Search

Grey mould caused by Botrytis cinerea is a devastating disease responsible for large losses to agricultural production, and B. cinerea is a necrotrophic model fungal plant pathogen. Membrane proteins are important targets of fungicides and hotspots in the research and development of fungicide products. Wuyiencin affects the permeability and pathogenicity of B. cinerea, parallel reaction monitoring revealed the association of membrane protein Bcsdr2, and the bacteriostatic mechanism of wuyiencin was elucidated. In the present work, we generated and characterised ΔBcsdr2 deletion and complemented mutant B. cinerea strains. The ΔBcsdr2 deletion mutants exhibited biofilm loss and dissolution, and their functional activity was illustrated by reduced necrotic colonisation on strawberry and grape fruits. Targeted deletion of Bcsdr2 also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted gene complementation. The roles of Bcsdr2 in biofilms and pathogenicity were also supported by quantitative real-time RT-PCR results showing that phosphatidylserine decarboxylase synthesis gene Bcpsd and chitin synthase gene BcCHSVII were downregulated in the early stages of infection for the ΔBcsdr2 strain. The results suggest that Bcsdr2 plays important roles in regulating various cellular processes in B. cinerea. Key points: • The mechanism of wuyiencin inhibits B. cinerea is closely associated with membrane proteins. • Wuyiencin can downregulate the expression of the membrane protein Bcsdr2 in B. cinerea. • Bcsdr2 is involved in regulating B. cinerea virulence, growth and development. [ABSTRACT FROM AUTHOR]

AbstractWheat stripe rust disease caused by Puccinia striiformis f. sp. tritici (Pst) seriously affects wheat yield. We studied the fungal infection process and host response between wheat ‘AvS’ and its near-isogenic line ‘AvS+Yr10’, which formed compatible and incompatible interactions, respectively, when challenged with Pst race CYR32. The difference in reactive oxygen species (ROS) accumulation between the compatible and incompatible interactions was analyzed by measuring the hydrogen peroxide (H2O2) concentration and observing H2O2 production. Hydrogen peroxide concentrations, H2O2 accumulation areas, necrotic areas and expression of several resistance-related genes in the AvS+Yr10/CYR32 interaction were higher than those in the AvS/CYR32 interaction during the entire infection process. Pst growth was effectively inhibited in the AvS+Yr10 all-stage resistant wheat line. Thus, ROS accumulation is crucial in wheat defence against pathogens. [ABSTRACT FROM AUTHOR]

BackgroundDuring the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear.ResultsWe analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type.ConclusionsWe show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions. [ABSTRACT FROM AUTHOR]

AbstractThe tolerance of the entomophagous fungus Lecanicillium muscarium to eight pesticides using a poisoned medium method in the laboratory was evaluated. The purpose was to identify compatible combinations between biopesticides based on pathogenic fungi and conventional pesticides. Pyridalyl slightly inhibited the germination of L. muscarium spores and very slightly inhibited hyphal growth at low concentrations. The maximum inhibition rate of pyridalyl was <30%. Difenoconazole very slightly inhibited the germination of L. muscarium of conidia, and thiamethoxam and dimethomorph both very slightly inhibited hyphal growth. In most treatments, the maximum inhibition rates of difenoconazole, dimethomorph, or thiamethoxam were <20%. Phoxim at a low concentration slightly inhibited both L. muscarium conidial germination and hyphal growth (maximum inhibition rate, <25%). The minimum inhibition rate of most chlorothalonil, triadimefon, or tetraconazole treatments exceeded 60%. At low concentrations, pyridalyl and phoxim showed weak inhibitory effects, and therefore, may be compatible with L. muscarium. These data will support recommendations for the use of pesticides in integrated pest management programmes that involve L. muscarium as the biocontrol agent. [ABSTRACT FROM AUTHOR]

The ADP-ribosylation factor 6 (Arf6), as the only member of the Arf family III protein, has been extensively studied for its diverse biological functions in animals. Previously, the Arf6 protein in Magnaporthe oryzae was found to be crucial for endocytosis and polarity establishment during asexual development. However, its role remains unclear in S. sclerotiorum. Here, we identified and characterized SsArf6 in S. sclerotiorum using a reverse genetic approach. Deletion of SsArf6 impaired hyphal growth and development and produced more branches. Interestingly, knockout of SsArf6 resulted in an augmented tolerance of S. sclerotiorum towards oxidative stress, and increased its sensitivity towards osmotic stress, indicative of the different roles of SsArf6 in various stress responses. Simultaneously, SsArf6 deletion led to an elevation in melanin accumulation. Moreover, the appressorium formation was severely impaired, and fungal virulence to host plants was significantly reduced. Overall, our findings demonstrate the essential role of SsArf6 in hyphal development, stress responses, appressorium formation, and fungal virulence to host plants. [ABSTRACT FROM AUTHOR]

Coccidioides is a dimorphic fungus responsible for Valley Fever and is the cause of severe morbidity and mortality in the infected population. Although there is some insight into the genes, pathways, and growth media involved in the parasitic to saprophytic growth transition, the exact determinants that govern this switch are largely unknown. In this work, we examined the growth and morphology of a Coccidioides posadasii strain (C. posadasii S/E) that efficiently produces spherules and endospores and persists in the parasitic life cycle at ambient CO2. We demonstrated that C. posadasii S/E remains virulent in an insect infection model. Surprisingly, under spherule-inducing conditions, the C. posadasii S/E culture was found to be completely hyphal. Differential interference contrast (DIC) and transmission electron microscopy (TEM) revealed unexpected cellular changes in this strain including cell wall remodeling and formation of septal pores with Woronin bodies. Our study suggests that the C. posadasii S/E strain is a useful BSL-2 model for studying mechanisms underlying the parasitic to saprophytic growth transition-a morphological switch that can impact the pathogenicity of the organism in the host.

Formation of hyphae is a key virulence trait of the fungal pathogen Candida albicans. Hypha morphogenesis depends upon the cyclin Hgc1, which acts together with cyclin-dependent protein kinase Cdc28 to phosphorylate effectors that drive polarized growth. Hgc1 has also been implicated in gene regulation through its effects on 2 transcription factors, Efg1 and Ume6. Here, we report RNA-sequencing (RNA-seq) analysis of 2 pairs of hgc1Δ/Δ mutants and their respective wild-type strains, which lie in 2 different genetic backgrounds. We find that hgc1Δ/Δ mutations alter expression of 271 genes in both genetic backgrounds and 266 of those genes respond consistently with regard to up- or down-regulation. Consistency is similar to what has been observed with efg1Δ/Δ mutations and greater than observed with nrg1Δ/Δ mutations in these 2 backgrounds. The gene expression response includes genes under Efg1 control, as expected from prior studies. Hgc1-responsive genes also include ergosterol biosynthetic genes and bud neck-related genes, which may reflect interactions between Hgc1 and additional transcription factors as well as effects of Hgc1 on cellular length-to-width ratios. [ABSTRACT FROM AUTHOR]

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions.

