Defects In Cell Wall Integrity Research Articles

N-acetylglucosamine kinase (BbHxk1) has pleiotropic effects on vegetative growth, cell wall integrity, morphological transition, cuticle infection, and metabolic modulation in the biological pesticide Beauveria bassiana

Beauveria bassiana is a popular and eco-friendly biopesticide. During its pathogen-pest interaction, both N-acetylglucosamine (GlcNAc) catabolism and anabolism are crucial for nutrient supply and cell-wall construction. The initiation of GlcNAc metabolism relies on the catalysis of GlcNAc kinase, which has been extensively studied in the human pathogen Candida albicans. However, the physiological function of GlcNAc kinase remains poorly understood in entomopathogenic fungi. In the present study, a GlcNAc kinase homolog was identified and designated as BbHxk1 in B. bassiana. Deletion of BbHxk1 resulted in viable but reduced vegetative growth on various carbon sources. ΔBbHxk1 mutants displayed severe defects in cell wall integrity, making them more susceptible to cell wall stress cues. Furthermore, the absence of BbHxk1 resulted in an increase in conidial yield and blastospore production, and a faster rate of germination and filamentation, potentially attributed to higher intracellular ATP levels. BbHxk1 deficiency led to a reduction in the activities of cuticle-degrading enzymes, which might contribute to the attenuated pathogenicity specifically through cuticle penetration rather than hemocoel infection towards Galleria mellonella larvae. Being different from C. albicans Hxk1, which facultatively acts as a catalyzing enzyme and transcriptional regulator, BbHxk1 primarily acts as a catalyzing enzyme and metabolic regulator. The altered metabolomic profiling correlated with the phenotypic defects in ΔBbHxk1 mutants, further implicating a potential metabolism-dependent mechanism of BbHxk1 in mediating physiologies of B. bassiana. These findings not only unveil a novel role for GlcNAc kinase in B. bassiana, but also provide a solid theoretical basis to guide metabolic reprogramming in order to maintain or even enhance the efficiency of fungi for practical applications.

Mitigating candidiasis with acarbose by targeting Candida albicans α-glucosidase: in-silico, in-vitro and transcriptomic approaches

Biofilm-associated candidiasis poses a significant challenge in clinical settings due to the limited effectiveness of existing antifungal treatments. The challenges include increased pathogen virulence, multi-drug resistance, and inadequate penetration of antimicrobials into biofilm structures. One potential solution to this problem involves the development of novel drugs that can modulate fungal virulence and biofilm formation, which is essential for pathogenesis. Resistance in Candida albicans is initiated by morphological changes from yeast to hyphal form. This transition triggers a series of events such as cell wall elongation, increased adhesion, invasion of host tissues, pathogenicity, biofilm formation, and the initiation of an immune response. The cell wall is a critical interface for interactions with host cells, primarily through various cell wall proteins, particularly mannoproteins. Thus, cell wall proteins and enzymes are considered potential antifungal targets. In this regard, we explored α-glucosidase as our potential target which plays a crucial role in processing mannoproteins. Previous studies have shown that inhibition of α-glucosidase leads to defects in cell wall integrity, reduced adhesion, diminished secretion of hydrolytic enzymes, alterations in immune recognition, and reduced pathogenicity. Since α-glucosidase, primarily converts carbohydrates, our study focuses on FDA-approved carbohydrate mimic drugs (Glycomimetics) with well-documented applications in various biological contexts. Through virtual screening of 114 FDA-approved carbohydrate-based drugs, a pseudo-sugar Acarbose, emerged as a top hit. Acarbose is known for its pharmacological potential in managing type 2 diabetes mellitus by targeting α-glucosidase. Our preliminary investigations indicate that Acarbose effectively inhibits C. albicans biofilm formation, reduces virulence, impairs morphological switching, and hinders the adhesion and invasion of host cells, all at very low concentrations in the nanomolar range. Furthermore, transcriptomic analysis reveals the mechanism of action of Acarbose, highlighting its role in targeting α-glucosidase.

Peroxin MoPex22 Regulates the Import of Peroxisomal Matrix Proteins and Appressorium-Mediated Plant Infection in Magnaporthe oryzae.

