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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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AtfA::GFP shows nuclear localization independently of sakA; SakA accumulates in the nucleus in response to hydrogen peroxide stress, where it interacts with transcription factor AtfA.A. Conidia from strains CLK43 (WT), TFL3 (AtfA::GFP), CFL9 (ΔsakA; AtfA::GFP) and TFL6 (SakA::GFP) were inoculated on coverslips submerged in liquid supplemented minimal medium, incubated for 12 h at 37°C and then fixed and stained with DAPI.B. Conidia from strain TFL6 were inoculated as in (A) and then treated with 30 mM H2O2 for 30 min. Arrowheads indicate SakA::GFP localization after stress treatment. Pictures were taken using Epifluorescence with a microscope NIKON Eclipse E600.C. BiFC analysis of SakA and AtfA. Strain TFL7 expressing YFP-N::SakA and YFP-C::AtfA was grown as in (B), transferred to minimal medium with 100 mM threonine for 3 h to induce the alcA promoter, and exposed to 30 mM H2O2 for 30 min. Yellow fluorescence was detected using a confocal microscope Olympus FV1000.Bar = 20 µm.
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fig04: AtfA::GFP shows nuclear localization independently of sakA; SakA accumulates in the nucleus in response to hydrogen peroxide stress, where it interacts with transcription factor AtfA.A. Conidia from strains CLK43 (WT), TFL3 (AtfA::GFP), CFL9 (ΔsakA; AtfA::GFP) and TFL6 (SakA::GFP) were inoculated on coverslips submerged in liquid supplemented minimal medium, incubated for 12 h at 37°C and then fixed and stained with DAPI.B. Conidia from strain TFL6 were inoculated as in (A) and then treated with 30 mM H2O2 for 30 min. Arrowheads indicate SakA::GFP localization after stress treatment. Pictures were taken using Epifluorescence with a microscope NIKON Eclipse E600.C. BiFC analysis of SakA and AtfA. Strain TFL7 expressing YFP-N::SakA and YFP-C::AtfA was grown as in (B), transferred to minimal medium with 100 mM threonine for 3 h to induce the alcA promoter, and exposed to 30 mM H2O2 for 30 min. Yellow fluorescence was detected using a confocal microscope Olympus FV1000.Bar = 20 µm.

Mentions: Our results above suggest a model in which the MAPK SakA regulates putative transcription factor AtfA, in response to different stress signals. To examine AtfA and SakA localization we generated strains TFL3 and TFL6, which carry atfA::gfp or sakA::gfp alleles replacing the wild-type genes respectively (confirmed by Southern blot analysis; not shown). As shown in Fig. S5, conidia from strains TFL3 and TFL6 were not sensitive to H2O2, indicating that GFP tagging did not interfere with AtfA or SakA functions. In addition, the atfA::gfp allele was introduced into a ΔsakA genetic background by sexual crosses, to generate strain CFL9, and the presence of both mutant alleles was confirmed by Southern analysis (not shown). When these strains were used to detect GFP localization, we found AtfA::GFP in the nucleus even in the absence of any experimentally induced stress. Furthermore, such nuclear localization was largely unaffected by the absence of the MAPK SakA (Fig. 4A). In contrast, SakA::GFP fluorescence was not restricted to DAPI-stained nuclei, showing a more uniform distribution in hyphae (Fig. 4A, right panels). These results supported AtfA function as transcription factor and suggested that to regulate AtfA, SakA would need to be translocated to the nucleus. Indeed, treatment with H2O2 induced a re-distribution of SakA::GFP fluorescence consistent with the nuclear localization of SakA in response to oxidative stress (Fig. 4B).


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)

AtfA::GFP shows nuclear localization independently of sakA; SakA accumulates in the nucleus in response to hydrogen peroxide stress, where it interacts with transcription factor AtfA.A. Conidia from strains CLK43 (WT), TFL3 (AtfA::GFP), CFL9 (ΔsakA; AtfA::GFP) and TFL6 (SakA::GFP) were inoculated on coverslips submerged in liquid supplemented minimal medium, incubated for 12 h at 37°C and then fixed and stained with DAPI.B. Conidia from strain TFL6 were inoculated as in (A) and then treated with 30 mM H2O2 for 30 min. Arrowheads indicate SakA::GFP localization after stress treatment. Pictures were taken using Epifluorescence with a microscope NIKON Eclipse E600.C. BiFC analysis of SakA and AtfA. Strain TFL7 expressing YFP-N::SakA and YFP-C::AtfA was grown as in (B), transferred to minimal medium with 100 mM threonine for 3 h to induce the alcA promoter, and exposed to 30 mM H2O2 for 30 min. Yellow fluorescence was detected using a confocal microscope Olympus FV1000.Bar = 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: AtfA::GFP shows nuclear localization independently of sakA; SakA accumulates in the nucleus in response to hydrogen peroxide stress, where it interacts with transcription factor AtfA.A. Conidia from strains CLK43 (WT), TFL3 (AtfA::GFP), CFL9 (ΔsakA; AtfA::GFP) and TFL6 (SakA::GFP) were inoculated on coverslips submerged in liquid supplemented minimal medium, incubated for 12 h at 37°C and then fixed and stained with DAPI.B. Conidia from strain TFL6 were inoculated as in (A) and then treated with 30 mM H2O2 for 30 min. Arrowheads indicate SakA::GFP localization after stress treatment. Pictures were taken using Epifluorescence with a microscope NIKON Eclipse E600.C. BiFC analysis of SakA and AtfA. Strain TFL7 expressing YFP-N::SakA and YFP-C::AtfA was grown as in (B), transferred to minimal medium with 100 mM threonine for 3 h to induce the alcA promoter, and exposed to 30 mM H2O2 for 30 min. Yellow fluorescence was detected using a confocal microscope Olympus FV1000.Bar = 20 µm.
Mentions: Our results above suggest a model in which the MAPK SakA regulates putative transcription factor AtfA, in response to different stress signals. To examine AtfA and SakA localization we generated strains TFL3 and TFL6, which carry atfA::gfp or sakA::gfp alleles replacing the wild-type genes respectively (confirmed by Southern blot analysis; not shown). As shown in Fig. S5, conidia from strains TFL3 and TFL6 were not sensitive to H2O2, indicating that GFP tagging did not interfere with AtfA or SakA functions. In addition, the atfA::gfp allele was introduced into a ΔsakA genetic background by sexual crosses, to generate strain CFL9, and the presence of both mutant alleles was confirmed by Southern analysis (not shown). When these strains were used to detect GFP localization, we found AtfA::GFP in the nucleus even in the absence of any experimentally induced stress. Furthermore, such nuclear localization was largely unaffected by the absence of the MAPK SakA (Fig. 4A). In contrast, SakA::GFP fluorescence was not restricted to DAPI-stained nuclei, showing a more uniform distribution in hyphae (Fig. 4A, right panels). These results supported AtfA function as transcription factor and suggested that to regulate AtfA, SakA would need to be translocated to the nucleus. Indeed, treatment with H2O2 induced a re-distribution of SakA::GFP fluorescence consistent with the nuclear localization of SakA in response to oxidative stress (Fig. 4B).

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