Filamentous fungi produce specialized cells called hyphae. These cells grow by polarized extension at their apex, which is maintained by the balance of endocytosis and exocytosis at the apex. Although endocytosis has been well characterized in other organisms, the details of endocytosis and its role in maintaining polarity during hyphal growth in filamentous fungi is comparatively sparsely studied. In recent years, a concentrated region of protein activity that trails the growing apex of hyphal cells has been discovered. This region, dubbed the "endocytic collar" (EC), is a dynamic 3-dimensional region of concentrated endocytic activity, the disruption of which results in the loss of hyphal polarity. Here, fluorescent protein–tagged fimbrin was used as a marker to map the collar during growth of hyphae in three fungi: Aspergillus nidulans, Colletotrichum graminicola, and Neurospora crassa. Advanced microscopy techniques and novel quantification strategies were then utilized to quantify the spatiotemporal localization and recovery rates of fimbrin in the EC during hyphal growth. Correlating these variables with hyphal growth rate revealed that the strongest observed relationship with hyphal growth is the distance by which the EC trails the apex, and that measured endocytic rate does not correlate strongly with hyphal growth rate. This supports the hypothesis that endocytic influence on hyphal growth rate is better explained by spatiotemporal regulation of the EC than by the raw rate of endocytosis. [ABSTRACT FROM AUTHOR]

Botrytis cinerea is a necrotrophic model fungal plant pathogen that causes grey mould, a devastating disease responsible for large losses in the agriculture sector. As important targets of fungicides, membrane proteins are hot spots in the research and development of fungicide products. We previously found that membrane protein Bcest may be closely related to the pathogenicity of Botrytis cinerea. Herein, we further explored its function. We generated and characterised ΔBcest deletion mutants of B. cinerea and constructed complemented strains. The ΔBcest deletion mutants exhibited reduced conidia germination and germ tube elongation. The functional activity of ΔBcest deletion mutants was investigated by reduced necrotic colonisation of B. cinerea on grapevine fruits and leaves. Targeted deletion of Bcest also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted-gene complementation. The role of Bcest in pathogenicity was also supported by reverse-transcriptase real-time quantitative PCR results indicating that melanin synthesis gene Bcpks13 and virulence factor Bccdc14 were significantly downregulated in the early infection stage of the ΔBcest strain. Taken together, these results suggest that Bcest plays important roles in the regulation of various cellular processes in B. cinerea. [ABSTRACT FROM AUTHOR]

Filamentous fungi grow through elongation of their apical region by exocytosis and secrete enzymes that can be of commercial or industrial importance. Their hyphae exhibit extensive branching, making it difficult to control hyphal growth for observation and analysis. Therefore, although hyphal morphology and productivity are closely related, the relationship between the two has not yet been clarified. Conventional morphology and productivity studies have only compared the results of macro imaging of fungal pellets cultured in bulk with the averaged products in the culture medium. Filamentous fungi are multicellular and their expression differs between different hyphae. To truly understand the relationship between morphology and productivity, it is necessary to compare the morphology and productivity of individual hyphae. To achieve this, we developed a microfluidic system that confines hyphae to individual channels for observation and investigated the relationship between their growth, morphology, and enzyme productivity. Furthermore, using Trichoderma reesei, a potent cellulaseproducing fungus, as a model, we developed a cellulase detection assay with 4-MUC substrate to detect hyphal growth and enzyme secretion in a microfluidic device in real time. Using a strain that expresses cellobiohydrolase I (CBH I) fused with AcGFP1, we compared fluorescence from the detection assay with GFP fluorescence intensity, which showed a strong correlation between the two. These results indicate that extracellular enzymes can be easily detected in the microfluidic device in real time because the production of cellulase is synchronized in T. reesei. This microfluidic system enables real-time visualization of the dynamics of hypha and enzymes during carbon source exchange and the quantitative dynamics of gene expression. This technology can be applied to many biosystems from bioenergy production to human health. [ABSTRACT FROM AUTHOR]

Candida lusitaniae is an emerging opportunistic pathogenic yeast capable of shifting from yeast to pseudohyphae form, and it is one of the few Candida species with the ability to reproduce sexually. In this study, we showed that a dpp3D mutant, inactivated for a putative pyrophosphatase, is impaired in cell separation, pseudohyphal growth and mating. The defective phenotypes were not restored after the reconstruction of a wild-type DPP3 locus, reinforcing the hypothesis of the presence of an additional mutation that we suspected in our previous study. Genetic crosses and genome sequencing identified an additional mutation in MED15, encoding a subunit of the mediator complex that functions as a general transcriptional co-activator in Eukaryotes. We confirmed that inactivation of MED15 was responsible for the defective phenotypes by rescuing the dpp3Δ mutant with a wildtype copy of MED15 and constructing a med15Δ knockout mutant that mimics the phenotypes of dpp3D in vitro. Proteomic analyses revealed the biological processes under the control of Med15 and involved in hyphal growth, cell separation and mating. This is the first description of the functions of MED15 in the regulation of hyphal growth, cell separation and mating, and the pathways involved in C. lusitaniae. [ABSTRACT FROM AUTHOR]

Communication is crucial for organismic interactions, from bacteria, to fungi, to humans. Humans may use the visual sense to monitor the environment before starting acoustic interactions. In comparison, fungi, lacking a visual system, rely on a cell-to-cell dialogue based on secreted signaling molecules to coordinate cell fusion and establish hyphal networks. Within this dialogue, hyphae alternate between sending and receiving signals. This pattern can be visualized via the putative signaling protein Soft (SofT), and the mitogen-activated protein kinase MAK-2 (MakB) which are recruited in an alternating oscillatory manner to the respective cytoplasmic membrane or nuclei of interacting hyphae. Here, we show that signal oscillations already occur in single hyphae of Arthrobotrys flagrans in the absence of potential fusion partners (cell monologue). They were in the same phase as growth oscillations. In contrast to the anti-phasic oscillations observed during the cell dialogue, SofT and MakB displayed synchronized oscillations in phase during the monologue. Once two fusion partners came into each other’s vicinity, their oscillation frequencies slowed down (entrainment phase) and transit into anti-phasic synchronization of the two cells’ oscillations with frequencies of 104±28 s and 117±19 s, respectively. Single-cell oscillations, transient entrainment, and anti-phasic oscillations were reproduced by a mathematical model where nearby hyphae can absorb and secrete a limited molecular signaling component into a shared extracellular space. We show that intracellular Ca2+ concentrations oscillate in two approaching hyphae, and depletion of Ca2+ from the medium affected vesicle-driven extension of the hyphal tip, abolished the cell monologue and the anti-phasic synchronization of two hyphae. Our results suggest that single hyphae engage in a ‘monologue’ that may be used for exploration of the environment and can dynamically shift their extracellular signaling systems into a ‘dialogue’ to initiate hyphal fusion.

