Formation Of Protoperithecia Research Articles

Influence of oxygenated derivatives of linoleic and linolenic acids on the formation of conidia and protoperithecia in wild-type and photoreceptor complex mutants of Neurospora crassa

The regulatory effect of two oxyderivatives of unsaturated fatty acids (oxylipins), 18-hydroxy-(9Z,12Z)-octadecadienoic acid (18-HODE) and 18-(9Z,12Z,15Z)-octadecatrienoic acid (18-HOTrE), on the sexual and asexual sporulation of wild-type Neurospora crassa strains and wc-1 and wc-1 mutants was studied. In the wild-type strain, 18-HODE, unlike 18-HOTrE, stimulated protoperithecia formation in the dark and in the light. In the same strain, the studied oxylipins influenced conidiagenesis only under illumination. 18-HODE stimulated and 18-HOTrE inhibited the conidia formation. Oxylipins had no effect on protoperithecia formation in photoreceptor complex mutants, which apparently indicated its involvement in signal transmission in N. crassa. The stimulating action of the studied oxylipins on conidiagenesis in wc-1 and the lack of action in wc-2 may indicate alternative signaling pathways of oxylipins in this process.

Occurrence of Phyllachora balansae in Toona ciliata in Southern Minas Gerais State, Brazil

)were noted to show black fruiting bodies in circular areas. At first,they appeared as minute discolored specks that grow and becomeyellow and brown distributed throughout the adaxial side of leafletsand petioles (Figure 1A, B and C) in the adult plant (Figure 1D)and on cedar bark (Figure 1E). Heavily infected seedlings shedtheir leaves and eventually die. Isolation of the causal agent fromleaves showing typical symptoms was done by disinfecting smallleaf fragments with 70% alcohol and 2% sodium hypochlorite,followed by transference to Petri dishes containing PDA culturemedium. Five days after incubation at 25oC, fungal colonies wereobserved; they spent 15 days each to reach the edge of the dishes(Figure 1G). Thirty days after incubation, there was formation ofprotoperithecia, which have no fertile structures inside.Pathogenicity was determined by growing five strains (UFLA01,UFLA02, UFLA03, UFLA04, and UFLA05) on PDA medium at25oC for 30 days. Koch postulates were adopted by placing nine-mm culture discs on healthy leaves of Australian cedar seedlingswithout injury (Figure 1H). Control plants were inoculated withPDA medium discs without fungal culture. After inoculation, plantswere incubated at 25±2°C for 15 days. Inoculated plants showedlesions similar to those observed in the field and the recoveredcolonies were similar to the inoculated colony (Figure 1I).Ascospores produced in the lesions were hyaline, spherical toelliptical, measuring 10-12 x 7-9 µm. Based on these characteristicsand on the literature (Chardon, C.E.; Miller, J.H.; Miller, A.S.Ascomycetes from the state of Minas Geraes (Brazil). Mycologia,v.32, n.2, p.172-204, 1940), the fungus isolated from

Effect of oxylipins on Neurospora crassa growth and differentiation

The effect of the natural oxylipins 3(R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE) and 18-hydroxy-(9Z,12Z)-octadecadienoic acids (18-HODE) on the growth and hypha aggregation, as well as on some light-depending processes, such as carotenoid biosynthesis, protoperithecia formation (sexual cycle), and conidiation (asexual cycle), of the ascomycete Neurospora crassa was studied. Hypha aggregation and growth slowdown were induced by 3-HETE, 18-HODE, and linoleic acid. At concentrations from 5 to 50 μM, these compounds had no significant effect on the light-induced carotenogenesis. At the same time, these 3-HETE and 18-HODE concentrations, unlike linoleic acid, induced the formation of protoperithecia in the dark. At the concentration of 5 μM, an additive effect of oxylipins and light was revealed. The studied oxylipins had different effects on the asexual reproduction of N. crassa: 3-HETE induced conidiation in the dark, whereas 18-HODE induced conidiation in the light. The possible involvement of oxylipins in the regulation of the processes of sexual and asexual reproduction of N. crassa is discussed.

