A velvet multisubunit complex was recently detected in the filamentous fungus Penicillium chrysogenum , the major industrial producer of the β-lactam antibiotic penicillin. Core components of this complex are P. chrysogenum VelA (PcVelA)... more
A velvet multisubunit complex was recently detected in the filamentous fungus Penicillium chrysogenum , the major industrial producer of the β-lactam antibiotic penicillin. Core components of this complex are P. chrysogenum VelA (PcVelA) and PcLaeA, which regulate secondary metabolite production, hyphal morphology, conidiation, and pellet formation. Here we describe the characterization of PcVelB, PcVelC, and PcVosA as novel subunits of this velvet complex. Using yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC), we demonstrate that all velvet proteins are part of an interaction network. Functional analyses using single- and double-knockout strains clearly indicate that velvet subunits have opposing roles in the regulation of penicillin biosynthesis and light-dependent conidiation. PcVelC, together with PcVelA and PcLaeA, activates penicillin biosynthesis, while PcVelB represses this process. In contrast, PcVelB and PcVosA promote conidiation, while PcVel...
Filamentous fungi are a large and ancient clade of microorganisms that occupy a broad range of ecological niches. The success of filamentous fungi is largely due to their elongate hypha, a chain of cells, separated from each other by... more
Filamentous fungi are a large and ancient clade of microorganisms that occupy a broad range of ecological niches. The success of filamentous fungi is largely due to their elongate hypha, a chain of cells, separated from each other by septa. Hyphae grow by polarized exocytosis at the apex, which allows the fungus to overcome long distances and invade many substrates, including soils and host tissues. Hyphal tip growth is initiated by establishment of a growth site and the subsequent maintenance of the growth axis, with transport of growth supplies, including membranes and proteins, delivered by motors along the cytoskeleton to the hyphal apex. Among the enzymes delivered are cell wall synthases that are exocytosed for local synthesis of the extracellular cell wall. Exocytosis is opposed by endocytic uptake of soluble and membrane-bound material into the cell. The first intracellular compartment in the endocytic pathway is the early endosomes, which emerge to perform essential additio...
ABSTRACT: Aim: Candida albicans is a pathogenic yeast, which forms a range of polarized and expanded cell shapes. We aimed to determine the correlation between honey extract (HFE) activity and changes in C. albicans cell cycle,... more
ABSTRACT: Aim: Candida albicans is a pathogenic yeast, which forms a range of polarized and expanded cell shapes. We aimed to determine the correlation between honey extract (HFE) activity and changes in C. albicans cell cycle, morphology and subcellular organelles. Materials & methods: HFE anticandidal properties were investigated using flow cytometry and scanning electron microscopy. Results: Flow cytometry and scanning electron microscopy analyses indicated that HFE may inhibit the growth of the three phenotypes displayed by C. albicans and reduce infection by affecting membrane integrity. HFE affects hyphal transition by reducing the G0/G1 phase and increasing the G2/M phase. Conversely, yeast and pseudohyphae do not appear to be affected. Modifications of vacuolization and mitochondrial activity, during yeast–hypha transition establish the involvement of vacuole and mitochondria. Conclusion: HFE improved mitochondrial functionality and reduced the vacuolization, modifying the ...
Blast disease destroys up to 30% of the rice crop annually and threatens global food security. The blast fungus invades plant tissue with hyphae that proliferate and grow from cell to cell, often through pit fields, where plasmodesmata... more
Blast disease destroys up to 30% of the rice crop annually and threatens global food security. The blast fungus invades plant tissue with hyphae that proliferate and grow from cell to cell, often through pit fields, where plasmodesmata cluster. We showed that chemical genetic inhibition of a single fungal mitogen-activated protein (MAP) kinase, Pmk1, prevents from infecting adjacent plant cells, leaving the fungus trapped within a single plant cell. Pmk1 regulates expression of secreted fungal effector proteins implicated in suppression of host immune defenses, preventing reactive oxygen species generation and excessive callose deposition at plasmodesmata. Furthermore, Pmk1 controls the hyphal constriction required for fungal growth from one rice cell to the neighboring cell, enabling host tissue colonization and blast disease.
