Next Article in Journal
The Characterization and the Biological Activity of Phytotoxin Produced by Paraphoma radicina
Next Article in Special Issue
The Toxicity of Salicylhydroxamic Acid and Its Effect on the Sensitivity of Ustilaginoidea virens to Azoxystrobin and Pyraclostrobin
Previous Article in Journal
Arbuscular Mycorrhizal Fungi Reduce Cadmium Leaching from Sand Columns by Reducing Availability and Enhancing Uptake by Maize Roots
Previous Article in Special Issue
Functional Analysis of the Plasma Membrane H+-ATPases of Ustilago maydis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JoF
Volume 8
Issue 8
10.3390/jof8080865
jof-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Enping Cai
1,2,†,
Meixin Yan
3,†,
Xian Sun
1,2,
Rong Zeng
1,2,
Wenqiang Zheng
1,2,
Yizhen Deng
1,2,
Zide Jiang
1 and
Changqing Chang
1,2,*
1
Integrate Microbiology Research Center, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China
2
Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China
3
Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2022, 8(8), 865; https://doi.org/10.3390/jof8080865
Submission received: 28 June 2022 / Revised: 13 August 2022 / Accepted: 15 August 2022 / Published: 17 August 2022
(This article belongs to the Special Issue Smut Fungi 2.0)
Browse Figures
Review Reports Versions Notes

Abstract

:
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.

1. 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 hog1Δ 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 smut disease [19,20]. MAP kinase Hog1 and cAMP/PKA signaling pathways were known to participate in virulence and mating/filamentation of S. scitamineum [21,22]. However, its role in haploid cell morphogenesis remains unknown.
Signal transduction is crucial for fungi and fungal MAPK pathways might interact with other signal transduction pathways during the morphogenetic process [5]. The antagonistic role of cAMP/PKA and Kpp2-MAPK pathways during morphogenesis has also been reported in U. maydis [23]. The cAMP-dependent protein kinase catalytic subunit Tpk1 was required in C. albicans for cell wall integrity and drug tolerance [24]. Contrarily, the deletion of the HOG1 gene conferred resistance to the cell wall perturbing agent Congo Red or Calcofluor White [11,25]. The Hog1-MAPK pathway was supposed to coordinate the chitin synthesis with other pathways [26]. Oppositely, the deletion of the HOG1 gene slightly increased the cell wall sensitivity to Congo Red or Calcofluor White in S. scitamineum during our previous study [27]. Similarly, the sakA (Hog1 orthologs) defective mutant was more sensitive to cell wall damaging agents in Aspergillus fumigatus [28].
The role of vacuoles in eukaryotes was important during cellular biochemical pathways including cellular homeostasis, nutrient storage, protein degradation, and growth regulation through the cell cycle and death [29,30,31,32,33]. Vacuoles were the largest plant cell organelles occupying 30–80% of the cell volume and their size increased with cell growth [34]. Hence, the vacuole size determined the cell elongation rates and size [34,35,36,37]. Contrarily, inhibition of vacuolar expansion restricted plant cell growth [34,36]. The characteristics of fungal vacuoles were similar to plant and mammalian lysosomes [31]. Different mechanisms regulate the size and shape of vacuoles [38]. Defective mutants in vacuole (vac8Δ and vac17Δ) inheritance depicted the increased vacuole scaling with replicative cell age [39]. Cyclin-dependent kinase Pho85/CDK5 could also activate the vacuolar signaling pathway for initiating rapid protective mechanisms against hyperosmotic stress before long-term adaptation through Hog1 [40].
The interrelating role of the signal pathway is important in fungi. However, the interlinking mechanism between Hog1-MAPK and cAMP/PKA signaling pathways is unknown in S. scitamineum. This study reveals that MAP kinase Hog1 and cAMP-dependent protein kinase catalytic subunit Adr1 antagonistically regulated the morphological alterations in S. scitamineum. However, SsHog1 and SsAdr1 did not interfere with each other. Further investigations demonstrated that SsHog1 and SsAdr1 antagonistically regulated the vacuolar size in S. scitamineum by balancing cell wall integrity and permeability. In short, the results suggest that SsHog1 and SsAdr1 maintained the S. scitamineum morphology by mediating the vacuole size and cell wall integrity and permeability.

2. Materials and Methods

2.1. 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, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ 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).

2.2. 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 ZeocinR (ZEOR) 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, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ as a template and the following primers: SsGPA3-inside-F/SsGPA3-inside-R, SsGPA3-outside-F/SsGPA3-outside-R, SsUAC1-inside-F/SsUAC1-inside-R, SsUAC1-outside-F/SsUAC1-outside-R, SsADR1-inside-F/SsADR1-inside-R, and SsADR1-outside-F/SsADR1-outside-R, individually. The primer sequence is shown in Table S1. In this study, transformants were constructed in the ss1hog1Δ background.