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 the conditions of nitrogen starvation, 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 conidiation (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 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.

The fle1 gene encoding a C2H2 zinc finger protein co-ordinates male and female sexual differentiation in Podospora anserina.

The flexuosa (fle1-1) mutant, isolated in Podospora anserina, displays vegetative defects and two antagonistic sexual phenotypes: it produces several 1000-fold fewer microconidia (male gametes) than the wild-type strain and, conversely, more abundant protoperithecia (female organs). Cloning and sequencing of the fle1 gene and of cDNA identified an open reading frame encoding a 382-amino-acid polypeptide with two C2H2 zinc finger motifs. The predicted FLE1 protein shares 46% identity with the FlbC protein of Aspergillus nidulans and 68% identity with a putative protein identified by a search in the Neurospora crassa database. The nuclear localization of FLE1 was demonstrated by fusion with the green fluorescent protein. Sequencing of the fle1-1 mutant allele revealed a frameshift mutation upstream of the zinc finger domain. The fle1-1 mutant was a null mutant, as targeted disruption of fle1 sequence led to the same pleiotropic phenotype. When fle1 was overexpressed by introduction of a transgenic copy of the native fle1 gene or a fusion with a strong promoter, formation of protoperithecia was impaired, leading to partial or complete female sterility. We propose that fle1 acts as a repressor of female sexual differentiation in order to maintain the balance between male and female sexual pathways.

The opposed effect of 5-azacytidine and light on the development of reproductive structures in Neurospora crassa.

Blue light inhibits the formation of asexual cycle spores (conidia) and stimulates the development of the sexual (female) reproductive structures (protoperithecia) in the nitrogen-starved mycelium of Neurospora crassa. The DNA methylation inhibitor, 5-azacytidine (3-300 microM), opposed the effect of light by suppressing the protoperithecia formation and stimulating a conidiation. The addition of 300 microM 5-azacytidine inhibited protoperithecia formation in the dark-cultivated mycelium by about two orders of magnitude and activated conidiation in the light-exposed mycelium by almost three orders of magnitude. Both in the dark-cultivated and the irradiated mycelium treated with various 5-azacytidine concentrations, the yield of conidia and protoperithecia demonstrated an inverse relationship. We suggest that DNA methylation and blue light are involved in the organism's selection of sexual or asexual reproductive cycle.

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 crassa was studied. Under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3-300 microM) 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 microM) and methotrexate (up to 3 microM) stimulated the light-induced carotenoid synthesis by 30%, whereas higher concentrations of these agents were toxic to carotenoid synthesis and growth.

Analysis of light signal transduction affecting morphogenesis of perithecia in Neurospora crassa

The development of perithecia in Neurospora crassa is under the control of light, nutritional condition such as nitrogen starvation and temperature. Blue light can stimulate(i)the formation of protoperithecia on nitrogen deficient media, (ii)positive bending of perithecial beak, and(iii)the ejection of ascospores from asci in the perithecia. We found that the position of the formation of perithecial beak was dependent on light.(iv)Light stimulated the positioning of perithecial beak on the upperward of it, which we defined as“ light induced polarity of perithecia”. We developed in vitro systems to analyse molecular mechanism of light signal transduction that used the membrane and soluble fractions of mycelial extract. UV-A light induced the binding of ATP/GTP to 58kDA, 77kDa, 83kDa, and 129kDa proteins in the membrane fraction. Blue light stimulated the ADP-ribosylation of 38kDa and 56kDa proteins in the soluble fraction. Blue light also stimulated the phosphorylation of 15kDa protein in the membrane fraction. We isolated a mutant missing completely the phosphorylation of 15kDa protein in the membrane fraction. The mutation designated as psp(phosphorylation of small protein)was mapped on LGVR between al-3and his-6. The psp mutats showed blindness in the light induced polarity of perithecia. The 15kDa protein was purified and the cDNA was cloned. It was identified as nucleoside diphosphate(NDP)kinase. The cDNA for NDP kinase prepared from psp mutants included a mutation of amino acid replacement from Pro 72to His. The putative function of NDP kinase in the light signal transduction to perithecial morphogenesis was discussed.