ABSTRACT Fungal dimorphism involves two morphologies: a unicellular yeast cell and a multicellular hyphal form. Invasion of hyphae into human cells causes severe opportunistic infections. The transition between yeast and hyphal forms is associated with the virulence of fungi; however, the mechanism is poorly understood. Therefore, we aimed to identify factors that induce hyphal growth of Trichosporon asahii, a dimorphic basidiomycete that causes trichosporonosis. T. asahii showed poor growth and formed small cells containing large lipid droplets and fragmented mitochondria when cultivated for 16 h in a nutrient-deficient liquid medium. However, these phenotypes were suppressed via the addition of yeast nitrogen base. When T. asahii cells were cultivated in the presence of different compounds present in the yeast nitrogen base, we found that magnesium sulfate was a key factor for inducing cell elongation, and its addition dramatically restored hyphal growth in T. asahii. In T. asahii hyphae, vacuoles were enlarged, the size of lipid droplets was decreased, and mitochondria were distributed throughout the cell cytoplasm and adjacent to the cell walls. Additionally, hyphal growth was disrupted due to treatment with an actin inhibitor. The actin inhibitor latrunculin A disrupted the mitochondrial distribution even in hyphal cells. Furthermore, magnesium sulfate treatment accelerated hyphal growth in T. asahii for 72 h when the cells were cultivated in a nutrient-deficient liquid medium. Collectively, our results suggest that an increase in magnesium levels triggers the transition from the yeast to hyphal form in T. asahii. These findings will support studies on the pathogenesis of fungi and aid in developing treatments. IMPORTANCE Understanding the mechanism underlying fungal dimorphism is crucial to discern its invasion into human cells. Invasion is caused by the hyphal form rather than the yeast form; therefore, it is important to understand the mechanism of transition from the yeast to hyphal form. To study the transition mechanism, we utilized Trichosporon asahii, a dimorphic basidiomycete that causes severe trichosporonosis since there are fewer studies on T. asahii than on ascomycetes. This study suggests that an increase in Mg2+, the most abundant mineral in living cells, triggers growth of filamentous hyphae and increases the distribution of mitochondria throughout the cell cytoplasm and adjacent to the cell walls in T. asahii. Understanding the mechanism of hyphal growth triggered by Mg2+ increase will provide a model system to explore fungal pathogenicity in the future.

The ADP-ribosylation factor 6 (Arf6), as the only member of the Arf family III protein, has been extensively studied for its diverse biological functions in animals. Previously, the Arf6 protein in Magnaporthe oryzae was found to be crucial for endocytosis and polarity establishment during asexual development. However, its role remains unclear in S. sclerotiorum. Here, we identified and characterized SsArf6 in S. sclerotiorum using a reverse genetic approach. Deletion of SsArf6 impaired hyphal growth and development and produced more branches. Interestingly, knockout of SsArf6 resulted in an augmented tolerance of S. sclerotiorum towards oxidative stress, and increased its sensitivity towards osmotic stress, indicative of the different roles of SsArf6 in various stress responses. Simultaneously, SsArf6 deletion led to an elevation in melanin accumulation. Moreover, the appressorium formation was severely impaired, and fungal virulence to host plants was significantly reduced. Overall, our findings demonstrate the essential role of SsArf6 in hyphal development, stress responses, appressorium formation, and fungal virulence to host plants.

Introduction: Biofungicides with low toxicity and high efficiency are a global priority for sustainable agricultural development. Phytohormone salicylic acid (SA) is an ancient medicine against various diseases in humans and activates the immune system in plants, but little is known of its function as a biofungicide. Objectives: Here, Fusarium oxysporum, the causal agent of devastating Fusarium wilt and immunodepressed patients, was used as a model system to explore whether SA can enter the pathogen cells and suppress key targets of the pathogen. Methods: Oxford Nanopore MinION sequencing and high-throughput chromosome conformation capture (Hi-C) sequencing were used to analyzed the genome of F. oxysporum. In addition, RNA-seq, qRT-PCR, and western blotting were conducted to detect gene and protein expression levels. Results: We isolated and sequenced the genome of F. oxysporum from potato dry rot, and the F. oxysporum included 12 chromosomes and 52.3 Mb genomic length. Pharmacological assays showed that exogenous application of SA can efficiently arrest hyphal growth, spore production, and pathogenicity of F. oxysporum, whereas endogenous salicylate hydroxylases significantly detoxify SA. The synergistic growth inhibition of F. oxysporum was observed when SA was combined with rapamycin. Kinase assays showed that SA inhibits FoTOR complex 1 (FoTORC1) by activating FoSNF1 in vivo. Transgenic potato plants with the interference of FoTOR1 and FoSAH1 genes inhibited the invasive growth of hyphae and significantly prevented the occurrence of Fusarium wilt. Conclusion: This study revealed the underlying mechanisms of SA against F. oxysporum and provided insights into SA in controlling various fungal diseases by targeting the SNF1-TORC1 pathway of pathogens.

Filamentous fungi grow through elongation of their apical region by exocytosis and secrete enzymes that can be of commercial or industrial importance. Their hyphae exhibit extensive branching, making it difficult to control hyphal growth for observation and analysis. Therefore, although hyphal morphology and productivity are closely related, the relationship between the two has not yet been clarified. Conventional morphology and productivity studies have only compared the results of macro imaging of fungal pellets cultured in bulk with the averaged products in the culture medium. Filamentous fungi are multicellular and their expression differs between different hyphae. To truly understand the relationship between morphology and productivity, it is necessary to compare the morphology and productivity of individual hyphae. To achieve this, we developed a microfluidic system that confines hyphae to individual channels for observation and investigated the relationship between their growth, morphology, and enzyme productivity. Furthermore, using Trichoderma reesei, a potent cellulase-producing fungus, as a model, we developed a cellulase detection assay with 4-MUC substrate to detect hyphal growth and enzyme secretion in a microfluidic device in real time. Using a strain that expresses cellobiohydrolase I (CBH I) fused with AcGFP1, we compared fluorescence from the detection assay with GFP fluorescence intensity, which showed a strong correlation between the two. These results indicate that extracellular enzymes can be easily detected in the microfluidic device in real time because the production of cellulase is synchronized in T. reesei. This microfluidic system enables real-time visualization of the dynamics of hypha and enzymes during carbon source exchange and the quantitative dynamics of gene expression. This technology can be applied to many biosystems from bioenergy production to human health.

