CDF family of zinc transporters ZRC-1, MSC-2, and ZRG-17 are involved in survival at high zinc conditions, vegetative development, and cellulase utilization in .

The cation diffusion facilitator (CDF) family of zinc transporters plays a crucial role in zinc homeostasis in eukaryotes, including fungi. Here, we investigated the cell functions and genetic interactions of CDF zinc transporters -1 and -2 in . The Δ-1 mutant could not grow in a high-zinc environment, indicating that the zinc transporter protein ZRC-1 was essential for growth in high-zinc conditions.

Calmodulin, Calcium/Calmodulin-Dependent Kinases-1 and 2 Regulate Expression of the Heat Shock Proteins for Heat Shock Tolerance and Pheromone Signaling Genes for Sexual Development in .

Unlabelled: Calmodulin (CaM) is a primary Ca sensor that binds and activates numerous target proteins and regulates several cellular processes in eukaryotes. CaM is essential in ; therefore, we generated a CaM mutant using repeat-induced point (RIP) mutation and investigated the mutant phenotypes. We also studied knockout mutants of four Ca/CaM kinases (, , , and ) for their role during stress conditions and sexual development.

The cell functions of phospholipase C-1, Ca/H exchanger-1, and secretory phospholipase A in tolerance to stress conditions and cellulose degradation in Neurospora crassa.

We investigated the cell functions of the Ca signaling genes phospholipase C-1 (plc-1), Ca/H exchanger (cpe-1), and secretory phospholipase A (splA) for stress responses and cellulose utilization in Neurospora crassa. The Δplc-1, Δcpe-1, and ΔsplA mutants displayed increased sensitivity to the alkaline pH and reduced survival during induced thermotolerance. The ΔsplA mutant also exhibited hypersensitivity to the DTT-induced endoplasmic reticulum (ER) stress, increased microcrystalline cellulose utilization, increased protein secretion, and glucose accumulation in the culture supernatants.

Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.

CRZ1 transcription factor is involved in cell survival, stress tolerance, and virulence in fungi.

The calcineurin-CRZ1 signaling pathway is conserved from yeasts to humans, and is involved in survival, tolerance to biotic and abiotic stress conditions, virulence, and drug resistance. The primary target of calcineurin is Crz1p in yeasts and nuclear factor of activated T-cells (NFAT) in mammals. Calcineurin regulates the expression of various genes involved in cell wall regeneration, ionic homeostasis, virulence, and other cellular processes.

Heat shock proteins and the calcineurin-crz1 signaling regulate stress responses in fungi.

The heat shock proteins (Hsps) act as a molecular chaperone to stabilize client proteins involved in various cell functions in fungi. Hsps are classified into different families such as HSP90, HSP70, HSP60, HSP40, and small HSPs (sHsps). Hsp90, a well-studied member of the Hsp family proteins, plays a role in growth, cell survival, and pathogenicity in fungi.

Calcium signaling is involved in diverse cellular processes in fungi.

Calcium (Ca) is a universal signalling molecule of life. The Ca signalling is an evolutionarily conserved process from prokaryotes to eukaryotes. Ca at high concentration is deleterious to the cell; therefore, cell maintains a low resting level of intracellular free Ca concentration ([Ca]).

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.

We studied a dominant mutant of the Neurospora crassa calcineurin catalytic (cna-1) subunit generated using the repeat-induced point mutation (RIP). The Cna-1 mutants showed defects in morphology, aerial hyphae development, carotenoid accumulation, and fertility. The Cna-1 mutants also showed sensitivity to osmotic stress and a reduction in acquisition of thermotolerance on exposure to lethal heat shock temperature.

Calcineurin responsive zinc-finger-1 binds to a unique promoter sequence to upregulate neuronal calcium sensor-1, whose interaction with MID-1 increases tolerance to calcium stress in Neurospora crassa.

We studied the molecular mechanism of neuronal calcium sensor-1 (NCS-1) signaling pathway for tolerance to Ca stress in Neurospora crassa. Increasing concentration of Ca increased the expression of ncs-1; however, the calcineurin inhibitor FK506 severely reduced ncs-1 mRNA transcript levels. Chromatin immunoprecipitation (ChIP) studies revealed that the transcription factor calcineurin responsive zinc finger-1 (CRZ-1) binds to the ncs-1 promoter, and CRZ-1 binding upregulated ncs-1 expression under high Ca concentrations.

Phospholipases play multiple cellular roles including growth, stress tolerance, sexual development, and virulence in fungi.

Phospholipases are ubiquitous enzymes that hydrolyze phospholipids. Based on the cleavage site of the ester linkage in the substrate phospholipids, phospholipases are classified into four major types, phospholipase A (PLA), phospholipase B (PLB), phospholipase C (PLC), and phospholipase D (PLD), which are further classified into various subtypes. Phospholipases hydrolyze phospholipids into various signaling products including phosphatidic acid (PA), diacylglycerol (DAG), free fatty acids (FFAs), and lyso-phospholipids (LPLs).

The NcZrg-17 gene of Neurospora crassa encodes a cation diffusion facilitator transporter required for vegetative development, tolerance to endoplasmic reticulum stress and cellulose degradation under low zinc conditions.