Magnaporthe oryzae, the pathogen responsible for rice blast disease, utilizes specialized infection structures known as appressoria to breach the leaf cuticle and establish intracellular, infectious hyphae. Our study demonstrates that the peroxin MoPex22 is crucial for appressorium function, specifically for the development of primary penetration hyphae. The ∆Mopex22 mutant exhibited slow growth, reduced aerial hyphae, and almost complete loss of virulence. Specifically, despite the mutant's capability to form appressoria, it showed abnormalities during appressorium development, including reduced turgor, increased permeability of the appressorium wall, failure to form septin rings, and significantly decreased ability to penetrate host cells. Additionally, there was a delay in the degradation of lipid droplets during conidial germination and appressorium development. Consistent with these findings, the ΔMopex22 mutant showed an inefficient utilization of long-chain fatty acids and defects in cell wall integrity. Moreover, our findings indicate that MoPex22 acts as an anchor for MoPex4, facilitating the localization of MoPex4 to peroxisomes. Together with MoPex4, it affects the function of MoPex5, thus regulating the import of peroxisomal matrix proteins. Overall, these results highlight the essential role of MoPex22 in regulating the transport of peroxisomal matrix proteins, which affect fatty acid metabolism, glycerol accumulation, cell wall integrity, growth, appressorium development, and the pathogenicity of M. oryzae. This study provides valuable insights into the significance of peroxin functions in fungal biology and appressorium-mediated plant infection.

The Gβ-like protein Bcgbl1 regulates development and pathogenicity of the gray mold Botrytis cinerea via modulating two MAP kinase signaling pathways.

The fungal Gβ-like protein has been reported to be involved in a variety of biological processes, such as mycelial growth, differentiation, conidiation, stress responses and infection. However, molecular mechanisms of the Gβ-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gβ-like protein gene Bcgbl1 in the gray mold fungus Botrytis cinerea plays a pivotal role in development and pathogenicity by regulating the mitogen-activated protein (MAP) kinases signaling pathways. The Bcgbl1 deletion mutants were defective in mycelial growth, sclerotial formation, conidiation, macroconidial morphogenesis, plant adhesion, and formation of infection cushions and appressorium-like structures, resulting in a complete loss of pathogenicity. Bcgbl1 interacted with BcSte50, the adapter protein of the cascade of MAP kinase (MAPK). Bcgbl1 mutants had reduced phosphorylation levels of two MAPKs, namely Bmp1 and Bmp3, thereby reducing infection. However, deletion of Bcgbl1 did not affect the intracellular cAMP level, and exogenous cAMP could not restore the defects. Moreover, Bcgbl1 mutants exhibited defects in cell wall integrity and oxidative stress tolerance. Transcriptional profiling revealed that Bcgbl1 plays a global role in regulation of gene expression upon hydrophobic surface induction. We further uncovered that three target genes encoding the hydrophobic surface binding proteins (HsbAs) contributed to the adhesion and virulence of B. cinerea. Overall, these findings suggest that Bcgbl1 had multiple functions and provided new insights for deciphering the Bcgbl1-mediated network for regulating development and pathogenicity of B. cinerea.

Transcription Factor VM1G_06867: A Requirement for Growth, Pathogenicity, Development, and Maintenance of Cell Wall Integrity in Valsa mali.

Apple canker disease, caused by Valsa mali, is one of the most serious apple tree diseases in China. VmSom1 is an important transcription factor that acts on the cyclic adenosine signaling pathway (cAMP/PKA), regulating the growth, development, morphological differentiation, and pathogenic forces of the pathogen. We perform transcriptome analysis of the VmSom1 deletion mutant and the wild-type strain 11-175 and identify a significantly differentially expressed gene, VM1G_06867, a zinc finger motif transcription factor in V. mali. In this study, we obtain the VM1G_06867 gene using the single deletion mutant via homologous recombination. To determine the relationship between VmSom1 and VM1G_06867, we also obtain a double deletion mutant ΔVmSom1/06867. Compared to the wild-type strain 11-175, the single deletion mutant VM1G_06867 shows a drastic reduction in growth rate and forms more pycnidia on the PDA medium. Additionally, the growth of the mutant is inhibited by SDS, Congo red, and fluorescent brighteners. In comparison to the single deletion mutant VmSom1, the double deletion mutant ΔVmSom1/06867 shows no significant change in growth or conidiation and is unable to produce conidia. The growth rate is significantly increased in Congo red, NaCl, and Sorbitol mediums. These results demonstrate that VM1G_06867 plays important roles in growth, pathogenicity, asexual development, and maintenance of cell wall integrity. VM1G_06867 can recover osmotic stress and cell wall integrity defects caused by the deletion of VmSom1, as well as restore the loss of pathogenicity caused by the deletion of the VmSom1 gene, but not completely.

