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1.
PLoS One ; 16(9): e0257823, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34587206

RESUMO

Fungal hyphal growth and branching are essential traits that allow fungi to spread and proliferate in many environments. This sustained growth is essential for a myriad of applications in health, agriculture, and industry. However, comparisons between different fungi are difficult in the absence of standardized metrics. Here, we used a microfluidic device featuring four different maze patterns to compare the growth velocity and branching frequency of fourteen filamentous fungi. These measurements result from the collective work of several labs in the form of a competition named the "Fungus Olympics." The competing fungi included five ascomycete species (ten strains total), two basidiomycete species, and two zygomycete species. We found that growth velocity within a straight channel varied from 1 to 4 µm/min. We also found that the time to complete mazes when fungal hyphae branched or turned at various angles did not correlate with linear growth velocity. We discovered that fungi in our study used one of two distinct strategies to traverse mazes: high-frequency branching in which all possible paths were explored, and low-frequency branching in which only one or two paths were explored. While the high-frequency branching helped fungi escape mazes with sharp turns faster, the low-frequency turning had a significant advantage in mazes with shallower turns. Future work will more systematically examine these trends.


Assuntos
Crowdsourcing/métodos , Fungos/crescimento & desenvolvimento , Técnicas Analíticas Microfluídicas/instrumentação , Ascomicetos/crescimento & desenvolvimento , Basidiomycota/crescimento & desenvolvimento , Fenômenos Biológicos , Fungos/classificação , Hifas/classificação , Hifas/crescimento & desenvolvimento , Especificidade da Espécie
2.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361746

RESUMO

New agricultural strategies aim to reduce the use of pesticides due to their damage to the environment and humans, and the caused resistance to pathogens. Therefore, alternative sources of antifungal compounds from plants are under investigation lately. Extracts from plants have a wide composition of chemical compounds which may complicate the development of pathogen resistance. Botrytis cinerea, causing grey mould, is an important horticultural and ornamental pathogen, responsible for the relevant yield and quality losses. B. cinerea isolated from a different plant host may differ in the sensitivity to antifungal substances from plants. Assessing the importance of research covering a wide range of pathogens for the rapid development of biopesticides, this study aims to determine the sensitivity of the B. cinerea isolate complex (10 strains) to plant extracts, describe morphological changes caused by the extract treatment, and detect differences between the sensitivity of different plant host isolates. The results showed the highest sensitivity of the B. cinerea isolates complex to cinnamon extract, and the lowest to laurel extract. In contrast, laurel extract caused the most changes of morphological attributes in the isolates. Five B. cinerea isolates from plant hosts of raspberry, cabbage, apple, bell pepper, and rose were grouped statistically according to their sensitivity to laurel extract. Meanwhile, the bell pepper isolate separated from the isolate complex based on its sensitivity to clove extract, and the strawberry and apple isolates based on their sensitivity to cinnamon extract.


Assuntos
Antifúngicos/farmacologia , Agentes de Controle Biológico/farmacologia , Botrytis/efeitos dos fármacos , Cinnamomum zeylanicum/química , Hifas/efeitos dos fármacos , Doenças das Plantas/prevenção & controle , Antifúngicos/isolamento & purificação , Agentes de Controle Biológico/isolamento & purificação , Botrytis/crescimento & desenvolvimento , Botrytis/isolamento & purificação , Brassica/microbiologia , Capsicum/microbiologia , Cinnamomum camphora/química , Fragaria/microbiologia , Humanos , Hifas/crescimento & desenvolvimento , Hifas/isolamento & purificação , Malus/microbiologia , Testes de Sensibilidade Microbiana , Doenças das Plantas/microbiologia , Extratos Vegetais/química , Syzygium/química , Vitis/microbiologia
3.
PLoS One ; 16(7): e0250064, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34329342

