Transposable Elements as Stress Adaptive Capacitors Induce Genomic Instability in Fungal Pathogen Magnaporthe oryzae

A fundamental problem in fungal pathogenesis is to elucidate the evolutionary forces responsible for genomic rearrangements leading to races with fitter genotypes. Understanding the adaptive evolutionary mechanisms requires identification of genomic components and environmental factors reshaping the genome of fungal pathogens to adapt. Herein, Magnaporthe oryzae, a model fungal plant pathogen is used to demonstrate the impact of environmental cues on transposable elements (TE) based genome dynamics. For heat shock and copper stress exposed samples, eight TEs belonging to class I and II family were employed to obtain DNA profiles. Stress induced mutant bands showed a positive correlation with dose/duration of stress and provided evidences of TEs role in stress adaptiveness. Further, we demonstrate that genome dynamics differ for the type/family of TEs upon stress exposition and previous reports of stress induced MAGGY transposition has underestimated the role of TEs in M. oryzae. Here, we identified Pyret, MAGGY, Pot3, MINE, Mg-SINE, Grasshopper and MGLR3 as contributors of high genomic instability in M. oryzae in respective order. Sequencing of mutated bands led to the identification of LTR-retrotransposon sequences within regulatory regions of psuedogenes. DNA transposon Pot3 was identified in the coding regions of chromatin remodelling protein containing tyrosinase copper-binding and PWWP domains. LTR-retrotransposons Pyret and MAGGY are identified as key components responsible for the high genomic instability and perhaps these TEs are utilized by M. oryzae for its acclimatization to adverse environmental conditions. Our results demonstrate how common field stresses change genome dynamics of pathogen and provide perspective to explore the role of TEs in genome adaptability, signalling network and its impact on the virulence of fungal pathogens.     

Evolutionary Genomics and Adaptive Evolution of the Hedgehog Gene Family (Shh,Ihh and Dhh) in Vertebrates

The Hedgehog (Hh) gene family codes for a class of secreted proteins composed of two active domains that act as signalling molecules during embryo development, namely for the development of the nervous and skeletal systems and the formation of the testis cord. While only one Hh gene is found typically in invertebrate genomes, most vertebrates species have three (Sonic hedgehog – Shh; Indian hedgehog – Ihh; and Desert hedgehog – Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. In this study, we used comparative genomic and adaptive evolutionary analyses to characterize the evolution of the Hh genes in vertebrates following the two major whole genome duplication (WGD) events. To overcome the lack of Hh-coding sequences on avian publicly available databases, we used an extensive dataset of 45 avian and three non-avian reptilian genomes to show that birds have all three Hh paralogs. We find suggestions that following the WGD events, vertebrate Hh paralogous genes evolved independently within similar linkage groups and under different evolutionary rates, especially within the catalytic domain. The structural regions around the ion-binding site were identified to be under positive selection in the signaling domain. These findings contrast with those observed in invertebrates, where different lineages that experienced gene duplication retained similar selective constraints in the Hh orthologs. Our results provide new insights on the evolutionary history of the Hh gene family, the functional roles of these paralogs in vertebrate species, and on the location of mutational hotspots.     

High efficiency, site-specific excision of a marker gene by the phage P1 cre–loxP system in the yellow fever mosquito, Aedes aegypti

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre–loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre–loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.     

Bidirectional-Genetics Platform,a Dual-Purpose Mutagenesis Strategy for Filamentous Fungi

Rapidly increasing fungal genome sequences call for efficient ways of generating mutants to translate quickly gene sequences into their functions. A reverse genetic strategy via targeted gene replacement (TGR) has been inefficient for many filamentous fungi due to dominant production of undesirable ectopic transformants. Although large-scale random insertional mutagenesis via transformation (i.e., forward genetics) facilitates high-throughput uncovering of novel genes of interest, generating a huge number of transformants, which is necessary to ensure the likelihood of mutagenizing most genes, is time-consuming. We propose a new strategy, entitled the Bidirectional-Genetics (BiG) platform, which combines both forward and reverse genetic strategies by recycling ectopic transformants derived from TGR as a source for random insertional mutants. The BiG platform was evaluated using the rice blast fungus Magnaporthe oryzae as a model. Over 10% of >1,000 M. oryzae ectopic transformants, generated during disruption of specific genes, displayed abnormality in vegetative growth, pigmentation, and/or asexual reproduction. In this pool of putative mutants, we isolated insertional mutants with mutations in three genes involved in histidine biosynthesis (MoHIS5), vegetative growth (MoVPS74), or conidiophore formation (MoFRQ) (where “Mo” indicates “M. oryzae”), supporting the utility of this platform for systematic gene function studies.     

