the wild type, the internalization rate of FITC-Dextran 70,000 [47] was measured in the MAT-1, ss1adr1∆, ss1hog1∆, and ss1hog1∆adr1∆ strains followed by flow cytometry. Internalized FITC-Dextran 70,000 was mostly found in the ss1hog1∆ mutant whereas the ss1adr1∆ mutant had its lowest presence. FITC-Dextran 70,000 in the ss1hog1∆adr1∆ mutant was similar to the wild type ( Figure 6C,D). Taken together, cell wall integrity and permeability were noted to be correlated with MAP kinase Hog1 and cAMP-dependent protein kinase catalytic subunit. Figure 6. Effects of SsHog1 and SsAdr1 on morphology cell wall integrity and permeability. (A) Transcriptional profile of cell wall integrity-and permeability-related genes in the MAT-1, ss1adr1∆, ss1hog1∆, and ss1hog1∆adr1∆ strains. Total RNA was extracted from the haploid sporidia which were allowed to grow on YePSA medium at 28 • C for 3 days, and then RT-qPCR was used to analyze the expression of genes related to cell wall integrity and permeability in the wild type and mutants. Relative gene expression level was calculated with the -∆∆Ct method with GAPDH as internal control. Bar chart depicts the statistical difference between the mean values ( p < 0.05, p < 0.01, p < 0.001). NS denotes a not statistically significant difference. The sample was independently performed with three independent biological repeats, each of which contained two replications. NS denotes a not statistically significant difference (p < 0.05). (B) Effects of Congo Red (CR) and Calcofluor White (CFW) on morphology of S. scitamineum. The fresh haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then 1.5 µL of cells was spotted onto YePSA medium with or without 1.0 mg/mL CR and 0.05 mg/mL CFW. Images were taken 5 days after cultivation. (C) Flow cytometry analyses of FITC-Dextran 70,000 internalized in the MAT-1, ss1adr1∆, ss1hog1∆, and ss1hog1∆adr1∆ strains. MAT-1, ss1adr1∆, ss1hog1∆, and ss1hog1∆adr1∆ haploid sporidia were allowed to grow till O.D. 600 = 1.0 and treated with FITC-Dextran 70,000 (1 µM) for 5 min. Then, cells were washed with PBS twice and re-suspended with PBS. Flow cytometry analysis was performed using the fluorescein isothiocyanate (FITC) filter. The sample was independently performed with three independent biological repeats, each of which contained two replications. Data analysis was performed using the FlowJo 10 software. (D) Bar chart depicting quantification of FITC fluorescence intensity as shown in (C). Bar chart depicts the statistical difference between the mean values ( p < 0.01, p < 0.001). NS denotes a not statistically significant difference. The sample was independently performed with three independent biological repeats, each of which contained two replications. NS denotes a not statistically significant difference (p < 0.05).

Discussion
MAPK and cAMP/PKA signaling pathways in fungi transmit the informational from the extracellular to the intracellular environment through important conservative mechanisms [5,16,48]. Morphogenesis facilitates the adaptation to environmental transformation by changing cellular dimensions [49,50]. The oversized cells exhibited slow cell division, cytoplasmic dilution, and altered transcriptome and proteome, suggesting that normal morphology maintenance is important for biological and physiological functions [51]. This study demonstrates that the SsHog1 and cAMP/PKA pathways regulate the S. scitamineum morphology by mediating the vacuole size, cell wall integrity, and permeability.