RESUMO

The ascomycete fungus Fusarium oxysporum f.sp. cucumerinum causes vascular wilt diseases in cucumber. However, few genes related to morphogenesis and pathogenicity of this fungal pathogen have been functionally characterized. BLASTp searches of the Aspergillus fumigatus UgmA and galatofuranosyltransferases (Galf-transferases) sequences in the F. oxysporum genome identified two genes encoding putative UDP-galactopyranose mutase (UGM), ugmA and ugmB, and six genes encoding putative Galf-transferase homologs. In this study, the single and double mutants of the ugmA, ugmB and gfsB were obtained. The roles of UGMs and GfsB were investigated by analyzing the phenotypes of the mutants. Our results showed that deletion of the ugmA gene led to a reduced production of galactofuranose-containing sugar chains, reduced growth and impaired conidiation of F. oxysporum f.sp. cucumerinum. Most importantly, the ugmA deletion mutant lost the pathogenicity in cucumber plantlets. Although deletion of the ugmB gene did not cause any visible phenotype, deletion of both ugmA and ugmB genes caused more severe phenotypes as compared with the ΔugmA, suggesting that UgmA and UgmB are redundant and they can both contribute to synthesis of UDP-Galf. Furthermore, the ΔgfsB exhibited an attenuated virulence although no other phenotype was observed. Our results demonstrate that the galactofuranose (Galf) synthesis contributes to the cell wall integrity, germination, hyphal growth, conidiation and virulence in Fusarium oxysporum f.sp. cucumerinum and an ideal target for the development of new anti-Fusarium agents.


Assuntos
Fusarium/genética , Galactose/metabolismo , Virulência/genética , Aspergillus nidulans/enzimologia , Cucumis sativus/microbiologia , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Galactose/análise , Hifas/genética , Hifas/crescimento & desenvolvimento , Transferases Intramoleculares/classificação , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Mananas/análise , Mananas/metabolismo , Mutagênese , Fenótipo , Filogenia , Doenças das Plantas/microbiologia
4.
Nat Commun ; 12(1): 3899, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162849

RESUMO

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans.


Assuntos
Candida albicans/metabolismo , Candidíase/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/metabolismo , Animais , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase/microbiologia , Divisão Celular/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Mutação , Neutrófilos/metabolismo , Virulência/genética
5.
Res Microbiol ; 172(4-5): 103849, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34089837

RESUMO

Co-infection with other microorganisms can promote the Candida albicans to be invasive. In this study, Escherichia coli and C. albicans were co-isolated from the women with candidiasis symptoms. The in vitro effects of E. coli on C. albicans hypha development, biofilm formation, antibiotic susceptibility, dispersion from the biofilm, expression of Als3, Hwp1, and Tup1 genes, and pathogenesis in Galleria mellonella were investigated. Electron microscopic images revealed that hypha induction was markedly increased in the bacteria-fungi co-culture. Biofilm formation was increased 2.2 fold in the presence of E. coli. The minimum inhibitory concentration of nystatin against Candida was increased from (µg mL-1) 25 to 50 in the dual biofilm. Candida dissemination was increased up to 2.7 fold from the mixed fungi/bacteria biofilm. The expression of ALS3 and HWP1 genes was increased (5.9 and 2.0 fold, respectively) while the TUP1 gene expression was decreased (0.4 fold) when C. albicans was incubated with E. coli. The simultaneous injection of C. albicans and E. coli to the insect larvae increased Galleria mortality up to 40%. This study demonstrated the effects of E. coli to promote fungi virulence factors, which suggest polymicrobial interaction should be considered during treatment of fungal infections.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candidíase Vulvovaginal/microbiologia , Coinfecção/microbiologia , Escherichia coli/fisiologia , Interações Microbianas , Fatores de Virulência , Animais , Candida albicans/genética , Feminino , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Larva/microbiologia , Mariposas/microbiologia
6.
Sci Rep ; 11(1): 11319, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059696

