Dominant mutants of the calcineurin catalytic subunit (CNA-1) showed developmental defects, increased sensitivity to stress conditions, and CNA-1 interacts with CaM and CRZ-1 in Neurospora crassa

2019 ◽  
Vol 202 (4) ◽  
pp. 921-934 ◽  
Author(s):  
Ajeet Kumar ◽  
Avishek Roy ◽  
Mandar V. Deshmukh ◽  
Ranjan Tamuli
Keyword(s):  
Neurospora Crassa ◽  
Catalytic Subunit ◽  
Stress Conditions ◽  
Developmental Defects ◽  
Increased Sensitivity ◽  
Dominant Mutants

The CRZ-1 Transcription Factor Regulates Hsp 80 Involves in the Acquisition of Thermotolerance, and NCA-2 for Calcium Stress Tolerance in Neurospora Crassa

2021 ◽  
Author(s):  
Avishek Roy ◽  
Ranjan Tamuli
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Neurospora Crassa ◽  
Stress Condition ◽  
Stress Conditions ◽  
Start Codon ◽  
Expression Levels ◽  
Heat Shock Stress ◽  
Shock Proteins ◽  
Shock Stress

Abstract Heat shock proteins (Hsps) are molecular chaperones and required for survival of organisms under heat stress conditions. In this study, we studied Hsp80, a member of the Hsp90 family, in Neurospora crassa. The expression of hsp80 was severely reduced in the N. crassa calcineurin B subunit RIP-mutant (cnb-1RIP) strains under the heat shock conditions. Furthermore, the expression levels of cnb-1, hsp60, hsp80, and the calcineurin-regulated transcription factor crz-1 were increased, but expression levels were reduced in the presence of the calcineurin inhibitor FK506 under the heat shock stress in the N. crassa wild type. Therefore, the calcineurin-crz-1 signaling pathway transcriptionally regulates hsp60 and hsp80 under the heat shock stress condition in N. crassa. In addition, the transcript levels of trm-9 and nca-2, a Ca2+ sensor and a Ca2+ ATPase, respectively, were increased under the heat shock stress condition. Moreover, the expression of the hsp80, but not the hsp60, was reduced in the Δtrm-9, Δnca-2, and the Δtrm-9 Δnca-2 double mutants. These results suggested that hsp80, trm-9, and nca-2 play a role in coping the heat shock stress in N. crassa. We found that CRZ-1 binds to 5ʹ-CCTTCACA-3ʹ and 5ʹ-AGCGGAGC-3ʹ 8 bp nucleotide sequences, located about 1075 bp and 679 bp upstream of the ATG start codon, respectively, of hsp80. We also found that CRZ-1 binds to an 8 bp nucleotide sequence 5ʹ-ACCGCGCC-3ʹ, located 234 bp upstream of the ATG start codon of nca-2 under Ca2+ stress condition. Thus, cnb-1, hsp60, hsp80, and crz-1 are involved in the heat shock stress response in N. crassa. Moreover, CRZ-1 upregulates the expressions of hsp80 and nca-2 under the heat shock stress and Ca2+ stress conditions, respectively, in N. crassa.

scholarly journals PhoY2 of Mycobacteria Is Required for Metabolic Homeostasis and Stress Response

2012 ◽  
Vol 195 (2) ◽  
pp. 243-252 ◽  
Author(s):  
Chuan Wang ◽  
Yi Mao ◽  
Jia Yu ◽  
Lin Zhu ◽  
Ming Li ◽  
...  
Keyword(s):  
Inorganic Phosphate ◽  
Stress Conditions ◽  
Nutrient Deprivation ◽  
Pho Regulon ◽  
Metabolic Homeostasis ◽  
Content Type ◽  
Redox States ◽  
Nadh Ratio ◽  
Increased Sensitivity

ABSTRACTThe ability of pathogenic mycobacteria to adapt to diverse environments is essential for their success as pathogens. Here we describe a transposon-inactivatedphoY2mutant ofMycobacterium marinum. PhoY2 of mycobacteria is a functional homologue of PhoU inEscherichia coliand an important component of the Pho regulon. We found that PhoY2 is required for maintaining intracellular inorganic phosphate (Pi) homeostasis and balanced energy and redox states. Disruption ofphoY2resulted in elevated levels of intracellular poly-Piand ATP and an elevated NAD+/NADH ratio, and the mutant strain exhibited increased sensitivity to environmental stress conditions, including nutrient deprivation as well as SDS and antibiotic treatments. Taken together, our results suggest that PhoY2 is required for maintaining metabolic homeostasis and adaptation to stress conditions, which may provide an explanation for the suggested role of PhoY2 in drug tolerance.

scholarly journals Asexual Development Is Increased in Neurospora crassa cat-3-Null Mutant Strains

2003 ◽  
Vol 2 (4) ◽  
pp. 798-808 ◽  
Author(s):  
Shaday Michán ◽  
Fernando Lledías ◽  
Wilhelm Hansberg
Keyword(s):  
Oxidative Stress ◽  
Neurospora Crassa ◽  
Mutant Strain ◽  
Protein Oxidation ◽  
Stress Conditions ◽  
Asexual Development ◽  
Air Exposure ◽  
Aerial Hyphae ◽  
Mutant Strains ◽  
Carotene Synthesis