Fungal pathogens need regulated mechanical and morphological fine-tuning for pushing through substrates to meet their metabolic and functional needs. Currently very little is understood on how coordinated colony level morphomechanical... more
Fungal pathogens need regulated mechanical and morphological fine-tuning for pushing through substrates to meet their metabolic and functional needs. Currently very little is understood on how coordinated colony level morphomechanical modifications regulate their behavior. This is due to an absence of a method that can simultaneously map, quantify, and correlate global fluctuations in physical properties of the expanding fungal colonies. Here, we show that three-dimensional ultrasonic reflections upon decoding can render acoustic contrast tomographs that contain information on material property and morphology in the same time scale of one important phytopathogen, Aspergillus parasiticus, at multiple length scales. By quantitative analysis of the changes in acoustic signatures collected as the A. parasiticus colony expands with time, we further demonstrate that the pathogen displays unique acoustic signatures during synthesis and release of its hepatocarcinogenic secondary metabolite, aflatoxin, suggesting an involvement of a multiscale morphomechanical reorganization of the colony in this process. Our studies illustrate for the first time, the feasibility of generating in any invading cell population, four-dimensional maps of global physical properties, with minimal physical perturbation of the specimens. Our developed method that we term quantitative acoustic contrast tomography (Q-ACT), provides a novel diagnostic framework for the identification of in-cell molecular factors and discovery of small molecules that may modulate pathogen invasion in a host.
The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production... more
The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and t...
Anthropogenic activities during the last century have increased levels of atmospheric CO2. Forest net primary productivity increases in response to elevated CO2, altering the quantity and quality of carbon supplied to the rhizosphere.... more
Anthropogenic activities during the last century have increased levels of atmospheric CO2. Forest net primary productivity increases in response to elevated CO2, altering the quantity and quality of carbon supplied to the rhizosphere. Ectomycorrhizal fungi form obligate symbiotic associations with the fine roots of trees that mediate improved scavenging for nutrients in exchange for a carbohydrate supply. Understanding how the community structure of ectomycorrhizal fungi is altered by climate change is important to further our understanding of ecosystem function. Betula pendula and Fagus sylvatica were grown in an elevated CO2 atmosphere delivered using free air carbon dioxide enrichment (FACE) under field conditions in the U.K., and Picea abies was grown under elevated CO2 in glass domes in the Czech Republic. We used morphotyping and sequencing of the internal transcribed spacer region of the fungal ribosomal operon to study ectomycorrhizal OPEN ACCESS Forests 2015, 5 1257 communi...
Ferroxidases are essential components of the high-affinity reductive iron assimilation pathway in fungi. Two ferroxidase genes, FET3-1 and FET3-2, have been identified in the genome of the maize anthracnose fungus Colletotrichum... more
Ferroxidases are essential components of the high-affinity reductive iron assimilation pathway in fungi. Two ferroxidase genes, FET3-1 and FET3-2, have been identified in the genome of the maize anthracnose fungus Colletotrichum graminicola. Complementation of growth defects of the ferroxidase-deficient Saccharomyces cerevisiae strain Δfet3fet4 showed that both Fet3-1 and Fet3-2 of C. graminicola represent functional ferroxidases. Expression of enhanced green fluorescent protein fusions in yeast and C. graminicola indicated that both ferroxidase proteins localize to the plasma membrane. Transcript abundance of FET3-1 increased dramatically under iron-limiting conditions but those of FET3-2 were hardly detectable. Δfet3-1 and Δfet3-2 single as well as Δfet3-1/2 double-deletion strains were generated. Under iron-sufficient or deficient conditions, vegetative growth rates of these strains did not significantly differ from that of the wild type but Δfet3-1 and Δfet3-1/2 strains showed i...