RESUMO

Target of rapamycin (TOR) is a conserved central growth regulator in eukaryotes that has a key role in maintaining cellular nutrient and energy status. Arbuscular mycorrhizal (AM) fungi are mutualistic symbionts that assist the plant in increasing nutrient absorption from the rhizosphere. However, the role of legume TOR in AM fungal symbiosis development has not been investigated. In this study, we examined the function of legume TOR in the development and formation of AM fungal symbiosis. RNA-interference-mediated knockdown of TOR transcripts in common bean (Phaseolus vulgaris) hairy roots notably suppressed AM fungus-induced lateral root formation by altering the expression of root meristem regulatory genes, i.e., UPB1, RGFs, and sulfur assimilation and S-phase genes. Mycorrhized PvTOR-knockdown roots had significantly more extraradical hyphae and hyphopodia than the control (empty vector) roots. Strong promoter activity of PvTOR was observed at the site of hyphal penetration and colonization. Colonization along the root length was affected in mycorrhized PvTOR-knockdown roots and the arbuscules were stunted. Furthermore, the expression of genes induced by AM symbiosis such as SWEET1, VPY, VAMP713, and STR was repressed under mycorrhized conditions in PvTOR-knockdown roots. Based on these observations, we conclude that PvTOR is a key player in regulating arbuscule development during AM symbiosis in P. vulgaris. These results provide insight into legume TOR as a potential regulatory factor influencing the symbiotic associations of P. vulgaris and other legumes.


Assuntos
Micorrizas/crescimento & desenvolvimento , Phaseolus/enzimologia , Phaseolus/microbiologia , Serina-Treonina Quinases TOR/metabolismo , Regulação da Expressão Gênica de Plantas , Hifas/crescimento & desenvolvimento , Especificidade de Órgãos , Plantas Geneticamente Modificadas , Simbiose
7.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066497

RESUMO

Autophagy is an intracellular process in all eukaryotes which is responsible for the degradation of cytoplasmic constituents, recycling of organelles, and recycling of proteins. It is an important cellular process responsible for the effective virulence of several pathogenic plant fungal strains, having critical impacts on important crop plants including potatoes. However, the detailed physiological mechanisms of autophagy involved in the infection biology of soil-borne pathogens in the potato crop needs to be investigated further. In this study, the autophagy-related gene, FoATG12, in potato dry rot fungus Fusarium oxysporum was investigated by means of target gene replacement and overexpression. The deletion mutant ∆FoATG12 showed reduction in conidial formation and exhibited impaired aerial hyphae. The FoATG12 affected the expression of genes involved in pathogenicity and vegetative growth, as well as on morphology features of the colony under stressors. It was found that the disease symptoms were delayed upon being inoculated by the deletion mutant of FoATG12 compared to the wild-type (WT) and overexpression (OE), while the deletion mutant showed the disease symptoms on tomato plants. The results confirmed the significant role of the autophagy-related ATG12 gene in the production of aerial hyphae and the effective virulence of F. oxysporum in the potato crop. The current findings provid an enhanced gene-level understanding of the autophagy-related virulence of F. oxysporum, which could be helpful in pathogen control research and could have vital impacts on the potato crop.


Assuntos
Proteína 12 Relacionada à Autofagia/genética , Autofagia/genética , Proteínas Fúngicas/genética , Fusarium/citologia , Fusarium/genética , Genes Fúngicos , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Hifas/crescimento & desenvolvimento , Mutação/genética , Fenótipo , Doenças das Plantas/genética , Esporos Fúngicos/crescimento & desenvolvimento , Estresse Fisiológico/genética
8.
mSphere ; 6(3)2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952658

