The so locus is required for vegetative cell fusion and postfertilization events in Neurospora crassa

The process of cell fusion is a basic developmental feature found in most eukaryotic organisms. In filamentous fungi, cell fusion events play an important role during both vegetative growth and sexual reproduction. We employ the model organism Neurospora crassa to dissect the mechanisms of cell fusion and cell-cell communication involved in fusion processes. In this study, we characterized a mutant with a mutation in the gene so, which exhibits defects in cell fusion. The so mutant has a pleiotropic phenotype, including shortened aerial hyphae, an altered conidiation pattern, and female sterility. Using light microscopy and heterokaryon tests, the so mutant was shown to possess defects in germling and hyphal fusion. Although so produces conidial anastomosis tubes, so germlings did not home toward wild-type germlings nor were wild-type germlings attracted to so germlings. We employed a trichogyne attraction and fusion assay to determine whether the female sterility of the so mutant is caused by impaired communication or fusion failure between mating partners. so showed no defects in attraction or fusion between mating partners, indicating that so is specific for vegetative hyphal fusion and/or associated communication events. The so gene encodes a protein of unknown function, but which contains a WW domain; WW domains are predicted to be involved in protein-protein interactions. Database searches showed that so was conserved in the genomes of filamentous ascomycete fungi but was absent in ascomycete yeast and basidiomycete species.     

Intimate Relationship between mtDNA, Plasmids, and the Fusion of Mitochondria

Physarum polycephalum. The conformation of Physarum mtDNA is currently thought to be circular. The inheritance of its mtDNA depends on the multiallelic mating type loci, matA. In a cross with ordinary matA combinations, the strain that has the higher matA status transmits its mtDNA to the progeny (uniparental inheritance). The mF plasmid promotes the fusion of mitochondria in the zygote and during sporulation. When it exists in a strain with a lower status matA, the mF plasmid overcomes the force of uniparental inheritance and is preferentially transmitted to the progeny via mitochondrial fusion. Moreover, the conformation of mtDNA is changed from circular to linear by recombination with the mF plasmid. Since biparental inheritance usually occurs in a cross involving a combination of matA1 and matA15, two types of inheritance of Physarum mtDNA exist. Considering the existence of the mF plasmid, there are four patterns of cytoplasmic inheritance in P. polycephalum: 1) uniparental inheritance of mtDNA, 2) uniparental inheritance of mtDNA and preferential transmission of the mF plasmid, 3) biparental inheritance of mtDNA, and 4) biparental inheritance of mtDNA and the mF plasmid. This article describes the events involved in each pattern. Finally, we discuss a hypothetical mechanism for mitochondrial fusion. The essential protein may be the ORF640 protein encoded in the mF plasmid. Received 8 March 2000/ Accepted in revised form 23 March 2000     

Mitochondria are inherited from the MATa parent in crosses of the basidiomycete fungus Cryptococcus neoformans

Previous studies demonstrated that mitochondrial DNA (mtDNA) was uniparentally transmitted in laboratory crosses of the pathogenic yeast Cryptococcus neoformans. To begin understanding the mechanisms, this study examined the potential role of the mating-type locus on mtDNA inheritance in C. neoformans. Using existing isogenic strains (JEC20 and JEC21) that differed only at the mating-type locus and a clinical strain (CDC46) that possessed a mitochondrial genotype different from JEC20 and JEC21, we constructed strains that differed only in mating type and mitochondrial genotype. These strains were then crossed to produce hyphae and sexual spores. Among the 206 single spores analyzed from six crosses, all but one inherited mtDNA from the MATa parents. Analyses of mating-type alleles and mtDNA genotypes of natural hybrids from clinical and natural samples were consistent with the hypothesis that mtDNA is inherited from the MATa parent in C. neoformans. To distinguish two potential mechanisms, we obtained a pair of isogenic strains with different mating-type alleles, mtDNA types, and auxotrophic markers. Diploid cells from mating between these two strains were selected and 29 independent colonies were genotyped. These cells did not go through the hyphal stage or the meiotic process. All 29 colonies contained mtDNA from the MATa parent. Because no filamentation, meiosis, or spore formation was involved in generating these diploid cells, our results suggest a selective elimination of mtDNA from the MATalpha parent soon after mating. To our knowledge, this is the first demonstration that mating type controls mtDNA inheritance in fungi.     