ABSTRACT We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3RIP colonies were sensitive to H2O2, indicating that wild-type (Wt) resistance to external H2O2 was due to CAT-3. cat-3RIP colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3RIP mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3RIP mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

scholarly journals The monothiol glutaredoxin Grx4 influences thermotolerance, cell wall integrity, and Mpk1 signaling in Cryptococcus neoformans

2021 ◽  
Author(s):  
Guanggan Hu ◽  
Linda Horianopoulos ◽  
Eddy Sánchez-León ◽  
Mélissa Caza ◽  
Wonhee Jung ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Iron Homeostasis ◽  
Elevated Temperatures ◽  
Stress Conditions ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Calcofluor White ◽  
Mitogen Activated Protein ◽  
Increased Sensitivity

Abstract Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fungal pathogen causes cryptococcal meningoencephalitis in immunocompromised individuals worldwide. Here, we demonstrate that Grx4 is required for proliferation at elevated temperatures (both 37°C and 39°C) and under stress conditions. In particular, the grx4Δ mutant was hypersensitive to SDS, calcofluor white (CFW), and caffeine, suggesting that Grx4 is required for membrane and cell wall integrity (CWI). In this context, we found that Grx4 regulated the phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) of the CWI pathway in cells grown at elevated temperature or upon treatment with CFW, caffeine, or SDS. The grx4Δ mutant also displayed increased sensitivity to FK506 and cyclosporin A, two inhibitors of the calcineurin pathway, indicating that Grx4 may influence growth at higher temperatures in parallel with calcineurin signaling. Upon thermal stress or calcium treatment, loss of Grx4 also caused partial mis-localization of Crz1, the transcription factor that is a calcineurin substrate. The phenotypes of the grx4Δ, crz1Δ, and cna1Δ (calcineurin) mutants suggest shared contributions to the regulation of temperature, cell wall, and other stresses. In summary, we show that Grx4 is also a key regulator of the responses to a variety of stress conditions in addition to its roles in iron homeostasis in C. neoformans.

scholarly journals The Heterotrimeric G-Protein Subunits GNG-1 and GNB-1 Form a Gβγ Dimer Required for Normal Female Fertility, Asexual Development, and Gα Protein Levels in Neurospora crassa

2005 ◽  
Vol 4 (2) ◽  
pp. 365-378 ◽  
Author(s):  
Svetlana Krystofova ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Magnaporthe Grisea ◽  
Cryphonectria Parasitica ◽  
Normal Female ◽  
Submerged Cultures ◽  
Heterotrimeric G Protein ◽  
Developmental Defects ◽  
Protein Levels

ABSTRACT We have identified a gene encoding a heterotrimeric G protein γ subunit, gng-1, from the filamentous fungus Neurospora crassa. gng-1 possesses a gene structure similar to that of mammalian Gγ genes, consisting of three exons and two introns, with introns present in both the open reading frame and 5′-untranslated region. The GNG-1 amino acid sequence displays high identity to predicted Gγ subunits from other filamentous fungi, including Giberella zeae, Cryphonectria parasitica, Trichoderma harzianum, and Magnaporthe grisea. Deletion of gng-1 leads to developmental defects similar to those previously characterized for Δgnb-1 (Gβ) mutants. Δgng-1, Δgnb-1, and Δgng-1 Δgnb-1 strains conidiate inappropriately in submerged cultures and are female sterile, producing aberrant female reproductive structures. Similar to previous results obtained with Δgnb-1 mutants, loss of gng-1 negatively influences levels of Gα proteins (GNA-1, GNA-2, and GNA-3) in plasma membrane fractions isolated from various tissues of N. crassa and leads to a significant reduction in the amount of intracellular cyclic AMP. In addition, we show that GNB-1 is essential for maintenance of normal steady-state levels of GNG-1, suggesting a functional interaction between GNB-1 and GNG-1. Direct evidence for a physical association between GNB-1 and GNG-1 in vivo was provided by coimmunoprecipitation.

scholarly journals THE ISOLATION OF MMS- AND HISTIDINE-SENSITIVE MUTANTS IN NEUROSPORA CRASSA

Genetics ◽  
1981 ◽  
Vol 97 (2) ◽  
pp. 247-259
Author(s):  
A M Delange ◽  
N C Mishra
Keyword(s):  
Neurospora Crassa ◽  
Methyl Methanesulfonate ◽  
Wild Type ◽  
Sensitive Mutant ◽  
Mendelian Segregation ◽  
Simple Method ◽  
Replica Plating ◽  
Increased Sensitivity ◽  
Two Temperatures ◽  
Mutant Phenotypes

ABSTRACT A simple method of replica plating has been used to isolate mutants of Neurospora crassa that have increased sensitivity to methyl methanesulfonate (MMS) and/or to histidine. Twelve mutants with increased sensitivity to MMS and one mutant with increased sensitivity to histidine showed Mendelian segregation of the mutant phenotypes. Three mutants were mapped to loci not previously associated with MMS sensitivity. Two others were allelic to the UV- and MMS-sensitive mutant, mei-3. Survival curves indicate that conidia (mutant or wild-type) survive on much higher concentrations of MMS at 25° than at 37°. In contrast, mycelial growth is more resistant to MMS at 37°. The possibility of qualitatively different repair processes at these two temperatures is discussed.

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