The Aspergillus nidulans MAPK Module AnSte11-Ste50-Ste7-Fus3 Controls Development and Secondary Metabolism

Bayram, Özgür; Ahmed, Yasar Luqman; Maruyama, Jun‐ichi; Valerius, Oliver; Rizzoli, Silvio O.; Ficner, Ralf; Irniger, Stefan; Braus, Gerhard H.

doi:10.1371/journal.pgen.1002816

Cited by 148 publications

(193 citation statements)

References 52 publications

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“…The 5,728-bp fusion PCR product obtained with primers 5NestSakA-GFP and 3=Nest-sakA was used to transform strain 1155. To label nuclei, transformants containing the SrkA::GFP construct were retransformed with the H2A::mRFP (monomeric red fluorescent protein) fragment bio-left-ORF-pyroA-gpdA-h2A-mrfpbio-right-ORF, derived from plasmid pOB340 (40). The SrkA::GFP/ ⌬sakA strain CRJ3, obtained as progeny from a cross between strains TRJ1 and TOL1, was transformed with the fragment ph2A::h2A::mrfpptrA, obtained from plasmid pON307 (40).…”

Section: Methodsmentioning

confidence: 99%

“…After this, the culture was treated with 10 mM H 2 O 2 for 10 min or left untreated. Protein purification was performed as reported previously (40). Briefly, samples were frozen with liquid nitrogen, ground, and resuspended in 5 ml of protein extraction buffer (50 mM Tris [pH 7.5], 100 mM KCl, 10 mM MgCl 2 , 0.1% NP-40, 10% glycerol, 20 mM b-glycerophosphate, 2 mM Na 3 VO 4 , 5 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM benzamidine, 1 mM EGTA, 1 mM dithiothreitol [DTT], and 2ϫ protease inhibitors [Roche]).…”

Section: S-tag Protein Purification and Identification By Lc-ms/msmentioning

confidence: 99%

“…Mycelia from strains 1155 (WT) and TRJ2 (SrkA::S-tag) were treated with 10 mM H 2 O 2 for the indicated times or left untreated, immediately frozen with liquid nitrogen, and processed for S-tag protein purification as described previously (40). Samples (30 g) were processed for immunoblotting using anti-phospho-P38 (␣P-SakA), anti-Hog1 (␣ Total SakA), and anti-S-tag (␣ S-tag) antibodies.…”

Section: Figmentioning

confidence: 99%

“…(A) SDS-polyacrylamide gel stained with silver reagent, using S-tag purified protein extracts from WT, SakA::S-tag, and SrkA::S-tag protein extracts. Mycelia from strains 1155 (WT), TFL22 (SakA::S-tag), and TRJ2 (SrkA::S-tag) were treated with 10 mM H 2 O 2 for 10 min or left untreated, immediately frozen with liquid nitrogen, and processed for S-tag protein purification as described previously (40). (B) Peptides identified by mass spectrometry using samples from lanes excised from the SDS gel shown in panel A. motic, oxidative, or cell wall stress and that SrkA does not mediate the sensitivity to the fungicide fludioxonil.…”

Section: Figmentioning

confidence: 99%

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The SrkA Kinase Is Part of the SakA Mitogen-Activated Protein Kinase Interactome and Regulates Stress Responses and Development in Aspergillus nidulans

et al. 2015

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cFungi and many other eukaryotes use specialized mitogen-activated protein kinases (MAPK) of the Hog1/p38 family to transduce environmental stress signals. In Aspergillus nidulans, the MAPK SakA and the transcription factor AtfA are components of a central multiple stress-signaling pathway that also regulates development. Here we characterize SrkA, a putative MAPK-activated protein kinase, as a novel component of this pathway. ⌬srkA and ⌬sakA mutants share a derepressed sexual development phenotype. However, ⌬srkA mutants are not sensitive to oxidative stress, and in fact, srkA inactivation partially suppresses the sensitivity of ⌬sakA mutant conidia to H 2 O 2 , tert-butyl-hydroperoxide (t-BOOH), and menadione. In the absence of stress, SrkA shows physical interaction with nonphosphorylated SakA in the cytosol. We show that H 2 O 2 induces a drastic change in mitochondrial morphology consistent with a fission process and the relocalization of SrkA to nuclei and mitochondria, depending on the presence of SakA. SakA-SrkA nuclear interaction is also observed during normal asexual development in dormant spores. Using SakA and SrkA S-tag pulldown and purification studies coupled to mass spectrometry, we found that SakA interacts with SrkA, the stress MAPK MpkC, the PPT1-type phosphatase AN6892, and other proteins involved in cell cycle regulation, DNA damage response, mRNA stability and protein synthesis, mitochondrial function, and other stress-related responses. We propose that oxidative stress induces DNA damage and mitochondrial fission and that SakA and SrkA mediate cell cycle arrest and regulate mitochondrial function during stress. Our results provide new insights into the mechanisms by which SakA and SrkA regulate the remodelling of cell physiology during oxidative stress and development. W e have proposed that when life was confronted with oxidative stress, cells evolved mechanisms not only to defend against reactive oxygen species (ROS) but also to use this ancestral form of stress to regulate their own growth and differentiation (1, 2). Indeed, the regulated production of ROS by enzymes of the NADPH oxidase family (NOX) is essential for sexual differentiation in Aspergillus nidulans (3) and Neurospora crassa and for polar growth and cell fusion in N. crassa (4), and NOX enzymes play multiple signaling functions in other fungal (5-10), animal, and plant species (11). However, little is known about ROS perception and the mechanisms by which ROS exert their signaling functions.The use of phosphorelay systems to perceive oxidative stress and other types of environmental stress is conserved in bacteria, plants (12), and fungi (13). The fission yeast Schizosaccharomyces pombe uses a multistep phosphorelay composed of the Mak2/3 sensor histidine kinases, Mpr1 HPt protein, and Mcs4 response regulator to transmit H 2 O 2 stress signals to the Spc1 (also known as StyI) mitogen-activated protein kinase (MAPK) cascade (14,15). Spc1 is homologous to Saccharomyces cerevisiae Hog1 and mammalian p38 MAPKs, which are also...

