Most molecular studies of plant stress tolerance have been performed with Arabidopsis thaliana, although it is not particularly stress tolerant and may lack protective mechanisms required to survive extreme environmental conditions. Thellungiella salsuginea has attracted interest as an alternative plant model species with high tolerance of various abiotic stresses. While the T. salsuginea genome has recently been sequenced, its annotation is still incomplete and transcriptomic information is scarce. In addition, functional genomics investigations in this species are severely hampered by a lack of affordable tools for genome-wide gene expression studies.
Here, we report the results of Thellungiella de novo transcriptome assembly and annotation based on 454 pyrosequencing and development and validation of a T. salsuginea microarray. ESTs were generated from a non-normalized and a normalized library synthesized from RNA pooled from samples covering different tissues and abiotic stress conditions. Both libraries yielded partially unique sequences, indicating their necessity to obtain comprehensive transcriptome coverage. More than 1 million sequence reads were assembled into 42,810 unigenes, approximately 50% of which could be functionally annotated. These unigenes were compared to all available Thellungiella genome sequence information. In addition, the groups of Late Embryogenesis Abundant (LEA) proteins, Mitogen Activated Protein (MAP) kinases and protein phosphatases were annotated in detail. We also predicted the target genes for 384 putative miRNAs. From the sequence information, we constructed a 44 k Agilent oligonucleotide microarray. Comparison of same-species and cross-species hybridization results showed superior performance of the newly designed array for T. salsuginea samples. The developed microarrays were used to investigate transcriptional responses of T. salsuginea and Arabidopsis during cold acclimation using the MapMan software.
This study provides the first comprehensive transcriptome information for the extremophile Arabidopsis relative T. salsuginea. The data constitute a more than three-fold increase in the number of publicly available unigene sequences and will greatly facilitate genome annotation. In addition, we have designed and validated the first genome-wide microarray for T. salsuginea, which will be commercially available. Together with the publicly available MapMan software this will become an important tool for functional genomics of plant stress tolerance.
Arabidopsis thaliana; Cold acclimation; Gene annotation; LEA proteins; MAP kinases; Microarray design; microRNAs; Protein phosphatases; Thellungiella salsuginea; Transcriptome sequencing
Deep RNA sequencing (RNAseq) has opened a new horizon for understanding global gene expression. The functional annotation of non-model mammalian genomes including bovines is still poor compared to that of human and mouse. This particularly applies to tissues without direct significance for milk and meat production, like skin, in spite of its multifunctional relevance for the individual. Thus, applying an RNAseq approach, we performed a whole transcriptome analysis of pigmented and nonpigmented bovine skin to describe the comprehensive transcript catalogue of this tissue.
A total of 39,577 unique primary skin transcripts were mapped to the bovine reference genome assembly. The majority of the transcripts were mapped to known transcriptional units (65%). In addition to the reannotation of known genes, a substantial number (10,884) of unknown transcripts (UTs) were discovered, which had not previously been annotated. The classification of UTs was based on the prediction of their coding potential and comparative sequence analysis, subsequently followed by meticulous manual curation. The classification analysis and experimental validation of selected UTs confirmed that RNAseq data can be used to amend the annotation of known genes by providing evidence for additional exons, untranslated regions or splice variants, by approving genes predicted in silico and by identifying novel bovine loci. A large group of UTs (4,848) was predicted to potentially represent long noncoding RNA (lncRNA). Predominantly, potential lncRNAs mapped in intergenic chromosome regions (4,365) and therefore, were classified as potential intergenic lncRNA. Our analysis revealed that only about 6% of all UTs displayed interspecies conservation and discovered a variety of unknown transcripts without interspecies homology but specific expression in bovine skin.
The results of our study demonstrate a complex transcript pattern for bovine skin and suggest a possible functional relevance of novel transcripts, including lncRNA, in the modulation of pigmentation processes. The results also indicate that the comprehensive identification and annotation of unknown transcripts from whole transcriptome analysis using RNAseq data remains a tremendous future challenge.
Transcriptome; RNAseq; Skin; Pigmentation; Noncoding RNA; Novel transcripts; Cattle
In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions INGenomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported.
In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries.
These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.