The cpc-2 gene of Neurospora crassa encodes a protein entirely composed of WD-repeat segments that is involved in general amino acid control and female fertility.

Phenotypic and molecular studies of the mutation U142 indicate that the cpc-2+ gene is required to activate general amino acid control under conditions of amino acid limitation in the vegetative growth phase, and for formation of protoperithecia in preparation for the sexual phase of the life cycle of Neurospora crassa. The cpc-2 gene was cloned by complementation of the cpc-2 mutation in a his-2ts bradytrophic background. Genomic and cDNA sequence analysis indicated a 1636 bp long open reading frame interrupted by four introns. The deduced 316 amino acid polypeptide reveals 70% positional identity over its full length with G-protein beta-subunit-related polypeptides found in humans, rat (RACK1), chicken, tobacco and Chlamydomonas. With the exception of RACK1 the function of these proteins is obscure. All are entirely made up of seven WD-repeats. Expression studies of cpc-2 revealed one abundant transcript in the wild type; in the mutant its level is drastically reduced. In mutant cells transformed with the complementing sequence, the transcript level, enzyme regulation and female fertility are restored. In the wild type the cpc-2 transcript is down-regulated under conditions of amino acid limitation. With cpc-2 a new element involved in general amino acid control has been identified, indicating a function for a WD-repeat protein that belongs to a class that is conserved throughout the evolution of eukaryotes.

Nitrogen and carbon starvation regulate conidia and protoperithecia formation in Neurospora crassa grown on solid media

Nitrogen and carbon starvation regulate conidia and protoperithecia formation in Neurospora crassa grown on solid media

Conidia induce the formation of protoperithecia in Neurospora crassa: further characterization of white collar mutants.

The treatment of undifferentiated mycelia with heavy suspensions of their own conidia triggers protoperithecial development. This effect was also observed with white collar (wc) mutants and suggests that the wc genes are not structural genes necessary for morphogenesis of protoperithecia but that they are probably involved in regulation.

Isolation of new white collar mutants of Neurospora crassa and studies on their behavior in the blue light-induced formation of protoperithecia.

White collar (wc) mutants of Neurospora crassa are thought to be regulatory mutants blocked in the photoinduction of carotenogenesis. Eight new wc mutants have been isolated after UV mutagenesis; their morphology and linear growth rate are not altered, although blue light-induced carotenogenesis is completely blocked. All of the wc mutations fall into two complementation groups corresponding to the already-known wc-1 and wc-2 loci. It is shown that the wc mutations impair another blue light effect, the photoinduction of protoperithecia formation, as well as the low constitutive production of protoperithecia in the dark. These effects are not due to the lack of carotenoids since the albino mutants show a normal sexual development. The pleiotropic effects of the mutations in the wc genes indicate that they play a key role in the mechanisms of regulation of the blue light-induced responses of N. crassa.

Podospora anserina mutations inhibiting several developmental alternatives and growth renewal

A mutation (modG) was selected on the basis of the suppression of a defect resulting from a mutation of modD gene. The modG mutation shows the same developmental consequences as modD mutations: the absence of protoperithecia and aerial hyphae, the non-renewal of growth from stationary cells and the non-germination of ascospores. Investigations of heterokaryotic mycelia showed that the action of modD and modG mutations on the formation of protoperithecia and aerial hyphae is autonomous. Furthermore, taking advantage of the thermosensitivity of a modD mutation, it was shown that the mutations have no incidence on cell differentiation prior to the achievement of stationary physiology. These findings lead to suggest the existence, in Podospora anserina, of a stage of cell totipotency, which we call the switch stage, that derives from the stationary state through the action of modD and mode genes. Accordingly, the switch stage would be the intermediate in the process of growth renewal from stationary cells and the basic stage for the selection of the developmental alternatives (aerial hyphae, protoperithecia ...) of Podospora mycelia.