In dimorphic fungi, the yeast‐to‐filament transition critical for cell survival under nutrient starvation is controlled by both activators and repressors. However, very few filamentation repressors are known. Here we report that, in the dimorphic yeast Yarrowia lipolytica, the conserved transcription factor YlNrg1 plays a minor role whereas Fts1, a newly identified Zn(II)2Cys6 zinc cluster transcription factor, plays a key role in filamentation repression. FTS1 deletion caused hyperfilamentation whereas Fts1 overexpression drastically reduced filamentation. The expression of FTS1 is downregulated substantially during the yeast‐to‐filament transition. Transcriptome sequencing revealed that Fts1 represses 401 genes, including the filamentation‐activating transcription factor genes MHY1, YlAZF1, and YlWOR4 and key cell wall protein genes. Tup1‐Ssn6, a general transcriptional corepressor, is involved in the repression of many cellular functions in fungi. We show that both YlTup1 and YlSsn6 strongly repress filamentation in Y. lipolytica. YlTup1 and YlSsn6 together repress 1383 genes, including a large number of transcription factor and cell wall protein genes, which overlap substantially with Fts1‐repressed genes. Fts1 interacts with both YlTup1 and YlSsn6, and LexA‐Fts1 fusion represses a lexAop‐promoter‐lacZ reporter in a Tup1‐Ssn6‐dependent manner. Our findings suggest that Fts1 functions as a transcriptional repressor, directing the repression of target genes through the Tup1‐Ssn6 corepressor. [ABSTRACT FROM AUTHOR]

Abstract Background During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during “overwintering.” Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. Results We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. Conclusions We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

[Display omitted] • Growth and sporulation rates do not correlate with virulence in Aphanomyces astaci. • Mitochondrial haplogroups cannot predict virulence in Aphanomyces astaci. • Aphanomyces astaci strains are characterised by unique combinations of phenotypes. • Aphanomyces astaci strains diverge due to local coevolution with the new host. Introduced into Europe from North America 150 years ago alongside its native crayfish hosts, the invasive pathogen Aphanomyces astaci is considered one of the main causes of European crayfish population decline. For the past two centuries, this oomycete pathogen has been extensively studied, with the more recent efforts focused on containing and monitoring its spread across the continent. However, after the recent introduction of new strains, the newly-discovered diversity of A. astaci in North America and several years of coevolution with its European host, a new assessment of the traits linked to the pathogen's virulence is much needed. To fill this gap, we investigated the presence of phenotypic patterns (i.e., in vitro growth and sporulation rates) possibly associated with the pathogen's virulence (i.e., induced mortality in crayfish) in a collection of 14 A. astaci strains isolated both in North America and in Europe. The results highlighted a high variability in virulence, growth rate and motile spore production among the different strains, while the total-sporulation rate was more similar across strains. Surprisingly, growth and sporulation rates were not significantly correlated with virulence. Furthermore, none of the analysed parameters, including virulence, was significantly different among the major A. astaci haplogroups. These results indicate that each strain is defined by a characteristic combination of pathogenic features, specifically assembled for the environment and host faced by each strain. Thus, canonical mitochondrial markers, often used to infer the pathogen's virulence, are not accurate tools to deduce the phenotype of A. astaci strains. As the diversity of A. astaci strains in Europe is bound to increase due to translocations of new carrier crayfish species from North America, there is an urgent need to deepen our understanding of A. astaci 's virulence variability and its ability to adapt to new hosts and environments. [ABSTRACT FROM AUTHOR]

ABSTRACT The yeast-to-filament transition is an important cellular response to environmental stimulations in dimorphic fungi. In addition to activators, there are repressors in the cells to prevent filament formation, which is important to keep the cells in the yeast form when filamentation is not necessary. However, very few repressors of filamentation are known so far. Here, we identify a novel repressor of filamentation in the dimorphic yeast Yarrowia lipolytica, Fts2, which is a C2H2-type zinc finger transcription factor. We show that fts2Δ cells exhibited increased filamentation under mild filament-inducing conditions and formed filaments under non-filament-inducing conditions. We also show that Fts2 interacts with YlSsn6, component of the Tup1-Ssn6 transcriptional corepressor, and Fts2-LexA represses a lexAop-PYlACT1-lacZ reporter in a Tup1-Ssn6-dependent manner, suggesting that Fts2 has transcriptional repressor activity and represses gene expression via Tup1-Ssn6. In addition, we show that Fts2 represses a large number of cell wall protein genes and transcription factor genes, some of which are implicated in the filamentation response. Interestingly, about two-thirds of Fts2-repressed genes are also repressed by Tup1-Ssn6, suggesting that Fts2 may repress the bulk of its target genes via Tup1-Ssn6. Lastly, we show that Fts2 expression is downregulated in response to alkaline pH and the relief of negative control by Fts2 facilitates the induction of filamentation by alkaline pH. IMPORTANCE The repressors of filamentation are important negative regulators of the yeast-to-filament transition. However, except in Candida albicans, very few repressors of filamentation are known in dimorphic fungi. More importantly, how they repress filamentation is often not clear. In this paper, we report a novel repressor of filamentation in Y. lipolytica. Fts2 is not closely related in amino acid sequence to CaNrg1 and Rfg1, two major repressors of filamentation in C. albicans, yet it represses gene expression via the transcriptional corepressor Tup1-Ssn6, similar to CaNrg1 and Rfg1. Using transcriptome sequencing, we determined the whole set of genes regulated by Fts2 and identified the major targets of Fts2 repression, which provide clues to the mechanism by which Fts2 represses filamentation. Our results have important implications for understanding the negative control of the yeast-to-filament transition in dimorphic fungi.

[Display omitted] • Isolating and sequencing the genome of F. oxysporum from potato tubers with dry rot symptoms. • SA efficiently arrests hyphal growth, sporular production and pathogenicity of F. oxysporum. • SA inhibits the activity of FoTORC1 via activating FoSNF1 in F. oxysporum. • Transgenic potato plants with interference of FoTOR1 and FoSAH1 genes prevent the occurrence of Fusarium wilt. • Providing insights SA into controlling various fungal diseases by targeting the SNF1-TORC1 pathway of pathogens. Biofungicides with low toxicity and high efficiency are a global priority for sustainable agricultural development. Phytohormone salicylic acid (SA) is an ancient medicine against various diseases in humans and activates the immune system in plants, but little is known of its function as a biofungicide. Here, Fusarium oxysporum , the causal agent of devastating Fusarium wilt and immunodepressed patients, was used as a model system to explore whether SA can enter the pathogen cells and suppress key targets of the pathogen. Oxford Nanopore MinION sequencing and high-throughput chromosome conformation capture (Hi-C) sequencing were used to analyzed the genome of F. oxysporum. In addition, RNA-seq, qRT-PCR, and western blotting were conducted to detect gene and protein expression levels. We isolated and sequenced the genome of F. oxysporum from potato dry rot, and the F. oxysporum included 12 chromosomes and 52.3 Mb genomic length. Pharmacological assays showed that exogenous application of SA can efficiently arrest hyphal growth, spore production, and pathogenicity of F. oxysporum , whereas endogenous salicylate hydroxylases significantly detoxify SA. The synergistic growth inhibition of F. oxysporum was observed when SA was combined with rapamycin. Kinase assays showed that SA inhibits FoTOR complex 1 (FoTORC1) by activating FoSNF1 in vivo. Transgenic potato plants with the interference of FoTOR1 and FoSAH1 genes inhibited the invasive growth of hyphae and significantly prevented the occurrence of Fusarium wilt. This study revealed the underlying mechanisms of SA against F. oxysporum and provided insights into SA in controlling various fungal diseases by targeting the SNF1-TORC1 pathway of pathogens. [ABSTRACT FROM AUTHOR]