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Section: Methodsmentioning

confidence: 99%

Section: S-tag Protein Purification and Identification By Lc-ms/msmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

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The SrkA Kinase Is Part of the SakA Mitogen-Activated Protein Kinase Interactome and Regulates Stress Responses and Development in Aspergillus nidulans

et al. 2015

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“…This conserved pathway also plays a role in sexual development and secondary metabolism and is required for the virulence of both plant and animal fungal pathogens (Roman et al 2007;Rispail and Di Pietro 2010;Bayram et al 2012). In S. cerevisiae, reception of a matingtype-specific pheromone signal results in the activation of Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK) (Bardwell 2005).…”

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confidence: 99%

Early Colony Establishment in Neurospora crassa Requires a MAP Kinase Regulatory Network

Leeder¹,

Jonkers²,

Li³

et al. 2013

Genetics

129

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Vegetative fusion is essential for the development of an interconnected colony in many filamentous fungi. In the ascomycete fungus Neurospora crassa, vegetative fusion occurs between germinated conidia (germlings) via specialized structures termed "conidial anastomosis tubes" (CATs) and between hyphae within a mature colony. In N. crassa, both CAT and hyphal fusion are under the regulation of a conserved MAP kinase cascade (NRC1, MEK2, and MAK2). Here we show that the predicted downstream target of the MAK2 kinase pathway, a Ste12-like transcription factor known as PP1, regulates elements required for CAT and hyphal fusion. The PP1 regulatory network was revealed by expression profiling of wild type and the Dpp-1 mutant during conidial germination and colony establishment. To identify targets required for cell fusion more specifically, expression-profiling differences were assessed via inhibition of MAK2 kinase activity during chemotropic interactions and cell fusion. These approaches led to the identification of new targets of the cell fusion pathway that, when mutated, showed alterations in chemotropic signaling and cell fusion. In particular, conidial germlings carrying a deletion of NCU04732 (Dham-11) failed to show chemotropic interactions and cell fusion. However, signaling (as shown by oscillation of MAK2 and SO to CAT tips), chemotropism, and cell fusion were restored in Dham-11 germlings when matched with wild-type partner germlings. These data reveal novel insights into the complex process of self-signaling, germling fusion, and colony establishment in filamentous fungi. CELL fusion between genetically identical cells is important in development (for example, myoblast fusion during muscle formation) and occurs in many multicellular organisms from simple ascomycete fungi to mammals (Chen et al. 2007;Aguilar et al. 2013). Cell fusion between genetically identical cells can be mediated by cells that have differentiated, but in some cases, also between cells in an identical developmental state, for example, cell fusion between germinating asexual spores (conidia) of filamentous fungi (Pandey et al. 2004;Roca et al. 2005a;Read et al. 2010). In filamentous fungi, these fusions are integral to the formation of an interconnected hyphal network, which mediates genetic mixing and the sharing of resources (Simonin et al. 2012;Roper et al. 2013). How this process is initiated and maintained and what proteins are involved are still mostly unknown.In filamentous ascomycete fungi, a conserved MAP kinase pathway that is involved in pheromone response and mating in Saccharomyces cerevisiae (Ste11, Ste7, and Fus3) (Bardwell 2005) is required for cell fusion and heterokaryon formation during vegetative growth (Hou et al. 2002;Wei et al. 2003;Pandey et al. 2004;Fu et al. 2011;Jun et al. 2011;Dettmann et al. 2012). This conserved pathway also plays a role in sexual development and secondary metabolism and is required for the virulence of both plant and animal fungal pathogens (Roman et al. 2007;Rispail and Di Piet...

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Advances in Synthetic Biology of Fungi and Contributions to the Discovery of New Molecules

Valiante

2023

ChemBioChem

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The sequencing of fungal genomes is becoming increasingly accessible, with a wealth of data already available. In parallel, the prediction of the putative biosynthetic pathways responsible for the synthesis of potential new natural products is also increasing. The difficulty of translating computational analyses into available compounds is becoming evident, slowing down a process that was thought to be faster with the advent of the genomic era. Advances in gene techniques made it possible to genetically modify a wider range of organisms, including fungi typically considered recalcitrant to DNA manipulation. However, the possibility of screening many gene cluster products for new activities in a high‐throughput manner remains unfeasible. Nonetheless, some updates on the synthetic biology of fungi could provide interesting insights that could help to achieve this goal in the future.

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The Aspergillus nidulans MAPK Module AnSte11-Ste50-Ste7-Fus3 Controls Development and Secondary Metabolism

Cited by 148 publications

References 52 publications

The SrkA Kinase Is Part of the SakA Mitogen-Activated Protein Kinase Interactome and Regulates Stress Responses and Development in Aspergillus nidulans

The SrkA Kinase Is Part of the SakA Mitogen-Activated Protein Kinase Interactome and Regulates Stress Responses and Development in Aspergillus nidulans

Early Colony Establishment in Neurospora crassa Requires a MAP Kinase Regulatory Network

Advances in Synthetic Biology of Fungi and Contributions to the Discovery of New Molecules

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