Mitochondrial DNA in Mon-Mon and Di-Mon Pairings of Pleurotus ostreatus

Based on enzymatically amplified regions of the mitochondrial DNA (mtDNA), stock-specific markers were obtained for two stocks of Pleurotus ostreatus. A length mutation was detected within a region encoding for DNA of the mitochondrial ribosomes. The inheritance of mtDNA was uniparental both in Mon-Mon and Di-Mon interstock pairings. Replacement of the recipient mtDNA to a large extent was finished 3 to 4 months after hyphal contact of paired mycelia. Dissolution of mitochondrial mosaics was somewhat faster in Di-Mon than in Mon-Mon pairings. As became evident in Di-Mon pairings, the spread of the dominating mtDNA is independent from nuclear migration, and these processes could go in opposite directions. While in compatible Di-Mon pairings two different types of nuclear background develop with respect to the mating type factors, only one certain mtDNA phenotype remained after dissolution of the mitochondrial mosaic. The spread of the mtDNA occurred at a distinctly slower rate than nuclear migration. Three weeks after hyphal contact the dominating mtDNA had not reached the growing edge of the recipient mycelium, whereas clamp formation was noted after 11 days at the latest. The data presented in this study indicate that no apparent correlation exists between mtDNA and nuclear background.     

Rapid,selective digestion of mitochondrial DNA in accordance with the matA hierarchy of multiallelic mating types in the mitochondrial inheritance of Physarum polycephalum

Although mitochondria are inherited uniparentally in nearly all eukaryotes, the mechanism for this is unclear. When zygotes of the isogamous protist Physarum polycephalum were stained with DAPI, the fluorescence of mtDNA in half of the mitochondria decreased simultaneously to give small spots and then disappeared completely approximately 1.5 hr after nuclear fusion, while the other mitochondrial nucleoids and all of the mitochondrial sheaths remained unchanged. PCR analysis of single zygote cells confirmed that the loss was limited to mtDNA from one parent. The vacant mitochondrial sheaths were gradually eliminated by 60 hr after mating. Using six mating types, the transmission patterns of mtDNA were examined in all possible crosses. In 39 of 60 crosses, strict uniparental inheritance was confirmed in accordance with a hierarchy of relative sexuality. In the other crosses, however, mtDNA from both parents was transmitted to plasmodia. The ratio of parental mtDNA was estimated to be from 1:1 to 1:10(-4). Nevertheless, the matA hierarchy was followed. In these crosses, the mtDNA was incompletely digested, and mtDNA replicated during subsequent plasmodial development. We conclude that the rapid, selective digestion of mtDNA promotes the uniparental inheritance of mitochondria; when this fails, biparental inheritance occurs.     

Germline Transformation Using a Prune Cdna Rescues Prune/Killer of Prune Lethality and the Prune Eye Color Phenotype in Drosophila

Filamentous fungi are capable of hyphal fusion, but heterokaryon formation between different isolates is controlled by specific loci termed het loci. Heterokaryotic cells formed between strains of different het genotype are rapidly destroyed or strongly inhibited in their growth. In Neurospora crassa, at least 11 loci, including the mating type locus, affect the capacity to form a heterokaryon between different isolates. In this report, we describe the molecular characterization of the vegetative incompatibility locus, het-C. The het-C(OR) allele was cloned by genetically identifying the het-C locus in a chromosome walk, and the activity of clones containing the het-C(OR) allele was tested in a functional transformation assay. The het-C(OR) allele encodes a 966-amino acid polypeptide with a putative signal peptide, a coiled-coil motif and a C-terminal glycine-rich domain, similar to glycine-rich domains detected in various extracellular and structural cell envelope proteins. Both the coiled-coil and one-third of the glycine-rich carboxyl terminal domains were required for full het-C(OR) activity. Mutants of het-C(OR) were obtained by repeat-induced point mutation (RIP); these mutants were indistinguishable from wild type during vegetative growth and sexual reproduction but displayed dual compatibility with both of two mutually incompatible het-C(OR) and het-c(PA) strains.     