The G Protein–Coupled Receptor Subset of the Chicken Genome

G protein–coupled receptors (GPCRs) are one of the largest families of proteins, and here we scan the recently sequenced chicken genome for GPCRs. We use a homology-based approach, utilizing comparisons with all human GPCRs, to detect and verify chicken GPCRs from translated genomic alignments and Genscan predictions. We present 557 manually curated sequences for GPCRs from the chicken genome, of which 455 were previously not annotated. More than 60% of the chicken Genscan gene predictions with a human ortholog needed curation, which drastically changed the average percentage identity between the human–chicken orthologous pairs (from 56.3% to 72.9%). Of the non-olfactory chicken GPCRs, 79% had a one-to-one orthologous relationship to a human GPCR. The Frizzled, Secretin, and subgroups of the Rhodopsin families have high proportions of orthologous pairs, although the percentage of amino acid identity varies. Other groups show large differences, such as the Adhesion family and GPCRs that bind exogenous ligands. The chicken has only three bitter Taste 2 receptors, and it also lacks an ortholog to human TAS1R2 (one of three GPCRs in the human genome in the Taste 1 receptor family [TAS1R]), implying that the chicken's ability and mode of detecting both bitter and sweet taste may differ from the human's. The chicken genome contains at least 229 olfactory receptors, and the majority of these (218) originate from a chicken-specific expansion. To our knowledge, this dataset of chicken GPCRs is the largest curated dataset from a single gene family from a non-mammalian vertebrate. Both the updated human GPCR dataset, as well the chicken GPCR dataset, are available for download.     

Multiple Chromosomal Rearrangements Structured the Ancestral Vertebrate Hox-Bearing Protochromosomes

While the proposal that large-scale genome expansions occurred early in vertebrate evolution is widely accepted, the exact mechanisms of the expansion—such as a single or multiple rounds of whole genome duplication, bloc chromosome duplications, large-scale individual gene duplications, or some combination of these—is unclear. Gene families with a single invertebrate member but four vertebrate members, such as the Hox clusters, provided early support for Ohno's hypothesis that two rounds of genome duplication (the 2R-model) occurred in the stem lineage of extant vertebrates. However, despite extensive study, the duplication history of the Hox clusters has remained unclear, calling into question its usefulness in resolving the role of large-scale gene or genome duplications in early vertebrates. Here, we present a phylogenetic analysis of the vertebrate Hox clusters and several linked genes (the Hox “paralogon”) and show that different phylogenies are obtained for Dlx and Col genes than for Hox and ErbB genes. We show that these results are robust to errors in phylogenetic inference and suggest that these competing phylogenies can be resolved if two chromosomal crossover events occurred in the ancestral vertebrate. These results resolve conflicting data on the order of Hox gene duplications and the role of genome duplication in vertebrate evolution and suggest that a period of genome reorganization occurred after genome duplications in early vertebrates.     

Molecular evolution of glycinin and β-conglycinin gene families in soybean (Glycine max L. Merr.)

There are two main classes of multi-subunit seed storage proteins, glycinin (11S) and β-conglycinin (7S), which account for approximately 70% of the total protein in a typical soybean seed. The subunits of these two protein classes are encoded by a number of genes. The genomic organization of these genes follows a complex evolutionary history. This research was designed to describe the origin and maintenance of genes in each of these gene families by analyzing the synteny, phylogenies, selection pressure and duplications of the genes in each gene family. The ancestral glycinin gene initially experienced a tandem duplication event; then, the genome underwent two subsequent rounds of whole-genome duplication, thereby resulting in duplication of the glycinin genes, and finally a tandem duplication likely gave rise to the Gy1 and Gy2 genes. The β-conglycinin genes primarily originated through the more recent whole-genome duplication and several tandem duplications. Purifying selection has had a key role in the maintenance of genes in both gene families. In addition, positive selection in the glycinin genes and a large deletion in a β-conglycinin exon contribute to the diversity of the duplicate genes. In summary, our results suggest that the duplicated genes in both gene families prefer to retain similar function throughout evolution and therefore may contribute to phenotypic robustness.     

Pioneering polyploids: the impact of whole-genome duplication on biome shifting in New Zealand Coprosma (Rubiaceae) and Veronica (Plantaginaceae)

The role of whole-genome duplication (WGD) in facilitating shifts into novel biomes remains unknown. Focusing on two diverse woody plant groups in New Zealand, Coprosma (Rubiaceae) and Veronica (Plantaginaceae), we investigate how biome occupancy varies with ploidy level, and test the hypothesis that WGD increases the rate of biome shifting. Ploidy levels and biome occupancy (forest, open and alpine) were determined for indigenous species in both clades. The distribution of low-ploidy (Coprosma: 2x, Veronica: 6x) versus high-ploidy (Coprosma: 4–10x, Veronica: 12–18x) species across biomes was tested statistically. Estimation of the phylogenetic history of biome occupancy and WGD was performed using time-calibrated phylogenies and the R package BioGeoBEARS. Trait-dependent dispersal models were implemented to determine support for an increased rate of biome shifting among high-ploidy lineages. We find support for a greater than random portion of high-ploidy species occupying multiple biomes. We also find strong support for high-ploidy lineages showing a three- to eightfold increase in the rate of biome shifts. These results suggest that WGD promotes ecological expansion into new biomes.     

