Survey of Early-Diverging Lineages of Fungi Reveals Abundant and Diverse Mycoviruses.

Mycoviruses are widespread and purportedly common throughout the fungal kingdom, although most are known from hosts in the two most recently diverged phyla, Ascomycota and Basidiomycota, together called Dikarya. To augment our knowledge of mycovirus prevalence and diversity in underexplored fungi, we conducted a large-scale survey of fungi in the earlier-diverging lineages, using both culture-based and transcriptome-mining approaches to search for RNA viruses. In total, 21.6% of 333 isolates were positive for RNA mycoviruses. This is a greater proportion than expected based on previous taxonomically broad mycovirus surveys and is suggestive of a strong phylogenetic component to mycoviral infection. Our newly found viral sequences are diverse, composed of double-stranded RNA, positive-sense single-stranded RNA (ssRNA), and negative-sense ssRNA genomes and include novel lineages lacking representation in the public databases. These identified viruses could be classified into 2 orders, 5 families, and 5 genera; however, half of the viruses remain taxonomically unassigned. Further, we identified a lineage of virus-like sequences in the genomes of members of Phycomycetaceae and Mortierellales that appear to be novel genes derived from integration of a viral RNA-dependent RNA polymerase gene. The two screening methods largely agreed in their detection of viruses; thus, we suggest that the culture-based assay is a cost-effective means to quickly assess whether a laboratory culture is virally infected. This study used culture collections and publicly available transcriptomes to demonstrate that mycoviruses are abundant in laboratory cultures of early-diverging fungal lineages. The function and diversity of mycoviruses found here will help guide future studies into mycovirus origins and ecological functions.IMPORTANCE Viruses are key drivers of evolution and ecosystem function and are increasingly recognized as symbionts of fungi. Fungi in early-diverging lineages are widespread, ecologically important, and comprise the majority of the phylogenetic diversity of the kingdom. Viruses infecting early-diverging lineages of fungi have been almost entirely unstudied. In this study, we screened fungi for viruses by two alternative approaches: a classic culture-based method and by transcriptome-mining. The results of our large-scale survey demonstrate that early-diverging lineages have higher infection rates than have been previously reported in other fungal taxa and that laboratory strains worldwide are host to infections, the implications of which are unknown. The function and diversity of mycoviruses found in these basal fungal lineages will help guide future studies into mycovirus origins and their evolutionary ramifications and ecological impacts.

Photomorphogenesis in Phycomyces: differential display of gene expression by PCR with arbitrary primers.

The zygomycete fungus Phycomyces blakesleeanus develops two types of fruiting bodies of very different size, macrophores and microphores. Blue light stimulates macrophorogenesis and inhibits microphorogenesis. I have adapted a method based on the polymerase chain reaction with arbitrary primers to investigate the role of differential gene expression during photophorogenesis in Phycomyces. Several cDNAs for genes induced in vegetative mycelium have been observed, but only one gene induced by blue light has been detected. I have demonstrated the feasibility of this approach by the isolation of a cDNA segment for the heat-shock protein HSP100 that is induced by blue light at the onset of sporangiophore development. The heat-shock protein HSP100 is an ATP-binding protein that has the ability to disassemble protein complexes. In plants, the gene for HSP100 is induced by light. The cDNA segment for HSP100 obtained from Phycomyces is 686 bp long and the predicted amino acid sequence contains one of the ATP-binding sites.

Genes for mevalonate biosynthesis in Phycomyces.

