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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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sakA is epistatic to atfA; the SakA–AtfA pathway regulates different antioxidant responses in spores versus mycelia.A. Conidia (1 × 104) from strains CLK43 (wild type; WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were inoculated on supplemented MM plates containing t-butylhydroperoxide (t-BOOH), menadione (Md), paraquat (Pq) or methylglyoxal (MG), at the indicated concentrations, and incubated at 37°C for 4 days.B. Conidia from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were inoculated as in (A) on plates containing 3 or 4 mM H2O2.C. Mycelial plugs cut from the growing edge of 5-day colonies from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were transferred to plates containing the indicated compounds and incubated at 37°C during 4 days.D. Conidia (1 × 104) and mycelial plugs from strains 11035 (WT), TFLΔsakA-03 (ΔsakA), TFLΔatfA-04 (ΔatfA) and TFL4 (ΔsakA ΔatfA) were inoculated on plates containing the indicated concentrations of H2O2 and t-butylhydroperoxide (t-BOOH) and incubated for 4 days at 37°C.See Table 2 for full strain genotypes.
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fig01: sakA is epistatic to atfA; the SakA–AtfA pathway regulates different antioxidant responses in spores versus mycelia.A. Conidia (1 × 104) from strains CLK43 (wild type; WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were inoculated on supplemented MM plates containing t-butylhydroperoxide (t-BOOH), menadione (Md), paraquat (Pq) or methylglyoxal (MG), at the indicated concentrations, and incubated at 37°C for 4 days.B. Conidia from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were inoculated as in (A) on plates containing 3 or 4 mM H2O2.C. Mycelial plugs cut from the growing edge of 5-day colonies from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were transferred to plates containing the indicated compounds and incubated at 37°C during 4 days.D. Conidia (1 × 104) and mycelial plugs from strains 11035 (WT), TFLΔsakA-03 (ΔsakA), TFLΔatfA-04 (ΔatfA) and TFL4 (ΔsakA ΔatfA) were inoculated on plates containing the indicated concentrations of H2O2 and t-butylhydroperoxide (t-BOOH) and incubated for 4 days at 37°C.See Table 2 for full strain genotypes.

Mentions: To evaluate atfA functions and possible connections to the SakA MAPK pathway, we first generated strains carrying complete deletions in either gene, as confirmed by Southern blot analysis (Figs S2 and S3A and B). ΔatfA and ΔsakA mutants were indistinguishable from the wild-type strain under high-temperature (42°C) or high-osmolarity (1 M NaCl or 1.2 M sorbitol) stress conditions (not shown). To test the mutant response to different types of oxidative stress, we incubated ΔatfA and ΔsakA strains in the presence of the redox-cycling compounds menadione and paraquat, the glutathione-depleting compound methylglyoxal and inorganic (H2O2) as well as organic (t-butylhydroperoxide; t-BOOH) peroxides. As shown in Fig. 1A, wild-type, ΔatfA and ΔsakA strains were similarly resistant to menadione and paraquat. In contrast, ΔatfA and ΔsakA mutants were hypersensitive to both t-butylhydroperoxide (Fig. 1A) and hydrogen peroxide (Fig. 1B), showing a slight sensitivity to methylglyoxal (Fig. 1A). Notably, ΔatfA and ΔsakA mutants were as sensitive to H2O2 as the ΔcatA mutant, which lacks the spore-specific catalase CatA (Navarro et al., 1996; Navarro and Aguirre, 1998). On the contrary, a mutant lacking the mycelial inducible catalase CatB (Kawasaki et al., 1997) showed a H2O2 resistance only slightly lower than the wild type (Fig. 1B). This and the fact that conidia from sakA mutants show decreased CatA activity (Kawasaki et al., 2002) suggested that under these conditions, mutant sensitivity to H2O2 could reflect low CatA activity in conidia. To explore this, we carried out similar oxidative stress plate assays but using mycelial plugs instead of conidia. As shown in Fig. 1C, mycelia from ΔatfA and ΔsakA mutants was resistant up to 6 mM H2O2 but resulted hypersensitive to t-BOOH. Notably, under these conditions, mycelia from ΔcatA, ΔcatB and wild-type strains showed similar resistance to H2O2 and t-BOOH. Compared with the wild-type, ΔatfA, ΔsakA and ΔcatB mutants were somewhat more sensitive to menadione. While all strains presented similar growth in paraquat, a brownish pigmentation and decreased conidiation was observed in ΔatfA and ΔsakA mutants (Fig. 1C). Results published during the course of this work show that conidia from ΔatfA (ΔsakA was not analysed) mutants were sensitive to 50 mM H2O2 (t-BOOH was not tested), while ΔatfA mycelia was resistant to 1.2 mM t-BOOH (Hagiwara et al., 2008). In agreement with our results, a more recent report shows that ΔatfA mycelium is indeed sensitive to t-BOOH (Balazs et al., 2010).


