Olivia Sánchez

In eukaryotic cells, environmental stress signals are transmitted by evolutionarily conserved MAPKs, such as Hog1 in the budding yeast Saccharomyces cerevisiae, Spc1 in the fission yeast Schizosaccharomyces pombe and p38/JNK in mammalian cells. Here, we report the identification of the Aspergillus nidulans sakA gene, which encodes a member of the stress MAPK family. The sakA gene is able to complement the S. pombe spc1- defects in both osmo-regulation and cell cycle progression. Moreover, SakA MAPK is activated in response to osmotic and oxidative stress in both S. pombe and A. nidulans. However, in contrast to hog1 and spc1 mutants, the sakA null mutant is not sensitive to high osmolarity stress, indicating a different regulation of the osmostress response in this fungus. On the other hand, the DeltasakA mutant shows development and cell-specific phenotypes. First, it displays premature steA-dependent sexual development. Second, DeltasakA mutant produces asexual spores that are highly sensitive to oxidative and heat shock stress and lose viability upon storage. Indeed, SakA is transiently activated early after induction of conidiation. Our results indicate that SakA MAPK is involved in stress signal transduction and repression of sexual development, and is required for spore stress resistance and survival.

Fungi utilize a phosphorelay system coupled to a MAP kinase module for sensing and processing environmental signals. In Aspergillus nidulans, response regulator SskA transmits osmotic and oxidative stress signals to the stress MAPK (SAPK) SakA. Using a genetic approach together with GFP tagging and molecular bifluorescence we show that SakA and ATF/CREB transcription factor AtfA define a general stress-signalling pathway that plays differential roles in oxidative stress responses during growth and development. AtfA is permanently localized in the nucleus, while SakA accumulates in the nucleus in response to oxidative or osmotic stress signals or during normal spore development, where it physically interacts with AtfA. AtfA is required for expression of several genes, the conidial accumulation of SakA and the viability of conidia. Furthermore, SakA is active (phosphorylated) in asexual spores, remaining phosphorylated in dormant conidia and becoming dephosphorylated during germination. SakA phosphorylation in spores depends on certain (SskA) but not other (SrrA and NikA) components of the phosphorelay system. 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.

Among eukaryotes, only slime molds, fungi, and plants contain signal transduction phosphorelay systems. In filamentous fungi, multiple sensor kinases appear to use a single histidine-containing phosphotransfer (HPt) protein to relay signals to two response regulators (RR). In Aspergillus nidulans, the RR SskA mediates activation of the mitogen-activated protein kinase SakA in response to osmotic and oxidative stress, whereas the functions of the RR SrrA were unknown. We used a genetic approach to characterize the srrA gene as a new member of the skn7/prr1 family and to analyze the roles of SrrA in the phosphorelay system composed of the RR SskA, the HPt protein YpdA, and the sensor kinase NikA. While mutants lacking the HPt protein YpdA are unviable, mutants lacking SskA (DeltasskA), SrrA (DeltasrrA), or both RR (DeltasrrA DeltasskA) are viable and differentially affected in osmotic and oxidative stress responses. Both RR are involved in osmostress resistance, but DeltasskA mutants are more sensitive to this stress, and only SrrA is required for H(2)O(2) resistance and H(2)O(2)-mediated induction of catalase CatB. In contrast, both RR are individually required for fungicide sensitivity and calcofluor resistance and for normal sporulation and conidiospore viability. The DeltasrrA and DeltasskA sporulation defects appear to be related to decreased mRNA levels of the key sporulation gene brlA. In contrast, conidiospore viability defects do not correlate with the activity of the spore-specific catalase CatA. Our results support a model in which NikA acts upstream of SrrA and SskA to transmit fungicide signals and to regulate asexual sporulation and conidiospore viability. In contrast, NikA appears dispensable for osmotic and oxidative stress signaling. These results highlight important differences in stress signal transmission among fungi and define a phosphorelay system involved in oxidative and osmotic stress, cell wall maintenance, fungicide sensitivity, asexual reproduction, and spore viability.

We report the transformation of swollen A. nidulans conidia by electroporation. With this method, transformation frequencies were similar to those obtained by using protoplast fusion. The methodology employed is simple, requiring no enzymes nor osmotic stabilizers. The effects of conidial age, DNA topology/concentration and electric field strength are presented

The redox-regulated transcription factors (TFs) of the bZIP AP1 family, such as yeast Yap1 and fission yeast Pap1, are activated by peroxiredoxin proteins (Prxs) to regulate the antioxidant response. Previously, Aspergillus nidulans mutants lacking the Yap1 ortholog NapA have been characterized as sensitive to H 2 O 2 and menadione. Here we study NapA roles in relation to TFs SrrA and AtfA, also involved in oxidant detoxification, showing that these TFs play different roles in oxidative stress resistance, catalase gene regulation and development, during A. nidulans life cycle. We also uncover novel NapA roles in repression of sexual development, normal conidiation, conidial mRNA accumulation, and carbon utilization. The phenotypic characterization of gpxA, tpxA, and tpxB single, double and triple peroxiredoxin mutants in wild type or napA backgrounds shows that none of these Prxs is required for NapA function in H 2 O 2 and menadione resistance. However, these Prxs participate in a minor NapA-independent H 2 O 2 resistance pathway and NapA and TpxA appear to regulate conidiation along the same route. Using transcriptomic analysis we show that during conidial development NapA-dependent gene expression pattern is different from canonical oxidative stress patterns. In the course of conidiation, NapA is required for regulation of at least 214 genes, including ethanol utilization genes alcR, alcA and aldA, and large sets of genes encoding proteins involved in transcriptional regulation, drug detoxification, carbohydrate utilization and secondary metabolism, comprising multiple oxidoreductases, membrane transporters and hydrolases. In agreement with this, napA mutants fail to grow or grow very poorly in ethanol, arabinose or fructose as sole carbon sources. Moreover, we show that NapA nuclear localization is induced not only by oxidative stress but also by growth in ethanol and by carbon starvation. Together with our previous work, these results show that SakA-AtfA, SrrA and NapA oxidative stress-sensing pathways regulate essential aspects of spore physiology (i.e., cell cycle arrest, dormancy, drug production and detoxification, and carbohydrate utilization).