RESUMO

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell's metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here, we studied the roles of three sirtuin deacetylases-Sir2, Hst1, and Hst2-in the hyphal growth of C. albicans We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hypha formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2 Moreover, the expression of hypha-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to the wild type. This regulation of hypha formation was likely dependent on the deacetylase activity of Sir2, as a similar defect in hypha formation was observed when an asparagine known to be required for deacetylation was mutated. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and nonredundant role in hyphal growth of C. albicans IMPORTANCE Candida albicans is one of the most common causes of hospital-acquired systemic fungal infections in the United States. It can switch between ovoid yeast and elongated hyphal growth forms in response to environmental cues. This morphological transition is essential for its survival in the host. Thus, identifying regulators involved in this process can lead to new therapies. In this study, we examined the contribution of three regulators called sirtuins (Sir2, Hst1, and Hst2) to the yeast-to-hypha transition of C. albicans We found that loss of Sir2 but not Hst1 or Hst2 hampered hypha formation. Moreover, the defect was caused by the loss of the catalytic activity of Sir2. Our study may lay the groundwork for discovering novel targets for antifungal therapies.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/genética , Regulação Fúngica da Expressão Gênica/genética , Hifas/crescimento & desenvolvimento , Sirtuínas/genética , Candida albicans/enzimologia , Nucléolo Celular , Hifas/genética , Sirtuína 2/genética , Sirtuína 2/metabolismo , Sirtuínas/classificação , Sirtuínas/metabolismo
9.
J Microbiol Biotechnol ; 31(6): 890-901, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34024892

RESUMO

The 11α-hydroxylation of canrenone can be catalyzed by Aspergillus ochraceus in bioreactors, where the geometry of the impeller greatly influences the biotransformation. In this study, the effects of the blade number and impeller diameter of a Rushton turbine on the 11α-hydroxylation of canrenone were considered. The results of fermentation experiments using a 50 mm four-blade impeller showed that 3.40% and 11.43% increases in the conversion ratio were achieved by increasing the blade number and impeller diameter, respectively. However, with an impeller diameter of 60 mm, the conversion ratio with a six-blade impeller was 14.42% lower than that with a four-blade impeller. Data from cold model experiments with a large-diameter six-blade impeller indicated that the serious leakage of inclusions and a 22.08% enzyme activity retention led to a low conversion ratio. Numerical simulations suggested that there was good gas distribution and high fluid flow velocity when the fluid was stirred by large-diameter impellers, resulting in a high dissolved oxygen content and good bulk circulation, which positively affected hyphal growth and metabolism. However, a large-diameter six-blade impeller created overly high shear compared to a large-diameter four-blade impeller, thereby decreasing the conversion ratio. The average shear rates of the former and latter cases were 43.25 s-1 and 35.31 s-1, respectively. We therefore concluded that appropriate shear should be applied in the 11α-hydroxylation of canrenone. Overall, this study provides basic data for the scaled-up production of 11α-hydroxycanrenone.


Assuntos
Reatores Biológicos , Canrenona/metabolismo , Aspergillus ochraceus/crescimento & desenvolvimento , Aspergillus ochraceus/metabolismo , Biotransformação , Canrenona/química , Meios de Cultura/química , Meios de Cultura/metabolismo , Fermentação , Hidroxilação , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Modelos Teóricos
10.
Sci Rep ; 11(1): 9418, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941791

RESUMO

Morels are some of the most highly prized edible and medicinal mushrooms, with great economic and scientific value. Outdoor cultivation has been achieved and expanded on a large scale in China in recent years. Sclerotial formation is one of the most important phases during the morel life cycle, and previous reports indicated that reactive oxygen species (ROS) play an important role. However, ROS response mechanisms at sclerotial initiation (SI) stage are poorly understood. In this study, comparative transcriptome analyses were performed with sclerotial and hyphal cells at different areas in the same plate at SI stage. Gene expression was significantly different at SI stage between sclerotial formation and mycelia growth areas. GO and KEGG analyses indicated more vigorous metabolic characteristics in the hyphae area, while transcription process, DNA repair, and protein processing were enriched in sclerotial cells. Gene expression related to H2O2 production was high in the hyphae area, while expression of H2O2-scavenging genes was high in sclerotial cells, leading to a higher H2O2 concentration in the hyphal region than in the sclerotium. Minor differences were observed in gene expression of H2O2-induced signaling pathway in sclerotial and hyphal cells; however, expression levels of the target genes of transcription factor MSN2, important in the H2O2-induced signaling pathways, were significantly different. MSN2 enhanced stress response regulation in sclerotia by regulating these target genes. Small molecular HSPs were also found upregulated in sclerotial cells. This study indicated that sclerotial cells are more resistant to ROS stress than hyphal cells through transcriptional regulation of related genes.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Ascomicetos/genética , Hifas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/genética , Perfilação da Expressão Gênica , Hifas/metabolismo , Micélio/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Transcrição Genética/genética
11.
Nat Commun ; 12(1): 2560, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33963193