A C. albicans TRAPP Complex-Associated Gene Contributes to Cell Wall Integrity, Hyphal and Biofilm Formation, and Tissue Invasion.

While endocytic and secretory pathways are well-studied cellular processes in the model yeast Saccharomyces cerevisiae, they remain understudied in the opportunistic fungal pathogen Candida albicans. We previously found that null mutants of C. albicans homologs of the S. cerevisiae early endocytosis genes ENT2 and END3 not only exhibited delayed endocytosis but also had defects in cell wall integrity, filamentation, biofilm formation, extracellular protease activity, and tissue invasion in an in vitro model. In this study, we focused on a potential C. albicans homolog to S. cerevisiae TCA17, which was discovered in our whole-genome bioinformatics approach aimed at identifying genes involved in endocytosis. In S. cerevisiae, TCA17 encodes a transport protein particle (TRAPP) complex-associated protein. Using a reverse genetics approach with CRISPR-Cas9-mediated gene deletion, we analyzed the function of the TCA17 homolog in C. albicans. Although the C. albicans tca17Δ/Δ null mutant did not have defects in endocytosis, it displayed an enlarged cell and vacuole morphology, impaired filamentation, and reduced biofilm formation. Moreover, the mutant exhibited altered sensitivity to cell wall stressors and antifungal agents. When assayed using an in vitro keratinocyte infection model, virulence properties were also diminished. Our findings indicate that C. albicans TCA17 may be involved in secretion-related vesicle transport and plays a role in cell wall and vacuolar integrity, hyphal and biofilm formation, and virulence. IMPORTANCE The fungal pathogen Candida albicans causes serious opportunistic infections in immunocompromised patients and has become a major cause of hospital-acquired bloodstream infections, catheter-associated infections, and invasive disease. However, due to a limited understanding of Candida molecular pathogenesis, clinical approaches for the prevention, diagnosis, and treatment of invasive candidiasis need significant improvement. In this study, we focus on identifying and characterizing a gene potentially involved in the C. albicans secretory pathway, as intracellular transport is critical for C. albicans virulence. We specifically investigated the role of this gene in filamentation, biofilm formation, and tissue invasion. Ultimately, these findings advance our current understanding of C. albicans biology and may have implications for the diagnosis and treatment of candidiasis.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens.

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

Transcriptome Analysis and Functional Characterization Reveal That Peclg Gene Contributes to the Virulence of Penicillium expansum on Apple Fruits.

Penicillium expansum is the causal agent of blue mold decay on apple fruits and is also known to be the major producer of patulin, a mycotoxin that represents serious hazard to human health. Several mechanisms have been suggested to explain the pathogenesis of P. expansum in host plants. Secreted effector proteins are vital for the pathogenicity of many fungal pathogens through manipulating their hosts for efficient colonization. In this study, we performed a RNA-Seq analysis followed by computational prediction of effector proteins from P. expansum during infection of the host apple fruits, and a total of 212 and 268 candidate effector protein genes were identified at 6 and 9 h after inoculation (hai), respectively. One of the candidate effector protein genes was identified as a concanavalin A-like lectin/glucanase (Peclg), which was dramatically induced during the pathogen-host interaction. Targeted knockout of Peclg resulted in significant reduction in conidial production and germination relative to the wild type. Further studies showed that in addition to salt stress, the mutant was much more sensitive to SDS and Congo red, suggesting a defect in cell wall integrity. Pathogenicity assays revealed that the ΔPeclg mutant showed significant decrease in virulence and infectious growth on apple fruits. All these results suggest that Peclg is required for fungal growth, stress response, and the virulence of P. expansum.