Selective sweeps of mitochondrial DNA can drive the evolution of uniparental inheritance

Although the uniparental (or maternal) inheritance of mitochondrial DNA (mtDNA) is widespread, the reasons for its evolution remain unclear. Two main hypotheses have been proposed: selection against individuals containing different mtDNAs (heteroplasmy) and selection against “selfish” mtDNA mutations. Recently, uniparental inheritance was shown to promote adaptive evolution in mtDNA, potentially providing a third hypothesis for its evolution. Here, we explore this hypothesis theoretically and ask if the accumulation of beneficial mutations provides a sufficient fitness advantage for uniparental inheritance to invade a population in which mtDNA is inherited biparentally. In a deterministic model, uniparental inheritance increases in frequency but cannot replace biparental inheritance if only a single beneficial mtDNA mutation sweeps through the population. When we allow successive selective sweeps of mtDNA, however, uniparental inheritance can replace biparental inheritance. Using a stochastic model, we show that a combination of selection and drift facilitates the fixation of uniparental inheritance (compared to a neutral trait) when there is only a single selective mtDNA sweep. When we consider multiple mtDNA sweeps in a stochastic model, uniparental inheritance becomes even more likely to replace biparental inheritance. Our findings thus suggest that selective sweeps of beneficial mtDNA haplotypes can drive the evolution of uniparental inheritance.     

Mitochondrial inheritance and the detection of non-parental mitochondrial DNA haplotypes in crosses of Agaricus bisporus homokaryons

This study evaluates mtDNA transmission in Agaricus bisporus, as well as the occurrence of non-parental haplotypes in heterokaryons produced by controlled crosses. Sixteen crosses were performed with blended liquid cultures, using different combinations of 13 homokaryotic strains. For each cross, different mtDNA haplotypes were present in each homokaryon. Heterokaryons generated from these crosses were subject to genetic analysis with RFLP markers to identify (i). karyotic status, (ii). mtDNA haplotype, and (iii). the occurrence of non-parental mtDNA haplotypes. These analyses generally supported the occurrence of uniparental mitochondrial (mt) inheritance in A. bisporus, with one mtDNA haplotype usually favoured in the new heterokaryon. The preponderance of one mtDNA haplotype in a new heterokaryon did not necessarily show a correlation with a greater mycelial growth rate for the parent homokaryon possessing that haplotype. Mixed mtDNA haplotypes and non-parental haplotypes were also identified in the heterokaryons from some crosses. Evidence for the occurrence of two mtDNA haplotypes in one heterokaryotic mycelium was observed in 8 of 16 crosses, suggesting the maintenance of true heteroplasmons after three successive subculturing steps. Non-parental mtDNA haplotypes were seen in heterokaryons produced from 7 of 16 crosses. The mating protocol described can be utilized to generate novel mtDNA haplotypes for strain improvement and the development of strain-specific markers. Mechanisms of mt selection and inheritance are discussed.     

Evolution of hierarchical cytoplasmic inheritance in the plasmodial slime mold Physarum polycephalum

A striking linear dominance relationship for uniparental mitochondrial transmission is known between many mating types of plasmodial slime mold Physarum polycephalum. We herein examine how such hierarchical cytoplasmic inheritance evolves in isogamous organisms with many self-incompatible mating types. We assume that a nuclear locus determines the mating type of gametes and that another nuclear locus controls the digestion of mitochondria DNAs (mtDNAs) of the recipient gamete after fusion. We then examine the coupled genetic dynamics for the evolution of self-incompatible mating types and biased mitochondrial transmission between them. In Physarum, a multiallelic nuclear locus matA controls both the mating type of the gametes and the selective elimination of the mtDNA in the zygotes. We theoretically examine two potential mechanisms that might be responsible for the preferential digestion of mitochondria in the zygote. In the first model, the preferential digestion of mitochondria is assumed to be the outcome of differential expression levels of a suppressor gene carried by each gamete (suppression-power model). In the second model (site-specific nuclease model), the digestion of mtDNAs is assumed to be due to their cleavage by a site-specific nuclease that cuts the mtDNA at unmethylated recognition sites. Also assumed is that the mtDNAs are methylated at the same recognition site prior to the fusion, thereby being protected against the nuclease of the same gamete, and that the suppressor alleles convey information for the recognition sequences of nuclease and methylase. In both models, we found that a linear dominance hierarchy evolves as a consequence of the buildup of a strong linkage disequilibrium between the mating-type locus and the suppressor locus, though it fails to evolve if the recombination rate between the two loci is larger than a threshold. This threshold recombination rate depends on the number of mating types and the degree of fitness reduction in the heteroplasmic zygotes. If the recombination rate is above the threshold, suppressor alleles are equally distributed in each mating type at evolutionary equilibrium. Based on the theoretical results of the site-specific nuclease model, we propose that a nested subsequence structure in the recognition sequence should underlie the linear dominance hierarchy of mitochondrial transmission.     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.     

The Effects of Natural Hybridization on the Regulation of Doubly Uniparental Mtdna Inheritance in Blue Mussels (Mytilus Spp.)

Blue mussels in the Mytilus edulis species complex have a doubly uniparental mode of mtDNA inheritance with separate maternal and paternal mtDNA lineages. Female mussels inherit their mtDNA solely from their mother, while males inherit mtDNA from both parents. In the male gonad the paternal mtDNA is preferentially replicated so that only paternal mtDNA is transmitted from fathers to sons. Hybridization is common among differentiated blue mussel taxa; whenever it involves M. trossulus, doubly uniparental mtDNA inheritance is disrupted. We have found high frequencies of males without and females with paternal mtDNA among hybrid mussels produced by interspecific matings between M. galloprovincialis and M. trossulus. In contrast, hybridization between M. galloprovincialis and M. edulis does not affect doubly uniparental inheritance, indicating a difference in the divergence of the mechanisms regulating mtDNA inheritance among the three blue mussel taxa. Our data indicate a high frequency of disrupted mtDNA transmission in F(1) hybrids and suggest that two separate mechanisms, one regulating the transmission of paternal mtDNA to males and another inhibiting the establishment of paternal mtDNA in females, act to regulate doubly uniparental inheritance. We propose a model for the regulation of doubly uniparental inheritance that is consistent with these observations.     

The a2 Mating-Type Locus Genes lga2 and rga2 Direct Uniparental Mitochondrial DNA (mtDNA) Inheritance and Constrain mtDNA Recombination During Sexual Development of Ustilago maydis

Uniparental inheritance of mitochondria dominates among sexual eukaryotes. However, little is known about the mechanisms and genetic determinants. We have investigated the role of the plant pathogen Ustilago maydis genes lga2 and rga2 in uniparental mitochondrial DNA (mtDNA) inheritance during sexual development. The lga2 and rga2 genes are specific to the a2 mating-type locus and encode small mitochondrial proteins. On the basis of identified sequence polymorphisms due to variable intron numbers in mitochondrial genotypes, we could demonstrate that lga2 and rga2 decisively influence mtDNA inheritance in matings between a1 and a2 strains. Deletion of lga2 favored biparental inheritance and generation of recombinant mtDNA molecules in combinations in which inheritance of mtDNA of the a2 partner dominated. Conversely, deletion of rga2 resulted in predominant loss of a2-specific mtDNA and favored inheritance of the a1 mtDNA. Furthermore, expression of rga2 in the a1 partner protected the associated mtDNA from elimination. Our results indicate that Lga2 in conjunction with Rga2 directs uniparental mtDNA inheritance by mediating loss of the a1-associated mtDNA. This study shows for the first time an interplay of mitochondrial proteins in regulating uniparental mtDNA inheritance.     

Maternal inheritance of mitochondria: multipolarity, multiallelism and hierarchical transmission of mitochondrial DNA in the true slime mold Physarum polycephalum

Direct evidence of digestion of paternal mitochondrial DNA (mtDNA) has been found in the true slime mold Physarum polycephalum. This is the first report on the selective digestion of mtDNA inside the zygote, and is striking evidence for the mechanism of maternal inheritance of mitochondria. Moreover, two mitochondrial nuclease activities were detected in this organism as candidates for the nucleases responsible for selective digestion of mtDNA. In the true slime mold, there is an additional feature of the uniparental inheritance of mitochondria. Although mitochondria are believed to be inherited from the maternal lineage in nearly all eukaryotes, the mating types of the true slime mold P. polycephalum is not restricted to two: there are three mating loci—matA, matB, and matC—and these loci have 16, 15, and 3 alleles, respectively. Interestingly, the transmission patterns of mtDNA are determined by the matA locus, in a hierarchical fashion (matA hierarchy) as follows: matA7 > matA2 > matA11 > matA12 > matA15/matA16 > matA1 > matA6. The strain possessing the higher status of matA would be the mtDNA donor in crosses. Furthermore, we have found that some crosses showed biparental inheritance of mitochondria. This review describes the phenomenon of hierarchical transmission of mtDNA in true slime molds, and discusses the presumed molecular mechanism of maternal and biparental inheritance.     

Doubly uniparental inheritance of mitochondrial DNA: Might it be simpler than we thought?

