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Flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi.

Delgado-Ramos L, Marcos AT, Ramos-Guelfo MS, Sánchez-Barrionuevo L, Smet F, Chávez S, Cánovas D - G3 (Bethesda) (2014)

Bottom Line: Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains.We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes.Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.

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Screening for fungal growth or fluorescence. Pictures of encapsulated A. nidulans spores after growing in different media and flow cytometry analysis of the particles. A pyrG89 strain (auxotrophic for uracil and uridine) carrying the histone H1-RFP fusion and a nonfluorescent wild-type strain were grown in minimal media in the absence or in the presence of U2 for 20 hr. Fungal growth in the microcapsules was visualized by light microscopy (A) and quantified by COPAS flow cytometry showing the extinction levels (B).
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fig4: Screening for fungal growth or fluorescence. Pictures of encapsulated A. nidulans spores after growing in different media and flow cytometry analysis of the particles. A pyrG89 strain (auxotrophic for uracil and uridine) carrying the histone H1-RFP fusion and a nonfluorescent wild-type strain were grown in minimal media in the absence or in the presence of U2 for 20 hr. Fungal growth in the microcapsules was visualized by light microscopy (A) and quantified by COPAS flow cytometry showing the extinction levels (B).

Mentions: COPAS flow cytometry allowed the analysis of these phenotypes across the microencapsulated population in a quantitative manner. Cytometric analysis detected a population of wild-type microcolonies with increased optical density that was missing in the microcapsules containing auxotrophic pyrG89 spores (Figure 4). In contrast, a prominent population of microcapsules with increased optical density was detected when the auxotrophic mutant was incubated in the presence of uracil and uridine (Figure 4). In the presence of U2, we were able to detect signal in the red channel in the case of the pyrG89 strain arising from all the H1-RFP−tagged nuclei. Despite of the wild-type growing, signal in the red channel was not observed (data not shown).


Flow cytometry of microencapsulated colonies for genetics analysis of filamentous fungi.

Delgado-Ramos L, Marcos AT, Ramos-Guelfo MS, Sánchez-Barrionuevo L, Smet F, Chávez S, Cánovas D - G3 (Bethesda) (2014)

Screening for fungal growth or fluorescence. Pictures of encapsulated A. nidulans spores after growing in different media and flow cytometry analysis of the particles. A pyrG89 strain (auxotrophic for uracil and uridine) carrying the histone H1-RFP fusion and a nonfluorescent wild-type strain were grown in minimal media in the absence or in the presence of U2 for 20 hr. Fungal growth in the microcapsules was visualized by light microscopy (A) and quantified by COPAS flow cytometry showing the extinction levels (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232552&req=5

fig4: Screening for fungal growth or fluorescence. Pictures of encapsulated A. nidulans spores after growing in different media and flow cytometry analysis of the particles. A pyrG89 strain (auxotrophic for uracil and uridine) carrying the histone H1-RFP fusion and a nonfluorescent wild-type strain were grown in minimal media in the absence or in the presence of U2 for 20 hr. Fungal growth in the microcapsules was visualized by light microscopy (A) and quantified by COPAS flow cytometry showing the extinction levels (B).
Mentions: COPAS flow cytometry allowed the analysis of these phenotypes across the microencapsulated population in a quantitative manner. Cytometric analysis detected a population of wild-type microcolonies with increased optical density that was missing in the microcapsules containing auxotrophic pyrG89 spores (Figure 4). In contrast, a prominent population of microcapsules with increased optical density was detected when the auxotrophic mutant was incubated in the presence of uracil and uridine (Figure 4). In the presence of U2, we were able to detect signal in the red channel in the case of the pyrG89 strain arising from all the H1-RFP−tagged nuclei. Despite of the wild-type growing, signal in the red channel was not observed (data not shown).

Bottom Line: Growth tests revealed that auxotrophic mutants required the appropriate nutrients and that pyrithiamine and glufosinate halted fungal growth of sensitive but not resistant strains.We used an Aspergillus nidulans, thermosensitive mutant in the cell-cycle regulator gene nimX(CDK1) as proof-of-concept to the detection and identification of genetic phenotypes.Sorting of the microparticles containing the clonal fungal mycelia proved the power of this method to perform positive and/or negative selection during genetic screenings.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío-CSIC-Universidad de Sevilla, Seville, Spain.

Show MeSH
Related in: MedlinePlus