TILLING; Fugu; ENU; HRM; Myostatin; Mutagenesis; Fish breeding
Ribosomes are essential ribonucleoprotein complexes that are engaged in translation and thus indispensable for growth. Arabidopsis thaliana ribosomes are composed of 80 distinct ribosomal proteins (RPs), each of which is encoded by two to seven highly similar paralogous genes. Little information is available on how RP genes respond to a shortage of essential mineral nutrients such as phosphate (Pi) or iron (Fe). In the present study, the expression of RP genes and the differential accumulation of RPs upon Pi or Fe deficiency in Arabidopsis roots were comprehensively analyzed.
Comparison of 3,106 Pi-responsive genes with 3,296 Fe-responsive genes revealed an overlap of 579 genes that were differentially expressed under both conditions in Arabidopsis roots. Gene ontology (GO) analysis revealed that these 579 genes were mainly associated with abiotic stress responses. Among the 247 RP genes retrieved from the TAIR10 release of the Arabidopsis genome (98 small subunit RP genes, 143 large subunit RP genes and six ribosome-related genes), seven RP genes were not detected in Arabidopsis roots by RNA sequencing under control conditions. Transcripts from 20 and 100 RP genes showed low and medium abundance, respectively; 120 RP genes were highly expressed in Arabidopsis roots. As anticipated, gene ontology (GO) analysis indicated that most RP genes were related to translation and ribosome assembly, but some of the highly expressed RP genes were also involved in the responses to cold, UV-B, and salt stress. Only three RP genes derived from three ‘sets’ of paralogous genes were differentially expressed between Pi-sufficient and Pi-deficient roots, all of which were induced by Pi starvation. In Fe-deficient plants, 81 RP genes from 51 ’sets’ of paralagous RP genes were significantly down-regulated in response to Fe deficiency. The biological processes ’translation’ (GO: 0006412), ’ribosome biogenesis (GO: 0042254), and ’response to salt (GO: 0009651), cold (GO: 0009409), and UV-B stresses (GO: 0071493)’ were enriched in this subset of RP genes. At the protein level, 21 and two RPs accumulated differentially under Pi- and Fe-deficient conditions, respectively. Neither the differentially expressed RP genes nor the differentially expressed RPs showed any overlap between the two growth types.
In the present study three and 81 differentially expressed RP genes were identified under Pi and Fe deficiency, respectively. At protein level, 21 and two RP proteins were differentially accumulated under Pi- and Fe-deficient conditions. Our study shows that the expression of paralogous genes encoding RPs was regulated in a stress-specific manner in Arabidopsis roots, presumably resulting in an altered composition of ribosomes and biased translation. These findings may aid in uncovering an unexplored mechanism by which plants adapt to changing environmental conditions.
Iron deficiency; Phosphate deficiency; Proteomics; Ribosomal proteins; RNA-seq; Translation
Thalassosaline waters produced by the concentration of seawater are widespread and common extreme aquatic habitats. Their salinity varies from that of sea water (ca. 3.5%) to saturation for NaCl (ca. 37%). Obviously the microbiota varies dramatically throughout this range. Recent metagenomic analysis of intermediate salinity waters (19%) indicated the presence of an abundant and yet undescribed gamma-proteobacterium. Two strains belonging to this group have been isolated from saltern ponds of intermediate salinity in two Spanish salterns and were named “Spiribacter”.
The genomes of two isolates of “Spiribacter” have been fully sequenced and assembled. The analysis of metagenomic datasets indicates that microbes of this genus are widespread worldwide in medium salinity habitats representing the first ecologically defined moderate halophile. The genomes indicate that the two isolates belong to different species within the same genus. Both genomes are streamlined with high coding densities, have few regulatory mechanisms and no motility or chemotactic behavior. Metabolically they are heterotrophs with a subgroup II xanthorhodopsin as an additional energy source when light is available.
This is the first bacterium that has been proven by culture independent approaches to be prevalent in hypersaline habitats of intermediate salinity (half a way between the sea and NaCl saturation). Predictions from the proteome and analysis of transporter genes, together with a complete ectoine biosynthesis gene cluster are consistent with these microbes having the salt-out-organic-compatible solutes type of osmoregulation. All these features are also consistent with a well-adapted fully planktonic microbe while other halophiles with more complex genomes such as Salinibacter ruber might have particle associated microniches.