Bacillus subtilis forms twisted cells with cell wall integrity defects upon removal of the molecular chaperones DnaK and trigger factor.

The protein homeostasis network ensures a proper balance between synthesis, folding, and degradation of all cellular proteins. DnaK and trigger factor (TF) are ubiquitous bacterial molecular chaperones that assist in protein folding, as well as preventing protein misfolding and aggregation. In Escherichia coli, DnaK and TF possess partially overlapping functions. Their combined depletion results in proteostasis collapse and is synthetically lethal at temperatures above 30°C. To increase our understanding on how proteostasis is maintained in Gram-positive bacteria, we have investigated the physiological effects of deleting dnaK and tig (encoding for DnaK and TF) in Bacillus subtilis. We show that combined deletion of dnaK and tig in B. subtilis is non-lethal, but causes a severe pleiotropic phenotype, including an aberrant twisted and filamentous cell morphology, as well as decreased tolerance to heat and to cell wall active antibiotics and hydrolytic enzymes, indicative of defects in cell wall integrity. In addition, cells lacking DnaK and TF have a much smaller colony size due to defects in motility. Despite these physiological changes, we observed no major compromises in important cellular processes such as cell growth, FtsZ localization and division and only moderate defects in spore formation. Finally, through suppressor analyses, we found that the wild-type cell shape can be partially restored by mutations in genes involved in metabolism or in other diverse cellular processes.

The cellular function of ROP GTPase prenylation is important for multicellularity in the moss Physcomitrium patens.

A complete picture of how signaling pathways lead to multicellularity is largely unknown. Previously, we generated mutations in a protein prenylation enzyme, GGB, and showed that it is essential for maintaining multicellularity in the moss Physcomitrium patens. Here, we show that ROP GTPases act as downstream factors that are prenylated by GGB and themselves play an important role in the multicellularity of P. patens. We also show that the loss of multicellularity caused by the suppression of GGB or ROP GTPases is due to uncoordinated cell expansion, defects in cell wall integrity and the disturbance of the directional control of cell plate orientation. Expressing prenylatable ROP in the ggb mutant not only rescues multicellularity in protonemata but also results in development of gametophores. Although the prenylation of ROP is important for multicellularity, a higher threshold of active ROP is required for gametophore development. Thus, our results suggest that ROP activation via prenylation by GGB is a key process at both cell and tissue levels, facilitating the developmental transition from one dimension to two dimensions and to three dimensions in P. patens.

Stress- and metabolic responses of Candida albicans require Tor1 kinase N-terminal HEAT repeats

Whether to commit limited cellular resources toward growth and proliferation, or toward survival and stress responses, is an essential determination made by Target of Rapamycin Complex 1 (TORC1) for a eukaryotic cell in response to favorable or adverse conditions. Loss of TORC1 function is lethal. The TORC1 inhibitor rapamycin that targets the highly conserved Tor kinase domain kills fungal pathogens like Candida albicans, but is also severely toxic to human cells. The least conserved region of fungal and human Tor kinases are the N-terminal HEAT domains. We examined the role of the 8 most N-terminal HEAT repeats of C. albicans Tor1. We compared nutritional- and stress responses of cells that express a message for N-terminally truncated Tor1 from repressible tetO, with cells expressing wild type TOR1 from tetO or from the native promoter. Some but not all stress responses were significantly impaired by loss of Tor1 N-terminal HEAT repeats, including those to oxidative-, cell wall-, and heat stress; in contrast, plasma membrane stress and antifungal agents that disrupt plasma membrane function were tolerated by cells lacking this Tor1 region. Translation was inappropriately upregulated during oxidative stress in cells lacking N-terminal Tor1 HEAT repeats despite simultaneously elevated Gcn2 activity, while activation of the oxidative stress response MAP kinase Hog1 was weak. Conversely, these cells were unable to take advantage of favorable nutritional conditions by accelerating their growth. Consuming oxygen more slowly than cells containing wild type TOR1 alleles during growth in glucose, cells lacking N-terminal Tor1 HEAT repeats additionally were incapable of utilizing non-fermentable carbon sources. They were also hypersensitive to inhibitors of specific complexes within the respiratory electron transport chain, suggesting that inefficient ATP generation and a resulting dearth of nucleotide sugar building blocks for cell wall polysaccharides causes cell wall integrity defects in these mutants. Genome-wide expression analysis of cells lacking N-terminal HEAT repeats showed dysregulation of carbon metabolism, cell wall biosynthetic enzymes, translational machinery biosynthesis, oxidative stress responses, and hyphal- as well as white-opaque cell type-associated genes. Targeting fungal-specific Tor1 N-terminal HEAT repeats with small molecules might selectively abrogate fungal viability, especially when during infection multiple stresses are imposed by the host immune system.