Characterization of the Oral Fungal Microbiome (Mycobiome) in Healthy Individuals

The oral microbiome–organisms residing in the oral cavity and their collective genome–are critical components of health and disease. The fungal component of the oral microbiota has not been characterized. In this study, we used a novel multitag pyrosequencing approach to characterize fungi present in the oral cavity of 20 healthy individuals, using the pan-fungal internal transcribed spacer (ITS) primers. Our results revealed the “basal” oral mycobiome profile of the enrolled individuals, and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. Among these genera, 39 were present in only one person, 16 genera were present in two participants, and 5 genera were present in three people, while 15 genera (including non-culturable organisms) were present in ≥4 (20%) participants. Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Four of these predominant genera are known to be pathogenic in humans. The low-abundance genera may represent environmental fungi present in the oral cavity and could simply be spores inhaled from the air or material ingested with food. Among the culturable genera, 61 were represented by one species each, while 13 genera comprised between 2 and 6 different species; the total number of species identified were 101. The number of species in the oral cavity of each individual ranged between 9 and 23. Principal component (PCO) analysis of the obtained data set followed by sample clustering and UniFrac analysis revealed that White males and Asian males clustered differently from each other, whereas both Asian and White females clustered together. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease.     

The Genomes of Oryza sativa: a history of duplications

We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000–40,000. Only 2%–3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.     

Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae

Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB structures of enzyme in R.oryzae are not known which were predicted using I-TASSER server and validated with PROCHECK. Peptide inhibitors, FMDP and ADGP previously used against enzyme of E.coli (PDBid: 1XFF), were used for docking studies of enzyme in R.oryzae by SchrödingerMaestro v9.1. To investigate binding between enzyme and inhibitors, Glide and Induced Fit docking were performed. IFD results of 1XFF with FMDP yielded C1, R73, W74, T76, G99 and D123 as the binding sites. C379 and Q427 appear to be vital for binding of R.oryzae enzymes to inhibitors. The comparison results of IFD scores of enzyme in R.oryzae and E.coli (PDBid: 2BPL) yield appreciable score, hinting at the probable effectiveness of inhibitors FMDP and ADGP against R.oryzae, with ADGP showing an improved enzyme affinity. Moreover, the two copies of gene G-6-P synthase due to extensive fungal gene duplication, in R. oryzae eliminating the problem of drug ineffectiveness could act as a potential antifungal drug target in R. oryzae with the application of peptide ligands.     

Changes in Mycobacterium tuberculosis Genotype Families Over 20 Years in a Population-Based Study in Northern Malawi

Background

Despite increasing interest in possible differences in virulence and transmissibility between different genotypes of M. tuberculosis, very little is known about how genotypes within a population change over decades, or about relationships to HIV infection.

Methods and Principal Findings

In a population-based study in rural Malawi we have examined smears and cultures from tuberculosis patients over a 20-year period using spoligotyping. Isolates were grouped into spoligotype families and lineages following previously published criteria. Time trends, HIV status, drug resistance and outcome were examined by spoligotype family and lineage. In addition, transmissibility was examined among pairs of cases with known epidemiological contact by assessing the proportion of transmissions confirmed for each lineage, on the basis of IS6110 RFLP similarity of the M tuberculosis strains. 760 spoligotypes were obtained from smears from 518 patients from 1986–2002, and 377 spoligotypes from cultures from 347 patients from 2005–2008. There was good consistency in patients with multiple specimens. Among 781 patients with first episode tuberculosis, the majority (76%) had Lineage 4 (“European/American”) strains; 9% had Lineage 3 (“East-African/Indian”); 8% Lineage 1 (“Indo-Oceanic”); and 2% Lineage 2 (“East-Asian”); others unclassifiable. Over time the proportion of Lineage 4 decreased from >90% to 60%, with an increase in the other 3 lineages (p<0.001). Lineage 1 strains were more common in those with HIV infection, even after adjusting for age, sex and year. There were no associations with drug resistance or outcome, and no differences by lineage in the proportion of pairs in which transmission was confirmed.

Conclusions

This is the first study to describe long term trends in the four M. tuberculosis lineages in a population. Lineage 4 has probably been longstanding in this population, with relatively recent introductions and spread of Lineages1–3, perhaps influenced by the HIV epidemic.     

