Molecular cloning of RNAs differentially expressed in monokaryons and dikaryons ofSchizophyllum commune in relation to fruiting

A complementary DNA library cloned in pBR327 was prepared from RNA of a fruiting dikaryon of the basidiomyceteSchizophyllum commune. An improved colony-lysis method led to the isolation of complementary DNA clones hybridizing to nine different RNAs abundantly present in the fruiting dikaryon but present in very low or undetectable levels in the monokaryons. One of these RNAs, measuring 580 nucleotides, was exceptional in contributing more than 3% to the mRNA mass of the fruiting dikaryon. A role of the detected dikaryon-specific RNAs in fruiting was suggested by (i) their absence or low concentration in vegetatively growing monokaryons or the dikaryon, (ii) their steep increase in concentration during fruit-body formation in the dikaryon, (iii) a higher concentration of most of these RNAs in the fruit-body initials than in the supporting mycelium, and (iv) their prevalence in full-grown fruit bodies. The “fruiting-specific” RNAs were also present in a suspension-grown dikaryon, in the absence of fruiting, but in concentrations less than 10% of those in surface-grown fruiting dikaryon. The level of expression of these RNAs in the monokaryons was even lower or undetectable. Since the dikaryon and the monokaryons are otherwise coisogenic, it appears that the presence of two different incompatibility genes in the dikaryon allows for a low level of expression of the “fruiting genes” even in the absence of fruiting, whereas the genes are fully expressed under conditions permitting fruit-body formation.     

Kraft Pulp Bleaching and Delignification by Dikaryons and Monokaryons of Trametes versicolor

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes (Coriolus) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.     

Genetical variability of glutamate dehydrogenase activity in monokaryotic and dikaryotic mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum

Summary Glutamate dehydrogenase (GDH) is the key enzyme of ammonium assimilation by ectomycorrhizal fungi. Its activity might be use as a criterion to select mycelia capable of enhancing the nitrogen nutrition of the host plants. Genetical variability of the GDH activity of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnési was studied in an attempt to determine if this enzyme activity could be improved by way of chromosomal genetics. The activity of 11 wild strains was compared with that of 70 mycelia obtained as the progeny of a laboratory fruiting strain HC1. These 70 mycelia were 20 monokaryons (5 for each mating type) and the 50 synthesized dikaryons obtained from all the compatible fusions between these monokaryons. The specific GDH activity of the 11 wild strains ranged from 1.5 to 11.6 nkat mg^-1 fungal protein. The activity of the monokaryotic progeny of the HC1 strain was, on average, three times lower (2.85 n kat mg^-1 fungal protein) than that of the parental dikaryon. In contrast, synthesized dikaryons originating from these monokaryons were very variable and had an average values similar to that of the parental dikaryon (9.1 nkat mg^-1 fungal protein). Eighteen of these synthesized dikaryons contained an activity higher than that of the original HC1 strain. The variation of the GDH activity of these dikaryons involves additive and non additive (interactive) components, each of them accounting for ca. 50% of the genetical variation. The non additive variation could not be explained by a model involving only dominance. These results are discussed with reference to the genetical improvement of mycorrhizal fungi in order to enhance nitrogen nutrition of the host plants.Abbreviations GDH glutamate dehydrogenase - IAA indole-3-acetic acid - NADP nicotimamide adenine dinucleotide phosphate     

Isolation and characterization of a sporeless mutant in Pleurotus eryngii

A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains.     

Life history and developmental processes in the basidiomycete Coprinus cinereus.

Coprinus cinereus has two main types of mycelia, the asexual monokaryon and the sexual dikaryon, formed by fusion of compatible monokaryons. Syngamy (plasmogamy) and karyogamy are spatially and temporally separated, which is typical for basidiomycetous fungi. This property of the dikaryon enables an easy exchange of nuclear partners in further dikaryotic-monokaryotic and dikaryotic-dikaryotic mycelial fusions. Fruiting bodies normally develop on the dikaryon, and the cytological process of fruiting-body development has been described in its principles. Within the specialized basidia, present within the gills of the fruiting bodies, karyogamy occurs in a synchronized manner. It is directly followed by meiosis and by the production of the meiotic basidiospores. The synchrony of karyogamy and meiosis has made the fungus a classical object to study meiotic cytology and recombination. Several genes involved in these processes have been identified. Both monokaryons and dikaryons can form multicellular resting bodies (sclerotia) and different types of mitotic spores, the small uninucleate aerial oidia, and, within submerged mycelium, the large thick-walled chlamydospores. The decision about whether a structure will be formed is made on the basis of environmental signals (light, temperature, humidity, and nutrients). Of the intrinsic factors that control development, the products of the two mating type loci are most important. Mutant complementation and PCR approaches identified further genes which possibly link the two mating-type pathways with each other and with nutritional regulation, for example with the cAMP signaling pathway. Among genes specifically expressed within the fruiting body are those for two galectins, beta-galactoside binding lectins that probably act in hyphal aggregation. These genes serve as molecular markers to study development in wild-type and mutant strains. The isolation of genes for potential non-DNA methyltransferases, needed for tissue formation within the fruiting body, promises the discovery of new signaling pathways, possibly involving secondary fungal metabolites.     

