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Aspergillus nidulans transcription factor AtfA interacts with the MAPK SakA to regulate general stress responses, development and spore functions.

Lara-Rojas F, Sánchez O, Kawasaki L, Aguirre J - Mol. Microbiol. (2011)

Bottom Line: Constitutive phosphorylation of SakA induced by the fungicide fludioxonil prevents both, germ tube formation and nuclear division.Similarly, Neurospora crassa SakA orthologue OS-2 is phosphorylated in intact conidia and gets dephosphorylated during germination.We propose that SakA-AtfA interaction regulates gene expression during stress and conidiophore development and that SAPK phosphorylation is a conserved mechanism to regulate transitions between non-growing (spore) and growing (mycelia) states.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510, México, D.F., México.

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Fungicide fludioxonil prevents conidia germination, SakA dephosphorylation and nuclear division in a SakA-dependent manner.A. Conidia from wild-type strain CLK43 were inoculated in MM in the absence (−) or the presence (+) of fludioxonil (2 µg ml−1) for up to 4 h. Samples were taken at indicated time points and examined by light microscopy.B. Fludioxonil-treated samples from (A) were collected, frozen with liquid nitrogen and processed for immunodetection as in Fig. 6.C. Conidia from strains CLK43 (WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were used to inoculate 50 ml of cultures plus and minus fludioxonil, incubated for 6 h and fixed. Nuclei were stained with DAPI and examined by phase-contrast (grey panels) and fluorescence microscopy (dark panels). Arrowheads indicate the presence of two nuclei in a single germinated spore.Bars = 10 µm.
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fig07: Fungicide fludioxonil prevents conidia germination, SakA dephosphorylation and nuclear division in a SakA-dependent manner.A. Conidia from wild-type strain CLK43 were inoculated in MM in the absence (−) or the presence (+) of fludioxonil (2 µg ml−1) for up to 4 h. Samples were taken at indicated time points and examined by light microscopy.B. Fludioxonil-treated samples from (A) were collected, frozen with liquid nitrogen and processed for immunodetection as in Fig. 6.C. Conidia from strains CLK43 (WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were used to inoculate 50 ml of cultures plus and minus fludioxonil, incubated for 6 h and fixed. Nuclei were stained with DAPI and examined by phase-contrast (grey panels) and fluorescence microscopy (dark panels). Arrowheads indicate the presence of two nuclei in a single germinated spore.Bars = 10 µm.

Mentions: To provide additional support to the idea that phosphorylated SakA maintains spore dormancy and/or prevents germination, we used the fungicide fludioxonil, which is known to result in constitutive phosphorylation of SakA orthologues (Zhang et al., 2002; Kojima et al., 2004). Results show that in the presence of fludioxonil wild-type conidia failed to produce germ tubes (Fig. 7A) and that under these conditions SakA remained phosphorylated (Fig. 7B). To test if SakA mediated this fludioxonil effect on spore germination and if such effect was related to nuclear division, we incubated spores (uninucleated) from wild-type, ΔsakA and ΔatfA strains with and without fludioxonil for up to 6 h. In the absence of the fungicide, conidia from all three strains formed germ tubes and two nuclei were detected by DAPI staining. In the presence of fludioxonil, wild-type conidia became swollen and none formed a germ tube, while DAPI staining of these spores produced a single, diffuse fluorescent signal that suggested the absence of nuclear division. In contrast, a high proportion of ΔsakA and ΔatfA conidia was able to develop a germ tube in the presence of the fungicide and showed two nuclei (Fig. 7C). Moreover, on solid medium containing fludioxonil, ΔsakA and ΔatfA conidia were able to form small colonies composed of short and aberrant hyphae (Fig. S6), while again wild-type conidia did swell but failed to generate any germ tube. As indicated, fludioxonil toxicity in A. nidulans is also mediated by the response regulator SrrA and full resistance is possible only if histidine kinase NikA or both response regulators (SrrA and SskA) are inactivated (Vargas-Perez et al., 2007).


