Biology subjects

Sharma, T.

Publications and source records attributed to Sharma, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Minor changes in electrostatics robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles

Ebola virus (EBOV) is a filamentous negative-sense RNA virus which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine (PS) and PI(4,5)P2 in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles (VLPs). In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

microbiology↗

bioRxiv2023

Fusarium graminearum Ste2 and Ste3 Receptors Undergo Peroxidase Induced Heterodimerization

F. graminearum FgSte2 and FgSte3 are G-protein coupled receptors (GPCRs) recently shown to play roles in mediating fungal hyphal chemotropism and plant pathogenesis in response to activity arising from host-released peroxidases. Here, we follow up on the previous observation that chemotropism is dependent on both FgSte2 and FgSte3 being present at the same time; testing the possibility that this effect might be due to formation of an FgSte2-FgSte3 heterodimer. Initially the recombinant cell-surface expression of the F. graminearum GPCRs was validated in S. cerevisiae by confocal immunofluorescence microscopy. Bioluminescence resonance energy transfer analyses were subsequently conducted, where the addition of horse radish peroxidase (HRP) was found to increase the transfer of energy from the inducibly-expressed FgSte3-Nano luciferase (FgSte3-NLuc) donor, to the constitutively-expressed FgSte2-yellow fluorescent protein (FgSte2-YFP) acceptor, compared to controls. A partial response was also detected when an HRP-derived ligand-containing extract was enriched from F. graminearum spores and applied to the S. cerevisiae BRET system directly. The selectivity of the interaction was demonstrated by comparison to treatment with pheromones as well as an unrelated bovine GPCR, rhodopsin, fused to YFP as acceptor, that yielded no response when co-expressed with FgSte3-NLuc. Finally, the peroxidase-stimulated heterodimerization was validated by affinity pulldown. Taken together these findings demonstrate the formation of HRP and HRP-derived ligand stimulated heterodimers between FgSte2 and FgSte3. Outcomes are discussed from the context of the roles of ligands and reactive oxygen species in GPCR dimerization.

biochemistry↗

bioRxiv2023

Proteomic profiling of regenerated urinary bladder tissue with stem cell seeded scaffold composites in a non-human primate bladder augmentation model

Urinary bladder insult can be caused by environmental, genetic, and developmental factors. Depending upon insult severity, the bladder may lose its ability to maintain capacity and intravesical pressures resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is employed to increase bladder capacity to preserve renal function using autologous bowel tissue as a "patch." To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) with CD34+ hematopoietic stem/progenitor cells (HSPCs) co-seeded onto a pliable synthetic scaffold [POCO; poly(1,8-octamethylene-citrate-co-octanol)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in a baboon bladder augmentation model. We set out to determine the protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogenous protein expression between the tissues following long-term engraftment. We posit that stem cell seeded scaffolds can recapitulate tissue that is almost indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE. Significance of StudyBladder augmentation enterocystoplasty has been used for decades as the gold-standard surgical procedure to treat severely dysfunctional bladders. Unfortunately, bowel (typically ileum) used for augmentation is an anatomical and physiological mismatch to bladder tissue and results in numerous complications including bladder perforation, secondary and tertiary redo surgeries, metabolic imbalances, excess mucus production, and increased risk of cancer. This is in part due to the intestinal protein expression that serves as a starting point and subsequent foundation in the optimization of pseudo bladder tissue. Within the context of this study, we demonstrate that autologous, bone marrow derived mesenchymal stem cells along with primitive hematopoietic stem/progenitor cells can used to regenerate bladder tissue in a large deficit, non-human primate bladder augmentation model. Data demonstrate that this synergistic cellular combination facilitates the promotion of a protein tissue landscape that is nearly identical to native bladder tissue.

bioengineering↗

bioRxiv2023

Hypothetical gene Rv0495c regulates redox homeostasis in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has evolved sophisticated surveillance mechanisms to regulate and neutralize redox imbalances and associated lethal consequences. Failing this, the accumulated ROS induces toxicity by oxidizing a variety of biological molecules including proteins, nucleic acids and lipids. In the present study we identified Mtbs Rv0495c gene as an important regulator of oxidized cytosolic environment. Compared to wild type Mtb strain lacking the Rv0495c gene, {Delta}Rv0495c, had increased ROS and NAD+/NADH ratio creating a highly oxidized intracellular environment. {Delta}Rv0495c strain demonstrated slow growth phenotype under in vitro and ex-vivo growth conditions and demonstrated enhanced susceptibility to drugs, oxidative, nitrosative and hypoxic growth conditions. In addition, the increase in the superoxide radicals triggered a Fenton-like reaction rendering the {Delta}Rv0495c susceptible to free iron. The increase in the intracellular ROS levels of the {Delta}Rv0495c was further corroborated by an increase in the expression of proteins involved in antioxidant defense and enhanced ROS-mediated oxidation and degradation of mycobacterial lipids. This superoxide-induced lipid degradation resulted in altered colony morphology and loss of membrane integrity in the {Delta}Rv0495c. Surprisingly, despite showing a growth defect phenotype in an ex-vivo macrophage infection model, the absence of the Rv0495c gene in Mtb enhanced the pathogenicity and augmented the ability of the Mtb to grow inside the host. Gene expression analysis revealed a Rv0495c mediated immunomodulation of the host controls inflammation and helps creates a favorable niche for long-term survival of Mtb inside the host. In summary, the current study underscores the fact that the truce in the war between the host and the pathogen favors long-term disease persistence in tuberculosis. We believe targeting Rv0495c could potentially be explored as a strategy to potentiate the current anti-TB regimen.