The constitution of incompatibility factor and mating characteristics of spore isolates in a bipolar mushroom, <Emphasis Type="Italic">Pholiota nameko</Emphasis>

Pholiota nameko is a wood-rotting edible mushroom that carries a bipolar A incompatibility factor gene. The linkage analysis of the multiple allelomorphic A factor gene demonstrated that sexual reproduction produced a monospore isolate carrying a new A factor gene in addition to two parental mating types of isolates. However, 10%–30% of the modified monospore isolates could not produce a dikaryon with both of the parental monokaryons by crossing. It is concluded that the bipolar A incompatibility factor gene of P. nameko is constituted of two functional subunits, Aα and Aβ, which might be successively located beside each other with an apparent genetic distance of 0.3 centi-Morgan between them on the same chromosome. Further, some monospore isolates that did not conjugate with both parental monokaryons could produce dikaryons with different monokaryotic stocks with either one of the parental mating types. This result suggests that the crossing capability of these isolates were essentially those for one of the mating types of the parental monokaryons, but that their function for mating activity was made partially by unequal crossing-over in the process of sexual recombination. Received: May 1, 2001 / Accepted: December 5, 2001     

Changes in isozyme patterns between monokaryons and dikaryons of a bipolar Coprinus

Proteins and isozymes of several different classes of enzymes in partially purified protein extracts of monokaryons, dikaryons, and monokaryon mixtures of a bipolar Coprinus sp. were separated on polyacrylamide gels by slab electrophoresis. Differences in protein and isozyme spectra were correlated with the operation of the incompatibility factors and with the results of Wang and Raper on Schizophyllum. It was concluded that the shift from monokaryon to dikaryon mediated a major change in the nature, quantity, or distribution of the proteins of this Coprinus sp.     

Fungal fidelity: Nuclear divorce from a dikaryon by mating or monokaryon regeneration

Basidiomycete fungi perform fertilizations by incorporation of nuclei into a monokaryotic mycelium to establish a dikaryon. The dikaryon cannot incorporate another type of nucleus, but can still act as a nucleus donor in a dikaryon–monokaryon (di–mon) mating, known as the Buller phenomenon. Previously, it has been observed that: (1) in a particular di–mon mating, one of the nuclear types of the dikaryon generally performs better as a donor than the other, and (2) when nuclei from a dikaryon are separated to form monokaryons again (dedikaryotisation), recovery of monokaryons of the two nuclear types is usually unequal. In this study, we investigated if these two observations of asymmetry are functionally related. We tested this hypothesis by performing both di–mon matings and dedikaryotisation of dikaryons derived from five different monokaryons. When a single mechanism controls both processes, the nucleus better at fertilizing a monokaryon in a Buller pairing should also be recovered upon dedikaryotisation with a higher frequency. The results showed a hierarchical structure for recovery among nuclei in dedikaryotisation, but this hierarchy did not correspond to the fertilization success during di–mon mating. These findings thus show that the mechanism causing asymmetric regeneration of nuclei, is most likely not the same as the mechanism responsible for increased chance of fertilization in di–mon matings. We discuss the complexity of the interactions that occur during di–mon matings with regards to the mating type loci.     

Protein Specificity and Sexual Morphogenesis in Schizophyllum commune

Soluble proteins of a number of strains of the basidiomycete Schizophyllum commune were separated on polyacrylamide gel by disc electrophoresis. The presence or absence, R(F) values, and relative intensity of protein bands, observed on gel columns and densitometric tracings, were used as criteria for analysis and comparison. Eight mycelial types were studied, namely, normal homokaryon, dikaryon, homokaryons carrying primary mutation(s) at one or both of the Abeta and Bbeta loci, and three mutant B homokaryons, each carrying a different type of modifier mutation. Similarities and dissimilarities in protein spectra among these mycelial types, observed with crude extracts, "total protein precipitates," and protein fractions, were consistent with predictions based on an earlier genetic model for the action of the two incompatibility factors known to control "sexual morphogenesis" in this fungus.     

