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. crassa strains expressing the A (cna-1) and B (cnb-1) subunit genes under the regulation of P_tcu-1, a copper-responsive promoter. In these strains, addition of bathocuproinedisulfonic acid (BCS), a copper chelator, results in induction of cna-1 and cnb-1, while excess Cu²⁺ represses gene expression. Through analysis of these strains under repressing and inducing conditions, we found that the calcineurin is required for normal growth, asexual development and female fertility in N. crassa. Moreover, we isolated and analyzed cnb-1 mutant alleles generated by repeat-induced point mutation (RIP), with the results further supporting roles for calcineurin in growth and fertility in N. crassa. We demonstrated a direct interaction between the CNA-1 and CNB-1 proteins using an assay system developed to study protein-protein interactions in N. crassa.     

Asexual transmission, non-suppressiveness and meiotic extinction of small plasmid-like derivatives of the mitochondrial DNA in Neurospora crassa

For reasons that are not obvious, sets of related plasmid-like elements that consist of short segments of DNA that overlap the 5' terminal region of the mitochondrial large-subunit rRNA gene sometimes appear spontaneously and become amplified in the mitochondria of some cytochrome-deficient and/or UV-sensitive mutants of Neurospora crassa. These elements are transmitted efficiently through hyphal anastomoses and appear to invade the mitochondria of recipient strains, but they do not cause senescence and at best cause only slight deficiencies in cytochromes a and b even though they are transcribed copiously. Hence, the small elements are not suppressive and, unlike large deletion derivatives of the mitochondrial chromosome, do not displace normal mtDNA molecules in vegetatively propagated mycelia. Unlike the mitochondrial chromosome, large plasmid-like mtDNA derivatives and true mitochondrial plasmids, the small plasmid-like mtDNA derivatives are rarely transmitted sexually even though they persist without selection in very high copy numbers in vegetative cells. The high copy numbers and high stability of these elements in vegetatively propagated cultures suggests that their monomers contain all the features required for their replication and transmission in the hyphae and conidia of Neurospora. However, the mt-rnl-derived molecules appear to lack a sequence or attribute required for the maintenance or transmission of mitochondrial genetic elements at some stage of the sexual reproductive cycle, including ascospore maturation and germination.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Developmental regulation of NAD+ kinase in Neurospora crassa

The specific activity of NAD⁺ kinase (ATP:NAD⁺ 2-phosphotransferase, EC 2.7.1.23) from Neurospora crassa shows sharp peaks when the organism enters a new developmental stage of the asexual life cycle: the peaks are observed during hydration and germination of conidia, at the transition from exponential to stationary growth and at the photostimulated conidiation. As stimulation of NAD⁺ kinase activity by light in conidiating mycelium is not sensitive to translation inhibitors, the activiation of pre-existing molecules, rather than induction of protein synthesis de novo may be supposed. Enzyme electrophoresis revealed the presence of four forms of NAD⁺ kinase having different apparent molecular weights (I=333,000; II=306,000; III=229,000 and IV=203,000). Manifestation of the activity of individual forms of NAD⁺ kinase is developmentally controlled: form III is most abundant during vegetative growth, forms I and II prevail in conidia. At the conidial germination the increase of NAD⁺ kinase activity is associated with the activation of form III, whereas during photostimulation of conidiation form II is the most activated one. Therefore, certain molecular forms of the enzyme may be regarded as biochemical markers for different developmental stages of N. crassa.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.     

Developmental regulation of choline sulfatase and aryl sulfatase in Neurospora crassa

Regulation of the synthesis of several enzymes of sulfur metabolism in Neurospora is a function of both metabolic regulation and the genetic control exerted by the cys-3 and scon regulatory genes. Additional control mechanisms appear to regulate the synthesis of choline sulfatase and aryl sulfatase in different developmental stages of the life cycle. The metabolic regulation of enzyme synthesis in conidia differs from that which occurs in the mycelial stage. During conidial germination and mycelial outgrowth, the synthesis of these enzymes is not coordinate but begins at different times and occurs at different rates. A rapid and early synthesis of choline sulfatase was observed during conidial germination under derepressing conditions; furthermore, synthesis of the enzyme also occurred for a brief period in germinating conidia even in the presence of repressing levels of sulfate. The results of this study suggest that several enzymes of sulfur metabolism are independently controlled by a developmental system which is superimposed upon the cys-3 regulatory mechanism. It was also found that choline sulfatase undergoes rapid turnover while aryl sulfatase is a stable species.     

Effect of 5-Azacytidine on the Light-Sensitive Formation of Sexual and Asexual Reproductive Structures in wc-1 and wc-2 Mutants of Neurospora crassa

Under nitrogen starvation conditions, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit condition (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in the light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.     

Effect of Inhibitors of Enzymatic DNA Methylation on the Formation of Reproductive Structures and Carotenoid Production in Neurospora crassa

The effect of inhibitors of DNA methylation on light-sensitive developmental stages of the filamentous fungus Neurospora crassawas studied. Under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3–300 M) inhibited protoperithecia formation and stimulated conidia formation (a 700-fold increase after light induction). After treatment of the mycelium with 5-azacytidine, the protoperithecia formation was accompanied by inversely proportional changes in the formation of conidia, both in the dark and after illumination. In the mycelium cultivated on the Vogel's medium, 5-azacytidine (up to 30 M) and methotrexate (up to 3 M) stimulated the light-induced carotenoid synthesis by 30%, whereas higher concentrations of these agents were toxic to carotenoid synthesis and growth.