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)

sakA is epistatic to atfA; the SakA–AtfA pathway regulates different antioxidant responses in spores versus mycelia.A. Conidia (1 × 104) from strains CLK43 (wild type; WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were inoculated on supplemented MM plates containing t-butylhydroperoxide (t-BOOH), menadione (Md), paraquat (Pq) or methylglyoxal (MG), at the indicated concentrations, and incubated at 37°C for 4 days.B. Conidia from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were inoculated as in (A) on plates containing 3 or 4 mM H2O2.C. Mycelial plugs cut from the growing edge of 5-day colonies from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were transferred to plates containing the indicated compounds and incubated at 37°C during 4 days.D. Conidia (1 × 104) and mycelial plugs from strains 11035 (WT), TFLΔsakA-03 (ΔsakA), TFLΔatfA-04 (ΔatfA) and TFL4 (ΔsakA ΔatfA) were inoculated on plates containing the indicated concentrations of H2O2 and t-butylhydroperoxide (t-BOOH) and incubated for 4 days at 37°C.See Table 2 for full strain genotypes.
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Show All Figures
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fig01: sakA is epistatic to atfA; the SakA–AtfA pathway regulates different antioxidant responses in spores versus mycelia.A. Conidia (1 × 104) from strains CLK43 (wild type; WT), TOL1 (ΔsakA) and TFLΔatfA-02 (ΔatfA) were inoculated on supplemented MM plates containing t-butylhydroperoxide (t-BOOH), menadione (Md), paraquat (Pq) or methylglyoxal (MG), at the indicated concentrations, and incubated at 37°C for 4 days.B. Conidia from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were inoculated as in (A) on plates containing 3 or 4 mM H2O2.C. Mycelial plugs cut from the growing edge of 5-day colonies from strains CLK43 (WT), TOL1 (ΔsakA), TFLΔatfA-02 (ΔatfA), TRN1 (ΔcatA) and TLK12 (ΔcatB) were transferred to plates containing the indicated compounds and incubated at 37°C during 4 days.D. Conidia (1 × 104) and mycelial plugs from strains 11035 (WT), TFLΔsakA-03 (ΔsakA), TFLΔatfA-04 (ΔatfA) and TFL4 (ΔsakA ΔatfA) were inoculated on plates containing the indicated concentrations of H2O2 and t-butylhydroperoxide (t-BOOH) and incubated for 4 days at 37°C.See Table 2 for full strain genotypes.
Mentions: To evaluate atfA functions and possible connections to the SakA MAPK pathway, we first generated strains carrying complete deletions in either gene, as confirmed by Southern blot analysis (Figs S2 and S3A and B). ΔatfA and ΔsakA mutants were indistinguishable from the wild-type strain under high-temperature (42°C) or high-osmolarity (1 M NaCl or 1.2 M sorbitol) stress conditions (not shown). To test the mutant response to different types of oxidative stress, we incubated ΔatfA and ΔsakA strains in the presence of the redox-cycling compounds menadione and paraquat, the glutathione-depleting compound methylglyoxal and inorganic (H2O2) as well as organic (t-butylhydroperoxide; t-BOOH) peroxides. As shown in Fig. 1A, wild-type, ΔatfA and ΔsakA strains were similarly resistant to menadione and paraquat. In contrast, ΔatfA and ΔsakA mutants were hypersensitive to both t-butylhydroperoxide (Fig. 1A) and hydrogen peroxide (Fig. 1B), showing a slight sensitivity to methylglyoxal (Fig. 1A). Notably, ΔatfA and ΔsakA mutants were as sensitive to H2O2 as the ΔcatA mutant, which lacks the spore-specific catalase CatA (Navarro et al., 1996; Navarro and Aguirre, 1998). On the contrary, a mutant lacking the mycelial inducible catalase CatB (Kawasaki et al., 1997) showed a H2O2 resistance only slightly lower than the wild type (Fig. 1B). This and the fact that conidia from sakA mutants show decreased CatA activity (Kawasaki et al., 2002) suggested that under these conditions, mutant sensitivity to H2O2 could reflect low CatA activity in conidia. To explore this, we carried out similar oxidative stress plate assays but using mycelial plugs instead of conidia. As shown in Fig. 1C, mycelia from ΔatfA and ΔsakA mutants was resistant up to 6 mM H2O2 but resulted hypersensitive to t-BOOH. Notably, under these conditions, mycelia from ΔcatA, ΔcatB and wild-type strains showed similar resistance to H2O2 and t-BOOH. Compared with the wild-type, ΔatfA, ΔsakA and ΔcatB mutants were somewhat more sensitive to menadione. While all strains presented similar growth in paraquat, a brownish pigmentation and decreased conidiation was observed in ΔatfA and ΔsakA mutants (Fig. 1C). Results published during the course of this work show that conidia from ΔatfA (ΔsakA was not analysed) mutants were sensitive to 50 mM H2O2 (t-BOOH was not tested), while ΔatfA mycelia was resistant to 1.2 mM t-BOOH (Hagiwara et al., 2008). In agreement with our results, a more recent report shows that ΔatfA mycelium is indeed sensitive to t-BOOH (Balazs et al., 2010).

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