Diversity and evolution of polyketide biosynthesis gene clusters in the Ceratocystidaceae

Polyketides are secondary metabolites with diverse biological activities. Polyketide synthases (PKS) are often encoded from genes clustered in the same genomic region. Functional analyses and genomic studies show that most fungi are capable of producing a repertoire of polyketides. We considered the potential of Ceratocystidaceae for producing polyketides using a comparative genomics approach. Our aims were to identify the putative polyketide biosynthesis gene clusters, to characterize them and predict the types of polyketide compounds they might produce. We used sequences from nineteen species in the genera, Ceratocystis, Endoconidiophora, Davidsoniella, Huntiella, Thielaviopsis and Bretziella, to identify and characterize PKS gene clusters, by employing a range of bioinformatics and phylogenetic tools. We showed that the genomes contained putative clusters containing a non-reducing type I PKS and a type III PKS. Phylogenetic analyses suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes appeared to have distinct evolutionary origins. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae.     

Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

Genomic analysis of the aggressive tree pathogen Ceratocystis albifundus

The overall goal of this study was to determine whether the genome of an important plant pathogen in Africa, Ceratocystis albifundus, is structured into subgenomic compartments, and if so, to establish how these compartments are distributed across the genome. For this purpose, the publicly available genome of C. albifundus was complemented with the genome sequences for four additional isolates using the Illumina HiSeq platform. In addition, a reference genome for one of the individuals was assembled using both PacBio and Illumina HiSeq technologies. Our results showed a high degree of synteny between the five genomes, although several regions lacked detectable long-range synteny. These regions were associated with the presence of accessory genes, lower genetic similarity, variation in read-map depth, as well as transposable elements and genes associated with host-pathogen interactions (e.g. effectors and CAZymes). Such patterns are regarded as hallmarks of accelerated evolution, particularly of accessory subgenomic compartments in fungal pathogens. Our findings thus showed that the genome of C. albifundus is made-up of core and accessory subgenomic compartments, which is an important step towards characterizing its pangenome. This study also highlights the value of comparative genomics for understanding mechanisms that may underly and influence the biology and evolution of pathogens.     

Growth arrest of Schizosaccharomyces pombe following overexpression of mouse type 1 protein phosphatases

The rice disease resistance gene Xa21, which encodes a receptor-like kinase, is a member of a multigene family. Based on comparisons of genomic?sequences of seven family members, seventeen transposon-like elements were identified in the 5′ and 3′ flanking regions and introns of these genes. Sequence characterization revealed that these elements are diverse, showing similarity to maize Ds, CACTA and miniature inverted repeat-like elements, as well as novel elements. Only two elements were located in presumed coding regions, indicating that integration of transposable elements at the Xa21 disease resistance locus occurred preferentially in noncoding regions.     

A new wilt and die-back disease of Acacia mangium associated with Ceratocystis manginecans and C. acaciivora sp. nov. in Indonesia

Species of Ceratocystis are well-known wound related pathogens of many tree species, including commercially planted Acacia spp. Recently, several Ceratocystis isolates were collected from wilting A. mangium in plantations in Indonesia. The aim of this study was to identify these Ceratocystis isolates and to investigate their ability to cause disease on two plantation-grown Acacia spp. using greenhouse and field inoculation experiments. For identification, morphological characteristics and comparisons of DNA sequence data for the ITS, β-tubulin and TEF 1-α gene regions, was used. Ceratocystis isolates were identified as C. manginecans, a serious pathogen of mango trees in Oman and Pakistan and a previously undescribed species, described here as C. acaciivora sp. nov. Both fungi produced significant lesions in inoculation experiments on A. mangium and A. crassicarpa, however, C. acaciivora was most pathogenic suggesting that this fungus is the primary cause of the death of trees under natural conditions.     

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.     

The MAT A locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes

The MAT A locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain. The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning. Sequence analysis revealed two ORFs in the MAT A locus. One of them (MAT A1) codes for a protein of 119 amino acids which is required to induce sporulation. The other (MAT A2) codes for a protein of 291 amino acids that is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. The product of MAT A1 shows no homology to any known protein and seems to represent a new class of mating-type genes. MAT A2 contains a HMG box with homology to other mating-type genes. Both MAT A1 and MAT A2 are mating-type specific. In cells of both mating types, the regions flanking the MAT A locus contain sequences with homology to either S. cerevisiae SLA2 and ORF YBB9, respectively. From hybridization and subcloning data we estimate that the MAT A region is approximately 2?kb long and is present only once in the genome.     

Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome

Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes, and, as a consequence, genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex-determining regions of the genome. Here, we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100-kilobase mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be ~20% between MAT and a flanking marker at 5, 10, 50, or 100 kilobases from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted ~10- to 50-fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. MAT heterozygosity was not required for enhanced recombination, implying that this process is not due to a physical distortion from the two non-paired alleles and could also occur during same-sex mating. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and pathogenic yeasts.     

Single mating type-specific genes and their 3′ UTRs control mating and fertility in Cochliobolus heterostrophus

To determine the number of proteins required for mating type (MAT) locus-regulated control of mating in Cochliobolus heterostrophus, MAT fragments of various sizes were expressed in MAT deletion strains. As little as 1.5?kb of MAT sequence, encoding a single unique protein in each mating type (MAT-1 and MAT-2), conferred mating ability, although an additional 160?bp of 3^′ UTR was needed for production of ascospores. No other mating type-specific genes involved in mating identity or fertility were found. Thus, although homologs of the C. heterostrophus MAT-1 and MAT-2 genes exist in the filamentous ascomycetes Neurospora crassa and Podospora anserina, C. heterostrophus does not appear to have mating type-specific homologs of two additional genes required by both N. crassa and P.?anserina for successful sexual reproduction. Three genes were identified in the common DNA flanking the MAT locus: a gene encoding a GTPase-activating protein and an ORF of unknown function lie 5^′ while a β-glucosidase encoding gene lies found 3^′. None of these genes appears to be involving in the mating process.     

The MAT A locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes

The MAT A locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain. The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning. Sequence analysis revealed two ORFs in the MAT A locus. One of them (MAT A1) codes for a protein of 119 amino acids which is required to induce sporulation. The other (MAT A2) codes for a protein of 291 amino acids that is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. The product of MAT A1 shows no homology to any known protein and seems to represent a new class of mating-type genes. MAT A2 contains a HMG box with homology to other mating-type genes. Both MAT A1 and MAT A2 are mating-type specific. In cells of both mating types, the regions flanking the MAT A locus contain sequences with homology to either S. cerevisiae SLA2 and ORF YBB9, respectively. From hybridization and subcloning data we estimate that the MAT A region is approximately 2 kb long and is present only once in the genome. Received: 25 January 1999 / Accepted: 16 April 1999     

Morphological and Genomic Characterization of Filobasidiella depauperata: A Homothallic Sibling Species of the Pathogenic Cryptococcus Species Complex

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MAT a alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MAT a locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MAT a locus appear to have a higher number of changes and substitutions than their MATα counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.     

Growth arrest of Schizosaccharomyces pombe following overexpression of mouse type 1 protein phosphatases

The rice disease resistance gene Xa21, which encodes a receptor-like kinase, is a member of a multigene family. Based on comparisons of genomic␣sequences of seven family members, seventeen transposon-like elements were identified in the 5′ and 3′ flanking regions and introns of these genes. Sequence characterization revealed that these elements are diverse, showing similarity to maize Ds, CACTA and miniature inverted repeat-like elements, as well as novel elements. Only two elements were located in presumed coding regions, indicating that integration of transposable elements at the Xa21 disease resistance locus occurred preferentially in noncoding regions. Received: 17 October 1997 / Accepted: 3 February 1998     

Mating Type Sequences in Asexually Reproducing Fusarium Species

To assess the potential for mating in several Fusarium species with no known sexual stage, we developed degenerate and semidegenerate oligonucleotide primers to identify conserved mating type (MAT) sequences in these fungi. The putative α and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The α box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria.     

Characterization of the mating-type genes in Leptographium procerum and Leptographium profanum

Leptographium procerum and the closely related species Leptographium profanum, are ascomycetes associated with root-infesting beetles on pines and hardwood trees, respectively. Both species occur in North America where they are apparently native. L. procerum has also been found in Europe, China New Zealand, and South Africa where it has most probably been introduced. As is true for many other Leptographium species, sexual states have never been observed in L. procerum or L. profanum. The objectives of this study were to clone and characterize the mating type loci of these fungi, and to develop markers to determine the mating types of individual isolates. To achieve this, a partial sequence of MAT1-2-1 was amplified using degenerate primers targeting the high mobility group (HMG) sequence. A complete MAT1-2 idiomorph of L. profanum was subsequently obtained by screening a genomic library using the HMG sequence as a probe. Long range PCR was used to amplify the complete MAT1-1 idiomorph of L. profanum and both the MAT1-1 and MAT1-2 idiomorphs of L. procerum. Characterization of the MAT idiomorphs suggests that the MAT genes are fully functional and that individuals of both these species are self-sterile in nature with a heterothallic mating system. Mating type markers were developed and tested on a population of L. procerum isolates from the USA, the assumed center of origin for this species. The results suggest that cryptic sexual reproduction is occurring or has recently taken place within this population.     

