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.     

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.     

THE BULLER PHENOMENON IN SCHIZOPHYLLUM COMMUNE: NUCLEAR SELECTION IN FULLY COMPATIBLE DIKARYOTIC-HOMOKARYOTIC MATINGS

Ellingboe , Albert H., and John R. Raper . (Harvard U., Cambridge, Mass.) The Buller Phenomenon in Schizophyllum commune: nuclear selection in fully compatible dikaryotic-homokaryotic matings. Amer. Jour. Bot. 49(5): 454–459. 1962.—Internuclear selection in fully compatible, dikaryotic-homokaryotic matings of isogenic strains is apparently controlled by the incompatibility factors and, in some cases, by extra-incompatibility factors. The relationship of the structure of the incompatibility factors to internuclear selection is not clear. Both types of nuclei from the dikaryon migrate into the homokaryon, and it appears that selection occurs in the final establishment of the new dikaryon.     

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。     

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.     

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.     

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.     

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。     

The A mating-type genes of the mushroom Coprinus cinereus are not differentially transcribed in monokaryons and dikaryons

Summary The A mating type factor of Coprinus cinereus regulates part of a developmental sequence that leads to the conversion of the asexual monokaryon into the fertile dikaryon. The A42 factor is a complex of seven genes, at least four of which are involved in determining the specificity of mating interactions. In this report we show that the A42 genes are constitutively expressed in both monokaryons and dikaryons. This has important implications with respect to intracellular recognition of a compatible mating, which requires an interaction between proteins already present within the cells of the mating partners, and for the subsequent maintenance of dikaryotic growth.     

Regulation of the pattern of protein synthesis in Schizophyllum commune by the incompatibility genes

The pattern of protein synthesis in various coisogenic mycelial types of Schizophyllum commune, viz. monokaryon, dikaryon, and homokaryons carrying primary mutations in the A and the B factors, was studied by two-dimensional gel electrophoresis. After pulse-labeling with ³⁵S-methionine, approximately 650 of 710 proteins analyzed were common to all mycelial types. Coisogenic monokaryons differed by only 2%, whereas the largest difference was found between these monokaryons and the dikaryon derived from them (6.6 and 7.7%). The majority of these differences fell into two about equally sized categories, i.e., proteins which were either specifically absent (“switched-off” proteins) or present (“switched-on” proteins) in the dikaryon. “Switched-on” proteins were on the average larger and slightly more acidic than “switched-off” proteins. The double factor mutant which best mimicked the dikaryon in morphology also best resembled the dikaryon in types of proteins synthesized. Unexpected, however, was the large overlap in proteins apparently controlled by each of the two incompatibility factors individually, despite the distinct morphological sequences directed by each of them.     

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。     

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.     

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.     

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 KINETICS OF INITIAL NUCLEAR EXCHANGE IN COMPATIBLE AND NONCOMPATIBLE MATINGS OF SCHIZOPHYLLUM COMMUNE

The effect of mating-type factors on exchange of nuclei between mycelial fragments mated in liquid culture was studied. Evidence for exchange of nuclei was based on three criteria for detecting nuclei from both mating partners in mycelial fragments of the developing dikaryon or hetero-karyon. The kinetics of nuclear exchange were shown to be relatively independent of the method used. It was shown that the kinetics of nuclear exchange in the first 96 hr are different in the compatible (A41 B41 × A42 B42), common-A (A42 B42 × A42 B41), common-B (A41 B42 × A42 B42), and common-AB (A41 B41 × A41 B41) matings. In all four types of matings, the percentage of fragments possessing both types of nuclei 12-24 hr after mating is nearly equal. After this time, significant differences appear in the patterns for compatible vs. the three non-compatible matings and the common-A vs. the common-B and common-AB matings. The percentage of mycelial fragments possessing both types of nuclei throughout the 96-hr test period is similar for both the common-B and common-AB matings. The kinetics of nuclear exchange were shown to be independent of the particular mating-type alleles and nutritional markers used. When the efficiency of nuclear exchange in complete and minimal media was compared, it was shown that nuclear exchange occurred more rapidly and synchronously in minimal medium. This difference is not due to growth differences in the two media. These data indicate that the earliest mating interactions, i.e., hyphal anastomosis and nuclear exchange, are independent of the mating-type factors but that subsequent events are determined by these genes.     

毛木耳A交配型位点的克隆和序列结构分析

以毛木耳Auricularia cornea杂交子APM2-16的亲本单核体APP7和M2S16为研究材料,经同源序列比对,从单核体APP7的基因组中获得了A交配型位点序列,并定位了mip基因的位置。根据APM2-16的转录组和单核体APP7基因组的相关信息设计引物,通过PCR扩增和克隆测序的方式获得单核体APP7和M2S16中的HD基因序列,通过生物信息学分析发现两者在核苷酸序列上没有同源性,都编码同源结构域转录因子。通过对毛木耳交配型位点的研究有助于进一步开展该物种的遗传学基础研究,为菌种遗传改良奠定基础。     

Nuclear selection and nuclear substitution in fully compatible di-mon matings inPleurotus ostreatus

Nuclear behavior in fully compatible dikaryon-monokaryon matings inPleurotus ostreatus was examined with esterase isozymes as markers, which are located at the EST1 locus independent of mating type factors A and B. Monokaryotic strains mMA91 and mOW89 were obtained from the stocks MA91 from Japan and OW89 from Thailand, respectively. The mMA91 strains represented genotype EST1 (100), and the mOW89 strains represented either EST1 (86) or EST1 (93). Most of the stocks derived from mMA91×OW89 represented the genotype EST1 (93)+EST1 (100). This result suggests that the donor nucleus of OW89 carrying EST1 (93) migrated preferentially into the monokaryon mMA91 to conjugate with the recipient nucleus of the latter. Some of the stocks from mOW89 × MA91 represented the genotype EST1 (100)+EST1 (100). This seems to have resulted from substitution of a nucleus of the recipient mOW89 by a pair of nuclei of the donor MA91. The variations in mycelial products, mycelial growth rate, and wood degrading ability among the D1 populations of stocks from mMA91 × OW89 were significantly smaller than those from mOW89 × MA91, reflecting the selective nuclear migration from OW89 to mMA91.     

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.     

ISSR标记在黑木耳单核体遗传分析中的应用

采用原生质体单核化和单孢分离法分别获得黑木耳Auricularia auricula栽培菌株新科5号两个亲本单核体(T1、T2)和33个F1代孢子单核体。从73条ISSR引物中筛选出13条可区分两个亲本单核体(T1、T2)的引物,对新科5号及F1代孢子单核体进行扩增,共扩增出70条带,其中63条在供试菌株间表现出多态性,多态位点百分率为90.0%。根据扩增结果采用软件NTsys2.10e计算菌株间的遗传相似系数并进行聚类分析,36个菌株间的遗传相似系数(GS值)的变化范围为0.2500～0.8382,其中T1和T2之间的GS值最小,遗传相似程度最低;F1代33个孢子单核体中24个与T1聚为一类,其余9个与T2聚为另一类,但并非严格按照交配型进行归类。试验表明黑木耳子实体上各个担子在减数分裂中染色体交换极具多样性,F1代孢子单核体表现出偏向其中一个亲本的现象,ISSR标记在黑木耳杂交育种中具有潜在的应用价值。     

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.