2.3. Nucleic Acid Related Manipulation

Genomic DNA of MAT-1, ss1hog1Δ, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ 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 ZEOR selection marker. For Southern blot analysis, Genomic DNA of MAT-1, ss1hog1Δ, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ, and pDAN vector were digested with the restriction enzyme HindIII and the ZEOR 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 -ΔΔCt method [45] and used cytoskeletal protein gene GAPDH as internal control. The experiment was conducted in triplicate and for three independent biological replicates.

2.4. 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).

2.5. 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).

2.6. Flow Cytometry Analysis

MAT-1, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ 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.

2.7. 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.

3. Results

3.1. 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 ss1gpa3Δ and ss1uac1Δ mutants as compared to wild type (Figure 1A). However, ss1adr1Δ 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 (ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ) 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 ss1gpa3Δ and ss1uac1Δ mutants except in the ss1adr1Δ 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.

3.2. MAP Kinase SsHog1 Is Required for Maintaining Haploid Cell Morphology of S. scitamineum

The hog1Δ mutant cells exhibited an irregular and larger cell morphology in S. cerevisiae under hypertonic stress [12]. However, the cell morphology of ss1hog1Δ mutants (with or without hypertonic stress) in S. scitamineum were not significantly different (unshown). Nevertheless, ss1hog1Δ mutant colonies appeared bigger with a folded surface in YePSA medium, compared with the wild-type and Ss1HOG1-COM strains (Figure 2A). Furthermore, the ss1hog1Δ 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.

3.3. 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 ss1hog1Δ 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 ss1hog1Δ mutants (Figure 3A). Moreover, PKA phosphorylation levels in ss1hog1Δ 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.

3.4. Deletion of SsHOG1 Recovers the Morphology of cAMP/PKA Defective Mutants

The causes of ss1hog1Δ and cAMP/PKA defective mutants’ contradictory impacts on morphology. The deletion strains of the cAMP/PKA pathway under the background of ss1hog1Δ mutants were individually generated by homologous recombination, and named as ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ. Target-sequence disruption in mutants was confirmed through PCR amplification (Figure S1A,B). Detection of double-deletion 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 glossy surface disappeared in double-deletion mutants as compared to cAMP/PKA defective mutants. The colony morphologies of ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ mutants were fully restored like ss1hog1Δ in the YePSA medium (Figure 1A and Figure 4A). However, the cell areas and cell lengths of double-deletion mutants returned to the wild type (Figure 4B–D). In short, the glossy surface of the cAMP/PKA defective mutants was recovered by deleting the SsHOG1 gene.

3.5. 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 ss1adr1Δ 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 ss1hog1Δ 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 ss1hog1Δadr1Δ 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.

3.6. 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 ss1adr1Δ, ss1hog1Δ, and wild-type sporidia grown in the YePSA medium. Differentially expressed genes (DEGs) were identified in the ss1adr1Δ and ss1hog1Δ 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 ss1hog1Δ mutant. Interestingly, “transport activity” was also a highly significant descriptor in the ss1adr1Δ 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 ss1adr1Δ mutant. Contrarily, gene expressions in the ss1hog1Δadr1Δ mutant were not significantly different from the wild type (Figure 6A). Moreover, the CDR99456.1 and CDS00122.1 genes were reduced in the ss1hog1Δ 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 ss1hog1Δ mutant and slightly down-regulated or remained undifferentiated in the ss1adr1Δ mutant as compared to the wild type (Figure 6A). The transcription levels of these genes decreased in the ss1hog1Δadr1Δ mutant than ss1hog1Δ 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, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ 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 ss1adr1Δ mutant could be fully restored by the exogenous addition of Congo Red or Calcofluor White, which returned the ss1hog1Δ 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, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ strains followed by flow cytometry. Internalized FITC-Dextran 70,000 was mostly found in the ss1hog1Δ mutant whereas the ss1adr1Δ mutant had its lowest presence. FITC-Dextran 70,000 in the ss1hog1Δadr1Δ 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.

4. 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 ss1gpa3Δ and ss1uac1Δ mutants’ morphology except for the ss1adr1Δ 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 ss1adr1Δ 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 ss1adr1Δ 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 ss1hog1Δ 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 ss1hog1Δ mutant returned to the level of the wild type after treatment with Congo Red or Calcofluor White. The reduced tolerance of ss1hog1Δ 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 ss1hog1Δ 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 ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ and ss1hog1Δadr1Δ mutants were generated according to the ss1hog1Δ 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 hog1Δ mutant background) restored melanin production, compared with the complete elimination of melanin formation in gpa1Δ, cac1Δ, or pka1Δ 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 ss1hog1Δ. 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof8080865/s1, Figure S1: Identification of mutants by PCR amplification, Southern blot, and RT-qPCR, Figure S2: GO enrichment analysis of differentially expressed genes (DEGs) in the MAT-1, ss1hog1Δ, and ss1adr1Δ strains, Table S1: The primers and sequences used in this study.