Polyphasic characterization of four soil-derived phenanthrene-degrading Acidovorax strains and proposal of Acidovorax carolinensis sp. nov.

Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C_16:1ω7c/C_16:1ω6c, C_16:0, and C_18:1ω7c, and were consistent with the genus. Genomic G + C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3^T as the type strain (=LMG 30136, =DSM 105008).     

Heterothallism revealed in the root rot fungi Berkeleyomyces basicola and B. rouxiae

Berkeleyomyces basicola and Berkeleyomyces rouxiae, two sister species previously treated collectively as Thielaviopsis basicola, reside in the Ceratocystidaceae (Microascales, Ascomycota). Both species are important root pathogens of many important agricultural crops and ornamental plants. Although T. basicola has been known for more than 150y, a sexual state has never been found and it has been assumed to be an asexual pathogen. The aim of this study was to determine the mating strategy of the two Berkeleyomyces species. Investigation of the genome sequences of two B. basicola isolates allowed for the complete characterization of the MATlocus, revealing that it has a typical heterothallic mating system with the MAT1-1andMAT1-2 idiomorphs occurring in different isolates. PCR amplification using mating type primers developed in this study, showed that the MAT1-1-1andMAT1-2-1 genes were also present in different isolates of B. rouxiae. Pairing of isolates representing the two mating types of both species,using a variety of techniques failed to produce sexual structures. Although we have found no direct evidence that they reproduce sexually, these fungi are clearly heterothallic with both mating types occurring in some countries suggesting that a cryptic sexual cycle could exist for them.     

The transposable elements of the Drosophila melanogaster euchromatin: a genomics perspective

Background

Transposable elements are found in the genomes of nearly all eukaryotes. The recent completion of the Release 3 euchromatic genomic sequence of Drosophila melanogaster by the Berkeley Drosophila Genome Project has provided precise sequence for the repetitive elements in the Drosophila euchromatin. We have used this genomic sequence to describe the euchromatic transposable elements in the sequenced strain of this species.

Results

We identified 85 known and eight novel families of transposable element varying in copy number from one to 146. A total of 1,572 full and partial transposable elements were identified, comprising 3.86% of the sequence. More than two-thirds of the transposable elements are partial. The density of transposable elements increases an average of 4.7 times in the centromere-proximal regions of each of the major chromosome arms. We found that transposable elements are preferentially found outside genes; only 436 of 1,572 transposable elements are contained within the 61.4 Mb of sequence that is annotated as being transcribed. A large proportion of transposable elements is found nested within other elements of the same or different classes. Lastly, an analysis of structural variation from different families reveals distinct patterns of deletion for elements belonging to different classes.

Conclusions

This analysis represents an initial characterization of the transposable elements in the Release 3 euchromatic genomic sequence of D. melanogaster for which comparison to the transposable elements of other organisms can begin to be made. These data have been made available on the Berkeley Drosophila Genome Project website for future analyses.     

Biogeography Revealed by Mariner-Like Transposable Element Sequences via a Bayesian Coalescent Approach

Genetic diversity of natural populations is useful in biogeographical studies. Here, we apply a Bayesian method based on the coalescent model to dating biogeographical events by using published DNA sequences of wild silkworms, Bombyx mandarina, and the domesticated model organisms B. mori, both of which categorized into the order of Lepidoptera, sampled from China, Korea, and Japan. The sequences consist of the BmTNML locus and the flanking intergenic regions. The BmTNML locus is composed of cecropia-type mariner-like element (MLE) with inverted terminal repeats, and three different transposable elements (TE), including L1BM, BMC1 retrotransposons, and BmamaT1, are inserted into the MLE. Based on the genealogy defined by TE insertions/deletions (indels), we estimated times to the most recent common ancestor and these indels events using the flanking, MLE, and indels sequences, respectively. These estimates by using MLE sequences strongly correlated with those by using flanking sequences, implying that cecropia-type MLEs can be used as a molecular clock. MLEs are thought to have transmitted horizontally among different species. By using a pair of published cecropia-type MLE sequences from lepidopteran insect, an emperor moth, and a coral in Ryukyu Islands, we demonstrated dating of horizontal transmission between species which are distantly related but inhabiting geographically close region.     

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio)

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 by long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 by inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.