Halophilic bacteria; Xanthorhodopsin; Hypersaline; Saltern; Spiribacter; Moderate halophile
The study of quantitative trait loci (QTL) in cotton (Gossypium spp.) is focused on traits of agricultural significance. Previous studies have identified a plethora of QTL attributed to fiber quality, disease and pest resistance, branch number, seed quality and yield and yield related traits, drought tolerance, and morphological traits. However, results among these studies differed due to the use of different genetic populations, markers and marker densities, and testing environments. Since two previous meta-QTL analyses were performed on fiber traits, a number of papers on QTL mapping of fiber quality, yield traits, morphological traits, and disease resistance have been published. To obtain a better insight into the genome-wide distribution of QTL and to identify consistent QTL for marker assisted breeding in cotton, an updated comparative QTL analysis is needed.
In this study, a total of 1,223 QTL from 42 different QTL studies in Gossypium were surveyed and mapped using Biomercator V3 based on the Gossypium consensus map from the Cotton Marker Database. A meta-analysis was first performed using manual inference and confirmed by Biomercator V3 to identify possible QTL clusters and hotspots. QTL clusters are composed of QTL of various traits which are concentrated in a specific region on a chromosome, whereas hotspots are composed of only one trait type. QTL were not evenly distributed along the cotton genome and were concentrated in specific regions on each chromosome. QTL hotspots for fiber quality traits were found in the same regions as the clusters, indicating that clusters may also form hotspots.
Putative QTL clusters were identified via meta-analysis and will be useful for breeding programs and future studies involving Gossypium QTL. The presence of QTL clusters and hotspots indicates consensus regions across cultivated tetraploid Gossypium species, environments, and populations which contain large numbers of QTL, and in some cases multiple QTL associated with the same trait termed a hotspot. This study combines two previous meta-analysis studies and adds all other currently available QTL studies, making it the most comprehensive meta-analysis study in cotton to date.
Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is a frequent resident of the rectovaginal tract in humans, and a major cause of neonatal infection. In addition, S. agalactiae is a known fish pathogen, which compromises food safety and represents a zoonotic hazard. The complete genome sequence of the piscine S. agalactiae isolate GD201008-001 was compared with 14 other piscine, human and bovine strains to explore their virulence determinants, evolutionary relationships and the genetic basis of host tropism in S. agalactiae.
The pan-genome of S. agalactiae is open and its size increases with the addition of newly sequenced genomes. The core genes shared by all isolates account for 50 ~ 70% of any single genome. The Chinese piscine isolates GD201008-001 and ZQ0910 are phylogenetically distinct from the Latin American piscine isolates SA20-06 and STIR-CD-17, but are closely related to the human strain A909, in the context of the clustered regularly interspaced short palindromic repeats (CRISPRs), prophage, virulence-associated genes and phylogenetic relationships. We identified a unique 10 kb gene locus in Chinese piscine strains.
Isolates from cultured tilapia in China have a close genomic relationship with the human strain A909. Our findings provide insight into the pathogenesis and host-associated genome content of piscine S. agalactiae isolated in China.
Streptococcus agalactiae; Tilapia; Pan-genome; China
Progress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery. Urine has become an ideal and rich source of biomarkers in clinical proteomics. Here we performed a proteomic analysis of urine samples from pregnant and non-pregnant patients using gel electrophoresis and high-resolution mass spectrometry. Furthermore, we also apply a non-prefractionation quantitative phosphoproteomic approach using mTRAQ labeling to evaluate the expression of specific phosphoproteins during pregnancy comparison with non-pregnancy.
In total, 2579 proteins (10429 unique peptides) were identified, including 1408 from the urine of pregnant volunteers and 1985 from the urine of non-pregnant volunteers. One thousand and twenty-three proteins were not reported in previous studies at the proteome level and were unique to our study. Furthermore, we obtained 237 phosphopeptides, representing 105 phosphoproteins. Among these phosphoproteins, 16 of them were found to be significantly differentially expressed, of which 14 were up-regulated and two were down-regulated in urine samples from women just before vaginal delivery.
Taken together, these results offer a comprehensive urinary proteomic profile of healthy women during before and after vaginal delivery and novel information on the phosphoproteins that are differentially regulated during the maintenance of normal pregnancy. Our results may provide a better understanding of the mechanisms of pregnancy maintenance, potentially leading to the development of biomarker-based sensitive assays for understanding pregnancy.