Terpenoids or isoprenoids constitute a vast family of organic compounds that includes sterols and carotenoids. The terpenoids in many organisms share early steps in their biosynthesis, including the synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) and its conversion to mevalonate. We have cloned and characterised the genes hmgS for HMG-CoA synthase and hmgR for HMG-CoA reductase from the Zygomycete Phycomyces blakesleeanus. Single copies of these genes are present in the Phycomyces genome. The predicted product of hmgS is largely hydrophilic and that of hmgR has eight putative transmembrane segments and a large hydrophilic domain. The hydrophilic domain suffices for catalytic activity, as shown by expressing it in Escherichia coli. Several features in the promoter of hmgS and in HMG-CoA reductase resemble motifs known to be involved in sterol-mediated regulation and sterol sensing. Carotene-overproducing mutants contain more hmgS mRNA than the wild type, possibly in response to an increased demand for HMG-CoA.

Genomic structure and sequence analysis of the valyl-tRNA synthetase gene of the Japanese pufferfish, Fugu rubripes.

The genomic sequence and exon-intron organisation of the valyl-tRNA synthetase gene in the Japanese pufferfish, Fugu rubripes, have been determined. This single-copy Fugu gene spans 8.5 kb, about 2.5 times smaller than that in man (21 kb). It contains 29 exons, with the largest intron being 1008 bp. The predicted polypeptide consists of 1217 amino acids, with a molecular weight of 138 kD and an isoelectric point of 7.27. It shares 40% identity in the overlapping region with its homolog in bacteria, 47% with yeast, and 67% with man. The Fugu gene has an additional N-terminal sequence which shows strong similarity to elongation factory-1gamma, a feature it shares only with the human sequence, but not with any other lower eukaryote or prokaryote studied so far. This N-terminal segment is encoded in the first six exons, suggesting their capture by a translocation through introns. Indeed, the acquisition of extra domains to perform related functions in RNA splicing and translation of polypeptides has already been observed in other aminoacyl-tRNA synthetases. Two cDNA sequences of human valyl-tRNA synthetase have been published, with discrepancies between them. Aided by comparisons with the Fugu gene, three of these discrepancies have been resolved, involving the elucidation of the sequence and positions of two introns. This compact vertebrate genome has demonstrated its value as a tool for the analysis of genes at the genomic level.

Genomic organization of the fungus Phycomyces.

The fungus Phycomyces blakesleeanus has a relatively small genome, 30 megabases (Mb), with a low guanine and cytosine (G + C) content, 35%; the coding sequences cloned to date all have a G + C content of about 50%. In order to investigate the organization of the genome of this fungus, we have cloned and sequenced 251 DNA fragments. One hundred and twenty-six clones were obtained by digestion with MspI (target sequence 5'-CCGG-3') and 125 random clones were obtained by sonication. The average length of sequence obtained was about 200 base pairs (bp) and the total length was about 50 kilobases (kb). The G + C content is not homogeneous throughout the genome: sequences obtained after digestion with MspI have an average of 5% more G + C content than the random fragments, and are enriched in coding sequences. Fourteen MspI fragments show similarities to known proteins and 21 encode ribosomal RNA (rRNA). By contrast, only three of the random fragments are similar to known proteins and only one to a rRNA. We conclude that the Phycomyces genome is composed of G + C-rich genes surrounded by G + C-poor areas. Two clones have similarities to the transposase of the transposon Tc1 from Caenorhabditis elegans. This result suggests the presence of a high copy number of a Tc1-like transposable element in the Phycomyces genome. Another clone was similar to the transposon Tx1 from Xenopus laevis. A novel repetitive nt sequence has been characterized; about 5% of the total genome is a repetition of any of two consensus sequences of 31 bp named PrA1 and PrA2.

Translocation events in the evolution of aminoacyl-tRNA synthetases.

We have characterized hisS, the gene encoding the histidyl-tRNA synthetase (HisRS) from the tetraodontoid fish Fugu rubripes. The hisS gene is about 3.5 kbp long and contains 13 exons and 12 introns of 172 bp, on average. The Fugu hisS gene encodes a putative protein of 519 amino acids with the three motifs identified as signatures of class 2 aminoacyl-tRNA synthetases. A model for the shifting of intron 8 between Fugu and hamster is proposed based on the successive appearance of a cryptic splicing site followed by an insertion mutation that created a new acceptor site. In addition, sequence comparisons suggest that the hisS gene has undergone a translocation through the first intron. As a result, the Fugu HisRS has an N-terminal sequence markedly different from that in the human and hamster enzymes. We propose that similar events have been responsible for variations at the N-terminal end of other aminoacyl-tRNA synthetases. Our analysis suggests that this involves exchanges through introns of two exons encoding an ancestral 32-amino acid motif.