Author Contributions

Conceptualization, C.C., E.C. and Z.J.; methodology, E.C., M.Y. and X.S.; formal analysis, E.C., M.Y. and X.S.; investigation, E.C., W.Z., R.Z. and X.S.; resources, C.C. and Z.J.; writing—original draft preparation, E.C., Y.D. and X.S.; writing—review and editing, C.C., Z.J. and E.C.; supervision, Z.J., C.C. and M.Y.; project administration, C.C. and Z.J.; funding acquisition, C.C., M.Y. and Z.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Key Realm R&D Program of Guang Dong Province (2020B0202090001), Key Projects of Guangzhou Science and Technology Plan (201904020010), and National Natural Science Foundation of China (31960521).

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.

Acknowledgments

We thank Xiaofan Zhou (South China Agricultural University) for the critical reading of our manuscript and helpful suggestions.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Dürrenberger, F.; Wong, K.; Kronstad, J.W. Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis. Proc. Natl. Acad. Sci. USA 1998, 95, 5684–5689. [Google Scholar] [CrossRef] [PubMed]
  2. Dambuza, I.M.; Drake, T.; Chapuis, A.; Zhou, X.; Correia, J.; Taylor-Smith, L.; LeGrave, N.; Rasmussen, T.; Fisher, M.C.; Bicanic, T.; et al. The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis. PLoS Pathog. 2018, 14, e1006978. [Google Scholar] [CrossRef] [PubMed]
  3. Maidan, M.M.; Thevelein, J.M.; Van Dijck, P. Carbon source induced yeast-to-hypha transition in Candida albicans is dependent on the presence of amino acids and on the G-protein-coupled receptor Gpr1. Biochem. Soc. Trans. 2005, 33, 291–293. [Google Scholar] [CrossRef] [PubMed]
  4. Maidan, M.M.; De Rop, L.; Serneels, J.; Exler, S.; Rupp, S.; Tournu, H.; Thevelein, J.M.; Van Dijck, P. The G protein-coupled receptor Gpr1 and the Galpha protein Gpa2 act through the cAMP-protein kinase A pathway to induce morphogenesis in Candida albicans. Mol. Biol. Cell 2005, 16, 1971–1986. [Google Scholar] [CrossRef] [PubMed]
  5. Martínez-Soto, D.; Ortiz-Castellanos, L.; Robledo-Briones, M.; León-Ramírez, C.G. Molecular Mechanisms Involved in the Multicellular Growth of Ustilaginomycetes. Microorganisms 2020, 8, 1072. [Google Scholar] [CrossRef]
  6. Caza, M.; Kronstad, J.W. The cAMP/Protein Kinase a Pathway Regulates Virulence and Adaptation to Host Conditions in Cryptococcus neoformans. Front. Cell. Infect. Microbiol. 2019, 9, 212. [Google Scholar] [CrossRef]
  7. Pan, X.; Harashima, T.; Heitman, J. Signal transduction cascades regulating pseudohyphal differentiation of Saccharomyces cerevisiae. Curr. Opin. Microbiol. 2000, 3, 567–572. [Google Scholar] [CrossRef]
  8. Thevelein, J.M.; Cauwenberg, L.; Colombo, S.; De Winde, J.H.; Donation, M.; Dumortier, F.; Kraakman, L.; Lemaire, K.; Ma, P.; Nauwelaers, D.; et al. Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast. Enzym. Microb. Technol. 2000, 26, 819–825. [Google Scholar] [CrossRef]
  9. Amigoni, L.; Colombo, S.; Belotti, F.; Alberghina, L.; Martegani, E. The transcription factor Swi4 is target for PKA regulation of cell size at the G1 to S transition in Saccharomyces cerevisiae. Cell Cycle 2015, 14, 2429–2438. [Google Scholar] [CrossRef]
  10. Cheetham, J.; Smith, D.A.; da Silva Dantas, A.; Doris, K.S.; Patterson, M.J.; Bruce, C.R.; Quinn, J. A single MAPKKK regulates the Hog1 MAPK pathway in the pathogenic fungus Candida albicans. Mol. Biol. Cell 2007, 18, 4603–4614. [Google Scholar] [CrossRef]
  11. Román, E.; Correia, I.; Prieto, D.; Alonso, R.; Pla, J. The HOG MAPK pathway in Candida albicans: More than an osmosensing pathway. Int. Microbiol. Off. J. Span. Soc. Microbiol. 2020, 23, 23–29. [Google Scholar] [CrossRef] [PubMed]
  12. Brewster, J.L.; Gustin, M.C. Hog1: 20 years of discovery and impact. Sci. Signal. 2014, 7, re7. [Google Scholar] [CrossRef] [PubMed]