The role of the cAMP/PKA pathway in fungal morphology regulation has been reported. The cAMP/PKA defective mutants exhibited glossy colony morphology and smaller cellular size. This phenotype is opposite to U. maydis, which disrupts cAMP/PKA pathways for constitutive pseudohypha growth [1,17,18]. Furthermore, the disruption of the SsPRF1 gene (located downstream of SsADR1) resulted in normal morphology similar to the wild type. The exogenous addition of cAMP partially restored the ss1gpa3∆ and ss1uac1∆ mutants' morphology except for the ss1adr1∆ mutants. These results indicate that SsGpa3 and SsUac1 regulate the SsAdr1 activity by cAMP, which further regulates morphogenesis in other target proteins rather than through the transcription factor SsPrf1. Interestingly, the glossy surface phenotype in the ss1adr1∆ mutant was fully restored after the exogenous addition of Congo Red or Calcofluor White. These findings suggest the involvement of SsAdr1 in cell wall integrity. The cAMP/PKA pathway participation in the cell wall integrity of C. neoformans has been reported [52]. We hypothesized that SsAdr1 regulates the biosynthesis or degradation of glucan and/or chitin but this requires further verification. Vacuolar size alteration is also an important factor affecting cellular morphogenesis. The vacuolization contributes to cell size heterogeneity and might resolve the conflicts regarding growth control at the cellular and organ levels [34][35][36][37]53]. The vacuolar size also increases with the cell size in fungi [38]. Further investigations showed that the vacuole-to-cell scaling ratio was diminished in the ss1adr1∆ mutant as compared to the wild type, suggesting that a smaller vacuole-to-cell scaling ratio is more likely to cause cell shrinkage. Taken together, these data indicated that SsAdr1 plays a crucial role in the regulation of morphology.
MAPK signaling pathways are also important in morphological regulation. In this study, the ss1hog1∆ mutants displayed a wrinkled colony morphology and a larger cell size under the ordinary culture. These results indicate that SsHog1 negatively regulates haploid cell morphogenesis, which is similar to the negative regulation of the HOG1 gene in S. cerevisiae under hypertonic stress [12]. Moreover, the large colony morphology of the ss1hog1∆ mutant returned to the level of the wild type after treatment with Congo Red or Calcofluor White. The reduced tolerance of ss1hog1∆ mutants to cell wall stress reagents might be the reason behind this phenomenon. The importance of MAP kinase Hog1 for the cell wall integrity in S. scitamineum has been reported [27]. This is an opposite phenotype to C. albicans, where deletion of the HOG1 gene enhanced the tolerance to Congo Red and Calcofluor White [25]. In addition, transcriptional expression of the glycosyl hydrolases genes (CDR88142.1 and CDU25158.1) and internalization of FITC-Dextran 70,000 were significantly (p < 0.05) increased in the ss1hog1∆ mutant as compared to the wild-type. It suggests that the change in cell wall permeability also affects morphogenesis.
What is the regulatory relationship between the Hog1-MAPK pathway and cAMP/PKA signaling pathways in the morphologies of S. scitamineum? To explore this question, the ss1hog1∆gpa3∆, ss1hog1∆uac1∆ and ss1hog1∆adr1∆ mutants were generated according to the ss1hog1∆ mutant. The results revealed that the morphology of cAMP/PKA defective mutants was recovered with the deletion of the SsHOG1 gene, which suggests that SsHog1 might be located downstream of the cAMP/PKA signaling pathway or is a parallel pathway. These findings are similar to C. neoformans, where the disruption of genes (GPA1, CAC1, or PKA1 in hog1∆ mutant background) restored melanin production, compared with the complete elimination of melanin formation in gpa1∆, cac1∆, or pka1∆ single mutants [16]. The abundance of ribosomal protein (RP) transcripts and the biosynthesis of ribosomes in C. albicans were maintained at mRNA stabilization by the dual and opposing actions of Hog1 and cAMP/PKA pathways [54]. Further investigations revealed that there were not significant changes in the transcription level of the SsHOG1 gene and phosphorylation level of SsHog1 in cAMP/PKA defective mutants, and vice versa, suggesting that MAP kinase Hog1 and SsAdr1 did not interfere with each other in S. scitamineum. These results indicated that SsHog1 is not located downstream of the cAMP/PKA signaling pathway. The next question is why deletion of SsHOG1 recovered the cAMP/PKA defective mutant morphology, even more like ss1hog1∆. We think that SsHog1 and SsAdr1 antagonistically regulated morphological development by mediating vacuolar size, cell wall integrity and permeability in S. scitamineum. Perhaps SsHog1 might have contributed more to maintaining the normal morphology than cAMP/PKA signaling pathways, or other factors.
To conclude, the study establishes that SsHog1 and the cAMP/PKA pathways are required for a particular morphology. The data further elaborated the antagonistic morphology regulation by mediating vacuolar size, cell wall integrity, and permeability in S. scitamineum.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable. Data Availability Statement: All data required to understand this article are presented in the study or the Supplementary Materials. Any raw data further requested will be provided by the corresponding authors.