Botrytis cinerea is a necrotrophic model fungal plant pathogen that causes grey mould, a devastating disease responsible for large losses in the agriculture sector. As important targets of fungicides, membrane proteins are hot spots in the research and development of fungicide products. We previously found that membrane protein Bcest may be closely related to the pathogenicity of Botrytis cinerea. Herein, we further explored its function. We generated and characterised ΔBcest deletion mutants of B. cinerea and constructed complemented strains. The ΔBcest deletion mutants exhibited reduced conidia germination and germ tube elongation. The functional activity of ΔBcest deletion mutants was investigated by reduced necrotic colonisation of B. cinerea on grapevine fruits and leaves. Targeted deletion of Bcest also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted-gene complementation. The role of Bcest in pathogenicity was also supported by reverse-transcriptase real-time quantitative PCR results indicating that melanin synthesis gene Bcpks13 and virulence factor Bccdc14 were significantly downregulated in the early infection stage of the ΔBcest strain. Taken together, these results suggest that Bcest plays important roles in the regulation of various cellular processes in B. cinerea.

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Filamentous fungi grow by adding cell wall and membrane exclusively at the apex of tubular structures called hyphae. Growth was previously believed to occur only through exocytosis at the Spitzenkörper, an organised body of secretory macro- and microvesicles found only in growing hyphae. More recent work has indicated that an area deemed the sub-apical collar is enriched for endocytosis and is also required for hyphal growth. It is now generally believed that polarity of filamentous fungi is achieved through the balancing of the processes of endocytosis and exocytosis at these two areas. This review is an update on the current progress and understanding surrounding the occurrence of endocytosis and its spatial regulation as they pertain to growth and pathogenicity in filamentous fungi.

Fusarium oxysporum is the causal agent of the devastating Fusarium wilt by invading and colonizing the vascular system in various plants, resulting in substantial economic losses worldwide. Target of rapamycin (TOR) is a central regulator that controls intracellular metabolism, cell growth, and stress responses in eukaryotes, but little is known about TOR signalling in F. oxysporum. In this study, we identified conserved FoTOR signalling pathway components including FoTORC1 and FoTORC2. Pharmacological assays showed that F. oxysporum is hypersensitive to rapamycin in the presence of FoFKBP12 while the deletion mutant strain ΔFofkbp12 is insensitive to rapamycin. Transcriptomic data indicated that FoTOR signalling controls multiple metabolic processes including ribosome biogenesis and cell wall‐degrading enzymes (CWDEs). Genetic analysis revealed that FoTOR1 interacting protein 4 (FoTIP4) acts as a new component of FoTOR signalling to regulate hyphal growth and pathogenicity of F. oxysporum. Importantly, transcript levels of genes associated with ribosome biogenesis and CWDEs were dramatically downregulated in the ΔFotip4 mutant strain. Electrophoretic mobility shift assays showed that FoTIP4 can bind to the promoters of ribosome biogenesis‐ and CWDE‐related genes to positively regulate the expression of these genes. These results suggest that FoTOR signalling plays central roles in regulating hyphal growth and pathogenicity of F. oxysporum and provide new insights into FoTOR1 as a target for controlling and preventing Fusarium wilt in plants. [ABSTRACT FROM AUTHOR]

Pyricularia oryzae causes the rice blast, which is one of the most devastating crop diseases worldwide, and is a model fungal pathogen widely used for dissecting the molecular mechanisms underlying fungal virulence/pathogenicity. Although the whole genome sequence of P. oryzae is publicly available, its current transcriptomes remain incomplete, lacking the information on non-protein coding genes and alternative splicing. Here, we performed and analyzed RNA-Seq of conidia and hyphae, resulting in the identification of 3,374 novel genes. Interestingly, the vast majority of these novel genes likely transcribed long non-coding RNAs (lncRNAs), and most of them were localized in the intergenic regions. Notably, their expressions were concomitant with the transcription of neighboring genes thereof in conidia and hyphae. In addition, 2,358 genes were found to undergo alternative splicing events. Furthermore, we exemplified that a lncRNA was important for hyphal growth likely by regulating the neighboring protein-coding gene and that alternative splicing of the transcription factor gene CON7 was required for appressorium formation. In summary, results from this study indicate that lncRNA transcripts and alternative splicing events are two important mechanisms for regulating the expression of genes important for conidiation, hyphal growth, and pathogenesis, and provide new insights into transcriptomes and gene regulation in the rice blast fungus. [ABSTRACT FROM AUTHOR]

Pyricularia oryzae causes the rice blast, which is one of the most devastating crop diseases worldwide, and is a model fungal pathogen widely used for dissecting the molecular mechanisms underlying fungal virulence/pathogenicity. Although the whole genome sequence of P. oryzae is publicly available, its current transcriptomes remain incomplete, lacking the information on non-protein coding genes and alternative splicing. Here, we performed and analyzed RNA-Seq of conidia and hyphae, resulting in the identification of 3,374 novel genes. Interestingly, the vast majority of these novel genes likely transcribed long non-coding RNAs (lncRNAs), and most of them were localized in the intergenic regions. Notably, their expressions were concomitant with the transcription of neighboring genes thereof in conidia and hyphae. In addition, 2,358 genes were found to undergo alternative splicing events. Furthermore, we exemplified that a lncRNA was important for hyphal growth likely by regulating the neighboring protein-coding gene and that alternative splicing of the transcription factor gene CON7 was required for appressorium formation. In summary, results from this study indicate that lncRNA transcripts and alternative splicing events are two important mechanisms for regulating the expression of genes important for conidiation, hyphal growth, and pathogenesis, and provide new insights into transcriptomes and gene regulation in the rice blast fungus.

Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts that have the potential to improve crop yield. These multinucleate organisms are either “homokaryotic” or “dikaryotic”. In AMF dikaryons, thousands of nuclei originating from two parental strains coexist in the same cytoplasm. In other fungi, homokaryotic and dikaryotic strains show distinct life history traits (LHTs), such as variation in growth rates and fitness. However, how such traits compare between dikaryons and homokaryons of AMF is unknown. To address this, we measured 20 LHT of four dikaryons and five homokaryons of the model fungus Rhizophagus irregularis across root organ cultures of three host plants (carrot, chicory, and tobacco). Our analyses show that dikaryons have clearly distinct life history strategies (LHSs) compared to homokaryons. In particular, spores of homokaryons germinate faster and to a higher proportion than dikaryons, whereas dikaryons grow significantly faster and create a more complex hyphal network irrespective of host plant species. Our study links AMF nuclear status with key LHT with possible implications for mycorrhizal symbiotic functioning.