  13. Cheetham, J.; MacCallum, D.M.; Doris, K.S.; da Silva Dantas, A.; Scorfield, S.; Odds, F.; Smith, D.A.; Quinn, J. MAPKKK-independent regulation of the Hog1 stress-activated protein kinase in Candida albicans. J. Biol. Chem. 2011, 286, 42002–42016. [Google Scholar] [CrossRef] [PubMed]
  14. Hommel, B.; Mukaremera, L.; Cordero, R.J.B.; Coelho, C.; Desjardins, C.A.; Sturny-Leclère, A.; Janbon, G.; Perfect, J.R.; Fraser, J.A.; Casadevall, A.; et al. Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators. PLoS Pathog. 2018, 14, e1006982. [Google Scholar] [CrossRef] [PubMed]
  15. Zaragoza, O.; García-Rodas, R.; Nosanchuk, J.D.; Cuenca-Estrella, M.; Rodríguez-Tudela, J.L.; Casadevall, A. Fungal cell gigantism during mammalian infection. PLoS Pathog. 2010, 6, e1000945. [Google Scholar] [CrossRef]
  16. Bahn, Y.S.; Kojima, K.; Cox, G.M.; Heitman, J. Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans. Mol. Biol. Cell 2005, 16, 2285–2300. [Google Scholar] [CrossRef]
  17. Gold, S.; Duncan, G.; Barrett, K.; Kronstad, J. cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Genes Dev. 1994, 8, 2805–2816. [Google Scholar] [CrossRef]
  18. Regenfelder, E.; Spellig, T.; Hartmann, A.; Lauenstein, S.; Bölker, M.; Kahmann, R. G proteins in Ustilago maydis: Transmission of multiple signals? EMBO J. 1997, 16, 1934–1942. [Google Scholar] [CrossRef]
  19. Que, Y.; Xu, L.; Wu, Q.; Liu, Y.; Ling, H.; Liu, Y.; Zhang, Y.; Guo, J.; Su, Y.; Chen, J.; et al. Genome sequencing of Sporisorium scitamineum provides insights into the pathogenic mechanisms of sugarcane smut. BMC Genom. 2014, 15, 996. [Google Scholar] [CrossRef]
  20. Yan, M.; Dai, W.; Cai, E.; Yi, Z.D.; Chang, C.; Jiang, Z.; Zhang, L.H. Transcriptome analysis of Sporisorium scitamineum reveals critical environmental signals for fungal sexual mating and filamentous growth. BMC Genom. 2016, 17, 354. [Google Scholar] [CrossRef]
  21. Chang, C.; Cai, E.; Deng, Y.Z.; Mei, D.; Qiu, S.; Chen, B.; Zhang, L.H.; Jiang, Z. cAMP/PKA signalling pathway regulates redox homeostasis essential for Sporisorium scitamineum mating/filamentation and virulence. Environ. Microbiol. 2019, 21, 959–971. [Google Scholar] [CrossRef] [PubMed]
  22. Cai, E.; Sun, S.; Deng, Y.; Huang, P.; Sun, X.; Wang, Y.; Chang, C.; Jiang, Z. Histidine Kinase Sln1 and cAMP/PKA Signaling Pathways Antagonistically Regulate Sporisorium scitamineum Mating and Virulence via Transcription Factor Prf1. J. Fungi 2021, 7, 610. [Google Scholar] [CrossRef] [PubMed]
  23. Garrido, E.; Pérez-Martín, J. The crk1 gene encodes an Ime2-related protein that is required for morphogenesis in the plant pathogen Ustilago maydis. Mol. Microbiol. 2003, 47, 729–743. [Google Scholar] [CrossRef]
  24. Fanning, S.; Xu, W.; Beaurepaire, C.; Suhan, J.P.; Nantel, A.; Mitchell, A.P. Functional control of the Candida albicans cell wall by catalytic protein kinase A subunit Tpk1. Mol. Microbiol. 2012, 86, 284–302. [Google Scholar] [CrossRef] [PubMed]
  25. Alonso-Monge, R.; Navarro-García, F.; Molero, G.; Diez-Orejas, R.; Gustin, M.; Pla, J.; Sánchez, M.; Nombela, C. Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans. J. Bacteriol. 1999, 181, 3058–3068. [Google Scholar] [CrossRef] [PubMed]
  26. Munro, C.A.; Selvaggini, S.; de Bruijn, I.; Walker, L.; Lenardon, M.D.; Gerssen, B.; Milne, S.; Brown, A.J.; Gow, N.A. The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans. Mol. Microbiol. 2007, 63, 1399–1413. [Google Scholar] [CrossRef] [PubMed]
  27. Cai, E.; Li, L.; Deng, Y.; Sun, S.; Jia, H.; Wu, R.; Zhang, L.; Jiang, Z.; Chang, C. MAP kinase Hog1 mediates a cytochrome P450 oxidoreductase to promote the Sporisorium scitamineum cell survival under oxidative stress. Environ. Microbiol. 2021, 23, 3306–3317. [Google Scholar] [CrossRef]
  28. Bruder Nascimento, A.C.; Dos Reis, T.F.; de Castro, P.A.; Hori, J.I.; Bom, V.L.; de Assis, L.J.; Ramalho, L.N.; Rocha, M.C.; Malavazi, I.; Brown, N.A.; et al. Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence. Mol. Microbiol. 2016, 100, 841–859. [Google Scholar] [CrossRef]