Fusarium verticillioides FvPex8 Is a Key Component of the Peroxisomal Docking/Translocation Module That Serves Important Roles in Fumonisin Biosynthesis but Not in Virulence.

Peroxisomes are ubiquitous organelles in eukaryotes that fulfill various important metabolic functions. In this study, we investigated the role of docking/translocation module (DTM) peroxins, mainly FvPex8, FvPex13, FvPex14, and FvPex33, in Fusarium verticillioides development, virulence, and fumonisin B1 (FB1) biosynthesis. Protein interaction experiments suggested that FvPex13 serves as the central DTM subunit in F. verticillioides. Notably, FvPex8 and FvPex14 did not show direct interaction in our experiments. We generated gene-deletion mutants (ΔFvpex8, ΔFvpex13, ΔFvpex14, ΔFvpex33, ΔFvpex33/14) and further examined the functional role of these peroxins. Deletion mutants exhibited disparity in carbon nutrient utilization and defect in cell-wall integrity when stress agents were applied. Under nutrient starvation, mutants also showed higher levels of lipid droplet accumulation. Particularly, ΔFvpex8 mutant showed significant FB1 reduction and altered expression of key FB1 biosynthesis genes. However, FvPex13 was primarily responsible for asexual conidia reproduction and virulence, while the ΔFvpex33/14 double mutant also showed a virulence defect. In summary, our study suggests that FvPex13 is the central component of DTM, with direct physical interaction with other DTM peroxins, and regulates peroxisome membrane biogenesis as well as PTS1- and PTS2-mediated transmembrane cargo transportation. Importantly, we also characterized FvPex8 as a key component in F. verticillioides DTM that affects peroxisome function and FB1 biosynthesis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The N-mannosyltransferase gene BbAlg9 contributes to cell wall integrity, fungal development and the pathogenicity of Beauveria bassiana

The mannosyltransferase Alg9 plays a vital role in N-linked protein glycosylation in Saccharomyces cerevisiae, but its function in most filamentous fungi is not clear. The present study characterized BbAlg9 (an ortholog of S. cerevisiae Alg9) in Beauveria bassiana to determine the roles of N-mannosyltransferase in biological control potential of the filamentous entomopathogenic fungus. The disruption of BbAlg9 led to slower fungal growth in media with various nutrition compositions. The conidiation of ΔBbAlg9 was less than that of the wild type from the third to the fifth day but showed no significant difference on the sixth day, suggesting that BbAlg9 affects the development of conidia rather than conidial yield of late stage. ΔBbAlg9 showed defects in conidial germination, multiple stress tolerances and the yield of blastospores, with altered size and density, and virulence in hosts infected via the immersion and injection methods. The deletion of BbAlg9 resulted in defects in cell wall integrity, including increased mannoprotein and glucan content and decreased chitin content, which were accompanied by transcriptional activation or suppression of genes related to cell wall component biosynthesis. Notably, deletion of the N-mannosyltransferase BbAlg9 altered the transcription levels of O-mannosyltransferase genes (Pmt and Ktr family). These data show that BbAlg9 is involved in the fungal development, conidial stress tolerance, cell wall integrity and virulence of B. bassiana.

Calcineurin-Responsive Transcription Factor CgCrzA Is Required for Cell Wall Integrity and Infection-Related Morphogenesis in Colletotrichum gloeosporioides.