Proteomic profile; Quantitative phosphoproteomic analysis; Human urine; Pregnancy and non-pregnancy; mTRAQ labeling
Adaptations to a new environment, such as a polluted one, often involve large modifications of the existing phenotypes. Changes in gene expression and regulation during critical developmental stages may explain these phenotypic changes. Embryos from a population of the teleost fish, Fundulus heteroclitus, inhabiting a clean estuary do not survive when exposed to sediment extract from a site highly contaminated with polycyclic aromatic hydrocarbons (PAHs) while embryos derived from a population inhabiting a PAH polluted estuary are remarkably resistant to the polluted sediment extract. We exposed embryos from these two populations to surrogate model PAHs and analyzed changes in gene expression, morphology, and cardiac physiology in order to better understand sensitivity and adaptive resistance mechanisms mediating PAH exposure during development.
The synergistic effects of two model PAHs, an aryl hydrocarbon receptor (AHR) agonist (β-naphthoflavone) and a cytochrome P4501A (CYP1A) inhibitor (α-naphthoflavone), caused significant developmental delays, impaired cardiac function, severe morphological alterations and failure to hatch, leading to the deaths of reference embryos; resistant embryos were mostly unaffected. Unexpectedly, patterns of gene expression among normal and moderately deformed embryos were similar, and only severely deformed embryos showed a contrasting pattern of gene expression. Given the drastic morphological differences between reference and resistant embryos, a surprisingly low percentage of genes, 2.24% of 6,754 analyzed, show statistically significant differences in transcript levels during late organogenesis between the two embryo populations.
Our study demonstrates important contrasts in responses between reference and resistant natural embryo populations to synergistic effects of surrogate model PAHs that may be important in adaptive mechanisms mediating PAH effects during fish embryo development. These results suggest that statistically significant changes in gene expression of relatively few genes contribute to the phenotypic changes and large morphological differences exhibited by reference and resistant populations upon exposure to PAH pollutants. By correlating cardiac physiology and morphology with changes in gene expression patterns of reference and resistant embryos, we provide additional evidence for acquired resistance among embryos whose parents live at heavily contaminated sites.
Development; Genomics; Embryos; Adaptation; Evolution; Fish; Transcriptomics
Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function.
AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change > |1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways.
This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.
Porphyromonas gingivalis; Aortic smooth muscle cells; Proliferation; Gene expression profiling
Intrinsically disordered proteins, found in all living organisms, are essential for basic cellular functions and complement the function of ordered proteins. It has been shown that protein disorder is linked to the G + C content of the genome. Furthermore, recent investigations have suggested that the evolutionary dynamics of the plant nucleus adds disordered segments to open reading frames alike, and these segments are not necessarily conserved among orthologous genes.
In the present work the distribution of intrinsically disordered proteins along the chromosomes of several representative plants was analyzed. The reported results support a non-random distribution of disordered proteins along the chromosomes of Arabidopsis thaliana and Oryza sativa, two model eudicot and monocot plant species, respectively. In fact, for most chromosomes positive correlations between the frequency of disordered segments of 30+ amino acids and both recombination rates and G + C content were observed.
These analyses demonstrate that the presence of disordered segments among plant proteins is associated with the rates of genetic recombination of their encoding genes. Altogether, these findings suggest that high recombination rates, as well as chromosomal rearrangements, could induce disordered segments in proteins during evolution.
Chromosome; Evolution; Intrinsically disordered proteins; Orthologues; Plant genome; Recombination rate
Previously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats.
Here we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed.
Using in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.
Drosophila virilis; in silico digestion; Medium size repeats; Evolution
The transport of sugars, hormones, amino acids, proteins, sugar alcohols, and other organic compounds from the sites of synthesis to the sites of use or storage occurs through the conducting cells of the phloem. To better understand these processes a comprehensive understanding of the proteins involved is required. While a considerable amount of data has been obtained from proteomic analyses of phloem sap, this has mainly served to identify the soluble proteins that are translocated through the phloem network.
In order to obtain more comprehensive proteomic data from phloem tissue we developed a simple dissection procedure to isolate phloem tissue from Brassica oleracea. The presence of a high density of phloem sieve elements was confirmed using light microscopy and fluorescently labeled sieve element-specific antibodies. To increase the depth of the proteomic analysis for membrane bound and associated proteins, soluble proteins were extracted first and subsequent extractions were carried out using two different detergents (SDS and CHAPSO). Across all three extractions almost four hundred proteins were identified and each extraction method added to the analysis demonstrating the utility of an approach combining several extraction protocols.
