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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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A. nidulans SakA and N. crassa OS-2 SAPKS are phosphorylated in intact conidia; SakA dephosphorylation occurs during germination.A. Conidia from strains CLK43 (WT), COSΔsrrA03 (ΔsrrA), COSΔsskA02 (ΔsskA), COSΔsrrA/ΔsskA02 (ΔsrrAΔsskA) and TOL1 (ΔsakA) were collected, immediately frozen with liquid nitrogen and processed for Western blot immunodetection of phosphorylated (SakA-P). ΔsakA strain TOL1 was included as negative control and a total SakA blot is included as protein loading control.B and C. (B) Conidia from wild-type strain CLK43 were used to inoculate MM and incubated at 37°C for up to 4 h. At indicated time points samples were collected, examined by light microscopy (arrowheads indicate some of the conidial germ tubes) and processed as in (A) for immunoblotting, to detect phosphorylated (SakA-P) and total SakA (C). Bar = 10 µm.D. Conidia from N. crassa or A. nidulans were processed for immunoblotting as in (A) to detect phosphorylated and non-phosphorylated levels of MAPK OS-2 (N. crassa) or SakA (A. nidulans). An anti-tubulin antibody was used as additional sample loading reference.See Table 2 for full strain genotypes.
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fig06: A. nidulans SakA and N. crassa OS-2 SAPKS are phosphorylated in intact conidia; SakA dephosphorylation occurs during germination.A. Conidia from strains CLK43 (WT), COSΔsrrA03 (ΔsrrA), COSΔsskA02 (ΔsskA), COSΔsrrA/ΔsskA02 (ΔsrrAΔsskA) and TOL1 (ΔsakA) were collected, immediately frozen with liquid nitrogen and processed for Western blot immunodetection of phosphorylated (SakA-P). ΔsakA strain TOL1 was included as negative control and a total SakA blot is included as protein loading control.B and C. (B) Conidia from wild-type strain CLK43 were used to inoculate MM and incubated at 37°C for up to 4 h. At indicated time points samples were collected, examined by light microscopy (arrowheads indicate some of the conidial germ tubes) and processed as in (A) for immunoblotting, to detect phosphorylated (SakA-P) and total SakA (C). Bar = 10 µm.D. Conidia from N. crassa or A. nidulans were processed for immunoblotting as in (A) to detect phosphorylated and non-phosphorylated levels of MAPK OS-2 (N. crassa) or SakA (A. nidulans). An anti-tubulin antibody was used as additional sample loading reference.See Table 2 for full strain genotypes.

Mentions: The fact that ΔsakA conidia lose their viability after prolonged storage (Kawasaki et al., 2002) prompted us to compare conidial viability in ΔsakA and ΔatfA mutants. Results in Fig. 5A show that both mutants produced conidia that gradually lost their viability with similar kinetics, while wild-type conidia retained full viability under the same conditions. Although Hawigara et al. have reported comparable results for ΔatfA conidia, it is not clear why under similar conditions they observed a complete loss of viability after 10 days (Hagiwara et al., 2008). When we examined SakA levels in wild-type and mutant conidia, we observed high levels of SakA in intact conidia from the wild-type strain, while SakA was almost undetectable in conidia from the ΔatfA mutant (Fig. 5B, left lanes). In contrast, AtfA was not required to maintain SakA levels or for SakA phosphorylation in response to H2O2, in mycelial samples (Fig. 5B, right lanes). Notably, our results also show that SakA is phosphorylated (active) in wild-type conidia (Fig. 5B). When atfA mRNA was examined in intact conidia and during germination we detected two transcripts with the probe used. However, only the larger transcript was missing in ΔatfA mutants (not shown) and therefore it corresponds to atfA. Both transcripts were accumulated in intact conidia and gradually decreased during spore germination. The atfA transcript showed lower levels after 5 h of germination (Fig. 5C), a time when all spores have germinated (see Fig. 6B). Our results suggest a connection between SakA phosphorylation and spore viability, show that AtfA is required for SakA accumulation in asexual spores but not in mycelia, and that SakA becomes phosphorylated during normal asexual development.