Light and developmental regulation of the gene con-10 of Neurospora crassa.

The gene con-10 of Neurospora crassa is expressed preferentially during conidiation and following illumination of vegetative mycelia with blue light. In this study we have examined the segmental locations of the genetic elements associated with con-10 that are responsible for light and developmental expression. A translational fusion was prepared between the initial segment of con-10 and Escherichia coli lacZ. Deletions were then introduced into the con-10 upstream region associated with this translational fusion. Each construct was integrated at the his-3 locus of N. crassa by transformation and homologous recombination. Photoinduction of mycelia containing the translational fusion with the intact upstream region revealed a two phase stimulus-response curve. Exposure to light for as little as 5 sec induced a transcriptional response. Following this initial induction, a period of 15 min in the dark or light was required for appearance of a second phase response. Only a brief light treatment was necessary for induction of the second phase response. Deletions within the upstream region altered normal light and developmental expression of constructs containing the con-10-lacZ translational fusion. The deleted segments appear to contain a mycelial repression site, two conidiation activation sites, and two dark repression sites. A repeated 17-bp sequence acted as a transcriptional enhancer. One copy of this enhancer is in the upstream region. The second copy, with the opposite orientation, is located in the first con-10 intron. The enhancer was required for proper mycelial and conidial expression of the con-10-lacZ fusion. The initial 10 bp of this enhancer sequence were sufficient to restore conidial expression to a deletion construct lacking both copies of the 17-bp repeat. Proteins were detected in extracts of mycelia and conidia that specifically bound to the enhancer sequence in vitro. Our findings suggest that conidiation-specific and mycelial-specific expression of con-10 requires the action of several factors acting independently and/or in concert at distinct sites located in the regulatory regions for con-10.

Sequence analysis of thymidine kinase-defective mutants of equine herpesvirus-1 (EHV-1).

We have amplified, cloned and sequenced the gene encoding the thymidine kinase (TK) of a wild-type strain (Ab4) of equine herpesvirus-1 (EHV-1) and two mutants with defective TK activity isolated for resistance to penciclovir (PCV). One of the mutants, PR1, has suffered a 879-bp deletion which reduces the size of TK to 180 bp. The other mutant, PR3, has an adenine to cytosine mutation resulting in a Lys38-->Thr change. This mutation modifies the amino acid sequence of a domain involved in binding ATP, leading to non-detectable enzymatic activity. Lys38 thus appears to be essential for the activity of the TK of EHV-1.

Sex, light and carotenes: the development of Phycomyces.

Phycomyces blakesleeanus is known for the elaborate behaviour of its sporangiophores (fruiting bodies). Sporangiophore development is exquisitely sensitive to blue light, easy to describe quantitatively, pliable to genetic and biochemical research, and reminiscent in many details of other photoresponses in the same and in other organisms. The developmental and behavioural processes of Phycomyces share a number of genes. A combinatorial use of gene expression appears to be the basis for the complexities observed in this fungus.

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.

A test of human cDNA synthesis by the polymerase chain reaction.

A fast and efficient method to test the quality of human cDNA synthesis based on the polymerase chain reaction (PCR) is described. An aliquot of the cDNA reaction is used as a template for PCR amplification of a segment of the human histidyl-tRNA synthetase gene. The presence of an intron of approximately 300 bp in that region of the gene permits the identification of any genomic contamination in the cDNA sample. The same protocol has also been used with other mammalian DNAs with similar results.