microbiology↗

bioRxiv2023

VapC12 ribonuclease toxin modulates host immune response during Mycobacterium tuberculosis infection

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with {Delta}vapC12 induced a proinflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with {Delta}vapC12 succumbed early to the disease. Finally, we reported that the above phenotype of {Delta}vapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggest that the inability of {Delta}vapC12 to resolve neutrophil-mediated inflammation reduced bacterial killing by altering the T-cell response. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the hosts innate immune response in ways that favor the long-term survival of the pathogen inside the host.

microbiology↗

bioRxiv2023

Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease

Somatic mutations in non-malignant tissues accumulate with age and insult, but whether these mutations are adaptive on the cellular or organismal levels is unclear. To interrogate mutations found in human metabolic disease, we performed lineage tracing in mice harboring somatic mosaicism subjected to non-alcoholic steatohepatitis (NASH). Proof-of-concept studies with mosaic loss of Mboat7, a membrane lipid acyltransferase, showed that increased steatosis accelerated clonal disappearance. Next, we induced pooled mosaicism in 63 known NASH genes, allowing us to trace mutant clones side-by-side. This in vivo tracing platform, which we coined MOSAICS, selected for mutations that ameliorate lipotoxicity, including mutant genes identified in human NASH. To prioritize new genes, additional screening of 472 candidates identified 23 somatic perturbations that promoted clonal expansion. In validation studies, liver-wide deletion of Bcl6, Tbx3, or Smyd2 resulted in protection against NASH. Selection for clonal fitness in mouse and human livers identifies pathways that regulate metabolic disease. HighlightsO_LIMosaic Mboat7 mutations that increase lipotoxicity lead to clonal disappearance in NASH. C_LIO_LIIn vivo screening can identify genes that alter hepatocyte fitness in NASH. C_LIO_LIMosaic Gpam mutations are positively selected due to reduced lipogenesis. C_LIO_LIIn vivo screening of transcription factors and epifactors identified new therapeutic targets in NASH. C_LI

cell biology↗

bioRxiv2023

Differential requirements for different subfamilies of the mammalian SWI/SNF chromatin remodeling enzymes in myoblast differentiation

Mammalian SWI/SNF (mSWI/SNF) complexes are ATP-dependent chromatin remodeling enzymes that are critical for normal cellular functions and that are mis-regulated in [~]20% of human cancers. These enzymes exhibit significant diversity in the composition of individual enzyme complexes. mSWI/SNF enzymes are classified into three general sub-families based on the presence or absence of specific subunit proteins. The three sub-families are called BAF (BRM or BRG1-associated factors), ncBAF (non-canonical BAF), and PBAF (Polybromo-associated BAF). The biological roles for the different subfamilies of mSWI/SNF enzymes are poorly described. We knocked down (KD) the expression of genes encoding subunit proteins unique to each of the three subfamilies, Baf250A, Brd9, and Baf180, which mark the BAF, ncBAF, and PBAF sub-families, respectively, and examined the requirement for each in myoblast differentiation. We found that BAF250A and the BAF complex was required to drive lineage-specific gene expression during myoblast differentiation. KD of Baf250A reduced the expression of the lineage determinant Myogenin and other differentiation markers, due to decreased binding of BAF250A to myogenic gene promoters. KD of Brd9 delayed myoblast differentiation. However, RNA-seq analysis revealed that while the Baf250A-dependent gene expression profile included genes involved in myogenesis, the Brd9-dependent gene expression profile did not. Moreover, no-colocalization of Baf250A and Brd9 was observed in differentiating cells, suggesting independent mechanisms of action for BAF and ncBAF complexes in myogenesis. The PBAF complex was dispensable for myoblast differentiation. The results distinguish between the roles of the mSWI/SNF enzyme subfamilies during myoblast differentiation.

molecular biology↗

bioRxiv2022

IGFBP2 expressing midlobular hepatocytes preferentially contribute to liver homeostasis and regeneration

