Asexual development is increased in Neurospora crassa cat-3-null mutant strains

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-3^RIP colonies were sensitive to H₂O₂, indicating that wild-type (Wt) resistance to external H₂O₂ was due to CAT-3. cat-3^RIP 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-3^RIP mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3^RIP 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.

     

Neurospora crassa Protein Arginine Methyl Transferases Are Involved in Growth and Development and Interact with the NDR Kinase COT1

The protein arginine methyltransferaseas (PRMTs) family is conserved from yeast to human, and regulates stability, localization and activity of proteins. We have characterized deletion strains corresponding to genes encoding for PRMT1/3/5 (designated amt-1, amt-3 and skb-1, respectively) in Neurospora crassa. Deletion of PRMT-encoding genes conferred altered Arg-methylated protein profiles, as determined immunologically. Δamt-1 exhibited reduced hyphal elongation rates (70% of wild type) and increased susceptibility to the ergosterol biosynthesis inhibitor voriconazole. In ▵amt-3, distances between branches were significantly longer than the wild type, suggesting this gene is required for proper regulation of hyphal branching. Deletion of skb-1 resulted in hyper conidiation (2-fold of the wild type) and increased tolerance to the chitin synthase inhibitor polyoxin D. Inactivation of two Type I PRMTs (amt-1 and amt-3) conferred changes in both asymmetric as well as symmetric protein methylation profiles, suggesting either common substrates and/or cross-regulation of different PRMTs. The PRMTs in N. crassa apparently share cellular pathways which were previously reported to be regulated by the NDR (Nuclear DBF2-related) kinase COT1. Using co-immunprecipitation experiments (with MYC-tagged proteins), we have shown that SKB1 and COT1 physically interacted and the abundance of the 75 kDa MYC::COT1 isoform was increased in a Δskb-1 background. On the basis of immunological detection, we propose the possible involvement of PRMTs in Arg-methylation of COT1.     

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.     

Growth patterns of adenine-3B; supersuppressor strains of Neurospora crassa.

The interaction between several suppressible ad-3B alleles and several supersuppressor genes had been examined both quantitatively and qualitatively in a tetrad analysis. Quantitatively, there is a good deal of variation in mean growth rates of cultures showing supersuppression: part of this variation can be attributed to variation in the suppressibility of ad-3B alleles, part to variation of suppression efficiency of ssu alleles, and part to the action of modifying genes. Qualitatively, ad-3B; ssu cultures nearly always show cyclic stop-start growth patterns. Escape from stop-start growth to wild-type growth is common.     

EMF Genes Interact with Late-Flowering Genes to Regulate Arabidopsis Shoot Development

To investigate the genetic mechanisms regulating the transitionfrom vegetative to reproductive phase in Arabidopsis, doublemutants between two embryonic flower (emf) and 12 differentlate-flowering mutants were constructed and analyzed. Doublemutants in all combinations displayed the emf phenotypes withoutforming rosettes during early development; however, clear variationsbetween different double mutants were observed during late development,fwa significantly enhanced the vegetative property of both emfmutants by producing a high number of sessile leaves withoutany further reproductive growth in emf1 fwa double mutants.It also produced numerous leaf-like flower structures similarto those in leafy ap1 double mutant in emf1 fwa double mutants.Nine late-flowering mutants, ft, fca, ld, fd, fpa, fe, fy, fha,and fve, caused different degrees of increase in the numberof sessile leaves, the size of inflorescence, and the numberof flowers only in weak emf1 and emf2 mutant alleles background.Two late-flowering mutants, co and gi, however, had no effecton either emf1 and emf2 mutant alleles in double mutants. Ourresults suggest that FWA function in distinct pathways fromboth EMF genes to regulate flower competence by activating geneswhich specify floral meristem identity. CO and GI negativelyregulate both EMF genes, whereas the other nine late-floweringgenes may interact with EMF genes directly or indirectly toregulate shoot maturation in Arabidopsis. ¹ To whom correspondence should be addressed.     

Two Novel Src Homology 2 Domain Proteins Interact to Regulate Dictyostelium Gene Expression during Growth and Early Development

There are 13 Dictyostelium Src homology 2 (SH2) domain proteins, almost 10-fold fewer than in mammals, and only three are functionally unassigned. One of these, LrrB, contains a novel combination of protein interaction domains: an SH2 domain and a leucine-rich repeat domain. Growth and early development appear normal in the mutant, but expression profiling reveals that three genes active at these stages are greatly underexpressed: the ttdA metallohydrolase, the abcG10 small molecule transporter, and the cinB esterase. In contrast, the multigene family encoding the lectin discoidin 1 is overexpressed in the disruptant strain. LrrB binds to 14-3-3 protein, and the level of binding is highest during growth and decreases during early development. Comparative tandem affinity purification tagging shows that LrrB also interacts, via its SH2 domain and in a tyrosine phosphorylation-dependent manner, with two novel proteins: CldA and CldB. Both of these proteins contain a Clu domain, a >200-amino acid sequence present within highly conserved eukaryotic proteins required for correct mitochondrial dispersal. A functional interaction of LrrB with CldA is supported by the fact that a cldA disruptant mutant also underexpresses ttdA, abcG10, and cinB. Significantly, CldA is itself one of the three functionally unassigned SH2 domain proteins. Thus, just as in metazoa, but on a vastly reduced numerical scale, an interacting network of SH2 domain proteins regulates specific Dictyostelium gene expression.     