The ascomycete fungus Colletotrichum gloeosporioides infects a wide range of plant hosts and causes enormous economic losses in the world. The transcription factors (TFs) play an important role in development and pathogenicity of many organisms. In this study, we found that the C2H2 TF CgCrzA is localized in both cytoplasm and nucleus under standard condition, and it translocated from cytoplasm to nucleus in a calcineurin-dependent manner. Moreover, the ΔCgCrzA was hypersensitive to cell wall perturbing agents and showed severe cell wall integrity defects. Deletion of the CgCRZA inhibited the development of invasive structures and lost pathogenicity to plant hosts. Our results suggested that calcineurin-responsive TF CgCrzA was not only involved in regulating cell wall integrity, but also in morphogenesis and virulence in C. gloeosporioides.

Analysis of the C‐terminal domain of MtlA and its role in cell wall metabolism in the fungus Aspergillus nidulans

The Mid2‐like protein MtlA is a putative sensor that affects cell wall integrity in the filamentous fungus Aspergillus nidulans. Plasmid‐based, overexpression of MtlA can suppress the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin‐binding agent Calcofluor White. In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity. MtlA is predicted to have a single transmembrane domain, an extracellular domain rich in serine and threonine amino acids residues, and a C‐terminal domain of 59 amino acids. A MtlA ‐GFP hybrid localizes to the plasma membrane of vegetative hyphae. In the cell wall integrity pathway in yeast, the C‐terminal domain of the stress receptors, Wsc1 and Mid2, have been shown to play an important role in the signal transduction pathway. We have shown that the C‐terminal domain of MtlA plays an important role in cell wall integrity. We have created and analyzed C‐terminal truncation mutants of MtlA and have shown that complete removal of the C‐terminal domain obliterates its function. We have also shown that amino acids 241–251 are critical for the function of MtlA. Site directed mutagenesis of key amino acids will be elucidated.Support or Funding InformationRhodes CollegeNational Science FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes.

Excess soluble salts in soil are harmful to the growth and development of most plants. Evidence is emerging that the plant cell wall is involved in sensing and responding to salt stress, but the underlying mechanisms are not well understood. We reveal that the histone acetyltransferase General control non-repressed protein 5 (GCN5) is required for the maintenance of cell wall integrity and salt stress tolerance. The levels of GCN5 mRNA are increased in response to salt stress. The gcn5 mutants exhibited severe growth inhibition and defects in cell wall integrity under salt stress conditions. Combining RNA sequencing and chromatin immunoprecipitation assays, we identified the chitinase-like gene CTL1, polygalacturonase involved in expansion-3 (PGX3) and MYB domain protein-54 (MYB54) as direct targets of GCN5. Acetylation of H3K9 and H3K14 mediated by GCN5 is associated with activation of CTL1, PGX3 and MYB54 under salt stress. Moreover, constitutive expression of CTL1 in the gcn5 mutant restores salt tolerance and cell wall integrity. In addition, the expression of the wheat TaGCN5 gene in Arabidopsis gcn5 mutant plants complemented the salt tolerance and cell wall integrity phenotypes, suggesting that GCN5-mediated salt tolerance is conserved between Arabidopsis and wheat. Taken together, our data indicate that GCN5 plays a key role in the preservation of salt tolerance via versatile regulation in plants.

The MAPKKK CgMck1 Is Required for Cell Wall Integrity, Appressorium Development, and Pathogenicity in Colletotrichum gloeosporioides.

Mitogen-activated protein kinase (MAPK) signaling pathway plays key roles in sensing extracellular signals and transmitting them from the cell membrane to the nucleus in response to various environmental stimuli. A MAPKKK protein CgMck1 in Colletotrichum gloeosporioides was characterized. Phenotypic analyses of the ∆Cgmck1 mutant showed that the CgMck1 was required for vegetative growth, fruiting body development, and sporulation. Additionally, the CgMCK1 deletion mutant showed significant defects in cell wall integrity, and responses to osmotic stresses. The mutant abolished the ability to develop appressorium, and lost pathogenicity to host plants. The ∆Cgmck1 mutant also exhibited a higher sensitivity to antifungal bacterium agent Bacillus velezensis. The deletion mutants of downstream MAPK cascades components CgMkk1 and CgMps1 showed similar defects to the ∆Cgmck1 mutant. In conclusion, CgMck1 is involved in the regulation of vegetative growth, asexual development, cell wall integrity, stresses resistance, and infection morphogenesis in C. gloeosporioides.