The phloem was found to be enriched in proteins associated with biotic and abiotic stress responses and structural proteins. Subsequent expression analysis identified a number of genes that appear to be expressed exclusively or at very high levels in phloem tissue, including genes that are known to express specifically in the phloem as well as novel phloem genes.
Brassica oleracea; Phloem; Proteome; Sieve-elements; Protein
Honeybee venom is a complicated defensive toxin that has a wide range of pharmacologically active compounds. Some of these compounds are useful for human therapeutics. There are two major forms of honeybee venom used in pharmacological applications: manually (or reservoir disrupting) extracted glandular venom (GV), and venom extracted through the use of electrical stimulation (ESV). A proteome comparison of these two venom forms and an understanding of the phosphorylation status of ESV, are still very limited. Here, the proteomes of GV and ESV were compared using both gel-based and gel-free proteomics approaches and the phosphoproteome of ESV was determined through the use of TiO2 enrichment.
Of the 43 proteins identified in GV, < 40% were venom toxins, and > 60% of the proteins were non-toxic proteins resulting from contamination by gland tissue damage during extraction and bee death. Of the 17 proteins identified in ESV, 14 proteins (>80%) were venom toxic proteins and most of them were found in higher abundance than in GV. Moreover, two novel proteins (dehydrogenase/reductase SDR family member 11-like and histone H2B.3-like) and three novel phosphorylation sites (icarapin (S43), phospholipase A-2 (T145), and apamin (T23)) were identified.
Our data demonstrate that venom extracted manually is different from venom extracted using ESV, and these differences may be important in their use as pharmacological agents. ESV may be more efficient than GV as a potential pharmacological source because of its higher venom protein content, production efficiency, and without the need to kill honeybee. The three newly identified phosphorylated venom proteins in ESV may elicit a different immune response through the specific recognition of antigenic determinants. The two novel venom proteins extend our proteome coverage of honeybee venom.
Honeybee; Venom; Proteome; Phosphoproteome
Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences.
Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations.
This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.
Seed-borne pathogen; Secretion system; Insertion sequence; Bean; Effector; Chemotaxis; Pseudogene
Loss of virulence is a phenotypic adaptation commonly seen in prokaryotic and eukaryotic pathogens. This mechanism is not well studied, especially in organisms with multiple host and life cycle stages such as Babesia, a tick-transmitted hemoparasite of humans and animals. B. bovis, which infects cattle, has naturally occurring virulent strains that can be reliably attenuated in vivo. Previous studies suggest the virulence loss mechanism may involve post-genomic modification. We investigated the transcriptome profiles of two geographically distinct B. bovis virulent and attenuated strain pairs to better understand virulence loss and to gain insight into pathogen adaptation strategies.
Expression microarray and RNA-sequencing approaches were employed to compare transcriptome profiles of two B. bovis strain pairs, with each pair consisting of a virulent parental and its attenuated derivative strain. Differentially regulated transcripts were identified within each strain pair. These included genes encoding for VESA1, SmORFs, undefined membrane and hypothetical proteins. The majority of individual specific gene transcripts differentially regulated within a strain were not shared between the two strains. There was a disproportionately greater number of ves genes upregulated in the virulent parental strains. When compared with their attenuated derivatives, divergently oriented ves genes were included among the upregulated ves genes in the virulent strains, while none of the upregulated ves genes in the attenuated derivatives were oriented head to head. One gene family whose specific members were consistently and significantly upregulated in expression in both attenuated strains was spherical body protein (SBP) 2 encoding gene where SBP2 truncated copies 7, 9 and 11 transcripts were all upregulated.
We conclude that ves heterodimer pair upregulation and overall higher frequency of ves gene expressions in the virulent strains is consistent with the involvement of this gene family in virulence. This is logical given the role of VESA1 proteins in cytoadherence of infected cells to endothelial cells. However, upregulation of some ves genes in the attenuated derivatives suggests that the consequence of upregulation is gene-specific. Furthermore, upregulation of the spherical body protein 2 gene family may play a role in the attenuated phenotype. Exactly how these two gene families may contribute to the loss or gain of virulence is discussed.
Babesia bovis; Apicomplexans; Virulence; Attenuation; Transcriptome; Microarray; RNA-sequencing
A hexaploidization event over 125 Mya underlies the evolutionary lineage of the majority of flowering plants, including very many species of agricultural importance. Half of these belong to the rosid subgrouping, containing severals whose genome sequences have been published. Although most duplicate and triplicate genes have been lost in all descendants, clear traces of the original chromosome triples can be discerned, their internal contiguity highly conserved in some genomes and very fragmented in others. To understand the particular evolutionary patterns of plant genomes, there is a need to systematically survey the fate of the subgenomes of polyploids, including the retention of a small proportion of the duplicate and triplicate genes and the reconstruction of putative ancestral intermediates between the original hexaploid and modern species, in this case the ancestor of the eurosid clade.
We quantitatively trace the fate of gene triples originating in the hexaploidy across seven core eudicot flowering plants, and fit this to a two-stage model, pre- and post-radiation. We also measure the simultaneous dynamics of duplicate orthologous gene loss in three rosids, as influenced by biological functional class. We propose a new protocol for reconstructing ancestral gene order using only gene adjacency data from pairwise genomic analyses, based on repeating MAXIMUM WEIGHT MATCHING at two levels of resolution, an approach designed to transcend limitations on reconstructed contig size, while still avoiding the ambiguities of a multiplicity of solutions. Applied to three high-quality rosid genomes without subsequent polyploidy events, our automated procedure reconstructs the ancestor of the eurosid clade.
The gene loss analysis and the ancestor reconstruction present complementary assessments of post-hexaploidization evolution, the first at the level of individual gene families within and across sister genomes and the second at the chromosome level. Despite the loss of more than 95% of gene duplicates and triplicates, and despite major structural rearrangement, our reconstructed eurosid ancestor clearly identifies the three regions corresponding to each of the seven original chromosomes of the earlier pre-hexaploid ancestor. Functional analysis confirmed trends reported for more recent plant polyploidy events: genes involved with regulation and responses were retained in multiple copies, while genes involved with metabolic processes were lost.
The identification of proteins based on analysis of tandem mass spectrometry (MS/MS) data is a valuable tool that is not fully realized because of the difficulty in carrying out automated analysis of large numbers of spectra. MS/MS spectra consist of peaks that represent each peptide fragment, usually b and y ions, with experimentally determined mass to charge ratios. Whether the strategy employed is database matching or De Novo sequencing, a major obstacle is distinguishing signal from noise. Improved ability to distinguish signal peaks of low intensity from background noise increases the likelihood of correctly identifying the peptide, as valuable information is preserved while extraneous information is not left to mislead.
This paper introduces an automated noise filtering method based on the construction of orthogonal polynomials. By subdividing the spectrum into a variable number (3 to 11) of bins, peaks that are considered "noise" are identified at a local level. Using a De Novo sequencing algorithm that we are developing, this filtering method was applied to a published dataset of more than 3000 mass spectra and an original dataset of more than 300 spectra. The samples were peptides from purified known proteins; therefore, the solutions could be compared to the correct sequences and the peaks corresponding to b, y and other fragments of significance could be identified. The same procedure was applied using two other published filtering methods. The ratios of the number of significant peaks that were preserved relative to the total number of peaks in each spectrum were determined. In the event that filtering out too many or too few signal peaks can lead to inaccuracy in sequence determination, the percentage of amino acid residues in the correct positions relative to the total number of amino acid residues in the correct sequence was also calculated for each sequence determined.
The results show that an orthogonal polynomial-based method of distinguishing signal peaks from background in mass spectra preserves a greater portion of signal peaks than compared methods, improving accuracy in sequence determination.
Flow cytometry has been widely used for the diagnosis of various hematopoietic diseases. Although there have been advances in the number of biomarkers that can be analyzed simultaneously and technologies that enable fast performance, the diagnostic data are still interpreted by a manual gating strategy. The process is labor-intensive, time-consuming, and subject to human error.
We used 80 sets of flow cytometry data from 44 healthy donors, 21 patients with chronic lymphocytic leukemia (CLL), and 15 patients with follicular lymphoma (FL). Approximately 15% of data from each group were used to build the profiles. Our approach was able to successfully identify 36/37 healthy donor cases, 18/18 CLL cases, and 12/13 FL cases.
This proof-of-concept study demonstrated that an automated diagnosis of CLL and FL can be obtained by examining the cell capture rates of a test case using the computational method based on the multi-profile detection algorithm. The testing phase of our system is efficient and can facilitate diagnosis of B-lymphocyte neoplasms.
Blunt snout bream (Megalobrama amblycephala) is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. MicroRNAs (miRNAs) play important roles in regulation of almost all biological processes in eukaryotes. Although previous studies have identified thousands of miRNAs from many species, little information is known for miRNAs of M. amblycephala. To investigate functions of miRNAs associated with growth of M. amblycephala, we adopted the Solexa sequencing technology to sequence two small RNA libraries prepared from four growth related tissues (brain, pituitary, liver and muscle) of M. amblycephala using individuals with relatively high and low growth rates.
In this study, we have identified 347 conserved miRNAs (belonging to 123 families) and 22 novel miRNAs in M. amblycephala. Moreover, we observed sequence variants and seed edits of the miRNAs. Of the 5,166 single nucleotide substitutions observed in two libraries, the most abundant were G-to-U (15.9%), followed by U-to-C (12.1%), G-to-A (11.2%), and A to G (11.2%). Subsequently, we compared the expression patterns of miRNAs in the two libraries (big-size group with high growth rate versus small-size group with low growth rate). Results indicated that 27 miRNAs displayed significant differential expressions between the two libraries (p < 0.05). Of these, 16 were significantly up-regulated and 11 were significantly down-regulated in the big-size group compared to the small-size group. Furthermore, stem-loop RT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in ten tissues, and the result revealed that the conserved miRNAs expressed at higher levels than the novel miRNAs, especially in brain, liver and muscle. Also, targets prediction of differentially expressed miRNAs and KEGG pathway analysis suggested that differentially expressed miRNAs are involved in growth and metabolism, signal transduction, cell cycle, neural development and functions.
The present study provides the first large-scale characterization of miRNAs in M. amblycephala and miRNA profile related to different growth performances. The discovery of miRNA resource from this study is expected to contribute to a better understanding of the miRNAs roles playing in regulating the growth biological processes and the study of miRNA function and phenotype-associated miRNA identification in fish.
The relationship between fish health and muscle growth is critical for continued expansion of the aquaculture industry. The effect of immune stimulation on the expression of genes related to the energy balance of fish is poorly understood. In mammals immune stimulation results in major transcriptional changes in muscle, potentially to allow a reallocation of amino acids for use in the immune response and energy homeostasis. The aim of this study was to investigate the effects of immune stimulation on fish muscle gene expression.
Atlantic salmon (Salmo salar) primary muscle cell cultures were stimulated with recombinant (r)IL-1β, a major proinflammatory cytokine, for 24 h in order to simulate an acute immune response. The transcriptomic response was determined by RNA hybridization to a 4 × 44 K Agilent Atlantic salmon microarray platform. The rIL-1β stimulation induced the expression of genes related to both the innate and adaptive immune systems. In addition there were highly significant changes in the expression of genes related to regulation of the cell cycle, growth/structural proteins, proteolysis and lipid metabolism. Of interest were a number of IGF binding proteins that were differentially expressed, which may demonstrate cross talk between the growth and immune systems.
We show rIL-1β modulates the expression of not only immune related genes, but also that of genes involved in processes related to growth and metabolism. Co-stimulation of muscle cells with both rIGF-I and rIL-1β demonstrates cross talk between these pathways providing potential avenues for further research. This study highlights the potential negative effects of inflammation on muscle protein deposition and growth in fish and extends our understanding of energy allocation in ectothermic animals.
Transcriptomics; Atlantic salmon (Salmo salar); Muscle cell culture; Inflammation; Catabolism; Cell cycle; IGF binding proteins
Single nucleotide polymorphisms (SNPs), the most abundant variations in a
genome, have been widely used in various studies. Detection and
characterization of citrus haplotype-based expressed sequence tag (EST) SNPs
will greatly facilitate further utilization of these gene-based
In this paper, haplotype-based SNPs were mined out of publicly available
citrus expressed sequence tags (ESTs) from different citrus cultivars
(genotypes) individually and collectively for comparison. There were a total
of 567,297 ESTs belonging to 27 cultivars in varying numbers and
consequentially yielding different numbers of haplotype-based quality SNPs.
