Orthologous Comparisons Research Articles

Identification of a novel and plant height-independent QTL for coleoptile length in barley and validation of its effect using near isogenic lines

Key messageThis study reported the identification and validation of novel QTL conferring coleoptile length in barley and predicted candidate genes underlying the largest effect QTL based on orthologous analysis and comparison of the whole genome assemblies for both parental genotypes of the mapping population.Coleoptile length (CL) is one of the most important agronomic traits in cereal crops due to its direct influence on the optimal depth for seed sowing which facilitates better seedling establishment. Varieties with longer coleoptiles are preferred in drought-prone areas where less moisture maintains at the top layer of the soil. Compared to wheat, genetic study on coleoptile length is limited in barley. Here, we reported a study on detecting the genomic regions associated with CL in barley by assessing a population consisting of 201 recombinant inbred lines. Four putative QTL conferring CL were consistently identified on chromosomes 1H, 5H, 6H, and 7H in each of the trials conducted. Of these QTL, the two located on chromosomes 5H and 6H (designated as Qcl.caf-5H and Qcl.caf-6H) are likely novel and Qcl.caf-5H showed the most significant effect explaining up to 30.9% of phenotypic variance with a LOD value of 15.1. To further validate the effect of this putative QTL, five pairs of near isogenic lines (NILs) were then developed and assessed. Analysis of the NILs showed an average difference of 21.0% in CL between the two isolines. Notably, none of the other assessed morphological characteristics showed consistent differences between the two isolines for each pair of the NILs. Candidate genes underlying the Qcl.caf-5H locus were also predicted by employing orthologous analysis and comparing the genome assemblies for both parental genotypes of the mapping population in the present study. Taken together, these findings expand our understanding on genetic basis of CL and will be indicative for further gene cloning and functional analysis underly this locus in barley.

Identification and gene expression analysis of serine proteases and their homologs in the Asian corn borer Ostrinia furnacalis

Serine proteases (SPs) and their homologs (SPHs) are among the best-characterized gene families. They are involved in several physiological processes, including digestion, embryonic development and immunity. In the current study, a total of 177 SPs-related genes were characterized in the genome of Ostrinia furnacalis. The activation site of SPs/SPHs and enzyme specificity of SPs were identified, and the findings showed that most of the SPs analyzed possessed trypsin substrate specificity. Several SPs/SPHs with similar simple gene structures had tandem repeat-like distributions on the scaffold, indicated that gene expansion has occurred in this large family. Furthermore, we constructed 30 RNA sequencing libraries including four with developmental stage and four middle larval stage tissues to study the transcript levels of these genes. Differentially upregulated and downregulated genes were obtained via data analysis. More than one-quarter of the genes were specifically identified as highly expressed in the midgut in compared to the other three tissues evaluated. In the current study, the domain structure, gene location and phylogenetic relationship of genes in O. furnacalis were explored. Orthologous comparisons of SPs/SPHs between model insects and O. furnacalis indicated their possible functions. This information provides a basis for understanding the functional roles of this large family.

The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes.

Protein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modifications in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster. Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells, ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24-mutant as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic. In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.

Revealing the species-specific genotype of the edible bird’s nest-producing swiftlet, Aerodramus fuciphagus and the proteome of edible bird’s nest

Only a few species of swiftlets in the Aerodramus and Collocalia genera can produce edible bird's nests (EBN). These saliva-cemented nests have been consumed as delicacies for centuries in Asia. Many researches have reported the aqueous extract of EBN has epidermal growth factor-like (EGF-like) activity. However, no standalone EGF has been identified in EBN. Moreover, proteome of EBN remained unclear due to lack of genomic data base of an EBN-producing swiftlet to support proteomic analysis of EBN. To address this, the first genome of the EBN-producing swiftlet, Aerodramus fuciphagus, was constructed. Orthology comparison of A. fuciphagus with 10 other avian species were conducted. The results revealed that the number of predicted paralogous coiled-coil domain-containing protein 63 (CCDC63) coding sequences (CDSs) in A. fuciphagus was found to be significantly expanded in comparison to Gallus gallus. There were 3 paralogous CCDC63 genes in the genome of A.fuciphagus. The CDSs predicted from the genome of A. fuciphagus were used to construct a database for proteomic analysis of EBN. In total, 398 proteins have been identified in EBN. The proteome of EBN was significant enriched with extracellular proteins as well as proteins related to extracellular matrix (ECM) organization and immune response. A few proteins with Ca2+-binding EGF-like domains were found in the proteome of EBN, like fibrillin-1, protocadherin fat 4 and coagulation factor X. No standalone EGF protein was identified. This indicated that the proteins with EGF-like domains might be responsible for the EGF-like activity of EBN. In addition, acidic mammalian chitinase and lysyl oxidase in EBN were found to be active when extracting with distilled water at room temperature. The current study has not just revealed the species-specific genotype of the EBN-producing swiftlet, A. fuciphagus, but also revealed the proteome of EBN. This established an important foundation for subsequently studies on efficacies of EBN.