RESUMO

The commensal fungus Candida albicans often causes life-threatening infections in patients who are immunocompromised with high mortality. A prominent but poorly understood risk factor for the C. albicans commensal‒pathogen transition is the use of broad-spectrum antibiotics. Here, we report that ß-lactam antibiotics cause bacteria to release significant quantities of peptidoglycan fragments that potently induce the invasive hyphal growth of C. albicans. We identify several active peptidoglycan subunits, including tracheal cytotoxin, a molecule produced by many Gram-negative bacteria, and fragments purified from the cell wall of Gram-positive Staphylococcus aureus. Feeding mice with ß-lactam antibiotics causes a peptidoglycan storm that transforms the gut from a niche usually restraining C. albicans in the commensal state to promoting invasive growth, leading to systemic dissemination. Our findings reveal a mechanism underlying a significant risk factor for C. albicans infection, which could inform clinicians regarding future antibiotic selection to minimize this deadly disease incidence.


Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Peptidoglicano/toxicidade , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , beta-Lactamas/efeitos adversos , Animais , Antibacterianos/efeitos adversos , Candida albicans/crescimento & desenvolvimento , Candidíase/complicações , Candidíase/tratamento farmacológico , Candidíase/patologia , Parede Celular/química , Parede Celular/efeitos dos fármacos , Cromatografia Líquida , Feminino , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/metabolismo , Humanos , Hifas/crescimento & desenvolvimento , Hifas/patogenicidade , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Peptidoglicano/química , Infecções Estafilocócicas/complicações , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo
12.
Biochem Biophys Res Commun ; 561: 106-112, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34022710

RESUMO

Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. The ability to switch between yeast and hyphal growth forms is critical for its pathogenesis. Hyphal development in C. albicans requires two temporally linked regulations for initiation and maintenance. Here, we performed transcriptome sequencing (RNA-Seq) to analyze the transcriptional consequences for the two different phases of hyphal development. Genome-wide transcription profiling reveals that the sets associated with hyphal initiation were significantly enriched in genes for hyphal cell wall, biofilm matrix and actin polarization. In addition to hypha-specific genes, numerous genes involved in iron acquisition, such as FTR1 and SEF1, are highly induced specifically during sustained hyphal development even when additional free iron is supplied in the medium. Therefore, iron uptake genes are induced by signals that can support prolonged hyphal development in an iron-independent manner. The induction of iron acquisition genes during hyphal elongation was further confirmed by quantitative reverse transcription-PCR under various hypha-inducing conditions. Remarkably, preventing C. albicans from acquiring iron blocks BRG1 activation, leading to impaired hyphal maintenance, and ectopically expressed BRG1 can sustain hyphal development bypassing the requirement of iron. Our study elucidates an underlying mechanism of how multiple virulence factors are interconnected and are induced simultaneously during infection.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Ferro/metabolismo , Candida albicans/genética , Proteínas Fúngicas/genética , Hifas/genética , Hifas/metabolismo , Virulência
13.
Fungal Biol ; 125(6): 485-494, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34024596

RESUMO

Some strains of Aspergillus niger have been previously reported to produce sclerotia under certain conditions. Sclerotia are aggregations of hyphae which can act either as survival or as sexual structures in species related to A. niger. In this study, we were able to induce the formation of sclerotia in the progenitor of the industrial citric acid producing strains of A. niger, ATCC 1015, and in pyrG mutants derived from it. Sclerotia can be stably formed by ATCC 1015 on malt extract agar medium supplemented with raisins, showing a spatial differentiation of the fungus dependent on the addition and on the position of the fruits into the medium. On other media, including malt extract agar, pyrG auxotrophs also form abundant sclerotia, while the complementation of this gene reverses this phenotype. Additionally, a macro- and microscopical analysis of the sclerotia is reported. Our results show that the sclerotia formed by A. niger are similar to those formed by other fungi, not only in their morphology but also in their ability to germinate and regenerate the organism.