Sweet orange (SO) had the most (213,830) ESTs, generating 11,182 quality
SNPs in 3,327 out of 4,228 usable contigs. Summed from all the individually
mining results, a total of 25,417 quality SNPs were discovered –
15,010 (59.1%) were transitions (AG and CT), 9,114 (35.9%) were
transversions (AC, GT, CG, and AT), and 1,293 (5.0%) were
insertion/deletions (indels). A vast majority of SNP-containing contigs
consisted of only 2 haplotypes, as expected, but the percentages of 2
haplotype contigs varied widely in these citrus cultivars. BLAST of the
25,417 25-mer SNP oligos to the Clementine reference genome scaffolds
revealed 2,947 SNPs had “no hits found”, 19,943 had 1 unique hit
/ alignment, 1,571 had one hit and 2+ alignments per hit, and 956 had 2+
hits and 1+ alignment per hit. Of the total 24,293 scaffold hits, 23,955
(98.6%) were on the main scaffolds 1 to 9, and only 338 were on 87 minor
scaffolds. Most alignments had 100% (25/25) or 96% (24/25) nucleotide
identities, accounting for 93% of all the alignments. Considering almost all
the nucleotide discrepancies in the 24/25 alignments were at the SNP sites,
it served well as in silico validation of these SNPs, in addition
to and consistent with the rate (81%) validated by sequencing and SNaPshot
High-quality EST-SNPs from different citrus genotypes were detected, and
compared to estimate the heterozygosity of each genome. All the SNP oligo
sequences were aligned with the Clementine citrus genome to determine their
distribution and uniqueness and for in silico validation, in
addition to SNaPshot and sequencing validation of selected SNPs.
Haplotype; Heterozygosity; Polymorphism; Transition; Transversion; Insertion/deletion; Non-synonymous; Synonymous
AtPAP2 is a purple acid phosphatase that is targeted to both chloroplasts and mitochondria. Over-expression (OE) lines of AtPAP2 grew faster, produced more seeds, and contained higher leaf sucrose and glucose contents. The present study aimed to determine how high energy status affects leaf and root transcriptomes.
ATP and ADP levels in the OE lines are 30-50% and 20-50% higher than in the wild-type (WT) plants. Global transcriptome analyses indicated that transcriptional regulation does play a role in sucrose and starch metabolism, nitrogen, potassium and iron uptake, amino acids and secondary metabolites metabolism when there is an ample supply of energy. While the transcript abundance of genes encoding protein components of photosystem I (PS I), photosystem II (PS II) and light harvesting complex I (LHCI) were unaltered, changes in transcript abundance for genes encoding proteins of LHCII are significant. The gene expressions of most enzymes of the Calvin cycle, glycolysis and the tricarboxylic acid (TCA) cycle were unaltered, as these enzymes are known to be regulated by light/redox status or allosteric modulation by the products (e.g. citrate, ATP/ADP ratio), but not at the level of transcription.
AtPAP2 overexpression resulted in a widespread reprogramming of the transcriptome in the transgenic plants, which is characterized by changes in the carbon, nitrogen, potassium, and iron metabolism. The fast-growing AtPAP2 OE lines provide an interesting tool for studying the regulation of energy system in plant.
Chloroplast; Mitochondria; LHC; Redox; Photosystem; Transcriptomes
Two sibling members of the Anopheles gambiae species complex display notable differences in female blood meal preferences. An. gambiae s.s. has a well-documented preference for feeding upon human hosts, whereas An. quadriannulatus feeds on vertebrate/mammalian hosts, with only opportunistic feeding upon humans. Because mosquito host-seeking behaviors are largely driven by the sensory modality of olfaction, we hypothesized that hallmarks of these divergent host seeking phenotypes will be in evidence within the transcriptome profiles of the antennae, the mosquito’s principal chemosensory appendage.
To test this hypothesis, we have sequenced antennal mRNA of non-bloodfed females from each species and observed a number of distinct quantitative and qualitative differences in their chemosensory gene repertoires. In both species, these gene families show higher rates of sequence polymorphisms than the overall rates in their respective transcriptomes, with potentially important divergences between the two species. Moreover, quantitative differences in odorant receptor transcript abundances have been used to model potential distinctions in volatile odor receptivity between the two sibling species of anophelines.
This analysis suggests that the anthropophagic behavior of An. gambiae s.s. reflects the differential distribution of olfactory receptors in the antenna, likely resulting from a co-option and refinement of molecular components common to both species. This study improves our understanding of the molecular evolution of chemoreceptors in closely related anophelines and suggests possible mechanisms that underlie the behavioral distinctions in host seeking that, in part, account for the differential vectorial capacity of these mosquitoes.
Anopheles; Mosquito; Antenna; Transcriptome; Olfaction; Malaria; Host-seeking; Odorant receptor; Molecular evolution; RNAseq