Arbuscular mycorrhizal fungi (AMF) are obligate plant symbionts that have the potential to improve crop yield. These multinucleate organisms are either "homokaryotic" or "dikaryotic". In AMF dikaryons, thousands of nuclei originating from two parental strains coexist in the same cytoplasm. In other fungi, homokaryotic and dikaryotic strains show distinct life history traits (LHTs), such as variation in growth rates and fitness. However, how such traits compare between dikaryons and homokaryons of AMF is unknown. To address this, we measured 20 LHT of four dikaryons and five homokaryons of the model fungus Rhizophagus irregularis across root organ cultures of three host plants (carrot, chicory, and tobacco). Our analyses show that dikaryons have clearly distinct life history strategies (LHSs) compared to homokaryons. In particular, spores of homokaryons germinate faster and to a higher proportion than dikaryons, whereas dikaryons grow significantly faster and create a more complex hyphal network irrespective of host plant species. Our study links AMF nuclear status with key LHT with possible implications for mycorrhizal symbiotic functioning. [ABSTRACT FROM AUTHOR]

When considering mold prevention strategies, the environmental conditions in which fungi grow need to be taken into consideration. This environment is often characterized by a time-dependent relative humidity, and porous substrate. Growth has mainly been investigated in steady-state experiments. Therefore, the goal of this study is to understand the hyphal growth of Penicillium rubens on porous gypsum, under dynamic humidity conditions. Spores of P. rubens were inoculated on porous gypsum containing nutrients, and placed in a small incubation chamber, allowing for microscopic hyphal observation. The relative humidity in this chamber varied multiple times between a high (close to 100%) and low value (35%, 55%, or 75%). The hyphae reacted to a lowered relative humidity by an immediate growth stop and dehydration. When the relative humidity was increased again, the hyphae re-hydrated and three responses were found: regrowing after approximately 4 h, after a time equal to the germination time, or no regrowth at all. No substantial regrowth was found for fluctuations faster than 4 h. This time-scale was found for multiple decreases in relative humidity, and has been reported for the first time. Key points: • Hyphae restart growth after a characteristic time of approximately 4 h. • Relative humidity fluctuations of 3 h can suppress hyphal growth. • Hyphae do not regrow after a severe desiccation and short periods of high humidity. [ABSTRACT FROM AUTHOR]

Pleurotus has a remarkable nutritional and nutraceutical profile due to mineral mobilization and accumulation abilities from the substrate. The present study aimed to observe the effect of single and dual supplementations Se and Zn on biochemical parameters of P. florida, P. sajor caju and P. djamor. Also, the bioaccumulation of the trace elements in fortified mushrooms was estimated. Biomass production and radial growth rate were observed on Se and Zn supplemented broth and agar based medium. Furthermore, the influence of Se and Zn supplementation was recorded on the fruit body yield. The colorimetric assays were employed to estimate total soluble protein, total phenol and total flavonoid contents. The antioxidant activity was assayed as DPPH radical scavenging test. While, ICP-AES was performed to estimate the variation in the Zn and Se content of the fruit bodies. The Se supplementation at low rate resulted in improvement in the radial growth rate and biomass production for P. sajor caju. For solid-state fermentation, a better yield was obtained with inorganic salt supplementation in comparison to organically enriched Se straw. The maximum total soluble protein content and total flavonoid content were observed in fruit bodies of P. sajor caju at 4 mg L -1 of Se and Se-Zn respectively. Pleurotus djamor exhibited the highest total phenolic content on Zn supplementation (10 mg L-1). Improved antioxidant potential was recorded with dual supplementations. Salt supplementations caused shrinkage, distortion of the fungal hyphae, and decreased basidiospores with significant amelioration in elemental composition in fortified mushrooms. The inorganic salt supplementation increased the biochemical potential of Pleurotus spp. in comparison to organically enriched substrate which could further be used for the development of dietary supplements. • Pleurotus spp., exhibit effective mobilization and bioaccumulation of trace elements. • Under submerged fermentation at lower concentrations, it showed enhanced radial growth and biomass production. • Single and dual supplementation of Inorganic salt increased total soluble proteins, and flavonoids/ phenolic content • Trace mineral fortification led to shrinkage in fungal hyphae with decreased basidiospores. [ABSTRACT FROM AUTHOR]

Candida tropicalis is one of the most important human fungal pathogens causing superficial infections in locations such as the oral mucosa and genital tract, as well as systemic infections with high mortality. In its sister species Candida albicans, the cyclic AMP/protein kinase A (cAMP/PKA) pathway regulates fungal adhesion and dimorphism, both of which correlate closely with virulence. CaTpk1 and CaTpk2, the catalytic subunits of PKA, not only share redundant functions in hyphal growth, adhesion, and biofilm formation, but also have distinct roles in stress responses and pathogenesis, respectively. However, studies on PKA in the emerging fungal pathogen C. tropicalis are limited. Our results suggest that Tpk1 is involved in cell wall integrity and drug tolerance. The tpk2/tpk2 mutants, which have no protein kinase A activity, have reduced hyphal growth and adhesion. In addition, the tpk1/tpk1 tpk2/tpk2 double deletion mutant demonstrated delayed growth and impaired hyphal formation. In a murine model of systemic infection, both TPK1 and TPK2 were required for full virulence. We further found that EFG1 and HWP1 expression is regulated by PKA, while BCR1, FLO8, GAL4, and RIM101 are upregulated in the tpk1/tpk1 tpk2/tpk2 mutant. This study demonstrates that Tpk1 is involved in drug tolerance and cell wall integrity, while Tpk2 serves as a key regulator in dimorphism and adhesion. Both Tpk1 and Tpk2 are required for growth and full virulence in C. tropicalis.

NDT80 -like family genes are highly conserved across a large group of fungi, but the functions of each Ndt80 protein are diverse and have evolved differently among yeasts and pathogens. The unique NDT80 gene in budding yeast is required for sexual reproduction, whereas three NDT80 -like genes, namely, NDT80 , REP1, and RON1 , found in Candida albicans exhibit distinct functions. Notably, it was suggested that REP1 , rather than RON1 , is required for N-acetylglucosamine (GlcNAc) catabolism. Although Candida tropicalis , a widely dispersed fungal pathogen in tropical and subtropical areas, is closely related to Candida albicans , its phenotypic, pathogenic and environmental adaptation characteristics are remarkably divergent. In this study, we focused on the Ron1 transcription factor in C. tropicalis. Protein alignment showed that C. tropicalis Ron1 (CtRon1) shares 39.7% identity with C. albicans Ron1 (CaRon1). Compared to the wild-type strain, the C. tropicalis ron1Δ strains exhibited normal growth in different carbon sources and had similar expression levels of several GlcNAc catabolic genes during GlcNAc treatment. In contrast, C. tropicalis REP1 is responsible for GlcNAc catabolism and is involved in GlcNAc catabolic gene expressions, similar to C. albicans Rep1. However, REP1 deletion strains in C. tropicalis promote hyphal development in GlcNAc with low glucose content. Interestingly, Ct RON1 , but not Ca RON1 , deletion mutants exhibited significantly impaired hyphal growth and biofilm formation. As expected, Ct RON1 was required for full virulence. Together, the results of this study showed divergent functions of CtRon1 compared to CaRon1; CtRon1 plays a key role in yeast-hyphal dimorphism, biofilm formation and virulence. Lay Abstract In this study, we identified the role of RON1, an NDT80-like gene, in Candida tropicalis. Unlike the gene in Candida albicans , our studies showed that RON1 is a key regulator of hyphal formation, biofilm development and virulence but is dispensable for N-acetylglucosamine catabolism in C. tropicalis. [ABSTRACT FROM AUTHOR]