Assuntos
Aspergillus niger , Hifas , Aspergillus niger/citologia , Aspergillus niger/genética , Aspergillus niger/metabolismo , Ácido Cítrico/metabolismo , Genes Fúngicos/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Mutação , Fenótipo
14.
Fungal Genet Biol ; 151: 103563, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33915282

RESUMO

The microscopic development of a mycelium is of importance in all aspects of fungal biology and biotechnology. However, the mechanics of three-dimensional (3D) hyphal growth has been not explored. Using light-sheet fluorescence microscopy, we follow the 3D growth of Trichoderma atroviride in liquid medium and observe two direct collision events among hyphae. In both cases, a hypha undergoing tip extension collides with the side of another hypha, causing mechanical deformation that remains after the collision. From these data we estimate that the force developed by hyphae during tip elongation is at least 260 pN.


Assuntos
Hifas/crescimento & desenvolvimento , Hypocreales/crescimento & desenvolvimento , Fenômenos Biomecânicos , Microscopia de Fluorescência
15.
Microbiol Res ; 248: 126748, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33752111

RESUMO

The hemibiotrophic pathogen Colletotrichum gloeosporioides is the causal agent of poplar anthracnose and causes considerable economic losses. This fungus infects its host through a specialized structure called an appressorium. In a previous study, we demonstrated that the mitogen-activated protein kinase (MAPK) CgMk1 plays a critical role in appressorium formation and pathogenicity. In this study, we identified three upstream components of CgMk1, the putative adaptor protein CgSte50, MAPKKK CgSte11, and MAPKK CgSte7, and showed that CgSte50, CgSte11, and CgSte7 positively regulate the phosphorylation of CgMk1. Deletion of CgSte50, CgSte11, and CgSte7 resulted in the loss of appressorium formation, penetration of the cellophane membrane, invasive growth and pathogenicity, similar to the defects observed in the CgMk1 mutant. CgSte50, CgSte11, CgSte7 and CgMk1 were also required for polarity during conidial germination. At the initial stage of appressorium formation, the accumulation of reactive oxygen species (ROS) was altered in the CgSte50, CgSte11, CgSte7 and CgMk1 deletion mutants compared with that in wild type (WT). Furthermore, the CgSte50, CgSte11, CgSte7 and CgMk1 deletion mutants manifested pleiotropic defects during vegetative growth; all mutants exhibited albino colonies, and the aerial hyphae had reduced hydrophobicity. In the mutants, autolysis was detected at the colony edge, and septum formation in the hyphae was elevated compared with that in the WT hyphae. Moreover, deletion of CgSte50, CgSte11, CgSte7 and CgMk1 affected vegetative growth under nitrogen-limiting and osmotic stress conditions. CgSte50, CgSte11, and CgSte7 but not CgMk1 were required for the oxidative stress response. Taken together, these results indicate that the CgMk1 MAPK cascade plays vital roles in various important functions in C. gloeosporioides.


Assuntos
Colletotrichum/enzimologia , Colletotrichum/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Doenças das Plantas/microbiologia , Populus/microbiologia , Colletotrichum/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Hifas/enzimologia , Hifas/genética , Hifas/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/genética , Morfogênese , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento
16.
J Microbiol Biotechnol ; 31(6): 815-822, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-33782223

