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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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Fungal phenotypes can be detected in microcapsules. Encapsulation allows one to screen for growth/no growth and/or fluorescence. In (A) the auxotroph mutant HA344: H1::RFP pyrG89 pyroA4 is not able to grow in the absence of pyridoxine or U2 (uracil + uridine). This screening can be extended to search for resistance to different compounds. For each case, we used a fungicide-resistant strain obtained by transformation of a resistance cassette in the genome and the corresponding isogenic strain. Capsules containing spores were incubated in minimal media in the presence or absence of pyrithiamine (B) and glufosinate (C) for 25−30 hr. (D) When nutritional requirements were added to the medium, the spores started to germinate and grow by apical elongation as shown by fluorescence microscopy.
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fig3: Fungal phenotypes can be detected in microcapsules. Encapsulation allows one to screen for growth/no growth and/or fluorescence. In (A) the auxotroph mutant HA344: H1::RFP pyrG89 pyroA4 is not able to grow in the absence of pyridoxine or U2 (uracil + uridine). This screening can be extended to search for resistance to different compounds. For each case, we used a fungicide-resistant strain obtained by transformation of a resistance cassette in the genome and the corresponding isogenic strain. Capsules containing spores were incubated in minimal media in the presence or absence of pyrithiamine (B) and glufosinate (C) for 25−30 hr. (D) When nutritional requirements were added to the medium, the spores started to germinate and grow by apical elongation as shown by fluorescence microscopy.

Mentions: To test the reliability of this method and its utility for genetic analysis, different assays were performed. First, we tested whether microcapsules were suitable systems for detecting common fungal phenotypes. Proliferation of an auxotrophic A. nidulans mutant (pyroA4 pyrG89) was only detected in the microcapsules when the corresponding supplements were present in the medium (Figure 3A). In agreement with previous observations (Osmani et al. 2006), the auxotrophic spores could germinate in the absence of U2, but growth soon stopped, resulting in very short hyphae. Similarly, strains resistant to two different fungicides (pyrithiamine and glufosinate) were capable of colonizing microcapsules in the presence of the drugs, whereas isogenic non-resistant strains were unable, as expected (Figure 3, B and C). Finally, strains expressing fluorescent-tagged proteins were visualized when growing inside the microcapsules (Figure 3D).


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)

Fungal phenotypes can be detected in microcapsules. Encapsulation allows one to screen for growth/no growth and/or fluorescence. In (A) the auxotroph mutant HA344: H1::RFP pyrG89 pyroA4 is not able to grow in the absence of pyridoxine or U2 (uracil + uridine). This screening can be extended to search for resistance to different compounds. For each case, we used a fungicide-resistant strain obtained by transformation of a resistance cassette in the genome and the corresponding isogenic strain. Capsules containing spores were incubated in minimal media in the presence or absence of pyrithiamine (B) and glufosinate (C) for 25−30 hr. (D) When nutritional requirements were added to the medium, the spores started to germinate and grow by apical elongation as shown by fluorescence microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Fungal phenotypes can be detected in microcapsules. Encapsulation allows one to screen for growth/no growth and/or fluorescence. In (A) the auxotroph mutant HA344: H1::RFP pyrG89 pyroA4 is not able to grow in the absence of pyridoxine or U2 (uracil + uridine). This screening can be extended to search for resistance to different compounds. For each case, we used a fungicide-resistant strain obtained by transformation of a resistance cassette in the genome and the corresponding isogenic strain. Capsules containing spores were incubated in minimal media in the presence or absence of pyrithiamine (B) and glufosinate (C) for 25−30 hr. (D) When nutritional requirements were added to the medium, the spores started to germinate and grow by apical elongation as shown by fluorescence microscopy.
Mentions: To test the reliability of this method and its utility for genetic analysis, different assays were performed. First, we tested whether microcapsules were suitable systems for detecting common fungal phenotypes. Proliferation of an auxotrophic A. nidulans mutant (pyroA4 pyrG89) was only detected in the microcapsules when the corresponding supplements were present in the medium (Figure 3A). In agreement with previous observations (Osmani et al. 2006), the auxotrophic spores could germinate in the absence of U2, but growth soon stopped, resulting in very short hyphae. Similarly, strains resistant to two different fungicides (pyrithiamine and glufosinate) were capable of colonizing microcapsules in the presence of the drugs, whereas isogenic non-resistant strains were unable, as expected (Figure 3, B and C). Finally, strains expressing fluorescent-tagged proteins were visualized when growing inside the microcapsules (Figure 3D).

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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Related in: MedlinePlus