  29. Klionsky, D.J.; Herman, P.K.; Emr, S.D. The fungal vacuole: Composition, function, and biogenesis. Microbiol. Rev. 1990, 54, 266–292. [Google Scholar] [CrossRef]
  30. Li, S.C.; Kane, P.M. The yeast lysosome-like vacuole: Endpoint and crossroads. Biochim. Et Biophys. Acta 2009, 1793, 650–663. [Google Scholar] [CrossRef]
  31. Richards, A.; Veses, V.; Gow, N.A.R. Vacuole dynamics in fungi. Fungal Biol. Rev. 2010, 24, 93–105. [Google Scholar] [CrossRef]
  32. Veses, V.; Gow, N.A. Vacuolar dynamics during the morphogenetic transition in Candida albicans. FEMS Yeast Res. 2008, 8, 1339–1348. [Google Scholar] [CrossRef] [PubMed]
  33. Weber, R.W.S. Vacuoles and the fungal lifestyle. Mycologist 2002, 16, 10–20. [Google Scholar] [CrossRef]
  34. Löfke, C.; Dünser, K.; Scheuring, D.; Kleine-Vehn, J. Auxin regulates SNARE-dependent vacuolar morphology restricting cell size. eLife 2015, 4, e05868. [Google Scholar] [CrossRef]
  35. Dünser, K.; Gupta, S.; Herger, A.; Feraru, M.I.; Ringli, C.; Kleine-Vehn, J. Extracellular matrix sensing by FERONIA and Leucine-Rich Repeat Extensins controls vacuolar expansion during cellular elongation in Arabidopsis thaliana. EMBO J. 2019, 38, e100353. [Google Scholar] [CrossRef]
  36. Kaiser, S.; Eisa, A.; Kleine-Vehn, J.; Scheuring, D. NET4 Modulates the Compactness of Vacuoles in Arabidopsis thaliana. Int. J. Mol. Sci. 2019, 20, 4752. [Google Scholar] [CrossRef]
  37. Scheuring, D.; Löfke, C.; Krüger, F.; Kittelmann, M.; Eisa, A.; Hughes, L.; Smith, R.S.; Hawes, C.; Schumacher, K.; Kleine-Vehn, J. Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression. Proc. Natl. Acad. Sci. USA 2016, 113, 452–457. [Google Scholar] [CrossRef]
  38. Chadwick, W.L.; Biswas, S.K.; Bianco, S.; Chan, Y.M. Non-random distribution of vacuoles in Schizosaccharomyces pombe. Phys. Biol. 2020, 17, 065004. [Google Scholar] [CrossRef]
  39. Chan, Y.H.; Reyes, L.; Sohail, S.M.; Tran, N.K.; Marshall, W.F. Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance. Curr. Biol. 2016, 26, 1221–1228. [Google Scholar] [CrossRef]
  40. Jin, N.; Jin, Y.; Weisman, L.S. Early protection to stress mediated by CDK-dependent PI3,5P(2) signaling from the vacuole/lysosome. J. Cell Biol. 2017, 216, 2075–2090. [Google Scholar] [CrossRef]
  41. Yan, M.; Zhu, G.; Lin, S.; Xian, X.; Chang, C.; Xi, P.; Shen, W.; Huang, W.; Cai, E.; Jiang, Z. The mating-type locus b of the sugarcane smut Sporisorium scitamineum is essential for mating, filamentous growth and pathogenicity. Fungal Genet. Biol. Fg. B 2015, 86, 1–8. [Google Scholar] [CrossRef] [PubMed]
  42. Zhu, G.; Deng, Y.; Cai, E.; Yan, M.; Cui, G.; Wang, Z.; Zou, C.; Zhang, B.; Xi, P.; Chang, C.; et al. Identification and Functional Analysis of the Pheromone Response Factor Gene of Sporisorium scitamineum. Front. Microbiol. 2019, 10, 2115. [Google Scholar] [CrossRef] [PubMed]
  43. Cai, E.P.; Mei, D.; Zhang, X.; Sun, X.; Li, L.; Wu, R.; Deng, Y.; Jiang, Z.; Chang, C. A gene knockout method based on protoplast transformation with two PCR fragments in Sporisorium scitamineum. Mycosystema 2021, 39, 1–14. [Google Scholar] [CrossRef]
  44. Góesneto, A.; Loguercioleite, C.; Guerrero, R.T. DNA extraction from frozen field-collected and dehydrated herbarium fungal basidiomata: Performance of SDS and CTAB-based methods. Biotemas 2005, 18, 19–32. [Google Scholar]
  45. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  46. Kaiser, S.; Scheuring, D. To Lead or to Follow: Contribution of the Plant Vacuole to Cell Growth. Front. Plant Sci. 2020, 11, 553. [Google Scholar] [CrossRef] [PubMed]
  47. Eriksson, I.; Öllinger, K.; Appelqvist, H. Analysis of Lysosomal pH by Flow Cytometry Using FITC-Dextran Loaded Cells. Methods Mol. Biol. 2017, 1594, 179–189. [Google Scholar] [CrossRef]
  48. Skalhegg, B.S.; Tasken, K. Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front. Biosci. A J. Virtual Libr. 2000, 5, D678–D693. [Google Scholar]