RESUMO

Indigenous fungus-feeding nematodes may adversely affect the growth and activity of introduced biocontrol fungi. Alginate pellets of the biocontrol fungus Trichoderma harzianum ThzID1-M3 and sclerotia of the fungal plant pathogen Sclerotinia sclerotiorum were added to nonsterile soil at a soil water potential of -50 or -1,000 kPa. The biomass of ThzID1-M3, nematode populations, and extent of colonization of sclerotia by ThzID1-M3 were monitored over time. The presence of ThzID1-M3 increased the nematode population under both moisture regimes (p < 0.05), and fungivores comprised 69-75% of the nematode population. By day 5, the biomass of ThzID1-M3b and its colonization of sclerotia increased and were strongly correlated (R2 = 0.98), followed by a rapid reduction, under both regimes. At -50 kPa (the wetter of the two environments), fungal biomass and colonization by ThzID1-M3 were less, in the period from 5 to 20 days, while fungivores were more abundant. These results indicate that ThzID1-M3 stimulated the population growth of fungivorous nematodes, which in turn, reduced the biocontrol ability of the fungus to mycoparasitize sclerotia. However, colonization incidence reached 100% by day 5 and remained so for the experimental period under both regimes, although hyphal fragments disappeared by day 20. Our results suggest that indigenous fungivores are an important constraint for the biocontrol activity of introduced fungi, and sclerotia can provide spatial refuge for biocontrol fungi from the feeding activity of fungivorous nematodes.


Assuntos
Hypocreales/crescimento & desenvolvimento , Nematoides/fisiologia , Controle Biológico de Vetores , Animais , Ascomicetos/crescimento & desenvolvimento , Biomassa , Hifas/crescimento & desenvolvimento , Nematoides/microbiologia , Solo/química , Solo/parasitologia , Microbiologia do Solo , Água/análise
17.
Fungal Genet Biol ; 150: 103549, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33675987

RESUMO

The evaluation of morphology is fundamental to comprehend how fungi grow, develop, and interact with the environment. Although fungal growth has been extensively studied associated to two-dimensional geometries, lack of appropriate experimental tools has limited exploration of the complex three-dimensional (3D) structures exhibited by mycelia in more general contexts. In this paper, we report the construction of a light-sheet fluorescence microscope (LSFM) capable of performing time-lapse visualization of 3D biological structures (4D microscopy), and the use of this instrument to follow the dynamics of fungal growth. LSFM uses scanning of selective plane illumination and digital reconstruction to provide 3D images of the specimen. We describe the optical, electronic, and computational means to implement two-color LSFM, and provide detailed procedures for aligning and testing the setup. We successfully demonstrate use of both autofluorescence and specific tagging to image Trichoderma atroviride and Neurospora crassa strains growing in liquid media, over extended times (~12 h) and volumes (~400 × 1500 × 800 µm3) at single-hypha resolution. The excellent image contrast provided by LSFM enables us to visualize the dynamics of mycelial architecture, interactions among hyphae, and measure rates of 3D apical extension. Altogether, our work shows a powerful imaging tool to perform 3D morphological analysis of fungi, from hyphae to mycelium.


Assuntos
Fungos/crescimento & desenvolvimento , Hifas/crescimento & desenvolvimento , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imageamento Tridimensional/instrumentação , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos
18.
mBio ; 12(2)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727355

RESUMO

Tip-growing fungal cells maintain cell polarity at the apical regions and elongate by de novo synthesis of the cell wall. Cell polarity and tip growth rate affect mycelial morphology. However, it remains unclear how both features act cooperatively to determine cell shape. Here, we investigated this relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 µm-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, any hypha growing through the channel must adapt its morphology. Live-cell imaging analyses revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels, or had lost apical polarity after emerging from the other end of the channel. Fluorescence live-cell imaging analyses of the Spitzenkörper, a collection of secretory vesicles and polarity-related proteins at the hyphal tip, in Neurospora crassa indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, including phytopathogenic fungi. This comparative approach revealed that the growth defects induced by the channels were not correlated with their taxonomic classification or with the width of hyphae, but, rather, correlated with the hyphal elongation rate. This report indicates a trade-off between morphological plasticity and velocity in mycelial growth and serves to help understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology, and pathogenicity.IMPORTANCE Cell morphology, which is controlled by polarity and growth, is fundamental for all cellular functions. However how polarity and growth act cooperatively to control cell shape remains unclear. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 µm-width channels of microfluidic devices. We found that most fast growing hyphae often lost the cell polarity after emerging from the channels, whereas slow growing hyphae retained polarity and continued growing, indicating a trade-off between plasticity and velocity in mycelial growth. These results serve to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.