PROTOPLASMIC INCOMPATIBILITY AND CELL LYSIS IN PODOSPORA ANSERINA. I. GENETIC INVESTIGATIONS ON MUTATIONS OF A NOVEL MODIFIER GENE THAT SUPPRESSES CELL DESTRUCTION

In Podospora anserina, protoplasmic incompatibility (a phenomenon that prevents heterokaryon formation because of the destruction of the fused cells) can be studied in homokaryotic strains that combine nonallelic incompatibility genes or carry mutations at the lys loci. In these strains cell destruction occurs early in development and is associated with an arrest of growth.-From the self-lysing strains lysA(1) and RV (R and V are nonallelic incompatibility genes) mutations have been selected that suppress the self-lysing trait, i.e., that prevent cell destruction and remove growth inhibition. Some of them were derived from a novel modifier locus, modC, located near the mating-type locus.-In C/D and C/E incompatibility systems, modC mutations, which per se have no obvious effect, were considered in addition to mutations in the previously identified modifier loci, modA and modB. The demonstration of a functional interdependence among the three mod genes suggested that modC is not the structural gene for the protease associated with cell lysis, but is involved, like modA and modB, in its control.-All three modC mutant strains investigated exhibit defects in the formation of protoperithecia, suggesting that the modC gene function is essential to the occurrence or development of the female organs. This is the third argument that supports the hypothesis ( Boucherie, Bégueret and Bernet 1976) that protoplasmic incompatibility and female organ formation might be related phenomena.

The molecular mechanism of protoplasmic incompatibility and its relationship to the formation of protoperithecia in Podospora anserina.

In Podospora anserina, protoplasmic incompatibility due to interactions between non-allelic genes was suppressed by the effect of mutations in two modifier genes, mod-I and mod-2. It is shown that mod-I and mod-2 are involved in the production of three specific proteins, a phenoloxidase and two previously identified proteases (Bégueret & Bernet 1973 a) which are associated with the phenomenon of protoplasmic disintegration. These enzymes, whose messengers are normallly latent during vegetative growth, appear at this stage of the life cycle only as a consequence of incompatible gene interactions. The mode-I and mod-2 genes and each of the five incompatibility loci involved in non-allelic incompatibility systems also participate in the formation of the protoperithecia. This pleiotropic effect suggests that protoplasmic incompatibility is a deviation in the normal physiological processes of protoperithecial formation.

Regulation of perithecium development and maturation in Pleospora herbarum by light and temperature

Exposure of growing colonies of Pleospora herbarum (Pers.) Rabenhorst (Imperfect stage = Stemphylium botryosum ) to UV radiation stimulated the formation of protoperithecia which matured only when subjected to long uninterrupted periods at low temperature (21 days at 5–10 °C). Equivalent periods of intermittent (diurnal) low temperature were not effective. Maturation was influenced by the temperature during near-UV induction, and protoperithecia formed at 24–27° were most likely to mature following cold treatment. The temperature following cold treatment also influenced maturation of protoperithecia: 10–15° was most favourable. Maturation was governed by an inverse relationship between the length of the cold treatment and of the terminal incubation. Light was essential for induction of protoperithecia but not for their subsequent maturation. Maturation once started by cold treatment was negated by relatively high temperatures (30°) Cold induction of protoperithecia in the dark, hitherto unrecorded, was observed when actively growing vegetative colonies were exposed to low temperatures (5–15°) for 1–35 days. An 8-day exposure to cold was necessary for maximum induction, although a few protoperithecia formed after a 24 h exposure. Maturation required another 35 days at low temperature (12–5°). The influence of low temperature on sexual reproduction of fungi is reviewed.