Increased resilience of pasture grasses mediated by fungal Epichloë endophytes is crucial to pastoral industries. The underlying mechanisms are only partially understood and likely involve very different activities of the endophyte in... more
Increased resilience of pasture grasses mediated by fungal Epichloë endophytes is crucial to pastoral industries. The underlying mechanisms are only partially understood and likely involve very different activities of the endophyte in different plant tissues and responses of the plant to these. We analyzed the transcriptomes of Epichloë festucae and its host Lolium perenne in host tissues of different function and developmental stages. The endophyte contributed approximately 10x more to the transcriptomes than to the biomass of infected tissues. Proliferating mycelium in growing host tissues highly expressed genes involved in hyphal growth. Non-proliferating mycelium in mature plant tissues, transcriptionally equally active, highly expressed genes involved in synthesizing anti-herbivore compounds. Transcripts from the latter accounted for 4% of fungal transcripts. Endophyte infection systemically but moderately increased transcription of L. perenne genes with roles in hormone biosyn...
Biofilm formation on implanted medical devices is a major source of lethal invasive infection by Candida albicans. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for... more
Biofilm formation on implanted medical devices is a major source of lethal invasive infection by Candida albicans. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A cak1 diminished expression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the tra...
Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitu-morals. They have a complex developmental cycle, in-cluding programmed cell death phenomena, that makes this bacterium a... more
Streptomyces species produce many clinically important secondary metabolites, including antibiotics and antitu-morals. They have a complex developmental cycle, in-cluding programmed cell death phenomena, that makes this bacterium a multicellular prokaryotic model. There are two differentiated mycelial stages: an early compart-mentalized vegetative mycelium (first mycelium) and a multinucleated reproductive mycelium (second mycelium) arising after programmed cell death processes. In the present study, we made a detailed proteomics analysis of the distinct developmental stages of solid confluent Streptomyces coelicolor cultures using iTRAQ (isobaric tags for relative and absolute quantitation) labeling and LC-MS/MS. A new experimental approach was developed to obtain homogeneous samples at each developmental
- ] complex reaching to a minimum values for the mycelium originating from inoculum exposed to the minimum inhibitory concentration (2%). Both aflatoxin (AFB 1 ) and citrinin were the most sensitive toxin.
Mycelial fungi play a central role in element cycling in nature by degrading dead organic material such as wood. Fungal colonization of a substrate starts with the invasion of exploring hyphae. These hyphae secrete enzymes that convert... more
Mycelial fungi play a central role in element cycling in nature by degrading dead organic material such as wood. Fungal colonization of a substrate starts with the invasion of exploring hyphae. These hyphae secrete enzymes that convert the organic material into small molecules that can be taken up by the fungus to serve as nutrients. Using green fluorescent protein (GFP) as a reporter, we show for the first time that exploring hyphae of Aspergillus niger differentiate with respect to enzyme secretion; some strongly express the glucoamylase gene glaA, while others hardly express it at all. When a cytoplasmic GFP was used, 27% of the exploring hyphae of a 5-day-old colony belonged to the low expressing hyphae. By fusing GFP to glucoamylase and by introducing an ER retention signal, this number increased to 50%. This difference is due to cytoplasmic streaming of the reporter in the former case, as was shown by using a photo-activatable GFP. Our findings indicate that a fungal mycelium ...
Resumen Los microorganismos del suelo son vitales para el correcto funcionamiento de los ecosistemas, principalmente por su papel en el ciclado de nutrientes. La intensificacion del uso del suelo y las practicas agricolas alteran... more
Resumen Los microorganismos del suelo son vitales para el correcto funcionamiento de los ecosistemas, principalmente por su papel en el ciclado de nutrientes. La intensificacion del uso del suelo y las practicas agricolas alteran negativamente la actividad microbiana. La biomasa fungica es uno de los parametros mas utilizados para estudiar el impacto de las actividades agricolas en la estructura y el funcionamiento del suelo. El objetivo del presente trabajo fue estimar la biomasa fungica en un suelo del sudoeste bonaerense con el fin de obtener valores de referencia que permitan usar este parametro como un indicador de cambios en el ecosistema y, por otro lado, demostrar que la metodologia empleada es sensible a las variaciones en las condiciones climaticas. Se colectaron muestras de suelos durante 2 anos consecutivos. Se prepararon frotis de suelo y se tineron con soluciones de distintas concentraciones de blanco de calcofluor y luego se estimo la biomasa fungica observando los fr...