Aspergillus nidulans transcription factor AtfA interacts with the MAPK SakA to regulate general stress responses, development and spore functions.

Lara-Rojas F, Sánchez O, Kawasaki L, Aguirre J - Mol. Microbiol. (2011)

Fungicide fludioxonil prevents conidia germination, SakA dephosphorylation and nuclear division in a SakA-dependent manner.A. Conidia from wild-type strain CLK43 were inoculated in MM in the absence (−) or the presence (+) of fludioxonil (2 µg ml−1) for up to 4 h. Samples were taken at indicated time points and examined by light microscopy.B. Fludioxonil-treated samples from (A) were collected, frozen with liquid nitrogen and processed for immunodetection as in Fig. 6.C. Conidia from strains CLK43 (WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were used to inoculate 50 ml of cultures plus and minus fludioxonil, incubated for 6 h and fixed. Nuclei were stained with DAPI and examined by phase-contrast (grey panels) and fluorescence microscopy (dark panels). Arrowheads indicate the presence of two nuclei in a single germinated spore.Bars = 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3108070&req=5

fig07: Fungicide fludioxonil prevents conidia germination, SakA dephosphorylation and nuclear division in a SakA-dependent manner.A. Conidia from wild-type strain CLK43 were inoculated in MM in the absence (−) or the presence (+) of fludioxonil (2 µg ml−1) for up to 4 h. Samples were taken at indicated time points and examined by light microscopy.B. Fludioxonil-treated samples from (A) were collected, frozen with liquid nitrogen and processed for immunodetection as in Fig. 6.C. Conidia from strains CLK43 (WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were used to inoculate 50 ml of cultures plus and minus fludioxonil, incubated for 6 h and fixed. Nuclei were stained with DAPI and examined by phase-contrast (grey panels) and fluorescence microscopy (dark panels). Arrowheads indicate the presence of two nuclei in a single germinated spore.Bars = 10 µm.
Mentions: To provide additional support to the idea that phosphorylated SakA maintains spore dormancy and/or prevents germination, we used the fungicide fludioxonil, which is known to result in constitutive phosphorylation of SakA orthologues (Zhang et al., 2002; Kojima et al., 2004). Results show that in the presence of fludioxonil wild-type conidia failed to produce germ tubes (Fig. 7A) and that under these conditions SakA remained phosphorylated (Fig. 7B). To test if SakA mediated this fludioxonil effect on spore germination and if such effect was related to nuclear division, we incubated spores (uninucleated) from wild-type, ΔsakA and ΔatfA strains with and without fludioxonil for up to 6 h. In the absence of the fungicide, conidia from all three strains formed germ tubes and two nuclei were detected by DAPI staining. In the presence of fludioxonil, wild-type conidia became swollen and none formed a germ tube, while DAPI staining of these spores produced a single, diffuse fluorescent signal that suggested the absence of nuclear division. In contrast, a high proportion of ΔsakA and ΔatfA conidia was able to develop a germ tube in the presence of the fungicide and showed two nuclei (Fig. 7C). Moreover, on solid medium containing fludioxonil, ΔsakA and ΔatfA conidia were able to form small colonies composed of short and aberrant hyphae (Fig. S6), while again wild-type conidia did swell but failed to generate any germ tube. As indicated, fludioxonil toxicity in A. nidulans is also mediated by the response regulator SrrA and full resistance is possible only if histidine kinase NikA or both response regulators (SrrA and SskA) are inactivated (Vargas-Perez et al., 2007).

Bottom Line: Constitutive phosphorylation of SakA induced by the fungicide fludioxonil prevents both, germ tube formation and nuclear division.Similarly, Neurospora crassa SakA orthologue OS-2 is phosphorylated in intact conidia and gets dephosphorylated during germination.We propose that SakA-AtfA interaction regulates gene expression during stress and conidiophore development and that SAPK phosphorylation is a conserved mechanism to regulate transitions between non-growing (spore) and growing (mycelia) states.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510, México, D.F., México.

Show MeSH
Related in: MedlinePlus