Comparative Single-Cell Transcriptomics Reveals Novel Genes Involved in Bivalve Embryonic Shell Formation and Questions Ontogenetic Homology of Molluscan Shell Types.

Mollusks are known for their highly diverse repertoire of body plans that often includes external armor in form of mineralized hardparts. Representatives of the Conchifera, one of the two major lineages that comprises taxa which originated from a uni-shelled ancestor (Monoplacophora, Gastropoda, Cephalopoda, Scaphopoda, Bivalvia), are particularly relevant regarding the evolution of mollusk shells. Previous studies have found that the shell matrix of the adult shell (teleoconch) is rapidly evolving and that the gene set involved in shell formation is highly taxon-specific. However, detailed annotation of genes expressed in tissues involved in the formation of the embryonic shell (protoconch I) or the larval shell (protoconch II) are currently lacking. Here, we analyzed the genetic toolbox involved in embryonic and larval shell formation in the quagga mussel Dreissena rostriformis using single cell RNA sequencing. We found significant differences in genes expressed during embryonic and larval shell secretion, calling into question ontogenetic homology of these transitory bivalve shell types. Further ortholog comparisons throughout Metazoa indicates that a common genetic biomineralization toolbox, that was secondarily co-opted into molluscan shell formation, was already present in the last common metazoan ancestor. Genes included are engrailed, carbonic anhydrase, and tyrosinase homologs. However, we found that 25% of the genes expressed in the embryonic shell field of D. rostriformis lack an ortholog match with any other metazoan. This indicates that not only adult but also embryonic mollusk shells may be fast-evolving structures. We raise the question as to what degree, and on which taxonomic level, the gene complement involved in conchiferan protoconch formation may be lineage-specific or conserved across taxa.

Mapping the Binding Sites of UDP and Prostaglandin E2 Glyceryl Ester in the Nucleotide Receptor P2Y6.

Cyclooxygenase‐2 catalyzes the biosynthesis of prostaglandins from arachidonic acid and the biosynthesis of prostaglandin glycerol esters (PG‐Gs) from 2‐arachidonoylglycerol. PG‐Gs are mediators of several biological actions such as macrophage activation, hyperalgesia, synaptic plasticity, and intraocular pressure. Recently, the human UDP receptor P2Y6 was identified as a target for the prostaglandin E2 glycerol ester (PGE2‐G). Here, we show that UDP and PGE2‐G are evolutionary conserved endogenous agonists at vertebrate P2Y6 orthologs. Using sequence comparison of P2Y6 orthologs, homology modeling, and ligand docking studies, we proposed several receptor positions participating in agonist binding. Site‐directed mutagenesis and functional analysis of these P2Y6 mutants revealed that both UDP and PGE2‐G share in parts one ligand‐binding site. Thus, the convergent signaling of these two chemically very different agonists has already been manifested in the evolutionary design of the ligand‐binding pocket.

Candidate Effectors of Plasmodiophora brassicae Pathotype 5X During Infection of Two Brassica napus Genotypes.

