Conidiation in Neurospora crassa

Summary Conidiation in Neurospora crassa has been studied in vivo by time-lapse microphotography and shown to be most generally (in aerial, dry conditions) a budding-fission process. Such a two-phase process is characterized by an initial basifugal budding of proconidial elements which are then secondarily separated as maturing conidia by interconidial septa. Dry macroconidia of Neurospora are thus blasto-arthrospores, i.e. blastospores basifugally budded on conidiophores and secondarily disarticulated from the proconidial chain as arthrosporal elements. Inception and median splitting of the interconidial septum have been electron microphotographed.In the vegetative hyphae, ethanol dehydrogenase has been cytochemically detected by oxidative assay and demonstrates a dense, uniform distribution of activity except at the hyphal tips. In the conidiating hyphae, the ethanol dehydro-genase becomes less dense in distribution, especially in the budding apices. Cytochrome oxidase activity, localized in the mitochondria, is confined in the subapical zone of vegetative hyphae while at the initiation of conidiation it becomes dispersed throughout the proconidial buds.     

Effects of Temperature on the Circadian Conidiation Rhythm of Temperature-Sensitive Mutants of Neurospora crassa

Period lengths at different temperatures and phase responsecurves at a high temperature (35°C) of circadian conidiationrhythms were examined in 13 temperature-sensitive (un) strainsof Neurospora crassa. Two strains, un-16 and un-18, had longerperiod lengths than the wild-type strain even at permissivetemperatures. Period lengths of six strains, un-4, un-11, un-16,un-18, un-19 and un-22, changed differently from that of thewild-type strain at restrictive temperatures. However, the shapeof phase response curves for high temperature (35°C) for3 h was almost the same for all un strains and the wild-typestrain. We isolated 97 temperature-sensitive mutants with periodlengths from 19.2 to 24.8 h and determined the dependence ontemperature of the period length of the conidiation rhythm foreach mutant. The mutants could be divided into four differentgroups in terms of their responses to changes in temperature. (Received September 8, 1993; Accepted March 10, 1994)     

Cold-sensitive Mutants of Neurospora crassa

Cold-sensitive mutants of the eucaryote Neurospora crassa have been isolated by a modification of the filtration-enrichment technique of Catcheside. The mutants include osmotic remedial, auxotrophic, transport, and incorporation deficient isolates.     

Mutants affecting thymidine metabolism in Neurospora crassa

When (14)C-thymidine labeled only in the ring is administered to Neurospora crassa, the majority of the recovered label is found in the ribonucleic acid (RNA). Three mutants were isolated in which different steps are blocked in the pathway that converts the pyrimidine ring of thymidine to an RNA precursor. Evidence from genetic, nutritional, and accumulation studies with the three mutants shows the pathway to proceed as follows: thymidine --> thymine --> 5-hydroxymethyluracil --> 5-formyluracil --> uracil --> uridylic acid. A mutant strain in which the thymidine to thymine conversion is blocked is unable to metabolize thymidine appreciably by any route, including entry into nucleic acids. This suggests that Neurospora lacks a thymidine phosphorylating enzyme. A second mutation blocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step, whereas a third prevents utilization of uracil and all compounds preceding it in the pathway. The mutant isolation procedures yielded three other classes of mutations which are proposed to be affecting, respectively, regulation of the thymidine degradative pathway, transport of pyrimidine free bases, and transport of pyrimidine nucleosides.     

Rhythms of Enzyme Activity Associated with Circadian Conidiation in Neurospora crassa

The mycelial growth front of the band strain of Neurospora grown on a solid surface exhibits a circadian rhythm of conidiation. Enzyme assays on extracts from that mycelium have shown that the activities of 6 of 13 enzymes (nicotinamide adenine dinucleotide nucleosidase, isocitrate lyase, citrate synthase, glyceraldehydephosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase) and soluble-protein content oscillate with the visible morphological change. The rhythmic enzymes associated with the Krebs and glyoxylate cycles are more active during conidiogenesis, whereas the activities of the rhythmic enzymes of glycolysis and the hexose monophosphate shunt are reduced during that phase. The absence of enzyme oscillations in wild-type and fluffy strains which do not form conidia under the conditions employed suggests that the enzyme fluctuations are associated with conidiogenesis itself. Oscillations of enzyme activity as a function of time are restricted to the growth front. A permanent record of rhythmicity associated with conidial and nonconidial regions does, however, exist in the mycelial mat behind the growth front. The activities of three enzymes (nicotinamide adenine dinucleotide nucleosidase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase) are not directly influenced by CO(2) concentration, but are correlated with the prescence or absence of conidiation which is controlled by CO(2) concentration. In contrast, citrate synthase and malate dehydrogenase activities are correlated with changes in CO(2) concentration.     

Selection of Respiratory Mutants of Neurospora crassa

A method is described that permits the isolation of mutants that are defective in mitochondrial respiration. The techniques of inositol-less death and overlay with 2,3,5-triphenyl-2H-tetrazolium chloride are utilized to select for mutant colonies. Colonies that survive inositol-less death and fail to reduce the tetrazolium dye are then tested polarographically for cyanide-sensitive respiration. A preliminary characterization of three mutants obtained by this method is presented. The mutants have been characterized by their cyanide-sensitive respiration rate, growth rate, the state III respiration rate of isolated mitochondria, inhibition of the respiration of isolated mitochondria by cyanide and antimycin A, and cytochrome spectra. All of the mutants described differ from the parent strain in some of these aspects.     