Filamentous fungi grow by adding cell wall and membrane exclusively at the apex of tubular structures called hyphae. Growth was previously believed to occur only through exocytosis at the Spitzenkörper, an organised body of secretory macro- and microvesicles found only in growing hyphae. More recent work has indicated that an area deemed the sub-apical collar is enriched for endocytosis and is also required for hyphal growth. It is now generally believed that polarity of filamentous fungi is achieved through the balancing of the processes of endocytosis and exocytosis at these two areas. This review is an update on the current progress and understanding surrounding the occurrence of endocytosis and its spatial regulation as they pertain to growth and pathogenicity in filamentous fungi. [ABSTRACT FROM AUTHOR]

Coccidioides is a dimorphic fungus responsible for Valley Fever and is the cause of severe morbidity and mortality in the infected population. Although there is some insight into the genes, pathways, and growth media involved in the parasitic to saprophytic growth transition, the exact determinants that govern this switch are largely unknown. In this work, we examined the growth and morphology of a Coccidioides posadasii strain (C. posadasii S/E) that efficiently produces spherules and endospores and persists in the parasitic life cycle at ambient CO2. We demonstrated that C. posadasii S/E remains virulent in an insect infection model. Surprisingly, under spherule-inducing conditions, the C. posadasii S/E culture was found to be completely hyphal. Differential interference contrast (DIC) and transmission electron microscopy (TEM) revealed unexpected cellular changes in this strain including cell wall remodeling and formation of septal pores with Woronin bodies. Our study suggests that the C. posadasii S/E strain is a useful BSL-2 model for studying mechanisms underlying the parasitic to saprophytic growth transition—a morphological switch that can impact the pathogenicity of the organism in the host.

The electron donor cytochrome b5 (CybE/Cyb5) fuels the activity of the ergosterol biosynthesis-related P450 enzymes (P450s) by providing electrons to P450s to promote ergosterol biosynthesis. Previous studies reported that a lack of Aspergillus fumigatus CybE reduces the proportion of ergosterol in total sterols and induces severe growth defects. However, the molecular characteristics of CybE and the underlying mechanism for CybE maintaining A. fumigatus growth remain poorly understood. Here, we found that the C terminus of CybE, with two transmembrane domains, is located at the endoplasmic reticulum. Therefore, a strain lacking the C terminus of CybE shows a defective growth phenotype similar to that of a cybE deletion strain. Notably, cybE deletion reduced the accumulation of the sterol-rich plasma membrane domains (SRDs; the assembly platform of polarity factors/cell end markers and growth machinery) in hyphal tips and decreased membrane fluidity, which corresponds to tardiness of hyphal extension and hypersensitivity to low temperature in the cybE deletion mutant. Additionally, overexpressing another electron donor, a heme-independent P450 reductase (cytochrome P450 reductase [CPR]), significantly rescued growth defects and recovered SRD accumulation in the cybE deletion mutant almost to the wild-type level, suggesting that CybE maintaining the growth and deposition of SRDs in hyphal tips is attributed to its nature as an electron donor. Protein pulldown assays revealed that CybE probably participates in the metabolism and transfer of lipids, construction of cytoskeleton, and mitochondria-associated energy metabolism to maintain the SRD accumulation in hyphal tips, membrane fluidity, and hyphal extension. The findings in this study give a hint that inhibition of CybE may be an effective strategy for resisting the infection of the human pathogen A. fumigatus.IMPORTANCE Investigating the knowledge of the growth regulation in the human opportunistic pathogen Aspergillus fumigatus is conducive to designing a new antifungal approach. The electron donor cytochrome b5 (CybE) plays a crucial role in maintaining the normal growth of A. fumigatus; however, the potential mechanism remains elusive. Here, we characterized the molecular features of CybE and found that the C terminus with two transmembrane domains is required for its endoplasmic reticulum (ER) localization and functions. In addition, we demonstrated that CprA, an electron donor-heme-independent P450 reductase, provides a reciprocal function for the missing cytochrome b5 protein-CybE in A. fumigatus. CybE maintains the normal growth probably via supporting two crucial physiological processes, the sterol-rich plasma membrane domain (SRD) accumulation in hyphal tips and membrane fluidity. Therefore, our findings reveal the mechanisms underlying the regulatory effect of CybE on A. fumigatus growth and indicate that inhibition of CybE might be an effective approach for alleviating A. fumigatus infection. [ABSTRACT FROM AUTHOR]

Glutathione reductase (Glr1) activity controls cellular glutathione and reactive oxygen species (ROS). We previously demonstrated two predominant methylglyoxal scavengers-NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase 1 (Adh1)-in glutathione-depleted γ-glutamyl cysteinyl synthetase-disrupted Candida albicans. However, experimental evidence for Candida pathophysiology lacking the enzyme activities of Mgd1 and Adh1 on glutathione-dependent redox regulation remains unclear. Herein, we have aimed to demonstrate that glutathione-dependent enzyme activities coupled with cellular ROS changes is regulated by methylglyoxal accumulation in Δmgd1/Δadh1 double disruptants. Δmgd1/Δadh1 showed severe growth defects and G1-phase cell cycle arrest. The observed complementary and reciprocal methylglyoxal-oxidizing and methylglyoxalreducing activities between Δmgd1 and Δadh1 were not always exhibited in Δmgd1/Δadh1. Although intracellular accumulation of methylglyoxal and pyruvate was shown in all disruptants, to a greater or lesser degree, methylglyoxal was particularly accumulated in the Δmgd1/Δadh1 double disruptant. While cellular ROS significantly increased in Δmgd1 and Δadh1 as compared to the wild-type, Δmgd1/Δadh1 underwent a decrease in ROS in contrast to Δadh1. Despite the experimental findings underlining the importance of the undergoing unbalanced redox state of Δmgd1/Δadh1, glutathione-independent antioxidative enzyme activities did not change during proliferation and filamentation. Contrary to the significantly lowered glutathione content and Glr1 enzyme activity, the activity staining-based glutathione peroxidase activities concomitantly increased in this mutant. Additionally, the enhanced GLR1 transcript supported our results in Δmgd1/Δadh1, indicating that deficiencies of both Adh1 and Mgd1 activities stimulate specific glutathione-dependent enzyme activities. This suggests that glutathione-dependent redox regulation is evidently linked to C. albicans pathogenicity under the control of methylglyoxal-scavenging activities. [ABSTRACT FROM AUTHOR]