Isolation and molecular analysis of the orotidine-5'-phosphate decarboxylase gene (pyrG) of Phycomyces blakesleeanus.

The pyrG gene of Phycomyces was isolated from a Phycomyces genomic library, constructed in the cosmid pHS255, by hybridization with a 170 bp fragment of the pyrG gene of Aspergillus niger. This fragment includes a consensus sequence found in almost all species in which the orotidine-5'-phosphate decarboxylase (OMPdecase) gene has been sequenced. The complete nucleotide sequence of the cloned pyrG gene from Phycomyces was determined and the transcription start sites mapped. In the predicted amino acid sequence there are regions of strong homology to the equivalent genes of Saccharomyces cerevisiae, A. niger, Schizophyllum commune and Homo sapiens. Analysis of the sequence revealed the presence of two introns. The precise length and location of these introns was determined by sequencing the pyrG cDNA and comparing it with the genomic clone. Non-coding flanking regions showed obvious homology to the consensus TATA and CAAT boxes, and the polyadenylation signal "AATAAA". The pyrG gene is the second Phycomyces gene that has been cloned and analysed. This is the first time that introns have been reported in Phycomyces.

Subliminal light control of dark adaptation kinetics in Phycomyces phototropism.

The dark adaptation kinetics of Phycomyces phototropism depend critically on the experimental protocol. When sporangiophores that had been light-adapted to a fluence rate of 1 W m-2 at 447 nm were exposed to dim unilateral light, the adaptation kinetics showed exponential decay (6 min time constant). However, when light-adapted sporangiophores were kept for variable intervals in darkness (i.e. in presence of traditional red safelight) and then exposed to dim unilateral test light, the decay kinetics of adaptation were biexponential with a rapid decay during the first minute (1 min time constant), followed by a slow recovery (11 min time constant). Thus, the dim subliminal light given after the sporangiophores had been adapted to 1 W m-2, was actually perceived, and exerted control over the dark-adaptation process. The observed acceleration of dark-adaptation kinetics constitutes a novel light effect of the sporangiophore. At wavelength 383 nm this effect was not observed. Because a beta-carotene lacking mutant, L91 (genotype carB), was unmodified in dark-adaptation kinetics measured in the presence or absence of subliminal light, it appears that beta-carotene is not involved in the photocontrol of adaptation.

Photomorphogenesis inPhycomyces: Dependence on environmental conditions.

The production of two kinds of vegetative reproductive structures, microphores and macrophores, byPhycomyces blakesleeanus Bgff. depends on plating density, ventilation, asparagine supply, and illumination. Quantitative determinations of these variables lead us to propose a new experimental system for developmental photobiology: standard plastic Petri plates containing 25 ml minimal medium are inoculated with 10(5) viable, heat-activated spores and incubated, unpiled and unsealed, at 22° C. After 4 d microphores are counted and macrophores are weighed. Both microphorogenesis and macrophorogenesis are governed by light. Photosensitivity is a developmental phenomenon which occurs 32 to 68 h after inoculation, just before the beginning of vegetative reproduction in the dark controls. The maximum photosensitivity occurs 48 h after inoculation.

Photomorphogenesis inPhycomyces: Fluence-response curves and action spectra.

Blue light regulates vegetative reproduction inPhycomyces blakesleeanus Bgff. by inhibiting the development of microphores and stimulating that of macrophores. Fluence-response curves were obtained at twelve different wavelengths. Each response exhibits a two-step ("biphasic") dependence on fluence, as if it resulted from the addition of two separate components with different thresholds, midpoints, and amplitudes. The absolute threshold is close to 10 photons·µm(2). The threshold fluence of the low-intensity component is about 10(4) times smaller than that of the high-intensity component. The action spectra for each of the two components of the two responses share general similarities, but exhibit significant differences that might be taken to favour four separate photosystems. Additional complexity is indicated by the wavelength dependence of the saturation levels.