  49. Brefort, T.; Doehlemann, G.; Mendoza-Mendoza, A.; Reissmann, S.; Djamei, A.; Kahmann, R. Ustilago maydis as a Pathogen. Annu. Rev. Phytopathol. 2009, 47, 423–445. [Google Scholar] [CrossRef]
  50. Jones, A.R.; Band, L.R.; Murray, J.A.H. Double or Nothing? Cell Division and Cell Size Control. Trends Plant Sci. 2019, 24, 1083–1093. [Google Scholar] [CrossRef]
  51. Veitia, R.A. DNA Content, Cell Size, and Cell Senescence. Trends Biochem. Sci. 2019, 44, 645–647. [Google Scholar] [CrossRef] [PubMed]
  52. Donlin, M.J.; Upadhya, R.; Gerik, K.J.; Lam, W.; VanArendonk, L.G.; Specht, C.A.; Sharma, N.K.; Lodge, J.K. Cross talk between the cell wall integrity and cyclic AMP/protein kinase A pathways in Cryptococcus neoformans. mBio 2014, 5, e01573-14. [Google Scholar] [CrossRef] [PubMed]
  53. D’Ario, M.; Sablowski, R. Cell Size Control in Plants. Annu. Rev. Genet. 2019, 53, 45–65. [Google Scholar] [CrossRef] [PubMed]
  54. Banerjee, D.; Bloom, A.L.; Panepinto, J.C. Opposing PKA and Hog1 signals control the post-transcriptional response to glucose availability in Cryptococcus neoformans. Mol. Microbiol. 2016, 102, 306–320. [Google Scholar] [CrossRef]
Jof 08 00865 g001 550
Figure 1. Effects of cAMP/PKA signaling pathways on morphology of S. scitamineum. (A) The colony morphology of MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains were measured. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains were measured. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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.
Figure 1. Effects of cAMP/PKA signaling pathways on morphology of S. scitamineum. (A) The colony morphology of MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains were measured. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ strains were measured. MAT-1, ss1gpa3Δ, ss1uac1Δ, ss1adr1Δ, Ss1ADR1-COM, and ss1prf1Δ 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.
Jof 08 00865 g001
Jof 08 00865 g002 550
Figure 2. Effects of SsHog1 on morphology of S. scitamineum. (A) The colony morphology of MAT-1, ss1hog1Δ, and Ss1HOG1-COM strains. The fresh haploid sporidia were spotted onto YePSA medium at 28 °C. Images were taken 4 days after cultivation. Scale bar = 5.0 mm. (B) The microscopic morphology of MAT-1, ss1hog1Δ, and Ss1HOG1-COM strains. MAT-1, ss1hog1Δ, and Ss1HOG1-COM haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then 5 μL of the sample was mounted on the slide and observed. Images were taken by Leica DMI8 Inverted Fluorescence Microscope. Scale bar = 10 µm. (C) The cell areas of MAT-1, ss1hog1Δ, and Ss1HOG1-COM strains were measured. MAT-1, ss1hog1Δ, 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, ss1hog1Δ, 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.
Figure 2. Effects of SsHog1 on morphology of S. scitamineum. (A) The colony morphology of MAT-1, ss1hog1Δ, and Ss1HOG1-COM strains. The fresh haploid sporidia were spotted onto YePSA medium at 28 °C. Images were taken 4 days after cultivation. Scale bar = 5.0 mm. (B) The microscopic morphology of MAT-1, ss1hog1Δ, and Ss1HOG1-COM strains. MAT-1, ss1hog1Δ, and Ss1HOG1-COM haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then 5 μL of the sample was mounted on the slide and observed. Images were taken by Leica DMI8 Inverted Fluorescence Microscope. Scale bar = 10 µm. (C) The cell areas of MAT-1, ss1hog1Δ, and Ss1HOG1-COM strains were measured. MAT-1, ss1hog1Δ, 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, ss1hog1Δ, 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.
Jof 08 00865 g002
Jof 08 00865 g003 550