Pleiotropic Roles of ChSat4 in Asexual Development, Cell Wall Integrity Maintenance, and Pathogenicity in Colletotrichum higginsianum.

Potassium has an important role to play in multiple cellular processes. In Saccharomyces cerevisiae, the serine/threonine (S/T) kinase Sat4/Hal4 is required for potassium accumulation, and thus, regulates the resistance to sodium salts and helps in the stabilization of other plasma membrane transporters. However, the functions of Sat4 in filamentous phytopathogenic fungi are largely unknown. In this study, ChSat4, the yeast Sat4p homolog in Colletotrichum higginsianum, has been identified. Target deletion of ChSAT4 resulted in defects in mycelial growth and sporulation. Intracellular K+ accumulation was significantly decreased in the ChSAT4 deletion mutant. Additionally, the ΔChsat4 mutant showed defects in cell wall integrity, hyperoxide stress response, and pathogenicity. Localization pattern analysis indicated ChSat4 was localized in the cytoplasm. Furthermore, ChSat4 showed high functional conservation with the homolog FgSat4 in Fusarium graminearum. Taken together, our data indicated that ChSat4 was important for intracellular K+ accumulation and infection morphogenesis in C. higginsianum.

Disruption of gul-1 decreased the culture viscosity and improved protein secretion in the filamentous fungus Neurospora crassa

BackgroundThe cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. However, large scale applications are hindered by its filamentous growth. Although previous investigations have shown that mycelial morphology in submerged culture can be controlled by altering physical factors, there is little knowledge available about the potential for morphology control by genetic modification.ResultsIn this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only two mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Transcriptional profiling showed that expression of genes encoding eight GPI-anchored cell wall proteins was lowered in Δgul-1 while expression of genes associated with two non-anchored cell wall proteins was elevated. Meanwhile, the expression levels of two hydrophobin genes were also significantly altered. These results suggested that GUL-1 affected the transcription of cell wall-related genes, thereby influencing cell wall structure and mycelial morphology. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical applications, deletion of gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth.ConclusionsUsing the model filamentous fungus N. crassa, genes that affect mycelial morphology in submerged culture were explored through systematic screening of morphological mutants. Disrupting several candidate genes altered viscosities in submerged culture. This work provides an example for controlling fungal morphology in submerged fermentation by genetic engineering, and will be beneficial for industrial fungal strain improvement.

Mannosylinositol phosphorylceramides and ergosterol coodinately maintain cell wall integrity in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group, and breakdown of their normal composition causes several cellular dysfunctions. Previously we found that loss of biosynthesis of mannosylinositol phosphorylceramide (MIPC) causes a defect in cell wall integrity (CWI). In this study, we screened for multicopy suppressor genes that rescue the defect in CWI in cells lacking MIPC synthases (Sur1 and Csh1), and found that the defect is partly suppressed by upregulation of ergosterol biosynthesis. In addition, repression of expression of ERG9, which encodes squalene synthase in the ergosterol biosynthesis pathway, in sur1∆ csh1∆ cells caused a strong growth defect and enhancement of the defect in CWI. The repression of ERG9 and/or the deletion of SUR1 and CSH1 caused an increase in the phosphorylated form of Slt2, a mitogen-activated protein kinase activated through impairment of CWI. Moreover, the deletion of SLT2 or WSC1/2, encoding a sensor protein recognizing CWI, enhanced the growth defect in the ERG9-repressed sur1∆ csh1∆ cells. On the other hand, the ERG9-repressed sur1∆ csh1∆ cells also exhibited an increase in the cell wall chitin level in a Slt2- and Wsc1/2-independent manner. These results suggested that MIPC and ergosterol are coordinately involved in maintenance of CWI, and the activation of Slt2 suppressed the CWI defect caused by these metabolic defects.