The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeastSaccharomyces cerevisiae, a dramatic polarization of sterol-rich domains to... more
The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeastSaccharomyces cerevisiae, a dramatic polarization of sterol-rich domains to the shmoo tip was observed in pheromone-induced cells (M. Bagnat and K. Simons, Proc. Natl. Acad. Sci. USA99:14183-14188, 2002). We therefore examined whether plasma membrane lipid polarization contributes to the ability of the fungal pathogenCandida albicansto grow in a highly polarized manner to form hyphae. Interestingly, staining with filipin revealed that membrane sterols were highly polarized to the leading edge of growth during all stages of hyphal growth. Budding and pseudohyphal cells did not display polarized staining. Filipin staining was also enriched at septation sites in hyphae, where colocalization with septin proteins was observed, suggesting a role for the septins in forming a boundary domain. Actin appeared to play a role in sterol...
Apical sterol-rich plasma membrane domains (SRDs), which can be viewed using the sterol-binding fluorescent dye filipin, are gaining attention for their important roles in polarized growth of filamentous fungi. The microdomain scaffolding... more
Apical sterol-rich plasma membrane domains (SRDs), which can be viewed using the sterol-binding fluorescent dye filipin, are gaining attention for their important roles in polarized growth of filamentous fungi. The microdomain scaffolding protein flotillin/reggie and related stomatin were thought to be good candidates involved in the formation of SRDs. Here, we show that the flotillin/reggie orthologue FloA tagged with GFP localized as stable dots along the plasma membrane except hyphal tips. Deletion of floA reduced the growth rate, often resulted in irregularly shaped hyphae and impaired SRDs. In contrast, the stomatin orthologue StoA, tagged with GFP, localized at the cortex of young branch tips and at the subapical cortex in long hyphal tips, and was transported bi-directionally along microtubules on endosomes. Deletion of stoA resulted in irregular hyphal morphology and increased branching especially in young hyphae, but did not obviously affect SRDs. Double deletion of floA an...
To better understand the molecular events underlying vulvovaginal candidiasis, we established anin vitrosystem. Immortalized vaginal epithelial cells were infected with live, yeast formC. albicansandC. albicanscultured in the same medium... more
To better understand the molecular events underlying vulvovaginal candidiasis, we established anin vitrosystem. Immortalized vaginal epithelial cells were infected with live, yeast formC. albicansandC. albicanscultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes inC. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in ourin vitrosystem revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cellsin vitro, thus indicating the key role for this pathway in the virulence ofC. albicansupon vulvovaginal candidiasis.
Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C.... more
Background Sporothrix schenckii is a pathogenic dimorphic fungus of worldwide distribution. It grows in the saprophytic form with hyaline, regularly septated hyphae and pyriform conidia at 25°C and as the yeast or parasitic form at 35°C. Previously, we characterized a calcium/calmodulin kinase in this fungus. Inhibitors of this kinase were observed to inhibit the yeast cell cycle in S. schenckii. Results The presence of RNA interference (RNAi) mechanism in this fungus was confirmed by the identification of a Dicer-1 homologue in S. schenckii DNA. RNAi technology was used to corroborate the role of calcium/calmodulin kinase I in S. schenckii dimorphism. Yeast cells were transformed with the pSilent-Dual2G (pSD2G) plasmid w/wo inserts of the coding region of the calcium/calmodulin kinase I (sscmk1) gene. Transformants were selected at 35°C using resistance to geneticin. Following transfer to liquid medium at 35°C, RNAi transformants developed as abnormal mycelium clumps and not as yea...