Elusive origins of the extra genes in Aspergillus oryzae

The genome sequence of Aspergillus oryzae revealed unexpectedly that this species has approximately 20% more genes than its congeneric species A. nidulans and A. fumigatus. Where did these extra genes come from? Here, we evaluate several possible causes of the elevated gene number. Many gene families are expanded in A. oryzae relative to A. nidulans and A. fumigatus, but we find no evidence of ancient whole-genome duplication or other segmental duplications, either in A. oryzae or in the common ancestor of the genus Aspergillus. We show that the presence of divergent pairs of paralogs is a feature peculiar to A. oryzae and is not shared with A. nidulans or A. fumigatus. In phylogenetic trees that include paralog pairs from A. oryzae, we frequently find that one of the genes in a pair from A. oryzae has the expected orthologous relationship with A. nidulans, A. fumigatus and other species in the subphylum Eurotiomycetes, whereas the other A. oryzae gene falls outside this clade but still within the Ascomycota. We identified 456 such gene pairs in A. oryzae. Further phylogenetic analysis did not however indicate a single consistent evolutionary origin for the divergent members of these pairs. Approximately one-third of them showed phylogenies that are suggestive of horizontal gene transfer (HGT) from Sordariomycete species, and these genes are closer together in the A. oryzae genome than expected by chance, but no unique Sordariomycete donor species was identifiable. The postulated HGTs from Sordariomycetes still leave the majority of extra A. oryzae genes unaccounted for. One possible explanation for our observations is that A. oryzae might have been the recipient of many separate HGT events from diverse donors.     

<Emphasis Type="Italic">Rhizopus homothallicus</Emphasis> Causing Invasive Infections: Series of Three Cases from a Single Centre in North India

Mucormycoses are opportunistic fungal infections with a high mortality rate. Rhizopus oryzae is the most common agent implicated in human infections. Although R. homothallicus has been previously reported to be a cause of pulmonary mucormycosis, it is the first time that we are reporting as a causative agent of rhino-orbital and cutaneous mucormycosis.     

Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene, xylA, from Aspergillus oryzae KBN616

β-Xylosidase secreted by the shoyu koji mold, Aspergillus oryzae, is the key enzyme responsible for browning of soy sauce. To investigate the role of β-xylosidase in the brown color formation, a major β-xylosidase, XylA, and its encoding gene were characterized. β-Xylosidase XylA was purified to homogeneity from culture filtrates of A. oryzae KBN616. The optimum pH and temperature of the enzyme were found to be 4.0 and 60°C, respectively, and the molecular mass was estimated to be 110 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The xylA gene comprises 2,397 bp with no introns and encodes a protein consisting of 798 amino acids (86,475 Da) with 14 potential N-glycosylation sites. The deduced amino acid sequence shows high similarity to Aspergillus nidulans XlnD (70%), Aspergillus niger XlnD (64%), and Trichoderma reesei BxII (63%). The xylA gene was overexpressed under control of the strong and constitutive A. oryzae TEF1 promoter. One of the A. oryzae transformants produced approximately 13 times more of the enzyme than did the host strain. The partial-length antisense xylA gene expressed under control of the A. oryzae TEF1 promoter decreased the β-xylosidase level in A. oryzae to about 20% of that of the host strain.     

Help or hindrance? The evolutionary impact of whole‐genome duplication on immunogenetic diversity and parasite load

Whole‐genome duplication (WGD) events occur in all kingdoms and have been hypothesized to promote adaptability. WGDs identified in the early history of vertebrates, teleosts, and angiosperms have been linked to the large‐scale diversification of these lineages. However, the mechanics and full outcomes of WGD regarding potential evolutionary impacts remain a topic of debate. The Corydoradinae are a diverse subfamily of Neotropical catfishes with over 170 species described and a history of WGDs. They are divided into nine mtDNA lineages, with species coexisting in sympatric—and often mimetic—communities containing representatives of two or more of the nine lineages. Given their similar life histories, coexisting species of Corydoras might be exposed to similar parasite loads and because of their different histories of WGD and genome size they provide a powerful system for investigating the impacts of WGD on immune diversity and function in an animal system. Here, we compared parasite counts and the diversity of the immune‐related toll‐like receptors (TLR) in two coexisting species of Corydoras catfish (C. maculifer and C. araguaiaensis), one diploid and one putative tetraploid. In the putative tetraploid C. araguaiaensis, we found significantly lower numbers of parasites and significantly higher diversity (measured by both synonymous and nonsynonymous SNP counts) in two TLR genes than in the diploid C. maculifer. These results provide insight into how WGD may impact evolution, in this case by providing greater immunogenetic diversity.