Growth rate in Neurospora crassa: Linkage of polygenes

Part of the polygenic system controlling the response to selection for fast linear growth rate in Neurospora crassa is linked to the aurescent locus on chromosome I. This genotypic material accounts for approximately 18 percent of the total response to selection. There was no evidence of linkage to the yellow locus on chromosome VI.Journal Series Paper No. 890. University of Georgia College of Agriculture Experiment Stations, College Station, Athens.     

A potassium-proton symport in Neurospora crassa

Combined ion flux and electrophysiological measurements have been used to characterized active transport of potassium by cells of Neurospora crassa that have been moderately starved of K+ and then maintained in the presence of millimolar free calcium ions. These conditions elicit a high-affinity (K1/2 = 1-10 microM) potassium uptake system that is strongly depolarizing. Current-voltage measurements have demonstrated a K+-associated inward current exceeding (at saturation) half the total current normally driven outward through the plasma membrane proton pump. Potassium activity ratios and fluxes have been compared quantitatively with electrophysiological parameters, by using small (approximately 15 micron diam) spherical cells of Neurospora grown in ethylene glycol. All data are consistent with a transport mechanism that carries K ions inward by cotransport with H ions, which move down the electrochemical gradient created by the primary proton pump. The stoichiometry of entry is 1 K ion with 1 H ion; overall charge balance is maintained by pumped extrusion of two protons, to yield a net flux stoichiometry of 1 K+ exchanging for 1 H+. The mechanism is competent to sustain the largest stable K+ gradients that have been measured in Neurospora, with no direct contribution from phosphate hydrolysis or redox processes. Such a potassium-proton symport mechanism could account for many observations reported on K+ movement in other fungi, in algae, and in higher plants.     

Correlation of Growth Inhibition Patterns to Nucleoside Transport Models in Neurospora crassa

Growth of inhibition patterns provide evidence for a common nucleoside transport or utilization system, a separate system or systems for adenine transport, and another adaptable mechanism of adenosine transport.     

Growth regulation in Neurospora crassa effects of nutrients and of temperature

Summary Mycelia of Neurospora crassa growing with quite different rates at a given temperature (30°C) are obtained by changing the composition of the culture medium: for instance a duplication time of 220 min is found when cultures are growing in Vogel's minimal medium supplemented with glycerol and one of 80 min when cultures are growing in Vogel's minimal medium supplemented with sucrose and casein hydrolysate. The kinetics of shifth-down and shift-up transitions of growth between culture conditions supporting different rates of growth are described. Temperatures above 40°C and below 20°C severely restrict growth in Vogel's minimal medium supplemented with sucrose. The Arrhenius plot of the constant of the rate of growth suggests that an inactivation process occurs at low temperatures: the molecular basis for such a process is discussed.Abbreviations used A₄₅₀ absorbance at 450 mm     

A nitrate-induced frq-less oscillator in Neurospora crassa

When nitrate is the only nitrogen source, Neurospora crassa's nitrate reductase (NR) shows endogenous oscillations in its nitrate reductase activity (NRA) on a circadian time scale. These NRA oscillations can be observed in darkness or continuous light conditions and also in a frq(9) mutant in which no functional FRQ protein is formed. Even in a white-collar-1 knockout mutant, NRA oscillations have been observed, although with a highly reduced amplitude. This indicates that the NRA oscillations are not a simple output rhythm of the white-collar-driven frq oscillator but may be generated by another oscillator that contains the nit-3 autoregulatory negative feedback loop as a part. In this negative feedback loop, a product in the reaction chain catalyzed by nitrate reductase, probably glutamine, induces repression of the nitrate reductase gene and thus downregulates its own production. This is the first example of an endogenous, nutritionally induced daily rhythm with known molecular components that is observed in the absence of an intact FRQ protein.     

A Novel Cryptochrome-Dependent Oscillator in Neurospora crassa

Several lines of evidence suggest that the circadian clock is constructed of multiple molecular feedback oscillators that function to generate robust rhythms in organisms. However, while core oscillator mechanisms driving specific behaviors are well described in several model systems, the nature of other potential circadian oscillators is not understood. Using genetic approaches in the fungus Neurospora crassa, we uncovered an oscillator mechanism that drives rhythmic spore development in the absence of the well-characterized FRQ/WCC oscillator (FWO) and in constant light, conditions under which the FWO is not functional. While this novel oscillator does not require the FWO for activity, it does require the blue-light photoreceptor CRYPTOCHROME (CRY); thus, we call it the CRY-dependent oscillator (CDO). The CDO was uncovered in a strain carrying a mutation in cog-1 (cry-dependent oscillator gate-1), has a period of ∼1 day in constant light, and is temperature-compensated. In addition, cog-1 cells lacking the circadian blue-light photoreceptor WC-1 respond to blue light, suggesting that alternate light inputs function in cog-1 mutant cells. We show that the blue-light photoreceptors VIVID and CRY compensate for each other and for WC-1 in CRY-dependent oscillator light responses, but that WC-1 is necessary for circadian light entrainment.     

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.     

Resistance to Azole Drugs in Neurospora crassa

Staben, C. 1995. Resistance to azole drugs in Neurospora crassa. Experimental Mycology 19: 163-165. Neurospora crassa was susceptible to azole drugs: ketoconazole (MIC 1 μg/ml), fluconazole (MIC 5 μg/ml), and SCH39304 (MIC 5 μg/ml). Mutants of N. crassa resistant to ketoconazole were selected and genetically characterized. The seven characterized resistance mutations represented at least four genetic loci. Some mutants, but not all, were also resistant to fluconazole and to SCH39304.