Assuntos
Polaridade Celular , Fungos/crescimento & desenvolvimento , Hifas/citologia , Hifas/crescimento & desenvolvimento , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Microtúbulos , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismo , Vesículas Secretórias/metabolismo
19.
Curr Genet ; 67(4): 613-630, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33683401

RESUMO

Aspergillus nidulans produces cleistothecia as sexual reproductive organs in a process affected by genetic and external factors. To gain a deeper insight into A. nidulans sexual development, we performed comparative proteome analyses based on the wild type developmental periods. We identified sexual development-specific proteins with a more than twofold increase in production during hypoxia or the sexual period compared to the asexual period. Among the sexual development-specific proteins analyzed by gene-deletion experiments and functional assays, MpdA, a putative mannitol-1-phosphate 5-dehydrogenase, plays multiple roles in growth and differentiation of A. nidulans. The most distinct mpdA-deletion phenotype was ascosporogenesis failure. Genetic mpdA deletion resulted in small cleistothecia with no functional ascospores. Transcriptional analyses indicated that MpdA modulates the expression of key development- and meiosis-regulatory genes during sexual development. The mpdA deletion increased hyphal branching and decreased conidial heat resistance. Mannitol production in conidia showed no difference, whereas it was decreased in mycelia and sexual cultures. Addition of mannitol during vegetative growth recovered the defects in conidial heat resistance and ascospore genesis. Taken together, these results indicate that MpdA plays an important role in sexual development, hyphal branching, and conidial heat resistance in Aspergillus nidulans.


Assuntos
Aspergillus nidulans/genética , Hifas/genética , Esporos Fúngicos/genética , Desidrogenase do Álcool de Açúcar/genética , Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/patogenicidade , Regulação Fúngica da Expressão Gênica/genética , Hifas/crescimento & desenvolvimento , Manitol/metabolismo , Meiose/genética , Desenvolvimento Sexual/genética , Esporos Fúngicos/metabolismo
20.
Fungal Genet Biol ; 149: 103530, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33561548

RESUMO

Sclerotinia sclerotiorum is a destructive necrotrophic fungal pathogen with worldwide distribution. The metabolism of reactive oxygen species (ROS) is critical for the development and infection process of this economically important pathogen. Hydrogen peroxide (H2O2) is converted into water and dioxygen by catalases, which are major ROS scavengers in cells. Several genes have been predicted to encode the catalases of S. sclerotiorum, but the critical ones that function in the ROS stress response are still unknown. In this research, a catalase gene called SsCat2 was found to contribute to the predominant catalase activity at the stages of hyphae growth and sclerotial development. SsCat2 transcripts were induced under oxidative stress, and the target deletion of SsCat2 led to significant sensitivity to H2O2, suggesting that SsCat2 is critical in dealing with the oxidative stress. SsCat2-deletion strains were sensitive to hyperosmotic stresses and cell membrane-perturbing agents, suggesting impairment in cell integrity due to the inactivation of SsCat2. The expression of the alternative oxidase-encoding gene was upregulated in the SsCat2-deletion strains, which showed decreased sensitivity to QoI fungicides. SsCat2-deletion strains showed impaired virulence in different hosts, and more H2O2 accumulation was detected during the infect processes. In summary, these results indicate that SsCat2 encodes a catalase that is related to the oxidative stress response, QoI fungicide sensitivity, and pathogenicity of S. sclerotiorum.


Assuntos
Ascomicetos/genética , Catalase/metabolismo , Fungicidas Industriais/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Catalase/genética , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/metabolismo , Hifas/crescimento & desenvolvimento , Pressão Osmótica , Estresse Oxidativo/efeitos dos fármacos , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Virulência
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