Figure 3. The relationship between MAP kinase Hog1 and cAMP/PKA signaling pathways. (A) Transcriptional profile of cAMP/PKA-pathway-related genes in MAT-1 and ss1hog1Δ. The transcription levels of genes SsGPA3, SsUAC1, and SsADR1 in the cAMP/PKA pathway were detected in the MAT-1 and ss1hog1Δ. 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, ss1adr1Δ, and ss1hog1Δ 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, ss1gpa3Δ, ss1uac1Δ, and ss1adr1Δ. 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, ss1hog1Δ, ss1gpa3Δ, ss1uac1Δ, and ss1adr1Δ 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.
Figure 3. The relationship between MAP kinase Hog1 and cAMP/PKA signaling pathways. (A) Transcriptional profile of cAMP/PKA-pathway-related genes in MAT-1 and ss1hog1Δ. The transcription levels of genes SsGPA3, SsUAC1, and SsADR1 in the cAMP/PKA pathway were detected in the MAT-1 and ss1hog1Δ. 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, ss1adr1Δ, and ss1hog1Δ 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, ss1gpa3Δ, ss1uac1Δ, and ss1adr1Δ. 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, ss1hog1Δ, ss1gpa3Δ, ss1uac1Δ, and ss1adr1Δ 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.
Jof 08 00865 g003
Jof 08 00865 g004 550
Figure 4. Morphology of SsHog1 and cAMP/PKA signaling pathways’ double-deletion mutants. (A) The colony morphology of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ haploid sporidia were spotted onto YePSA medium at 28 °C. Images were taken 4 days after cultivation. Scale bar = 5.0 mm. (B) The microscopic morphology of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then 5 μL of the sample was mounted on the slide and observed. Images were taken by Leica DMI8 Inverted Fluorescence Microscope. Scale bar = 10 µm. (C) The cell areas of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains were measured. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ 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. The sample was independently performed with three independent biological repeats, each of which contained twenty replications. (D) The cell lengths of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains were measured. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ haploid sporidia were allowed to grow till O.D. 600 = 1.0, and the length of the cell was measured. Images were taken by Leica DMI8 Inverted Fluorescence Microscope. The unit is µm. Bar chart depicts statistical difference between the mean values (p < 0.05). NS denotes a not statistically significant difference. The sample was independently performed with three independent biological repeats, each of which contained twenty replications.
Figure 4. Morphology of SsHog1 and cAMP/PKA signaling pathways’ double-deletion mutants. (A) The colony morphology of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ haploid sporidia were spotted onto YePSA medium at 28 °C. Images were taken 4 days after cultivation. Scale bar = 5.0 mm. (B) The microscopic morphology of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ haploid sporidia were allowed to grow till O.D. 600 = 1.0, and then 5 μL of the sample was mounted on the slide and observed. Images were taken by Leica DMI8 Inverted Fluorescence Microscope. Scale bar = 10 µm. (C) The cell areas of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains were measured. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ 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. The sample was independently performed with three independent biological repeats, each of which contained twenty replications. (D) The cell lengths of MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ strains were measured. MAT-1, ss1hog1Δgpa3Δ, ss1hog1Δuac1Δ, and ss1hog1Δadr1Δ haploid sporidia were allowed to grow till O.D. 600 = 1.0, and the length of the cell was measured. Images were taken by Leica DMI8 Inverted Fluorescence Microscope. The unit is µm. Bar chart depicts statistical difference between the mean values (p < 0.05). NS denotes a not statistically significant difference. The sample was independently performed with three independent biological repeats, each of which contained twenty replications.