The Neurospora crassa gene NcZrg-17 encodes a membrane protein with homology to the cation diffusion facilitator (CDF) family of transporters. We analyzed the phenotypic and functional characteristics of ΔNcZrg-17 and the implications of these characteristics in vivo. The ΔNcZrg-17 mutant showed several phenotypes that are zinc suppressible such as reduced growth rate, short aerial hyphae, increased hyphal branching, early and enhanced conidiation and delayed conidial germination.

The pleiotropic vegetative and sexual development phenotypes of Neurospora crassa arise from double mutants of the calcium signaling genes plc-1, splA2, and cpe-1.

We investigated phenotypes of the double mutants of the calcium (Ca) signaling genes plc-1, splA2, and cpe-1 encoding for a phospholipase C1 (PLC-1), a secretory phospholipase A (sPLA), and a Ca/H exchanger (CPE-1), respectively, to understand the cell functions regulated by their genetic interactions. Mutants lacking plc-1 and either splA2 or cpe-1 exhibited numerous defects including reduced colonial growth, stunted aerial hyphae, premature conidiation on plates with delayed germination, inappropriate conidiation in submerged culture, and lesser mycelial pigmentation. Moreover, the ∆plc-1; ∆splA2 and ∆plc-1; ∆cpe-1 double mutants were female-sterile when crossed with wild type as the male parent.

The calmodulin gene in Neurospora crassa is required for normal vegetative growth, ultraviolet survival, and sexual development.

We isolated a Neurospora crassa mutant of the calmodulin (cmd) gene using repeat-induced point mutation and studied its phenotypes. The cmd mutant showed a defect in growth, reduced aerial hyphae, decreased carotenoid accumulation, a severe reduction in viability upon ultraviolet (UV) irradiation, and a fertility defect. Moreover, meiotic silencing of the cmd gene resulted in a barren phenotype.

Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N.

Calcineurin Subunits A and B Interact to Regulate Growth and Asexual and Sexual Development in Neurospora crassa.

Calcineurin is a calcium/calmodulin dependent protein phosphatase in eukaryotes that consists of a catalytic subunit A and a regulatory subunit B. Previous studies in the filamentous fungus Neurospora crassa had suggested that the catalytic subunit of calcineurin might be an essential protein. We generated N.

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance.

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N.

Calcium/calmodulin-dependent kinases are involved in growth, thermotolerance, oxidative stress survival, and fertility in Neurospora crassa.

Calcium/calmodulin-dependent kinases (Ca(2+)/CaMKs) are Ser/Thr protein kinases that respond to change in cytosolic free Ca(2+) ([Ca(2+)]c) and play multiple cellular roles in organisms ranging from fungi to humans. In the filamentous fungus Neurospora crassa, four Ca(2+)/CaM-dependent kinases, Ca(2+)/CaMK-1 to 4, are encoded by the genes NCU09123, NCU02283, NCU06177, and NCU09212, respectively. We found that camk-1 and camk-2 are essential for full fertility in N.

Neurospora crassa homologue of Neuronal Calcium Sensor-1 has a role in growth, calcium stress tolerance, and ultraviolet survival.

NCU04379 gene encodes a conserved Ca(2+) and/or calmodulin binding protein that possesses a consensus signal for N-terminal myristoylation and four EF-hands, characteristics of Neuronal Calcium Sensor-1proteins. The NCU04379.2 knockout mutant shows slow growth rate, increased sensitivity to calcium and ultraviolet (UV) irradiation, and a wild-type fragment carrying NCU04379 gene complements the mutant.

Cellular roles of neuronal calcium sensor-1 and calcium/calmodulin-dependent kinases in fungi.

The neuronal calcium sensor-1 (NCS-1) possesses a consensus signal for N-terminal myristoylation and four EF-hand Ca(2+)-binding sites, and mediates the effects of cytosolic Ca(2+). Minute changes in free intracellular Ca(2+) are quickly transformed into changes in the activity of several kinases including calcium/calmodulin-dependent protein kinases (Ca(2+)/CaMKs) that are involved in regulating many eukaryotic cell functions. However, our current knowledge of NCS-1 and Ca(2+)/CaMKs comes mostly from studies of the mammalian enzymes.

Dominant suppression of repeat-induced point mutation in Neurospora crassa by a variant catalytic subunit of DNA polymerase zeta.

Crosses involving the Adiopodoumé strain of Neurospora crassa are defective for repeat-induced point mutation (RIP), a genome defense mechanism of fungi. We show here that the Adiopodoumé strain possesses an incompletely penetrant and variably expressive dominant suppressor of RIP (Srp) that maps to an approximately 34-kbp genome segment that is approximately 26 kbp proximal to mat on linkage group IL. Gene disruption experiments revealed that Srp is the upr-1 allele of Adiopodoumé (upr-1(Ad)) that is contained within this segment.

Translesion DNA polymerases Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa.

Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases.

Genetic transformation of Neurospora tetrasperma, demonstration of repeat-induced point mutation (RIP) in self-crosses and a screen for recessive RIP-defective mutants.

The pseudohomothallic fungus Neurospora tetrasperma is naturally resistant to the antibiotic hygromycin. We discovered that mutation of its erg-3 (sterol C-14 reductase) gene confers a hygromycin-sensitive phenotype that can be used to select transformants on hygromycin medium by complementation with the N. crassa erg-3+ and bacterial hph genes.