Altered Fatty Acid Distribution in Mutants of Neurospora crassa

Morphological mutants of Neurospora with decreased levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide ad enine dinucleotide (NADH) contained only 20% as much of a polyunsaturated fatty acid (linolenic acid) as the wild type in both the phospholipid and neutral lipid fractions. There was an excellent correlation between linolenic acid levels and morphological appearance as a function of total NADPH content, but no correlation with NADH content. The linolenic acid deficiency was balanced by a relative increase in the amounts of the less unsaturated fatty acids (oleic and linoleic acids), but the level of three other fatty acids did not appear to be changed. This accumulation of these two precursors suggests that the NADPH deficiency preferentially affected the final desaturation step, i.e., the conversion of linoleic to linolenic acid. The NADPH needed for this reaction in vivo was probably generated by the pentose phosphate shunt, since mutations affecting the shunt lead to the decreased levels of linolenic acid. It is not clear whether the changes in fatty acid distribution affect the morphogenesis of Neurospora, or if these changes are just part of the NADPH-deficiency syndrome.     

Malonate Metabolism, Stimulation of Conidiation and Succinate Dehydrogenase Activity in Neurospora crassa

Malonate was studied for its effect on succinate dehydrogenase activity and conidiation. It Was found to stimulate the succinate dehydrogenase activity and also conidiation of Neurospora crassa. The efficiency of sucrose metabolization for cellular synthesis was improved in malonate supplemented cultures. High Concentration (0.5 M) had a distinct toxic effect on conidiation and economic efficiency. Teratological structures were observed at 0.5 M malonate concentration. A stimulation of the glyoxylate cycle is considered to be one of the factors responsible for the conidiogenic effect.     

Conidiation of Neurospora crassa induced by treatment with natrium fluoride in submerged culture

Several strains of Rhizobium meliloti which have been subcultured for 23–33 years have changed from being markedly specific in their somatic agglutination reactions to become widely cross-reactive. On the other hand a fresh collection of the same species obtained from naturally nodulated, field-grown plants revealed the high degree of agglutinating specificity which had previously characterised the old-cultures. Attempts to reselect a specific substrain from old cross-agglutinating cultures by six plant passages, or to detect change to cross reactivity by ten successive subcultures of recent isolates were unsuccessful. However, one strain, isolated in 1939, was recently found to contain both specific and cross-reactive substrains. Practically all the cultures, both old and recent, showed considerable mutability in colony characteristics but none of these was consistently correlated with cross agglutinability. Instability in 6.4% (w/v) NaCl was characteristic of the cross-agglutinating cultures. Cross reactivity was associated with a shared lipopolysaccharide antigen (LPS) which appeared to be obscured by an outer antigen in most strains still showing specific agglutinability. In the exceptional case of strain SU27, agglutination could be attributed to its specific LPS.     

Isolation and Characterization of Low-Kynureninase Mutants of Neurospora crassa

Two kynureninase activities are known in Neurospora crassa, one of which (kynureninase I) is inducible, the other (kynureninase II) being constitutive. A method is described for the isolation of low-kynureninase mutants of N. crassa. When grown on an inducer, the mutants show significantly less kynureninase I activity compared with wild type, whereas constitutive kynureninase II activity is unaffected. Since a low level of kynureninase I activity remains in the mutants examined, the mutations may be in a regulatory gene or genes. Other experiments are described concerning the molecular weights of the two enzymes and the intracellular localization and specificity of kynureninase II.     

Conidiation of Neurospora crassa induced by treatment with natrium fluoride in submerged culture.

A transient treatment of pregerminated conidia of Neurospora crassa with NaF induced young, submerged cultures to prematurely differentiate conidia. The inductive treatment decreased the rate of respiration (with lower RQ), reduced the relative concentration of nucleoside triphosphates, and inhibited leucine incorporation into protein and adenosine incorporation into RNA.     

Photoreceptor Pigment for Blue Light in Neurospora crassa

Irradiating the mycelium of Neurospora crassa with moderate intensities of blue light causes a reversible photoreduction of a b-type cytochrome. The action spectrum for the photoreduction of cytochrome b is very similar to the absorption spectrum of flavin pigments. Prolonged irradiation of the mycelium with strong blue light irreversibly bleaches flavin-like pigments and as these pigments are bleached the photoresponse of cytochrome b is lost. We conclude from these and other data that a flavin is the photoreceptor pigment for the photoreduction of cytochrome b. The close similarity between the action spectrum for the photoreduction of cytochrome b and action spectra for a number of physiological photoresponses suggests that this photoreceptor pigment controls a wide variety of photobiological processes in a wide diversity of organisms.     

Respiration of Wild Type and Extrachromosomal Mutants of Neurospora crassa

The specific rates of respiration of cells of wild type and four extrachromosomal mutants of Neurospora crassa were measured throughout the vegetative growth cycle. Two forms of respiration were observed: (i) cyanide sensitive; and (ii) cyanide resistant, salicyl hydroxamate sensitive. These two forms are called terminal and alternate, respectively. The former proceeds by the mitochondrial electron transfer chain and involves the cytochromes; the latter apparently proceeds by the initial portion of the electron transfer chain and does not involve cytochromes. Large and rapid changes of both the terminal and alternate respiratory activities occurred during the vegetative growth cycle. The kinetics of these changes in wild type were compared under some conditions which inhibit protein synthesis and others in which the nitrogen source was varied. The kinetics of the changes of the two forms of respiration of mutants differed from those normally exhibited by wild type, but with varied experimental conditions wild type could be made to resemble the mutants. The results of these studies are discussed in terms of a dynamic model of regulation of mitochondrial biogenesis in the coordination of the synthesis of mitochondrial proteins encoded by nuclear and mitochondrial genomes.