Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants.

The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11. The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway. The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis. Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A. thaliana. Yeast cells expressing the A. thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells. Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival. Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival. Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway. In A. thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress. This is the same time frame for osmoticum-induced effects on the electrical properties of A. thaliana cells, both an immediate response and adaptation. Therefore, we propose that the A. thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.     

Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen‐activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the high‐affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen‐activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1–MoMkk1–MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)‐induced UPR pathway in M. oryzae.     

CWI和HOG信号通路基因在斑玉蕈不同发育阶段的表达模式

为了更清楚地了解MAPK信号通路中的细胞壁完整性信号通路（cell wall integrity,CWI）和高渗透压甘油（high-osmolarity glycerol pathway,HOG）信号通路对斑玉蕈菌丝成熟、原基形成和子实体发育过程的影响及调节作用,对MAPK信号通路中的CWI和HOG信号通路基因在斑玉蕈不同菌丝培养时间（40、60、80和100d）和不同生长发育关键时期（24h、菌丝恢复期、菌丝转色期、原基期和子实体期）的表达模式进行分析,以期揭示这两条信号通路基因参与调节斑玉蕈菌丝的生长、子实体的形成和发育的作用。在斑玉蕈的CWI和HOG信号通路中经分析鉴定一共获得了15个关键基因。CWI信号通路基因表达分析表明：在菌丝培养的40-100d的过程中,大部分CWI信号通路基因在第60天时表达量最高,其中rho1、ssk1、ssk2和ste20的基因表达量上调了2-5倍,在第80-100天时出现持续下降。在HOG信号通路中的大部分基因也在菌丝培养的第60天表达量达到最高。其中sho1、ste20、ssk1和ssk2基因的表达量上调最为显著,而hog1基因的表达量在菌丝培养的第40-100天呈持续下降。子实体形成过程中两条通路的大部分基因在原基形成时期表达量最高,而在子实体时期表达量下调。其中HOG信号通路中的ssk2基因表达量上调最为显著。以上结果说明在菌丝生长过程中第60天时菌丝细胞生长增殖最为旺盛,而在第80天开始菌丝细胞基本开始停止生长,菌丝也逐渐达到成熟。同时在菌丝增殖生长过程中,斑玉蕈持续地上调CWI信号通路基因的表达来调控菌丝细胞壁的完整性,从而控制菌丝细胞壁的形成。其中bck1、mkk1和slt2基因可能对斑玉蕈菌丝细胞的分裂增殖和细胞壁的形成以及诱导子实体形成起到关键作用。     

Mitogen activated protein kinases SakAHOG1 and MpkC collaborate for Aspergillus fumigatus virulence

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen‐activated protein kinases of the high‐osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild‐type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.     

Cohesin dysfunction results in cell wall defects in budding yeast

Cohesin is a conserved chromatin-binding multisubunit protein complex involved in diverse chromosomal transactions such as sister-chromatid cohesion, chromosome condensation, regulation of gene expression, DNA replication, and repair. While working with a budding yeast temperature-sensitive mutant, mcd1-1, defective in a cohesin subunit, we observed that it was resistant to zymolyase, indicating an altered cell wall organization. The budding yeast cell wall is a strong but elastic structure essential for maintenance of cell shape and protection from extreme environmental challenges. Here, we show that the cohesin complex plays an important role in cell wall maintenance. Cohesin mutants showed high chitin content in the cell wall and sensitivity to multiple cell wall stress-inducing agents. Interestingly, temperature-dependent lethality of cohesin mutants was osmoremedial, in a HOG1-MAPK pathway-dependent manner, suggesting that the temperature sensitivity of these mutants may arise partially from cell wall defects. Moreover, Mpk1 hyper-phosphorylation indicated activation of the cell wall integrity (CWI) signaling pathway in cohesin mutants. Genetic interaction analysis revealed that the CWI pathway is essential for survival of mcd1-1 upon additional cell wall stress. The cell wall defect was independent of the cohesion function and accompanied by misregulation of expression of several genes having cell wall-related functions. Our findings reveal a requirement of cohesin in maintenance of CWI that is independent of the CWI pathway, and that may arise from cohesin’s role in regulating the expression of multiple genes encoding proteins involved in cell wall organization and biosynthesis.     

Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions

We have isolated several Saccharomyces cerevisiae mutants resistant to calcofluor that contain mutations in the PBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.     

Ssk1p-Independent Activation of Ssk2p Plays an Important Role in the Osmotic Stress Response in Saccharomyces cerevisiae: Alternative Activation of Ssk2p in Osmotic Stress

In Saccharomyces cerevisiae, external high osmolarity activates the HOG MAPK pathway, which controls various aspects of osmoregulation. MAPKKK Ssk2 is activated by Ssk1 in the SLN1 branch of the osmoregulatory HOG MAPK pathway under hyperosmotic stress. We observed that Ssk2 can be activated independent of Ssk1 upon osmotic shock by an unidentified mechanism. The domain for the Ssk1p-independent activation was identified to be located between the amino acids 177∼240. This region might be involved in the binding of an unknown regulator to Ssk2 which in turn activates Ssk2p without Ssk1p under hyperosmotic stress. The osmotic stress response through the Ssk1p-independent Ssk2p activation is strong, although its duration is short compared with the Ssk1p-dependent activation. The alternative Ssk2p activation is also important for the salt resistance.     

Low temperature highlights the functional role of the cell wall integrity pathway in the regulation of growth in Saccharomyces cerevisiae

Unlike other stresses, the physiological significance and molecular mechanisms involved in the yeast cold response are largely unknown. In the present study, we show that the CWI (cell wall integrity) pathway plays an important role in the growth of Saccharomyces cerevisiae at low temperatures. Cells lacking the Wsc1p (wall integrity and stress response component 1) membrane sensor or the MAPKs (mitogen-activated protein kinases) Bck1p (bypass of C kinase 1), Mkk (Mapk kinase) 1p/Mkk2p or Slt2p (suppressor of lyt2) exhibited cold sensitivity. However, there was no evidence of either a cold-provoked perturbation of the cell wall or a differential cold expression program mediated by Slt2p. The results of the present study suggest that Slt2p is activated by different inputs in response to nutrient signals and mediates growth control through TORC1 (target of rapamycin 1 complex)-Sch9p (suppressor of cdc25) and PKA (protein kinase A) at low temperatures. We found that absence of TOR1 (target of rapamycin 1) causes cold sensitivity, whereas a ras2Δ mutant shows increased cold growth. Lack of Sch9p alleviates the phenotype of slt2Δ and bck1Δ mutant cells, as well as attenuation of PKA activity by overexpression of BCY1 (bypass of cyclase mutations 1). Interestingly, swi4Δ mutant cells display cold sensitivity, but the phenotype is neither mediated by the Slt2p-regulated induction of Swi4p (switching deficient 4)-responsive promoters nor influenced by osmotic stabilization. Hence, cold signalling through the CWI pathway has distinct features and might mediate still unknown effectors and targets.