Hydrophobins Sc3 and Sc4 gene expression in mounds, fruiting bodies and vegetative hyphae of Schizophyllum commune

An abnormal growth form called mound has been hypothesized to be a neoplasm in the filamentous fungus Schizophyllum commune. An alternative hypothesis is that mounds represent some unusual developmental form in the fruiting body morphogenetic pathway. Hydrophobin proteins have been found in fruiting bodies where they line the surface of gas exchange pores and function to keep the pores hydrophobic. To further determine possible relationships between mounds and fruiting bodies, mound tissue was examined for gas exchange pores and the presence of hydrophobins. Cryoscanning electron microscopic images revealed the presence of channels in mound tissue and presumptive hydrophobin rodlets similar to the air channels in fruiting bodies. Hydrophobin gene expression was also measured in mound tissue using quantitative real-time PCR and showed both monokaryotic and dikaryotic mound tissue exhibited high expression of the dikaryotic specific Sc4 hydrophobin gene. In contrast, Sc4 hydrophobin expression was barely detectable in monokaryotic fruiting bodies. The expression of Sc4 hydrophobin genes in mounds suggests mound development uses this aspect of the dikaryotic fruiting developmental pathway.     

SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune

Two monokaryons of Schizophyllum commune can form a fertile dikaryon when the mating-type genes differ. Monokaryons form sterile aerial hyphae, while dikaryons also form fruiting bodies that function in sexual reproduction. The SC3 hydrophobin gene is expressed both in monokaryons and in dikaryons. The SC4 hydrophobin is dikaryon specific. In the monokaryon, SC3 lowers the water surface tension, coats aerial hyphae with a hydrophobic layer and mediates attachment of hyphae to hydrophobic surfaces. The SC4 protein lines gas channels within fruiting bodies with a hydrophobic membrane. Using gene disruptions, in this study, we show that in dikaryons SC3 fulfils the same roles as in monokaryons. SC4, on the other hand, has a role within fruiting bodies. In contrast to gas channels in fruiting bodies of the wild type, those of a DeltaSC4 strain easily filled with water. Thus, SC4 prevents gas channels filling with water under wet conditions, probably serving uninterrupted gas exchange. Other dikaryon-specific hydrophobin genes, SC1 and SC6, apparently do not substitute for the SC4 gene. In addition, by expressing the SC4 gene behind the SC3 promoter in a DeltaSC3 monokaryon, it was shown that SC4 cannot fully substitute for SC3, indicating that both hydrophobins evolved to fulfil specific functions.     

Monokariotic fruiting body and clamp cell formation in Mycoleptodonoides aitchisonii (Bunaharitake)

The ability to produce monokaryotic fruiting bodies and clamp cells in culture was examined in monokaryotic strain isolated from several dikaryotic parental strains of the edible mushroom, Mycoleptodonoides aitchisonii (Bunaharitake). We describe a single dikaryotic M. aitchisonii strain, TUFC50005, and 20 monokaryons derived from it, which exhibited a wide spectrum of monokaryotic fruiting types. Most strains formed primordia, or young fruiting body-like structures, but only one of the monokaryons, strain TUFC50005-4, formed a fruiting body, even though it had only one nucleus and produced only two spores after meiosis. We demonstrated that dikariotization was not required for clamp cell formation, fruiting body formation, or meiosis, in this mushroom.     

Dikaryotic fruiting body development in a single dikaryon of <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and the spectrum of monokaryotic fruiting types in its monokaryotic progeny

Using monokaryotic offspring from several dikaryotic parental strains, the phenomenon of monokaryotic fruiting has been previously analysed in the commercially cultivated high-quality edible mushroom Agrocybe aegerita, revealing a variety of monokaryotic fruiting types. Here, we report a single dikaryotic A. aegerita strain, A. aegerita AAE-3, and 40 monokaryons derived from it, which exhibit a wide spectrum of monokaryotic fruiting types, including a rare, previously unknown type. Advantageously, the selected parental strain A. aegerita AAE-3 completes its life cycle within three weeks by the formation of dikaryotic fruiting bodies of typical agaric morphology on malt extract agar plates. In order to morphologically compare normal dikaryotic fruiting to monokaryotic fruiting, histology was performed from all dikaryotic fruiting body development stages and all fruiting types of monokaryotic origin. No clamp connections or dikaryotic hyphae were observed within the plectenchyma of monokaryotic fruiting stages. Among the monokaryotic fruiting types of the A. aegerita AAE-3-derived monokaryons, we also characterised the rare ‘stipe type’ here described as ‘elongated initials type’ as no differentiation into a future cap and stipe was seen. The two mating-compatible monokaryotic strains representing the extremes of the fruiting type spectrum observed, A. aegerita AAE-3-13 (‘mycelium type’) and A. aegerita AAE-3-32 (‘abortive?+?true homokaryotic fruiting fruiter type, AHF?+?THF fruiter type’), were also found to readily produce oidia (arthrospores). In order to obtain a set of mating-compatible monokaryons covering the whole observed spectrum of monokaryotic fruiting, the two monokaryons A. aegerita AAE-3-40 (‘initials type’) and A. aegerita AAE-3-37 (‘elongated initials type’) have been selected for their mating compatibility with A. aegerita AAE-3-32 and A. aegerita AAE-3-13, respectively. Together with the parental dikaryotic strain A. aegerita AAE-3, this set of standard monokaryons could prove useful for studies exploring the factors regulating monokaryotic fruiting in comparison to dikaryotic mushroom formation.     

Carbohydrate Metabolism During Morphogenesis of Coprinus lagopus (sensu Buller)

The occurrence and properties of enzymes of carbohydrate metabolism were studied during dikaryotic fruiting of the mushroom Coprinus lagopus. Enzymes of hexose monophosphate catabolism, sugar alcohol (polyol) dehydrogenases (DH), and trehalase occurred throughout development. The ratio of xylitol DH to sorbitol DH was greater than unity in both monokaryotic mycelium and dikaryotic fruit body caps, whereas this ratio decreased in the stipe (stalk) tissue. Xylitol DH and sorbitol DH were both dependent upon nicotinamide adenine dinucleotide (NAD) and showed maximal activity at pH 9. Two separate enzymes were suspected on the basis of preferential utilization of the NAD analogue, thionicotinamide-NAD, by xylitol DH, and this feature was consistent throughout development. An appraisal of the carbohydrate pool revealed trehalose and glucose, with the former predominant in the stipe and the latter in excess in the cap of dikaryotic fruit bodies. Trehalase activity in dialyzed enzyme extracts showed pH optima at acid and alkaline pH levels in monokaryotic mycelium, dikaryotic stipes, and cap tissues.     

Relationship between deficiency of phosphoglucose isomerase in Coprinus macrorhizus and fruiting body formation.

A mutant (pgi) of Coprinus macrorhizus deficient in phosphoglucose isomerase did not grow on fructose and grew poorly on glucose. The pgi mutation inhibited the formation of monokaryotic and dikaryotic fruiting bodies.     

Chemical and genetical control of induction of monokaryotic fruiting bodies in Coprinus macrorhizus

Summary Fruiting-inducing substance (FIS), which is effective to induce fruiting bodies in monokaryotic mycelia of a strain of the Basidiomycete, Coprinus macrorhizus, has been detected in cell-free extracts of fruiting bodies and dikaryotic mycelia of C. macrorhizus itself and fruiting bodies of several other Basidiomycete species. Inducibility by FIS was controlled by genetic and environmental factors. Constitutive mutants which form monokaryotic fruiting bodies without addition of FIS were isolated. Fruiting bodies and monokaryotic mycelia of these mutants also contained FIS.FIS was stable to heat, acid and alkaline hydrolysis, and several enzymes degradating proteins and nucleic acids. Two fractions of FIS were obtained after Sephadex G-25 chromatography. One of them contained protein and the other appears to have similar chemical nature as adenosine-3, 5-monophosphate or adenosine-3-monophosphate which were active to induce monokaryotic fruiting.     

Purification and Identification of the Fruiting-Inducing Substances in Coprinus macrorhizus

Substances which are effective in inducing fruiting bodies in monokaryotic mycelia of the fis(+) strain of Coprinus macrorhizus were purified and characterized. The active components of fruiting-inducing substances were identified as adenosine-3'-monophosphate, adenosine 3',5'-cyclic monophosphate (cyclic AMP), and a protein which is bound with the cyclic AMP. Cyclic AMP was synthesized from adenine within mycelia of the mutant strains which form monokaryotic fruiting bodies without the addition of fruiting-inducing substances, but not in those of the strains which do not form monokaryotic fruiting bodies. The proteins which bind with cyclic AMP were detected in crude extracts of mycelia of those strains which form monokaryotic fruiting bodies and of the dikaryon, but not in those of the strains which do not form monokaryotic fruiting bodies.     

Identification, Characterization, and In Situ Detection of a Fruit-Body-Specific Hydrophobin of Pleurotus ostreatus

Hydrophobins are small (length, about 100 ± 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium. Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae. We identified and isolated a hydrophobin (fruit body hydrophobin 1 [Fbh1]) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom Pleurotus ostreatus. In order to study the temporal and spatial expression of the fbh1 gene, we determined the N-terminal amino acid sequence of Fbh1. We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments. Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.     

Sporulation timing in Myxococcus xanthus is controlled by the espAB locus

The fruiting body development of Myxococcus xanthus consists of two separate but interacting pathways: one for aggregation of many cells to form raised mounds and the other for sporulation of individual cells into myxospores. Sporulation of individual cells normally occurs after mound formation, and is delayed at least 30 h after starvation under our laboratory conditions. This suggests that M. xanthus has a mechanism that monitors progress towards aggregation prior to triggering sporulation. A null mutation in a newly identified gene, espA (early sporulation), causes sporulation to occur much earlier compared with the wild type (16 h earlier). In contrast, a null mutation in an adjacent gene, espB, delays sporulation by about 16 h compared with the wild type. Interestingly, it appears that the espA mutant does not require raised mounds for sporulation. Many mutant cells sporulate outside the fruiting bodies. In addition, the mutant can sporulate, without aggregation into raised mounds, under some conditions in which cells normally do not form fruiting bodies. Based on these observations, it is hypothesized that EspA functions as an inhibitor of sporulation during early fruiting body development while cells are aggregating into raised mounds. The aggregation-independent sporulation of the espA mutant still requires starvation and high cell density. The espA and espB genes are expressed as an operon and their translations appear to be coupled. Expression occurs only under developmental conditions and does not occur during vegetative growth or during glycerol-induced sporulation. Sequence analysis of EspA indicates that it is a histidine protein kinase with a fork head-associated (FHA) domain at the N-terminus and a receiver domain at the C-terminus. This suggests that EspA is part of a two-component signal transduction system that regulates the timing of sporulation initiation.     

长根小奥德蘑双孢菌株与四孢菌株核相变化的比较

用双苯并咪唑（Hoechst 33258）染色法分别对长根小奥德蘑Oudemansiella radicata双孢菌株和四孢菌株的菌丝、子实体、担孢子进行染色观察,结果表明：双孢长根小奥德蘑菌丝细胞多为单核,无锁状联合;原担子中单核进行一次有丝分裂形成两个横向或纵向排列的子核,这2个子核分别进入2个担孢子中,留下无核的空担子;成熟担孢子具有一个核。四孢长根小奥德蘑菌丝细胞大多数为双核,具有锁状联合;进入原担子中的两个单倍性细胞核先发生核配,形成一个二倍性的核,再经过减数分裂形成四个染色体减半的单倍性子核,     

双向核迁移在香菇遗传和育种中的应用研究

在来源于香菇Lentinula edodes不同双核菌株的单核菌株间进行单—单杂交,并利用双向核迁移现象获得两对双核菌株.之后采用挑取菌落尖端单根菌丝的方法纯化所获得的双核菌株并通过双—单杂交进行单向核迁移,进一步纯化所获得的双核菌株.从菌落形态、显微镜镜检、出菇实验和分子标记等4个方面对两对配对单核和所获得的双核菌株...     

The architecture of termite mounds: a result of a trade-off between thermoregulation and gas exchange?

We examined the influence of gas exchange on the architectureof termite mounds. In Comoé National Park (Côted'Ivoire), Macrotermes bellicosus builds, as an adaptation toambient temperature conditions, differently shaped mounds inthe shrub savanna and the gallery forest. Previous studies suggestedthat there might be a constraint that limits the degree of thermalinsulation of the interior (i.e., nest) of the mounds in environmentswith relatively low ambient temperatures. This factor causes,in proximate terms, suboptimal low nest temperatures and ultimatelyleads to reduced reproductive success in the gallery forest.In this study, we examined whether the necessity for gas exchangemight constrain mound architecture. We measured CO₂ concentrationsin the air channels of mounds in different habitats and undermanipulated temperature regimes. During both the dry and therainy season we found higher CO₂ concentrations in mounds ofthe gallery forest than in mounds of the savanna. Additional measurementsin forest mounds, architecturally resembling those of the savanna dueto an experimental increase in ambient temperatures, revealedlower CO₂ concentrations than unmanipulated mounds in this habitat. Generally,concentrations were higher during the rainy season comparedto the dry season and lower during day than during night. Summarizingthese results we present a model that illustrates this trade-offbetween thermoregulation and gas exchange under different temperatureregimes. Both factors together result in different mound architecturesunder different environmental temperatures and may finally limitthe distribution of this species.     

Uncontrolled growth associated with novel somatic recombination in the fungus Schizophyllum

In the bracket mushroom, Schizophyllum commune, a recessive genetic alteration, mnd, causes abnormally hyperplastic three-dimensional mounds of hyphae to rise from the surface of both haploid and dikaryotic mycelia. mnd, although not a genetic block in the fruiting body developmental pathway, is at least partially epistatic to fruiting. Within dikaryons containing both mutant and wild-type nuclei, [mnd + mnd⁺], a nonreciprocal somatic recombination event can lead to stable conversion of the mnd⁺ region of the wild-type nucleus to mnd. This transformation to the homoallelic [mnd + mnd] condition involves no genomic areas other than the mnd region and permanently prevents any further fruiting. Studies relating to the recombination mechanism have ruled out a diploid intermediate state and other concomitants of orthodox somatic recombination, as well as whole chromosome transfer. Instead, a novel form of internuclear genetic transfer is postulated whereby a nearby locus, mob⁺, controls the mobilization of the mnd chromosomal region alone from one nucleus to the other within the binucleate cells of dikaryotic mycelia.     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

Insertional mutagenesis combined with an inducible filamentation phenotype reveals a conserved STE50 homologue in Cryptococcus neoformans that is required for monokaryotic fruiting and sexual reproduction

Cryptococcus neoformans typically grows in a yeast-like morphology; however, under specific conditions the fungus can produce hyphae that are either dikaryotic or monokaryotic. In this study, we developed a simple method for inducing robust monokaryotic fruiting and combined the assay with Agrobacterium tumefaciens insertional mutagenesis to screen for hyphal mutants. A C. neoformans homologue of the Saccharomyces cerevisiae STE50 gene was identified and characterized. STE50 was found to be required for sexual reproduction and monokaryotic fruiting. Ste50p has conserved SAM and RA domains, as well as two SH3 domains specific to basidiomycetous Ste50 proteins. Analysis of protein-protein interaction showed that Ste50p can interact with Ste11p and Ste20p, and epistasis experiments placed STE50 between STE20 and STE11. Genetic analysis of the role of STE50 in sexual reproduction showed that it was required for all steps, from response to pheromone to production of hyphae. Analysis of the effect of individual Ste50p domains on sexual reproduction and monokaryotic fruiting revealed domain-specific effects for both processes. This study revealed that the C. neoformans STE50 gene has both conserved and novel functions during sexual reproduction and monokaryotic fruiting, and these functions are domain-dependent.     

A common genetic control of dikaryotic and monokaryotic fruiting in the basidiomycete Agrocybe aegerita

Summary In the edible mushroom Agrocybe aegerita (Agaricales) fruit bodies may be formed in both the sexual and asexual cycle. The major difference between the two types of fruit bodies is that the latter are smaller and contain only two spores on each basidium. Sexual fruiting requires the establishment of a dikaryon which is under the control of the well known incompatibility factors A and B. Asexual fruiting starts directly from a monokaryon. In both dikaryotic and monokaryotic fruiting the same two genes (fi ⁺ , fb ⁺ )are responsible for the initiation and differentiation of fruit bodies respectively. This shows that the morphogenetic procedures leading to fruit body formation in higher basidiomycetes are not necessarily correlated with the sexual cycle. These findings are significant for basic and applied research.