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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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 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 in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora , with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. 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 genus 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 described as genus Neocosmospora were recombined in genus 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.

Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unlabelled: 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 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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.

Mitochondrial DNA rearrangements and deletions are a prevailing feature of filamentous fungal cultures that undergo senescence. In Neurospora spp., strains containing the Mauriceville and Varkud mitochondrial retroplasmids routinely senesce at elevated temperatures, a process that is initiated by the integration of variant forms of the plasmids into the mitochondrial genome. Here, we describe a strain that is phenotypically distinguishable from previously characterized senescent strains and show that senescence can occur in the absence of plasmid integration and associated alterations in mitochondrial DNA. The MS4416 strain contains a unique variant of the Mauriceville retroplasmid, and undergoes senescence at highly predictable frequencies at 37 degrees, 25 degrees and 18 degrees C. Decline in vegetative growth rate correlates with increased levels of the variant plasmid and alterations in the synthesis of mitochondrially encoded proteins, suggesting that plasmid over-replication interferes with mitochondrial translation. We also report the isolation of a mutant strain that escapes senescence yet still maintains high levels of the variant plasmid. Its ability to tolerate a growth-suppressive retroplasmid suggests that there are mechanisms in Neurospora which compensate for the deleterious effects that plasmid over-replication has on mitochondrial function.

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 nearby flanking 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 Zn2+ 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. A cis-dominant splicing-defective mutant of aI2 continues to synthesize cDNAs containing the introns but is completely defective in both 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.

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.

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 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.

The mitochondrial gene T-urf13 in T cytoplasm maize is associated with sensitivity to disease toxins and with cytoplasmic male sterility. T-urf13 is co-transcribed with an open reading frame designated orf221. We have detected alterations in the transcription of the T-urf13/orf221 region that are affected by nuclear genotype. There are multiple mRNA transcripts generated from the T-urf13/orf221 region, one of which is a processed 1538-nucleotide (nt) transcript. This 1538-nt transcript is present in Wf9 (T), but was not found in mitochondrial RNAs (mtRNAs) from maize inbreds B14A (T) and 33-16 (T). For B14A (T) a 1500-nt transcript was detected and for 33-16 (T) a 1400-nt transcript was detected. In F1 progeny of the cross of Wf9 (T) x 33-16 (N), only the 1400-nt transcript was present. Genetic analyses revealed this processing event is nuclear controlled with dominant gene action and is independent of nuclear restorer gene Rf1-associated processing events. T-urf13/orf221 transcriptional patterns were shown to vary in both sterile and fertile states. Segregation analysis of a 1100-nt orf221-specific transcript indicated that the genetic basis of nuclear control for the presence of this transcript was relatively simple. Analysis of the A188 (T4) tissue culture mutant, which has reverted to male fertility but displays the same T-urf13/orf221 transcript pattern as A188 (T), indicated no DNA sequence differences between T4-orf221 and T-orf221. Presence of the nuclear gene Rf2 was not necessary for expression of the T4 cytoplasm-associated malefertile phenotype.

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.

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.

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.