香菇交配型因子次级重组体的鉴定

对13个香菇菌株的担孢子后代进行了交配型分析,其中8个菌株非亲和反应与亲和反应之比与预期的3∶1的比例无显著差异。另外5个菌株非亲和反应与亲和反应之比不符合3∶1,其中4个菌株在0.05显著水平的X2值仅略高于理论值,而另一菌株HL01具有特殊的表现,其单核体132个随机配对的非亲和反应与亲和反应之比为82∶50,X2值显著偏离3∶1的临界值。用4个标准测试菌株鉴定了来自HL01同一子实体的189个孢子单核体的交配型,在189个单核体中,161个单核体归于4种正常交配型(A1B1,A2B2,A1B2,A2B1)之一。而另外28个可能源于次级重组的单核体可分成另外4个类群。通过以所有可能的组合进行配对杂交,进一步分析了28个单核体的交配型。结果表明,次级重组同时在A因子和B因子中发生,重组值分别为8.5%和11.6%。A因子至少由2个亚基组成而B因子可能由不止2个亚基组成。随后的出菇试验表明,至少含有1个重组体的所有可亲和配对均具有结实能力。     

Comparison of protein patterns in cockroach muscles innervated by fast or slow neurons

The protein composition of six of the coxal depressor muscles of the leg of the cockroach, Periplaneta americana, was analyzed by one and two dimensional SDS—polyacrylamide gel electrophoresis in conjunction with a sensitive silver stain. The proteins in each muscle were fractionated according to their solubilities in either low salt buffer, 1%NP-40, or 2%SDS. The gel patterns resulting from the electrophoresis of each of the fractions from “fast”, “slow”, and “mixed” type muscle were compared with reference to the innervation of the muscle by a particular motor neuron. More than 50 proteins were detected which were not common to all three types of muscle. These proteins were found to be distributed fairly equally amongst each of the three fractions. The large number of differentially expressed proteins observed, are not likely to be involved in specific neuromuscular recognition, but instead are probably correlated with the physiological state of the muscle.     

Development of SCAR Markers to Determine the Mating Types of Lepista nuda Protoplast Monokaryons

Lepista nuda (Bull. ex Fr.) Cooke belongs to Tricholomataceae and is an edible fungus with both economic and medical value. Mycelia were isolated from the fruiting bodies of L. nuda and were used to prepare the protoplast monokaryons. One hundred and fifteen monokaryons were obtained and their mating types were determined using somatic incompatibility tests. Protoplast monokaryons segregated into either the A₁B₁ or the A₂B₂ mating types. Inter-simple sequence repeats and sequence-related amplified polymorphism fingerprinting were used to analyse the mating types of these protoplast monokaryons and 16 sequence-characterised amplified region primers were developed to efficiently differentiate between the monokaryon mating types. Multiplex PCR analyses were also established. The data presented here outline a method for the precise and rapid identification of protoplast monokaryon mating types, which has the promise to shorten the period required for conventional crossbreeding.     

Sexual selection in mushroom-forming basidiomycetes

We expect that sexual selection may play an important role in the evolution of mushroom-forming basidiomycete fungi. Although these fungi do not have separate sexes, they do play female and male roles: the acceptance and the donation of a nucleus, respectively. The primary mycelium (monokaryon) of basidiomycete fungi, growing from a germinating sexual spore, is hermaphroditic, but it loses female function upon the acceptance of a second nucleus. The resulting dikaryon with two different nuclei in each cell retains a male potential as both nuclei can fertilize receptive mycelia. We tested the occurrence of sexual selection in the model species of mushroom-forming basidiomycetes, Schizophyllum commune, by pairing monokaryons with fully compatible dikaryons. In most pairings, we found a strong bias for one of the two nuclei although both were compatible with the monokaryon when paired alone. This shows that sexual selection can occur in mushroom-forming basidiomycetes. Since the winning nucleus of a dikaryon occasionally varied depending on the receiving monokaryon, we infer that sexual selection can operate through choosiness of the receiving individual (analogous to female choice). However, in other cases the same nucleus won, irrespective of the receiving monokaryon, suggesting that competition between the two nuclei of the donating mycelium (analogous to male–male competition) might also play a role.     