Jof 08 00865 g004
Jof 08 00865 g005 550
Figure 5. Effect of SsHog1 and SsAdr1 on vacuolar size. (A) Neutral Red staining in the MAT-1, ss1adr1Δ, Ss1ADR1-COM, ss1hog1Δ, Ss1HOG1-COM, and ss1hog1Δadr1Δ strains. The Neutral Red was mixed with the fresh haploid sporidia, and then 5 μL of the sample was mounted on the slide and observed (Scale bar = 10 μm). (B) The vacuole-to-cell size scaling ratio of MAT-1, ss1adr1Δ, Ss1ADR1-COM, ss1hog1Δ, Ss1HOG1-COM, and ss1hog1Δadr1Δ strains. Bar chart depicts statistical difference between the mean values (★ p < 0.05 and ★★ p < 0.01). The sample was independently performed with three independent biological repeats. For each instance n > 50, samples were assessed.
Figure 5. Effect of SsHog1 and SsAdr1 on vacuolar size. (A) Neutral Red staining in the MAT-1, ss1adr1Δ, Ss1ADR1-COM, ss1hog1Δ, Ss1HOG1-COM, and ss1hog1Δadr1Δ strains. The Neutral Red was mixed with the fresh haploid sporidia, and then 5 μL of the sample was mounted on the slide and observed (Scale bar = 10 μm). (B) The vacuole-to-cell size scaling ratio of MAT-1, ss1adr1Δ, Ss1ADR1-COM, ss1hog1Δ, Ss1HOG1-COM, and ss1hog1Δadr1Δ strains. Bar chart depicts statistical difference between the mean values (★ p < 0.05 and ★★ p < 0.01). The sample was independently performed with three independent biological repeats. For each instance n > 50, samples were assessed.
Jof 08 00865 g005
Jof 08 00865 g006 550
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, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ 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 -ΔΔCt 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, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ strains. MAT-1, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ 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).
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, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ 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 -ΔΔCt 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, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ strains. MAT-1, ss1adr1Δ, ss1hog1Δ, and ss1hog1Δadr1Δ 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).
Jof 08 00865 g006
Table
Table 1. Details of strains shown in this study.
Table 1. Details of strains shown in this study.
StrainsAccession Number for ProteinGenotype (Resistance)Reference or Source
MAT-1wild typea1, b1[41]
ss1gpa3ΔCDU22142.1a1, b1 Δgpa3 (HYGR)[21]
ss1uac1ΔCDU22142.1a1, b1 Δuac1 (HYGR)[21]
ss1adr1ΔCDU22142.1a1, b1 Δadr1 (HYGR)[21]
Ss1ADR1-COMCDU22142.1a1, b1 Δadr1 adr1(ZEOR)[21]
ss1prf1ΔCDU21933.1a1, b1 Δprf1 (HYGR)[42]
ss1hog1ΔCDU21933.1a1, b1 Δhog1 (HYGR)[27]
Ss1HOG1-COMCDU21933.1a1, b1 Δhog1 hog1 (ZEOR)[27]
ss1hog1Δgpa3ΔCDU22378.1a1, b1 Δhog1 Δgpa3 (HYGR ZEOR)This study
ss1hog1Δuac1ΔCDU25762.1a1, b1 Δhog1 Δuac1 (HYGR ZEOR)This study
ss1hog1Δadr1ΔCDU22361.1a1, b1 Δhog1 Δadr1 (HYGR ZEOR)This study
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Cai, E.; Yan, M.; Sun, X.; Zeng, R.; Zheng, W.; Deng, Y.; Jiang, Z.; Chang, C. Kinase Hog1 and Adr1 Opposingly Regulate Haploid Cell Morphology by Controlling Vacuole Size in Sporisorium scitamineum. J. Fungi 2022, 8, 865. https://doi.org/10.3390/jof8080865

AMA Style

Cai E, Yan M, Sun X, Zeng R, Zheng W, Deng Y, Jiang Z, Chang C. Kinase Hog1 and Adr1 Opposingly Regulate Haploid Cell Morphology by Controlling Vacuole Size in Sporisorium scitamineum. Journal of Fungi. 2022; 8(8):865. https://doi.org/10.3390/jof8080865

Chicago/Turabian Style

Cai, Enping, Meixin Yan, Xian Sun, Rong Zeng, Wenqiang Zheng, Yizhen Deng, Zide Jiang, and Changqing Chang. 2022. "Kinase Hog1 and Adr1 Opposingly Regulate Haploid Cell Morphology by Controlling Vacuole Size in Sporisorium scitamineum" Journal of Fungi 8, no. 8: 865. https://doi.org/10.3390/jof8080865

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop