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International audience; Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. Previously (Geiser et al. 2013; Phytopathology 103:400-408. 2013), the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani Species Complex (FSSC). Subsequently, this concept was challenged by one research group (Lombard et al. 2015 Studies in Mycology 80: 189-245) who proposed dividing Fusarium into seven genera, including the FSSC as the genus Neocosmospora, with subsequent justification based on claims that the Geiser et al. (2013) concept of Fusarium is polyphyletic (Sandoval-Denis et al. 2018; Persoonia 41:109-129). Here we test this claim, and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species recently described as Neocosmospora were recombined in Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural and practical taxonomic option available.

Isolation of purified mitochondria is an essential technique for the analysis of metabolic and cellular functions associated with this vital organelle. Filamentous fungi, such as Neurospora crassa, have been proven to be highly amenable to the analysis of mitochondria, in part due to their rapid growth rate and relative ease of isolation. Here we describe a step-by-step procedure for the isolation of mitochondria from Fusarium species via differential centrifugation and density step-gradient centrifugation, and include methods to overcome potential complications. Mitochondria purified by flotation gradient procedures remain active for functional assays and can be further fractionated for isolation of nucleic acids or ribonucleoprotein particles that retain enzymatic activity.

Isolation of purified mitochondria is an essential technique for the analysis of metabolic and cellular functions associated with this vital organelle. Filamentous fungi, such as Neurospora crassa, have been proven to be highly amenable to the analysis of mitochondria, in part due to their rapid growth rate and relative ease of isolation. Here we describe a step-by-step procedure for the isolation of mitochondria from Fusarium species via differential centrifugation and density step-gradient centrifugation, and include methods to overcome potential complications. Mitochondria purified by flotation gradient procedures remain active for functional assays and can be further fractionated for isolation of nucleic acids or ribonucleoprotein particles that retain enzymatic activity.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the

Mitochondrial genome variation and its effects on phenotypes have been widely analyzed in higher eukaryotes but less so in the model eukaryote Saccharomyces cerevisiae. Here, we describe mitochondrial genome variation in 96 diverse S. cerevisiae strains and assess associations between mitochondrial genotype and phenotypes as well as nuclear-mitochondrial epistasis. We associate sensitivity to the ATP synthase inhibitor oligomycin with SNPs in the mitochondrially encoded ATP6 gene. We describe the use of iso-nuclear F1 pairs, the mitochondrial genome equivalent of reciprocal hemizygosity analysis, to identify and analyze mitochondrial genotype-dependent phenotypes. Using iso-nuclear F1 pairs, we analyze the oligomycin phenotype-ATP6 association and find extensive nuclear-mitochondrial epistasis. Similarly, in iso-nuclear F1 pairs, we identify many additional mitochondrial genotype-dependent respiration phenotypes, for which there was no association in the 96 strains, and again find extensive nuclear-mitochondrial epistasis that likely contributes to the lack of association in the 96 strains. Finally, in iso-nuclear F1 pairs, we identify novel mitochondrial genotype-dependent nonrespiration phenotypes: resistance to cycloheximide, ketoconazole, and copper. We discuss potential mechanisms and the implications of mitochondrial genotype and of nuclear-mitochondrial epistasis effects on respiratory and nonrespiratory quantitative traits.

The mitochondrial (mt) genomes of Fusarium verticillioides, Fusarium solani and Fusarium graminearum were annotated and found to be 53.7, 63.0 and 95.7 kb in length, respectively. The genomes encode all genes typically associated with mtDNAs of filamentous fungi yet are considerably larger than the mt genome of F. oxysporum. Size differences are largely due to the number of group I introns. Surprisingly, the genomes contain a highly variable region of 7-9 kb that encodes an exceptionally large, unidentified open reading frame (uORF). The region has the hallmarks of a horizontally transmitted DNA and was likely acquired prior to the divergence of Fusarium species. Two additional uORFs were detected that are also under positive selection. DNA repeats associated with the uORFs suggest that 3' gene duplication may be an adaptive mechanism to modify coding regions or generate new ORFs. The acquisition of these new genes contrasts to the wide-scale size reduction experienced by fungal mt genomes.

The pFOXC mitochondrial retroplasmids are small, autonomously replicating linear DNAs that have a telomere-like repeat of a 5-bp sequence at their termini. The plasmids are possible evolutionary precursors of the ribonucleoprotein complex telomerase, as they encode an active reverse transcriptase (RT) that is involved in plasmid replication. Using an in vitro system to study reverse transcription, we show that the pFOXC RT is capable of copying in vitro-synthesized RNAs by use of cDNA primers or extension of snapped-back RNA templates. The ability of the pFOXC RT to use base-paired primers distinguishes it from the closely related RTs encoded by the Mauriceville and Varkud mitochondrial retroplasmids of Neurospora spp. Reaction products are similar, but not identical, to those obtained with conventional RTs, and differences reflect the ability of the pFOXC RT to initiate cDNA synthesis with loosely associated primers. The pFOXC RT can also copy DNA templates and extend 3′ mismatched DNA oligonucleotide primers. Analysis of pFOXC in vivo replication intermediates suggests that telomeric repeats are added during reverse transcription, and the ability to extend loosely associated primers could play a role in repeat formation by mechanisms similar to those associated with telomerase and certain non-long-terminal-repeat retrotransposons.

This chapter covers the functions of mitochondria in filamentous fungi. Mitochondrial respiration occurs via transfer of electrons from reduced electron carriers to molecular oxygen. This is accomplished using an electron transport chain housed in the mitochondrial inner membrane (MIM). Fungal mitochondrial genomes are AT-rich DNAs that map as circular molecules. However, several studies suggest that fungal mtDNAs are actually long, concatenated linear molecules. The study of fungal mitochondrial introns has been an active area of research that has yielded a number of significant findings. There are numerous reports of RNA elements being associated with mitochondria. Though they are formally classified as mitoviruses, they can also be considered plasmids based on their structural similarities to linear mt plasmids and lack of an extracellular form. The large number of different proteins that are found in mitochondria (probably about 1,000 for fungi) and the limited coding capacity of mtDNA emphasize the fact that most mitochondrial proteins are encoded by nuclear genes, translated in the cytosol, and imported into the organelle. The chapter attempts to define the basic import process and summarizes the literature while pointing out contributions that have involved filamentous fungi-almost entirely N. crassa. Filamentous fungi are generally considered immortal. Podospora anserina is an exception to this rule, as it has a defined vegetative- growth life span.

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum , a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e.g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci., IMPORTANCE Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

The mitochondria of certain natural isolates of Neurospora contain both the Varkud plasmid, which encodes a reverse transcriptase, and a small unrelated RNA (VS RNA) that performs RNA-mediated self-cleavage and ligation reactions. Here, we show that VS RNA is transcribed from a VS plasmid DNA template by the Neurospora mitochondrial RNA polymerase using a promoter located immediately upstream of the RNA self-cleavage site that generates monomeric transcripts. VS RNA is then reverse transcribed by the Varkud plasmid reverse transcriptase to yield a full-length (-) strand cDNA, a predicted replication intermediate. Combined with previous genetic evidence, our results indicate that the VS plasmid replicates by reverse transcription as a satellite of the Varkud plasmid. This mode of replication, unprecedented for a satellite RNA, likely reflects the promiscuity of the Varkud plasmid reverse transcriptase, which does not require a specific primer to initiate cDNA synthesis. Our findings indicate how primitive reverse transcriptases with similar relaxed specificity could have facilitated the evolution of new retroelements.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.

The Mauriceville plasmid and the closely related Varkud plasmid of Neurospora spp. are retroelements that propagate in mitochondria. Replication appears to occur by a novel mechanism in which a monomer-length plasmid transcript having a 3' tRNA-like structure ending in CCA is reverse transcribed to give a full-length minus-strand cDNA beginning at or near the 3' end of the RNA. Here, we show that the plasmids are transcribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site approximately 260 bp upstream of the 5' end of the plasmid transcript. The location of the transcription start site suggests that the monomer-length transcripts are generated by transcription around the plasmid combined with a site-specific RNA cleavage after the 3'-CCA sequence. The 5' ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A major class of minus-strand cDNAs begins opposite C2 of the 3'-CCA sequence, the same site used for de novo initiation of cDNA synthesis by the plasmid reverse transcriptase in vitro. A second class of minus-strand cDNAs begins with putative primer sequences that correspond to cDNA copies of the plasmid or mitochondrial transcripts. These findings are consistent with the possibility that the plasmid reverse transcriptase initiates minus-strand cDNA synthesis in vivo both by de novo initiation and by a novel template-switching mechanism in which the 3' OH of a previously synthesized cDNA is used to prime the synthesis of a new minus-strand cDNA directly at the 3' end of the plasmid transcript.

Background The pFOXC retroplasmids are small, autonomously replicating DNA molecules found in mitochondria of certain strains of the filamentous fungus Fusarium oxysporum and are among the first linear genetic elements shown to replicate via reverse transcription. The plasmids have a unique clothespin structure that includes a 5'-linked protein and telomere-like terminal repeats, with pFOXC2 and pFOXC3 having iterative copies of a 5 bp sequence. The plasmids contain a single large open reading frame (ORF) encoding an active reverse transcriptase (RT). The pFOXC-RT is associated with the plasmid transcript in a ribonucleoprotein (RNP) complex and can synthesize full-length (-) strand cDNA products. In reactions containing partially purified RT preparations with exogenous RNAs, the pFOXC3-RT has been shown to initiate cDNA synthesis by use of snapped-back RNAs, as well as loosely associated DNA primers. Results The complete sequence of the distantly related pFOXC1 plasmid was determined and found to terminate in 3-5 copies of a 3 bp sequence. Unexpectedly, the majority of (-) strand cDNA molecules produced from endogenous pFOXC1 transcripts were attached to protein. In vitro experiments using partially purified pFOXC3-RT preparations having a single radiolabeled deoxyribonucleotide triphosphate (dNTP) generated a nucleotide-labeled protein that migrated at the size of the pFOXC-RT. The nucleotide preference of deoxynucleotidylation differed between pFOXC3 and pFOXC1 and showed complementarity to the respective 3' terminal repeats. In reactions that include exogenous RNA templates corresponding to the 3' end of pFOXC1, a protein-linked cDNA product was generated following deoxynucleotidylation, suggesting that reverse transcription initiates with a protein primer. Conclusions The finding that reverse transcription is protein primed suggests the pFOXC retroplasmids may have an evolutionary relationship with hepadnaviruses, the only other retroelement family known to initiate reverse transcription via a protein primer. Moreover, the similarity to protein-primed linear DNA elements supports models in which the terminal repeats are generated and maintained by a DNA slideback mechanism. The ability of the pFOXC-RT to utilize RNA, DNA and protein primers is unique among polymerases and suggests that the pFOXC plasmids may be evolutionary precursors of a broad range of retroelements, including hepadnaviruses, non-long terminal repeat (non-LTR) retrotransposons and telomerase.

Group II introns al1 and al2 of the yeast mtDNA cox1 gene encode reverse transcriptase-like proteins that function in RNA splicing and may play a role in intron mobility and excision. We find that ribonucleoprotein particles from yeast mitochondria contain a reverse transcriptase activity that is likely encoded by al1 and al2 and is highly specific for the introns and their flanking exons. Using a mutant strain with elevated activity, we show that the reverse transcriptase uses either excised intron RNA or cox1 pre-mRNA as template and initiates cDNA synthesis near the 3′ end of al2 and immediately downstream in E3. Our results suggest that introns al1 and al2 are retroelements, which encode reverse transcriptases that have adapted to function in RNA splicing.

The LaBelle mitochondrial plasmid hybridizes to a small region of the mtDNA of different Neurospora species. Here, we show that the region of homology encompasses 1385 bp of plasmid sequence and 1649 bp of mtDNA sequence. Several findings--that the region of homology is not found in the mtDNAs of other organisms, that it includes the C-terminus of the ORF encoding the plasmid DNA polymerase, and that the ORF sequence in the mtDNA is interrupted by insertions--suggest that the region was part of the plasmid that integrated into mtDNA prior to the divergence of Neurospora species. Since the LaBelle plasmid has been found in only one Neurospora strain, we infer that the plasmid was lost subsequently from most strains. The LaBelle plasmid is transcribed by the host Neurospora mitochondrial RNA polymerase and the major promoter is located upstream of the long ORF, within the region of homology to mtDNA. A promoter used for the transcription of the mitochondrial small rRNA is found at a corresponding position in Neurospora mtDNA and may have been acquired via integration of the plasmid sequence. Our results provide evidence that an autonomous infectious element may contribute to sequences that functionally constitute an organism's mtDNA.

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the “Fusarium solani species complex”. Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches., Author Summary Nectria haematococca MPVI occurs as a saprophyte in diverse habitats and as a plant and animal pathogen. It also was the first fungus shown to contain supernumerary chromosomes with unique habitat-defining genes. The current study reveals that it has one of the largest fungal genomes (15,707 genes), which may be related to its habitat diversity, and describes two additional supernumerary chromosomes. Two classes of genes were identified that have contributed to gene expansion: 1) specific genes that are not found in other fungi, and 2) genes that are present as multiple copies in N. haematococca but commonly occur as a single copy in other fungi. Some of these genes have properties suggesting their acquisition by horizontal gene transfer. We show that the three supernumerary chromosomes are different from the normal chromosomes; they contain more repeat sequences, are particularly enriched in unique and duplicated genes, and have a lower G+C content. Additionally, the biochemical functions of genes on these chromosomes suggest they may be involved in niche adaptation. The dispensable nature and possession of habitat-determining genes by these chromosomes make them the biological equivalent of bacterial plasmids. We believe they contribute to microbial diversity and have been overlooked in models of fungal evolution.

Using an in vitro transcription assay, we previously identified promoters in Neurospora mtDNA at the 5' ends of the genes encoding the mitochondrial (mt) small and large rRNAs and cob pre-mRNA. Here, we identified two additional promoters in mtDNA restriction fragment EcoRI-6, 3.8 and 5.5 kilobases upstream of the 5' end of the mt small rRNA. By comparing the two new promoters with the three identified previously, we derived a modified promoter consensus sequence (AT-rich)15-27TTAG(A/T)RR(G/T)(G/C)N(A/T). The mt small rRNA in Neurospora is transcribed from at least two promoters, a major promoter at the 5' end of the small rRNA and one or both of the newly identified promoters in EcoRI-6. The latter gives a series of putative pre-rRNAs that contain 5' end extensions of various sizes. The 5' ends of a number of these RNAs map at or near hairpin structures. The [poky] mutant, which is grossly deficient in the mt small rRNA, has a 4-base pair deletion in the major promoter at the 5' end of the mt small rRNA. The residual small rRNAs in [poky] appear to be synthesized via the upstream promoter(s), but are missing 37-44 nucleotides from their 5' ends, indicating either that pre-rRNAs are processed abnormally or that abnormal 5' RNA ends are unstable. The effect of the promoter mutation in [poky] on other transcripts suggests that the mt small rRNA is cotranscribed with downstream genes encoding tRNAs, coIII and ND6. Seven nonallelic nuclear suppressors of [poky] result in increased concentrations of the mt small rRNA and pre-rRNAs, but do not restore the ability to synthesize small rRNAs having the correct 5' ends. The suppressor mutations could act by increasing transcription, processing, or stability of the mt small rRNA or its precursors. The suppressors provide a genetic approach for identifying components that affect transcription and processing of the mt small rRNA.

Retroplasmids are a unique group of genetic elements that replicate via reverse transcription. They are small, double-stranded DNAs that reside in mitochondria of certain filamentous fungal species. Retroplasmids are divided into two groups based on structure – circular and linear – and each group has distinct replication mechanisms. The reverse transcriptases (RTs) encoded by retroplasmids are deeply rooted in RT phylogeny and studies of retroplasmid replication have revealed unique mechanisms of reverse transcription that collectively suggest that retroplasmids are related to primitive genetic elements. Here, we review the types of retroplasmids that have been reported to date and focus on the studies of the replication cycle of the Mauriceville plasmid of Neurospora crassa, a prototype of circular retroplasmids, and studies of the pFOXC plasmids of Fusarium oxysporum, which are the first linear genetic elements shown to replicate via reverse transcription. The structural and mechanistic features of circular and linear retroplasmids suggest that at some point they could have been common ancestors of all known contemporary elements that replicate via reverse transcription and may hold clues to events associated with the transition of the RNA to DNA/protein world. While most mitochondrial plasmids appear to be benign, circular retroplasmids can integrate into mitochondrial DNAs and cause senescence. The significance of host–retroplasmid interactions are discussed in regard to the potential influence retroplasmids may have had on the evolution of mitochondrial DNAs and eukaryotic cells in general.

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Mitochondrial plasmids are autonomously replicating genetic elements commonly associated with fungal and plant species. Analysis of several plant and fungal mitochondrial genomes has revealed regions that show significant homology to mitochondrial plasmids, suggesting that plasmids have had a long-term association with their mitochondrial hosts. To assess the degree to which plasmids have invaded fungal mitochondrial genomes, BLAST search parameters were modified to identify plasmid sequences within highly AT-rich mtDNAs, and output data were parsed by E value, score, and sequence complexity. High scoring hits were evaluated for the presence of shared repetitive elements and location within plasmids and mtDNAs. Our searches revealed multiple sites of sequence similarity to four distinct plasmids in the wild-type mtDNA of Neurospora crassa, which collectively comprise more than 2% of the mitochondrial genome. Regions of plasmid similarity were not restricted to plasmids known to be associated with senescence, indicating that all mt plasmids can potentially integrate into mitochondrial DNA. Unexpectedly, plasmid-related sequences were found to be clustered in regions that have disproportionately low numbers of PstI palindromic sequences, suggesting that these repetitive elements may play a role in eliminating foreign DNA. A separate class of GC-rich palindromes was identified that appear to be mobile, as indicated by their occurrence within regions of plasmid homology. Sites of sequence similarity to mitochondrial plasmids were also detected in other filamentous fungi, but to a lesser degree. The tools developed here will be useful in assessing the contribution plasmids have made to mitochondrial function and in understanding the co-evolution of mitochondrial plasmids and their hosts.

Diverse types of linear RNA and DNA autonomously replicating genetic elements exist in prokaryotic and eukaryotic hosts, yet linear elements that replicate by reverse transcription have not been identified. Here, we report the sequence and organization of two linear mitochondrial plasmids of the fungal plant pathogen F. oxysporum and the characterization of a plasmid-associated reverse transcriptase activity. Plasmids pFOXC2 and pFOXC3 are 1.9 kb in length and have a "clothespin" genomic structure, which includes a terminal hairpin and a telomere-like iteration of a 5 bp sequence at the other terminus. The retroplasmid replication cycle involves novel strategies for copying terminal sequences, which may provide clues concerning the origin of telomerase as well as the evolution of linear DNAs.

Group II introns aI1 and aI2 of the yeast mitochondrial COXI gene are mobile elements that encode an intron-specific reverse transcriptase (RT) activity. We show here that the introns of Saccharomyces cerevisiae ID41-6/161 insert site specifically into intronless alleles. The mobility is accompanied by efficient, but highly asymmetric, coconversion of nearbyflanking exon sequences. Analysis of mutants shows that the aI2 protein is required for the mobility of both aI1 and aI2. Efficient mobility is dependent on both the RT activity of the aI2-encoded protein and a separate function, a putative DNA endonuclease, that is associated with the Zn 21 finger-like region of the intron reading frame. Surprisingly, there appear to be two mobility modes: the major one involves cDNAs reverse transcribed from unspliced precursor RNA; the minor one, observed in two mutants lacking detectable RT activity, appears to involve DNA level recombination. Acis-dominant splicingdefectivemutantofaI2continuestosynthesizecDNAscontainingtheintronsbutiscompletelydefectiveinboth mobility modes, indicating that the splicing or the structure of the intron is required. Our results demonstrate that the yeast group II intron aI2 is a retroelement that uses novel mobility mechanisms. Some group I and group II introns are mobile genetic elements. Mobile group I introns are widespread, being found in organelle, bacteriophage, and nuclear genomes (reviewed in reference25).ThebasicmechanismofgroupIintronmobility, or homing, appears to be the same in all cases examined. Each mobile group I intron encodes a site-specific DNA endonuclease that cleaves intronless alleles, initiating intron movement by a double-strand DNA break gap repair process (47, 53). As a consequence of this process, there is extensive coconversion of markers flanking the mobile intron, often extending up to 500 bp on either side of the intron insertion site (25). Neither splicing nor the structure of the intron plays a role in group I intron mobility (4).

The Mauriceville plasmid and the closely related Varkud plasmid of Neurospora spp. are retroelements that propagate in mitochondria. Replication appears to occur by a novel mechanism in which a monomer-length plasmid transcript having a 3' tRNA-like structure ending in CCA is reverse transcribed to give a full-length minus-strand cDNA beginning at or near the 3' end of the RNA. Here, we show that the plasmids are transcribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site approximately 260 bp upstream of the 5' end of the plasmid transcript. The location of the transcription start site suggests that the monomer-length transcripts are generated by transcription around the plasmid combined with a site-specific RNA cleavage after the 3'-CCA sequence. The 5' ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A major class of minus-strand cDNAs begins opposite C2 of the 3'-CCA sequence, the same site used for de novo initiation of cDNA synthesis by the plasmid reverse transcriptase in vitro. A second class of minus-strand cDNAs begins with putative primer sequences that correspond to cDNA copies of the plasmid or mitochondrial transcripts. These findings are consistent with the possibility that the plasmid reverse transcriptase initiates minus-strand cDNA synthesis in vivo both by de novo initiation and by a novel template-switching mechanism in which the 3' OH of a previously synthesized cDNA is used to prime the synthesis of a new minus-strand cDNA directly at the 3' end of the plasmid transcript.

The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3' end of the plasmid transcript, which has a 3' tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.

Megasporogenesis and megagametogenesis were examined in a male-sterile, female-fertile mutant (ms1) of soybean (Glycine max (L.) Merr.). Multinucleate (more than eight) megagametophytes resulted from failure of postmeiotic cytokinesis that leads to a coenomegaspore of four haploid nuclei. Fusion of some nuclei may occur. Subsequent development results in mature megagametophytes with up to four eggs, or often abortion. These results support field data that show the male-sterile ms1 gene is associated with increased frequencies of polyembryony, polyploidy, haploidy, and reduced female fertility.Key words: Glycine max, female sterility, polyembryony, polyploidy, megasporogenesis, megagametogenesis.

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

Megasporogenesis and megagametogenesis were examined in Glycine max with light, fluorescence, and electron microscopy. Megasporogenesis results in a lineartetrad offour megaspores. Megagametophyte development is ofthe Polygonum type, with the functional chalazal megaspore undergoing three successive mitotic divisions to produce an eight-nucleate, seven-celled mature megagametophyte. The central cell becomes packed with starch. At fertilization, the antipodals are degenerate, the polar nuclei have fused, starch is diminished, and the egg occupies most of the micropylarportion ofthe megagametophyte. Several pollen tubes were occasionally observed at each micropyle, yet only one was involved in fertilization. Pollen tube entry occurs through a slightly reduced, viable synergid cell. Endosperm development precedes embryo growth. These results describing normal development allow important comparison with genetic mutants of soybean that affect female fertility. In the interest of comparing the influence of male-sterile genes on female fertility in certain genetic mutants of soybean (Kennell and Horner, 1985), it was important to document normal development using procedures that provided optimum preservation and resolution, as has been demonstrated with other species (e.g., maize; Russell, 1978). The present paper reports cytological and anatomical aspects of the soybean megagametophyte from the archesporial cell to the young embryo by utilizing an ovule-clearing technique and resin-embedded material. The latter material also allows for ultrastructural observations by using a reembedding procedure of selected thick sections. MATERIALS AND METHODS Plants of Glycine max (L.) Merr. (genetic type collection numbers T260H, T266H, T267H, and T268H) were grown in the field, glasshouse, or growth chamber. Whole-ovule clearings-Gynoecia were dissected from flower buds and fixed in chromium-acetic acid-formaldehyde overnight. The gynoecia were sliced open to expose the ovules, bulk stained with Mayer's Hemalum for 2030 min, and destained in 2% acetic acid for 2 min. Dehydration was carried out in an ethanol (EtOH) series to 100% EtOH and gradually replaced with methyl salicylate (Stelly et al., 1984). Ovules then were removed from the gynoecia and mounted on Raj slides (Raj and Herr, 1970) in methyl salicylate. Observations and photomicrographs were made by using interference-contrast optics. HAVING UNDERGONE a sevenfold production increase in the past 40 yr, soybeans (Glycine max L. Merr.) are currently second in value amongallU.S. crops (Anonymous, 1984). Their increased importance has led to a great number of genetic and physiological studies; however, few reports exist concerning the morphological and anatomical nature of the soybean flower particularly the female reproductive system. There are relatively few studies characterizing the various developmental stages of the soybean gynoecium (Table 1). These studies employed diffierent procedures associated with microscopy. The first and most complete study, including both megasporogenesis and megagametogenesis, was by Pamplin (1963), but these observations of paraffin-embedded material were never published. Other studies (Table 1) have concentrated on a particular stage or were limited by the resolution associated with paraffin-embedded material (Prakash and Chan, 1976). 1 Received for publication 23 November 1984; revision accepted 7 May 1985. Journal Paper No. J11688 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2539. This study was supported by a USDA Competitive Grant 59-2191 1 1 -760-0 awarded to H. T. Horner and R. G. Palmer, and the work was carried out in the Bessey Microscopy Facility. The authors thank Drs. R. A. Graybosch, N. R. Lersten, and R. G. Palmer for their helpful criticisms of the manuscript. We also thank Lillian Johnson for typing the manuscript. Address all correspondence to: Professor H. T. Horner Department of Botany, 3A Bessey Hall, Iowa State University, Ames, IA 50011; telephone 515/294-8635 or -3872. 2 Present address: Department of Plant Pathology, University of Florida, Gainesville, FL 32611.

The mitochondrial gene T-urf13 is implicated in cytoplasmic male sterility and maternal inheritance of susceptibility to disease in T cytoplasm maize. The organization of the T-urf13 region is a consequence of multiple recombinational events, one of which has duplicated a 5 kb region that is 5′ to atp6. Transcription initiation and processing sites for atp6 were identified within the 5 kb region and found to serve T-urf13 and the cotranscribed gene ORF221. Differences in atp6 transcripts from N and T cytoplasms were associated with two small insertions in the T mitochondrial genome that create a unique processing site. Fertility restoration is associated with alterations of T-urf13 transcripts and a significant reduction in the gene product, a 13 kDa polypeptide. Specifically, the abundance of major T-urf13 transcripts is slightly reduced and a novel transcript is detected in T cytoplasm plants containing the dominant nuclear restorer gene Rf1. The 5′ terminus of the restorer-specific transcript mapped internal to T-urf13 and the reduction of major transcripts in plants with Rf1 is not as marked as the reduction in the 13 kDa polypeptide. This raises the possibility that the primary action of the Rf1 gene is associated with translational control, rather than RNA processing.