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)

A. nidulans SakA and N. crassa OS-2 SAPKS are phosphorylated in intact conidia; SakA dephosphorylation occurs during germination.A. Conidia from strains CLK43 (WT), COSΔsrrA03 (ΔsrrA), COSΔsskA02 (ΔsskA), COSΔsrrA/ΔsskA02 (ΔsrrAΔsskA) and TOL1 (ΔsakA) were collected, immediately frozen with liquid nitrogen and processed for Western blot immunodetection of phosphorylated (SakA-P). ΔsakA strain TOL1 was included as negative control and a total SakA blot is included as protein loading control.B and C. (B) Conidia from wild-type strain CLK43 were used to inoculate MM and incubated at 37°C for up to 4 h. At indicated time points samples were collected, examined by light microscopy (arrowheads indicate some of the conidial germ tubes) and processed as in (A) for immunoblotting, to detect phosphorylated (SakA-P) and total SakA (C). Bar = 10 µm.D. Conidia from N. crassa or A. nidulans were processed for immunoblotting as in (A) to detect phosphorylated and non-phosphorylated levels of MAPK OS-2 (N. crassa) or SakA (A. nidulans). An anti-tubulin antibody was used as additional sample loading reference.See Table 2 for full strain genotypes.
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fig06: A. nidulans SakA and N. crassa OS-2 SAPKS are phosphorylated in intact conidia; SakA dephosphorylation occurs during germination.A. Conidia from strains CLK43 (WT), COSΔsrrA03 (ΔsrrA), COSΔsskA02 (ΔsskA), COSΔsrrA/ΔsskA02 (ΔsrrAΔsskA) and TOL1 (ΔsakA) were collected, immediately frozen with liquid nitrogen and processed for Western blot immunodetection of phosphorylated (SakA-P). ΔsakA strain TOL1 was included as negative control and a total SakA blot is included as protein loading control.B and C. (B) Conidia from wild-type strain CLK43 were used to inoculate MM and incubated at 37°C for up to 4 h. At indicated time points samples were collected, examined by light microscopy (arrowheads indicate some of the conidial germ tubes) and processed as in (A) for immunoblotting, to detect phosphorylated (SakA-P) and total SakA (C). Bar = 10 µm.D. Conidia from N. crassa or A. nidulans were processed for immunoblotting as in (A) to detect phosphorylated and non-phosphorylated levels of MAPK OS-2 (N. crassa) or SakA (A. nidulans). An anti-tubulin antibody was used as additional sample loading reference.See Table 2 for full strain genotypes.
Mentions: The fact that ΔsakA conidia lose their viability after prolonged storage (Kawasaki et al., 2002) prompted us to compare conidial viability in ΔsakA and ΔatfA mutants. Results in Fig. 5A show that both mutants produced conidia that gradually lost their viability with similar kinetics, while wild-type conidia retained full viability under the same conditions. Although Hawigara et al. have reported comparable results for ΔatfA conidia, it is not clear why under similar conditions they observed a complete loss of viability after 10 days (Hagiwara et al., 2008). When we examined SakA levels in wild-type and mutant conidia, we observed high levels of SakA in intact conidia from the wild-type strain, while SakA was almost undetectable in conidia from the ΔatfA mutant (Fig. 5B, left lanes). In contrast, AtfA was not required to maintain SakA levels or for SakA phosphorylation in response to H2O2, in mycelial samples (Fig. 5B, right lanes). Notably, our results also show that SakA is phosphorylated (active) in wild-type conidia (Fig. 5B). When atfA mRNA was examined in intact conidia and during germination we detected two transcripts with the probe used. However, only the larger transcript was missing in ΔatfA mutants (not shown) and therefore it corresponds to atfA. Both transcripts were accumulated in intact conidia and gradually decreased during spore germination. The atfA transcript showed lower levels after 5 h of germination (Fig. 5C), a time when all spores have germinated (see Fig. 6B). Our results suggest a connection between SakA phosphorylation and spore viability, show that AtfA is required for SakA accumulation in asexual spores but not in mycelia, and that SakA becomes phosphorylated during normal asexual development.

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