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of canola (Brassica napus) in Canada. Disease management relies heavily on planting clubroot resistant (CR) cultivars, but in recent years, new resistance-breaking pathotypes of P. brassicae have emerged. Current efforts against the disease are concentrated in developing host resistance using traditional genetic breeding, omics and molecular biology. However, because of its obligate biotrophic nature, limited resources have been dedicated to investigating molecular mechanisms of pathogenic infection. We previously performed a transcriptomic study with the cultivar resistance-breaking pathotype 5X on two B. napus hosts presenting contrasting resistance/susceptibility, where we evaluated the mechanisms of host response. Since cultivar-pathotype interactions are very specific, and pathotype 5X is one of the most relevant resistance-breaking pathotypes in Canada, in this study, we analyze the expression of genes encoding putative secreted proteins from this pathotype, predicted using a bioinformatics pipeline, protein modeling and orthologous comparisons with effectors from other pathosystems. While host responses were found to differ markedly in our previous study, many common effectors are found in the pathogen while infecting both hosts, and the gene response among biological pathogen replicates seems more consistent in the effectors associated with the susceptible interaction, especially at 21 days after inoculation. The predicted effectors indicate the predominance of proteins with interacting domains (e.g., ankyrin), and genes bearing kinase and NUDIX domains, but also proteins with protective action against reactive oxygen species from the host. Many of these genes confirm previous predictions from other clubroot studies. A benzoic acid/SA methyltransferase (BSMT), which methylates SA to render it inactive, showed high levels of expression in the interactions with both hosts. Interestingly, our data indicate that E3 ubiquitin proteasome elements are also potentially involved in pathogenesis. Finally, a gene with similarity to indole-3-acetaldehyde dehydrogenase is a promising candidate effector because of its involvement in indole acetic acid synthesis, since auxin is one of the major players in clubroot development.

Large-scale phylogenomic analysis provides new insights into the phylogeny of the class Oligohymenophorea (Protista, Ciliophora) with establishment of a new subclass Urocentria nov. subcl

The class Oligohymenophorea is one of the most diverse assemblage of ciliated protists, which are particularly important in fundamental biological studies including understanding the evolutionary relationships among the lineages. Phylogenetic relationships within the class remain largely elusive, especially within the subclass Peniculia, which contains the long-standing problematic taxa Urocentrum and Paranassula. In the present study, we sequenced the genomes and/or transcriptomes of six non-culturable oligohymenophoreans using single-cell sequencing techniques. Phylogenomic analysis was performed based on expanded taxon sampling of 85 taxa, including 157 nuclear genes encoding 36,953 amino acids. The results indicate that: (1) urocentrids form an independent branch that is sister to the clade formed by Scuticociliatia and Hymenostomatia, which, together with the morphological data, supports the establishment of a new subclass, Urocentria n. subcl., within Oligohymenophorea; (2) phylogenomic analysis and ortholog comparison reveal a close relationship between Paranassula and peniculines, providing corroborative evidence for removing Paranassula from Nassulida and elevating it as an order, Paranassulida, within the subclass Peniculia; (3) based on the phylogenomic analyses and morphological data, we hypothesize that Peritrichia is the earliest diverging clade within Oligohymenophorea while Scuticociliatia and Hymenostomatia share the most common ancestor, followed successively by Urocentria and Peniculia. In addition, stop codon analyses indicate that oligohymenophoreans widely use UGA as the stop codon, while UAR are reassigned to glutamate (peritrichs) or glutamine (others), supporting the evolutionary hypothesis.

Proteome-Scale Detection of Differential Conservation Patterns at Protein and Subprotein Levels with BLUR.

In the multiomics era, comparative genomics studies based on gene repertoire comparison are increasingly used to investigate evolutionary histories of species, to study genotype–phenotype relations, species adaptation to various environments, or to predict gene function using phylogenetic profiling. However, comparisons of orthologs have highlighted the prevalence of sequence plasticity among species, showing the benefits of combining protein and subprotein levels of analysis to allow for a more comprehensive study of genotype/phenotype correlations. In this article, we introduce a new approach called BLUR (BLAST Unexpected Ranking), capable of detecting genotype divergence or specialization between two related clades at different levels: gain/loss of proteins but also of subprotein regions. These regions can correspond to known domains, uncharacterized regions, or even small motifs. Our method was created to allow two types of research strategies: 1) the comparison of two groups of species with no previous knowledge, with the aim of predicting phenotype differences or specializations between close species or 2) the study of specific phenotypes by comparing species that present the phenotype of interest with species that do not. We designed a website to facilitate the use of BLUR with a possibility of in-depth analysis of the results with various tools, such as functional enrichments, protein–protein interaction networks, and multiple sequence alignments. We applied our method to the study of two different biological pathways and to the comparison of several groups of close species, all with very promising results. BLUR is freely available at http://lbgi.fr/blur/.

Evolutionary Study of Disorder in Protein Sequences.

Intrinsically disordered proteins (IDPs) contain regions lacking intrinsic globular structure (intrinsically disordered regions, IDRs). IDPs are present across the tree of life, with great variability of IDR type and frequency even between closely related taxa. To investigate the function of IDRs, we evaluated and compared the distribution of disorder content in 10,695 reference proteomes, confirming its high variability and finding certain correlation along the Euteleostomi (bony vertebrates) lineage to number of cell types. We used the comparison of orthologs to study the function of disorder related to increase in cell types, observing that multiple interacting subunits of protein complexes might gain IDRs in evolution, thus stressing the function of IDRs in modulating protein-protein interactions, particularly in the cell nucleus. Interestingly, the conservation of local compositional biases of IDPs follows residue-type specific patterns, with E- and K-rich regions being evolutionarily stable and Q- and A-rich regions being more dynamic. We provide a framework for targeted evolutionary studies of the emergence of IDRs. We believe that, given the large variability of IDR distributions in different species, studies using this evolutionary perspective are required.

Chromosome-level de novo assembly of the pig-tailed macaque genome using linked-read sequencing and HiC proximity scaffolding.

BackgroundMacaque species share >93% genome homology with humans and develop many disease phenotypes similar to those of humans, making them valuable animal models for the study of human diseases (e.g., HIV and neurodegenerative diseases). However, the quality of genome assembly and annotation for several macaque species lags behind the human genome effort.ResultsTo close this gap and enhance functional genomics approaches, we used a combination of de novo linked-read assembly and scaffolding using proximity ligation assay (HiC) to assemble the pig-tailed macaque (Macaca nemestrina) genome. This combinatorial method yielded large scaffolds at chromosome level with a scaffold N50 of 127.5 Mb; the 23 largest scaffolds covered 90% of the entire genome. This assembly revealed large-scale rearrangements between pig-tailed macaque chromosomes 7, 12, and 13 and human chromosomes 2, 14, and 15. We subsequently annotated the genome using transcriptome and proteomics data from personalized induced pluripotent stem cells derived from the same animal. Reconstruction of the evolutionary tree using whole-genome annotation and orthologous comparisons among 3 macaque species, human, and mouse genomes revealed extensive homology between human and pig-tailed macaques with regards to both pluripotent stem cell genes and innate immune gene pathways. Our results confirm that rhesus and cynomolgus macaques exhibit a closer evolutionary distance to each other than either species exhibits to humans or pig-tailed macaques.ConclusionsThese findings demonstrate that pig-tailed macaques can serve as an excellent animal model for the study of many human diseases particularly with regards to pluripotency and innate immune pathways.

Cellular and Molecular Targets of Waterbuck Repellent Blend Odors in Antennae of Glossina fuscipes fuscipes Newstead, 1910.

Insects that transmit many of the world’s deadliest animal diseases, for instance trypanosomosis, find their suitable hosts and avoid non-preferred hosts mostly through olfactory cues. The waterbuck repellent blend (WRB) comprising geranylacetone, guaiacol, pentanoic acid, and δ-octalactone derived from waterbuck skin odor is a repellent to some savannah-adapted tsetse flies and reduces trap catches of riverine species. However, the cellular and molecular mechanisms associated with detection and coding of the repellent odors remain to be elucidated. Here, we demonstrated that WRB inhibited blood feeding in both Glossina pallidipes Austen, 1903 and Glossina fuscipes fuscipes Newstead, 1910. Using the DREAM (Deorphanization of Receptors based on Expression Alterations in odorant receptor mRNA levels) technique, combined with ortholog comparison and molecular docking, we predicted the putative odorant receptors (ORs) for the WRB in G. f. fuscipes, a non-model insect. We show that exposure of G. f. fuscipes in vivo to WRB odorant resulted in up- and downregulation of mRNA transcript of several ORs. The WRB component with strong feeding inhibition altered mRNA transcript differently as compared to an attractant odor, showing these two odors of opposing valence already segregate at the cellular and molecular levels. Furthermore, molecular dynamics simulations demonstrated that the predicted ligand–OR binding pockets consisted mostly of hydrophobic residues with a few hydrogen bonds but a stable interaction. Finally, our electrophysiological response showed the olfactory sensory neurons of G. f. fuscipes tuned to the tsetse repellent components in different sensitivity and selectivity.

Details of the Conformational Cycle of Hsp90 Probed using Optical Tweezers

Heat shock protein 90 (Hsp90) is an ATP-driven molecular chaperone which is essential in eukaryotes. It functions as a part of a number of complexes with many different co-chaperone proteins, which regulate its conformational cycle [1]. Using a custom-built single molecule optical trapping experimental setup, we have previously elucidated the mechanism by which this large protein folds [2], characterised the role of its flexible charged linker region [3], performed a detailed comparison of Hsp90 orthologs [4] and elucidated details of its dimerization dynamics [5]. Hsp90 is known to exist in two forms in yeast – the constitutively expressed Hsc82 and the form that is upregulated under cellular stress conditions, Hsp82 [6]. We recently performed a detailed comparison of these two isoforms, using a broad range of biophysical techniques to compare their function, structural properties and interactomes. Despite a 97% sequence similarity, we find significant differences in their stabilities, folding and conformational regulation [7]. We are currently working towards developing a complete quantitative description of the conformational cycle of Hsp82, as driven by co-chaperone proteins, nucleotides and clients using single molecule optical tweezers combined with standard biochemical techniques. [1] F. H. Schopf, M. M. Biebl & J. Buchner, Nat. Rev. Mol. Cell Biol. 18, 345 – 360, (2017). [2] M. Jahn, et al. Proc. Nat. Acad. Sci. 113, 1232–1237, (2016). [3] M. Jahn, et al. Proc. Nat. Acad. Sci. 111, 17881–17886, (2014). [4] M. Jahn and K. Tych, et al. Structure 26, 96 – 105, (2018). [5] K. Tych et al. J. Phys. Chem. B 122, 11373-11380 (2018). [6] K.A. Borkovic et al. Mol. Cell. Biol. 9, 3919 – 3930, (1989) [7] H. Girstmair and F. Tippel, et al. Nature Comms. 10, 1 – 15 (2019).

De novo Assembly and Annotation of the Blood Transcriptome of the Southern Elephant Seal Mirounga leonina from the South Shetland Islands, Antarctica

The southern elephant seal Mirounga leonina is the largest member of the phocid seals and is a highly sexually dimorphic predator at circumpolar regions. In this study, we generated the first high-quality de novo assembly of the blood transcriptome of M. leonina using the Illumina MiSeq platform. A total of 40.5 million raw reads were obtained and assembled using the Oases assembly platform, resulting in 46,445 contigs with an N50 value of 634 bp. We performed functional gene annotations through pathway analyses of the Gene Ontology (GO), Eukaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Ortholog comparison revealed that a high proportion of the M. leonina blood transcriptome had significant sequence homology within pinnipeds. M. leonine, a deep and long-diving seal, is routinely exposed to progressive hypoxia during dives. KEGG analyses detected an intact hypoxia-inducible factor-1 (HIF-1) signaling pathway, which is a key metabolism pathway in the adaptive homeostatic responses to hypoxia. In the blood transcriptome, many essential genes involved in innate immunity were detected, which suggests that the blood serves as a host defense modulator against exogenous infections in M. leonina. This genomic resource will be useful for understanding adaptive metabolism upon repeated breath-hold dives and determining the health status of southern elephant seals without the need to sacrifice them in experiments.

Blood transcriptome resources of chinstrap (Pygoscelis antarcticus) and gentoo (Pygoscelis papua) penguins from the South Shetland Islands, Antarctica.

The chinstrap (Pygoscelis antarcticus) and gentoo (P. papua) penguins are distributed throughout Antarctica and the sub-Antarctic islands. In this study, high-quality de novo assemblies of blood transcriptomes from these penguins were generated using the Illumina MiSeq platform. A total of 22.2 and 21.8 raw reads were obtained from chinstrap and gentoo penguins, respectively. These reads were assembled using the Oases assembly platform and resulted in 26,036 and 21,854 contigs with N50 values of 929 and 933 base pairs, respectively. Functional gene annotations through pathway analyses of the Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed for each blood transcriptome, resulting in a similar compositional order between the two transcriptomes. Ortholog comparisons with previously published transcriptomes from the Adélie (P. adeliae) and emperor (Aptenodytes forsteri) penguins revealed that a high proportion of the four penguins’ transcriptomes had significant sequence homology. Because blood and tissues of penguins have been used to monitor pollution in Antarctica, immune parameters in blood could be important indicators for understanding the health status of penguins and other Antarctic animals. In the blood transcriptomes, KEGG analyses detected many essential genes involved in the major innate immunity pathways, which are key metabolic pathways for maintaining homeostasis against exogenous infections or toxins. Blood transcriptome studies such as this may be useful for checking the immune and health status of penguins without sacrifice.

Multiple model species selection for transcriptomics analysis of non-model organisms

BackgroundTranscriptomic sequencing (RNA-seq) related applications allow for rapid explorations due to their high-throughput and relatively fast experimental capabilities, providing unprecedented progress in gene functional annotation, gene regulation analysis, and environmental factor verification. However, with increasing amounts of sequenced reads and reference model species, the selection of appropriate reference species for gene annotation has become a new challenge.MethodsWe proposed a novel approach for finding the most effective reference model species through taxonomic associations and ultra-conserved orthologous (UCO) gene comparisons among species. An online system for multiple species selection (MSS) for RNA-seq differential expression analysis was developed, and comprehensive genomic annotations from 291 reference model eukaryotic species were retrieved from the RefSeq, KEGG, and UniProt databases.ResultsUsing the proposed MSS pipeline, gene ontology and biological pathway enrichment analysis can be efficiently achieved, especially in the case of transcriptomic analysis of non-model organisms. The results showed that the proposed method solved problems related to limitations in annotation information and provided a roughly twenty-fold reduction in computational time, resulting in more accurate results than those of traditional approaches of using a single model reference species or the large non-redundant reference database.ConclusionsSelection of appropriate reference model species helps to reduce missing annotation information, allowing for more comprehensive results than those obtained with a single model reference species. In addition, adequate model species selection reduces the computational time significantly while retaining the same order of accuracy. The proposed system indeed provides superior performance by selecting appropriate multiple species for transcriptomic analysis compared to traditional approaches.

Unravelling the Mechanics of a Molecular Chaperone

The large molecular chaperone, heat shock protein 90 (Hsp90), is a dimeric ATP-driven molecular machine. It is one of the most abundant proteins found in eukaryotic cells, and is known to function together in complexes with many different co-chaperones. These complexes are involved, amongst other things, in the regulation of cell division and signalling [1]. As such, Hsp90 has emerged as an important target for the development of cancer therapeutics [2]. The exact mechanisms by which Hsp90 undergoes conformational changes, the roles of nucleotides, different domain orientations and how these influence the function of the chaperone in complex with its co-chaperones are mostly unknown. Using a custom-built single molecule optical trapping experimental setup, we have so far elucidated the mechanism by which this large protein folds [3], characterised the role of its flexible charged linker region [4] and performed a detailed comparison of Hsp90 orthologs [5]. Current research efforts are building on these findings to characterise the dimerization of Hsp90, and to study the effect of nucleotide binding and co-chaperones on the conformational cycle and single molecule mechanics of Hsp90. [1] A. J. McClellan, et al. Cell 131, 121-135, (2007). [2] J. S. Isaacs, et al. Cancer cell 3, 213-217, (2003). [3] M. Jahn, et al. Proc. Nat. Acad. Sci. 113, 1232-1237, (2016). [4] M. Jahn, et al. Proc. Nat. Acad. Sci. 111, 17881-17886, (2014). [5] M. Jahn and K. Tych, et al. Structure (under revision) (2017).

Multiplex quantitative SILAC for analysis of archaeal proteomes: a case study of oxidative stress responses.