More than 100 species of bivalve mollusks are currently known to carry two highly diverged mitochondrial DNA (mtDNA) molecules, one of which is transmitted through the egg and the other through the sperm generation after generation, faithfully and uninterruptedly. This mtDNA system, which has become known as doubly uniparental inheritance (DUI), is most likely unique in eukaryotes and constitutes a striking deviation from the strictly maternal inheritance (SMI) of mtDNA that is the rule in the animal kingdom. Here, I present a model of how the paternal mtDNA may escape the mitochondrial destruction that occurs prior to sperm formation and enter the male germ line in the newly formed embryo. In essence, the model treats the sperm-transmitted mtDNA as a molecule that takes a ride with the sperm. The model can be easily tested and, if passed the tests, may open the way for the understanding of DUI at the molecular level and throw light on the mechanisms and evolution of mtDNA transmission in general. In addition, the model shifts attention from nuclear control of paternal mtDNA inheritance, whether systematic (as DUI) or leaky (as the cases reported in a wide variety of animal species), to the mtDNA itself as the protagonist of its own transmission. This possibility has been, so far, ignored in studies of paternal mtDNA transmission in other species including humans.     

Differential segregation patterns of sperm mitochondria in embryos of the blue mussel (Mytilus edulis)

In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two- and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.     

Uniparental Inheritance of Mitochondrial Genes in Yeast: Dependence on Input Bias of Mitochondrial DNA and Preliminary Investigations of the Mechanism

In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes, which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsp5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These classes of models are distinguished genetically by the fact that the first predicts that the output frequency of a given allele among the progeny of a large number of zygotes will approximately equal the average input frequency of that allele, while the second class predicts that any input bias will be amplified in the output. The data suggest that bias amplification does occur. We hypothesize that maternal inheritance of mitochondrial or chloroplast genes in many organisms may depend upon a biased input of organelle DNA molecules, which usually favors the maternal parent, followed by failure of the minority (paternal) molecules to replicate in many or all zygotes.     

What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations

The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is relaxed. Furthermore, because mtDNA replication is not strictly regulated, within-cell selection may favour mtDNA variants with a replication advantage, but a deleterious effect on cell fitness. The opportunities for selfish mtDNA mutations to spread are restricted by various organism-level adaptations, such as uniparental transmission, germline mtDNA bottlenecks, germline selection and, during somatic growth, regular alternation between fusion and fission of mitochondria. These mechanisms are all hypothesized to maintain functional mtDNA. However, the strength of selection for maintenance of functional mtDNA progressively declines with age, resulting in age-related diseases. Furthermore, organismal adaptations that most probably evolved to restrict the opportunities for selfish mtDNA create secondary problems. Owing to predominantly maternal mtDNA transmission, recombination among mtDNA from different individuals is highly restricted or absent, reducing the scope for repair. Moreover, maternal inheritance precludes selection against mtDNA variants with male-specific effects. We finish by discussing the consequences of life-history differences among taxa with respect to mtDNA evolution and make a case for the use of microorganisms to experimentally manipulate levels of selection.     

Paternal inheritance of mitochondria in Chlamydomonas

To analyze mitochondrial DNA (mtDNA) inheritance, differences in mtDNA between Chlamydomonas reinhardtii and Chlamydomonas smithii, respiration deficiency and antibiotic resistance were used to distinguish mtDNA origins. The analyses indicated paternal inheritance. However, these experiments raised questions regarding whether paternal inheritance occurred normally. Mitochondrial nucleoids were observed in living zygotes from mating until 3 days after mating and then until progeny formation. However, selective disappearance of nucleoids was not observed. Subsequently, experimental serial backcrosses between the two strains demonstrated strict paternal inheritance. The fate of mt+ and mt− mtDNA was followed using the differences in mtDNA between the two strains. The slow elimination of mt+ mtDNA through zygote maturation in darkness was observed, and later the disappearance of mt+ mtDNA was observed at the beginning of meiosis. To explain the different fates of mtDNA, methylation status was investigated; however, no methylation was detected. Variously constructed diploid cells showed biparental inheritance. Thus, when the mating process occurs normally, paternal inheritance occurs. Mutations disrupting mtDNA inheritance have not yet been isolated. Mutations that disrupt maternal inheritance of chloroplast DNA (cpDNA) do not disrupt inheritance of mtDNA. The genes responsible for mtDNA inheritance are different from those of chloroplasts.