中国白灵侧耳自然群体的交配型因子分析

为了研究我国新疆白灵侧耳自然种群的遗传多样性,估测自然种群的遗传丰度,对51株新疆野生白灵侧耳的84个原生质体单核体的交配型因子进行分析。在84个单核体中存在54个不同的A因子和59个不同的B因子。x2检测表明交配型因子A和B的系列因子均为等概率分布。据此估算我国白灵侧耳自然群体中有79个A因子,100个B因子,自然群体中的单核交配型总数79×100=7,900个,双核交配型总数31,201,050个。可见,新疆白灵侧耳遗传多样性丰富,新疆是我国白灵侧耳的野生种质资源宝库。     

Haemonchus contortus: New data on its genetic constitution

Studies on the genetic constitution of single smooth-bodied females, appearing from time to time in the homozygous (aa) linguiform strain “C,” revealed that these worms have the constitution of the linguiform (aa) morph but the character “flap” in them has failed to develop because of the undetermined effect of some additional factors. Experiments were also performed to check the dominance to recessiveness ratios of the characters “flap,” “knob,” and “smooth.” With the Haemonchus types present in Bulgaria these ratios have persisted over seven or eight years. It is stated that they are the same in populations whose morphologic types in female specimens show different percentage distributions.     

Isolation and characterization of a sporeless mutant in <Emphasis Type="Italic">Pleurotus eryngii</Emphasis>

 A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains. Received: September 5, 2002 / Accepted: October 16, 2002 Acknowledgments We are grateful to Mrs. Motoe Masuda for her skillful technical assistance. Contribution no. 358 from the Tottori Mycological Institute Correspondence to:Y. Obatake     

The evolution of non-reciprocal nuclear exchange in mushrooms as a consequence of genomic conflict

Heterothallic mushrooms accomplish sex by exchanging nuclei without cytoplasm. Hyphal fusions occur between haploid mycelia resulting from germinated spores and subsequent reciprocal nuclear exchange without cytoplasmic mixing. The resulting dikaryon is therefore a cytoplasmic mosaic with uniformly distributed nuclei (two in each cell). Cytoplasmic inheritance is doubly uniparental: both mated monokaryons can potentially transmit their cytoplasm to the sexual spores, but normally only a single type per spore is found.Intracellular competition between mitochondria is thus limited, but at the dikaryon level, the two types of mitochondria compete over transmission. This creates the conditions for genomic conflict: within the dikaryon, a selfish mitochondrial mutant with increased relative transmission can be favoured, but selection between dikaryons will act against such a mitochondrial mutant. Moreover, because nuclear fitness is directly dependent on dikaryon fitness, a reduction in dikaryon fitness directly conflicts with nuclear interests. We propose that genomic conflict explains the frequent occurrence of non-reciprocal nuclear exchange in mushrooms. With non-reciprocal exchange, one monokaryon donates a nucleus and the other accepts it, but not vice versa as in the typical life cycle. We propose a model where non-reciprocal nuclear exchange is primarily driven by mitochondria inducing male sterility and the evolution of nuclear suppressors.     

Internuclear Genetic Transfer in Vegetative Dikaryons of SCHIZOPHYLLUM COMMUNE: I. Di-Mon Mating Analysis

A series of hemi-compatible dikaryon x monokaryon (di-mon) matings was designed to determine whether there was any genetic interaction between the dikaryotic nuclei. One of the nuclei in each dikaryon was known to carry a recessive gene (mnd) that promoted the development of an abnormal growth form, mound. Dikaryons containing both mnd ⁺ and mnd nuclei produced mosaic colonies that consisted of three distinct kinds of hyphae: mound, fruiting body, and vegetative (devoid of any multihyphal structure). When dikaryotic hyphae from each of these morphological regions were used in di-mon matings, the genetic and developmental characteristics of the selected nuclear types were examined in the resulting derived dikaryons. The results showed that fruiting-body and vegetative cells contained the expected mnd and mnd⁺ nuclei. Dikaryotic mound hyphae, however, contained only mnd nuclei. In a manner as yet unresolved, but clearly dependent on the presence of the mnd allele, the mnd ⁺ allele of a wild nucleus was altered to, or acquired, the mnd allele. A number of hypotheses were considered to explain the genetic event(s) that generates [mnd + mnd*] dikaryotic cells from [mnd⁺ + mnd] cells, but none was found to be entirely satisfactory.     

基于F2群体的香菇遗传图谱构建及其在QTL定位中的应用

以171个F2双核体菌株为作图群体,通过相互配对的2个单核体的基因型推断双核体基因型,构建了第一张基于双核体群体的香菇遗传图谱。该图谱包含分布于15个连锁群的459个标记,覆盖长度为989.7cM,平均标记间隔为2.2cM。此外,以此双核体群体作为表型分离群体,定位了6个与香菇双核体菌丝生长速度相关的QTLs,位于5个连锁群上。采用全同胞单核体随机交配策略,易于构建相对大的双核体群体,用于连锁图构建和QTL定位。研究表明,在食用菌连锁图谱构建及QTL定位研究中,利用F2群体,可能为提高遗传作图效率,解决作图群体与表型分离群体间不一致问题提供新的途径。