The evolution of multicellularity has been one of the major transitions in the history of life. In contrast to animals and plants, how multicellularity evolved in fungi and how it compares to the general principles distilled from the study of more widely studied model systems, has received little attention. This review broadly discusses multicellular functioning and evolution in fungi. We focus on how fungi solved some of the common challenges associated with the evolution of multi-celled organisms and what unique challenges follow from the peculiar, filamentous growth form of fungi. We identify and discuss seven key challenges for fungal multicellular growth: apical growth, compartmentalization, long-distance mass transport, controlling mutational load, cell-to-cell communication, differentiation and adhesion. Some of these are characteristic of all multicellular transitions, whereas others are unique to fungi. We hope this review will facilitate the interpretation of fungal multicellularity in comparison with that of other multicellular lineages and will prompt further research into how fungi solved fundamental challenges in one of the major transitions in their evolutionary history. - Fungi evolved multicellularity via unique mechanisms compared to other lineages - Fungi had to overcome 7 key obstacles during the evolution of multicellularity - Fungal hyphae are different from most other forms of multicellularity - Both simple and complex multicellularity occur in fungi [ABSTRACT FROM AUTHOR]

Heterotrimeric G-proteins are crucial for fungal growth and differentiation. The α subunits of heterotrimeric G-proteins play an essential role in controlling signal transduction. However, the function of G-protein α subunits in entomopathogenic fungi remains poorly understood. Two group II Gα subunits (MrGPA2 and MrGPA4) were characterized in the entomopathogenic fungus, Metarhizium robertsii. Bioinformatics analysis showed that the relationship between MrGPA2 and MrGPA4 was closer than that of other MrGPAs. Both green fluorescent protein (GFP)-tagged MrGPA2 and MrGPA4 were localized at the cytoplasm. Furthermore, ∆MrGpa2∆MrGpa4 double mutants showed remarkably reduced vegetative growth compared to the wild-type and single-mutant strains, which was accompanied by the downregulation of several growth-related genes, such as ssk2, pbs2, stuA, hog1, and ac. Only the ∆MrGpa2∆MrGpa4 double mutant was sensitive to Congo red stress. The insect bioassay demonstrated significantly attenuated virulence for the ∆MrGpa2∆MrGpa4 double mutant compared to the wild-type and single-mutant strains. Further analysis indicated that double deletion of MrGpa2 and MrGpa4 had no effect on appressorium formation but suppressed the expression levels of several virulence-related genes in the insect hemocoel. These findings demonstrate that MrGpa2 and MrGpa4 exhibit functional redundancy and contribute to the vegetative growth, stress tolerance, and pest control potential in M. robertsii.

Fusarium solani and F. tricinctum are important root rot pathogens of soybean in the northern Great Plains of North America. The role of temperature on their growth was investigated in vitro at temperatures between 0 to 35ºC, while disease development in the soybean cultivar 'Barnes' was tested at 15°C, 20°C, 25°C and 30°C using several methods over 1 to 4 weeks. All experiments were conducted in a growth chamber. The effect of temperature on mycelial growth was modelled using an asymmetric logistic single peak model, which described fungal growth rate at temperatures of 8.5–34.3°C for F. solani and 3.1–32.2°C for F. tricinctum. The non-linear model estimated 29.3°C as the optimum temperature for growth of F. solani, and 24.4°C for F. tricinctum. Temperature had a significant effect on disease development in cotyledons and roots after 1-week incubation in rolled germination paper. The most favourable temperature for disease in cotyledons was 30°C for F. solani and 25°C for F. tricinctum. For roots, the optimal temperature for F. solani was also 30°C, but root disease by F. tricinctum was highest at 15°C. In infested soil after 4 weeks, lesion lengths on roots were longer and disease incidence was greater for F. solani at 30°C versus lower temperatures, whereas for F. tricinctum the optimum temperature for root disease was 15°C. The thermal specific characteristics of these pathogens implied disease development by F. solani is favoured by warmer temperatures, while in F. tricinctum it is favoured by cooler temperatures. [ABSTRACT FROM AUTHOR]

Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth. Deletion of the Arf GTPase Arl1 results in hyphae that are shorter as well as reduced virulence. How Arl1 is regulated during hyphal growth, a process characteristic of filamentous fungi, yet absent in S. cerevisiae, is unknown. Here, we investigated the importance of the Rab6 homolog, Ypt6, in Arl1-dependent hyphal growth and determined that YPT6 overexpression specifically rescued the hyphal growth defect of an arl1 mutant, but not the converse. Furthermore, we show that deletion of ARL1 results in an alteration of the distribution of the Rab8 homolog, Sec4, in hyphal cells and that this defect is restored upon YPT6 overexpression. [ABSTRACT FROM AUTHOR]

The ability of the human pathogenic fungus Candida albicans to switch between yeast-like and filamentous forms of growth has long been linked to pathogenesis. Numerous environmental conditions, including growth at high temperatures, nutrient limitation, and exposure to serum, can trigger this morphological switch and are frequently used in in vitro models to identify genes with roles in filamentation. Previous work has suggested that differences exist between the various in vitro models both in the genetic requirements for filamentation and transcriptional responses to distinct filamentation-inducing media, but these differences had not been analyzed in detail. We compared 10 in vitro models for filamentation and found broad genetic and transcriptomic differences between model systems. The comparative analysis enabled the discovery of novel media-independent genetic requirements for filamentation as well as a core filamentation transcriptional profile. Our data also suggest that the physical environment drives distinct programs of filamentation in C. albicans, which has significant implications for filamentation in vivo.

The ADP-ribosylation factor 6 (Arf6), as the only member of the Arf family III protein, has been extensively studied for its diverse biological functions in animals. Previously, the Arf6 protein in Magnaporthe oryzae was found to be crucial for endocytosis and polarity establishment during asexual development. However, its role remains unclear in S. sclerotiorum . Here, we identified and characterized SsArf6 in S. sclerotiorum using a reverse genetic approach. Deletion of SsArf6 impaired hyphal growth and development and produced more branches. Interestingly, knockout of SsArf6 resulted in an augmented tolerance of S. sclerotiorum towards oxidative stress, and increased its sensitivity towards osmotic stress, indicative of the different roles of SsArf6 in various stress responses. Simultaneously, SsArf6 deletion led to an elevation in melanin accumulation. Moreover, the appressorium formation was severely impaired, and fungal virulence to host plants was significantly reduced. Overall, our findings demonstrate the essential role of SsArf6 in hyphal development, stress responses, appressorium formation, and fungal virulence to host plants.