Stable isotope labelling of amino acids in cell culture (SILAC) is a quantitative proteomic method that can illuminate new pathways used by cells to adapt to different lifestyles and niches. Archaea, while thriving in extreme environments and accounting for ∼20%-40% of the Earth's biomass, have not been analyzed with the full potential of SILAC. Here, we report SILAC for quantitative comparison of archaeal proteomes, using Haloferax volcanii as a model. A double auxotroph was generated that allowed for complete incorporation of 13 C/15 N-lysine and 13 C-arginine such that each peptide derived from trypsin digestion was labelled. This strain was found amenable to multiplex SILAC by case study of responses to oxidative stress by hypochlorite. A total of 2565 proteins was identified by LC-MS/MS analysis (q-value ≤ 0.01) that accounted for 64% of the theoretical proteome. Of these, 176 proteins were altered at least 1.5-fold (p-value < 0.05) in abundance during hypochlorite stress. Many of the differential proteins were of unknown function. Those of known function included transcription factor homologs related to oxidative stress by 3D-homology modelling and orthologous group comparisons. Thus, SILAC is found to be an ideal method for quantitative proteomics of archaea that holds promise to unravel gene function.

Comparative Genomics of Ralstonia solanacearum Identifies Candidate Genes Associated with Cool Virulence.

Strains of the Ralstonia solanacearum species complex in the phylotype IIB group are capable of causing Bacterial Wilt disease in potato and tomato at temperatures lower than 24°C. The capability of these strains to survive and to incite infection at temperatures colder than their normally tropical boundaries represents a threat to United States agriculture in temperate regions. In this work, we used a comparative genomics approach to identify orthologous genes linked to the lower temperature virulence phenotype. Six R. solanacearum cool virulent (CV) strains were compared to six strains non-pathogenic at low temperature (NPLT). CV strains can cause Bacterial Wilt symptoms at temperatures below 24°C, while NPLT cannot. Four R. solanacearum strains were sequenced for this work in order to complete the comparison. An orthologous genes comparison identified 44 genes present only in CV strains and 19 genes present only in NPLT strains. Gene annotation revealed a high percentage of genes compared with whole genomes in the transcriptional regulator and transport categories. A single nucleotide polymorphism (SNP) analysis identified 265 genes containing conserved non-synonymous SNPs in CV strains. Ten genes in the pathogenicity category were identified in this group. Comparisons of type 3 secretion system, type 6 secretion system (T6SS) clusters, and associated effectors did not indicate a correlation with the CV phenotype except for one T6SS VGR effector potentially associated with the CV phenotype. This is the first R. solanacearum genomic comparative analysis of multiple strains with different temperature related virulence. The candidate genes identified by this comparison are potential factors involved in virulence at low temperatures that need to be investigated. The high percentage of transcriptional regulators among the genes present only in CV strains supports the hypothesis that temperature dependent regulation of virulence genes explains the differential virulence phenotype at low temperatures. This comparison contributes to find new possible connections of temperature dependent virulence to the previously described complex regulatory system involving quorum-sensing, phenotype conversion (phcA), acyl-HSL production and responses to SA. It also added novel candidate T6SS effectors and useful detailed information about the T6SS in R. solanacearum.

Evolutionary expansion and structural functionalism of the ancient family of profilin proteins

Structure and functional similarities of a recent protein's orthologs with its ancient counterpart are largely determined by the configuration of evolutionary preservation of amino acids. The emergence of genome sequencing databases allowed dissecting the evolutionarily important gene families at a comprehensive and genome-wide scale. The profilin multi-gene family is an ancient, universal, and functionally diverged across kingdoms, which regulates various aspects of cellular development in both prokarya and eukarya, especially cell-wall maintenance through actin sequestering, nucleation and cytokinesis. We performed a meta-analysis of the evolutionary expansion, structural conservation, evolution of function motifs, and transcriptional biases of profilin proteins across kingdoms. An exhaustive search of various genome databases of cyanobacteria, fungi, animalia and plantae kingdoms revealed 172 paralogous/orthologous profilins those were phylogenetically clustered in various groups. Orthologous gene comparisons indicated that segmental and tandem duplication events under strong purifying selection are predominantly responsible for their convoluted structural divergences. Evidently, structural divergences were more prevalent in the paralogs than orthologs, and evolutionary variations in the exon/intron architecture were accomplished by ‘exon/intron-gain’ and insertion/deletion during sequence-exonization. Remarkably, temporal expression evolution of profilin paralogs/homeologs during cotton fiber domestication provides evolutionary impressions of the selection of highly diverged transcript abundance notably in the fiber morpho-evolution. These results provide global insights into the profilin evolution, their structural design across taxa; and their future utilization in translational research.