While midlobular hepatocytes in zone 2 are a recently identified cellular source for liver homeostasis and regeneration, these cells have not been exclusively fate mapped. We generated a Igfbp2-CreER knockin strain, which specifically labels midlobular hepatocytes. During homeostasis over 1 year, zone 2 hepatocytes increased in abundance from occupying 21% to 41% of the lobular area. After either pericentral injury with carbon tetrachloride or periportal injury with DDC, IGFBP2+ cells replenished lost hepatocytes in zones 3 and 1, respectively. IGFBP2+ cells also preferentially contributed to regeneration after 70% partial hepatectomy, as well as liver growth during pregnancy. Because IGFBP2 labeling increased substantially with fasting, we used single nuclear transcriptomics to explore zonation as a function of nutrition, revealing that the zonal division of labor shifts dramatically with fasting. These studies demonstrate the contribution of IGFBP2-labeled zone 2 hepatocytes to liver homeostasis and regeneration. HighlightsO_LILineage tracing showed that midlobular hepatocytes proliferate during homeostasis. C_LIO_LIZone 2 cells were protected from portal and centrilobular injuries and replaced lost hepatocytes. C_LIO_LIFasting induced significant changes in liver zonation. C_LI eTOC statementThe liver consists of different zones with spatial heterogeneity in their metabolic functions. Here, a new Igfbp2-CreER line enabled direct tracing of midlobular hepatocytes, which showed that IGFBP2+ cells serve as a source of new hepatocytes during normal homeostasis and regeneration.

developmental biology↗

bioRxiv2022

Ab-AMR: A Comprehensive Repository of Acinetobacter baumannii to Understand the Molecular Landscape of Antimicrobial Resistance

Ab-AMR is a comprehensive repository of drug resistance mechanisms in Acinetobacter baumannii. The current version of Ab-AMR provides a drug resistance profile of 788 genomes. In order to ensure that the datasets in Ab-AMR have relevance both to the research and clinical community, standards of defining MIC breakpoints, whole genome sequence quality metrics as defined by EUCAST/CLSI and classification of isolates into susceptible, MDR, XDR and PDR as defined by CDC/ECDC are implemented. As of now, 364 DR determinants associated with antibiotic inactivation ({beta}-lactamases, aminoglycoside modification, chloramphenicol acetyltransferase), efflux, protein modulating permeability and alteration of target site are comprehensively annotated. In addition, data from pangenome analysis across 788 genomes is also provided for identification of core and accessory DR determinants. AB ATCC 17978 (Accession-CP000521.1), the reference strain, was annotated on January 1, 2014 but subsequently the same strain was re-annotated on March 21, 2017 (NZ_CP018664.1) due to incorrect assembly. Therefore, the genome comparison of both, 2014 and 2017 versions were performed for maintaining the correct annotations as most of the literature data referred to the earlier version of the reference genome. In Ab-AMR, the reference coordinates of the revised reference genome are used to represent manually curated and comprehensively annotated data on 614 essential genes, 1334 genes mapped to pathways, 221 PDB structures, 81 reported drug targets, 364 genes with reported resistance mechanism, 118 transcription factors, 4 sigma factors and 14 two component systems. Ab-AMR is made using the standard php-mysql framework and offers various search tools including a query builder that facilitates query on over 60 different features for addressing complex questions like core genes which are also essential and have a role to play in drug resistance with no known human homolog, etc. Ab-AMR offers a centralized data resource for systematic mapping of DR determinants, both plasmid and chromosomal mediated, along with deep annotation of clinical isolates. Database URL: https://datascience.imtech.res.in/anshu/ab-amr/

bioinformatics↗

bioRxiv2022

Significance of the RBD mutations in the SARS-CoV-2 Omicron: from spike opening to antibody escape and cell attachment

We computationally investigated the role of the Omicron RBD mutations on its structure and interactions with ACE2. Our results suggest that, compared to the WT and Delta, the mutations in the Omicron RBD facilitate a more efficient RBD opening and ACE2 attachment. These effects, combined with antibody evasion, may contribute to its dominance over Delta. While the Omicron RBD escapes most antibodies from prior infections, epitope analysis shows that it harbors sequences with significantly improved antigenicity compared to other variants, suggesting more potent Omicron-specific neutralizing antibodies.

biophysics↗

bioRxiv2021

Anisotropic interaction and motion states of locusts in a hopper band

Swarming locusts present a quintessential example of animal collective motion. Juvenile locusts march and hop across the ground in coordinated groups called hopper bands. Composed of up to millions of insects, hopper bands exhibit aligned motion and various collective structures. These groups are well-documented in the field, but the individual insects themselves are typically studied in much smaller groups in laboratory experiments. We present the first trajectory data that detail the movement of individual locusts within a hopper band in a natural setting. Using automated video tracking, we derive our data from footage of four distinct hopper bands of the Australian plague locust, Chortoicetes terminifera. We reconstruct nearly twenty-thousand individual trajectories composed of over 3.3 million locust positions. We classify these data into three motion states: stationary, walking, and hopping. Distributions of relative neighbor positions reveal anisotropies that depend on motion state. Stationary locusts have high-density areas distributed around them apparently at random. Walking locusts have a low-density area in front of them. Hopping locusts have low-density areas in front and behind them. Our results suggest novel insect interactions, namely that locusts change their motion to avoid colliding with neighbors in front of them.

animal behavior and cognition↗

bioRxiv2021

A Model for DHX15 Mediated Disassembly of A-Complex Spliceosomes

A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation and several RNA-dependent-ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNPs engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 can have an expanded role in splicing.

molecular biology↗

bioRxiv2021

A kiwellin protein-like fold containing rust effector protein localizes to chloroplast and suppress cell death in plants

The effector proteins expressed by plant pathogens are one of the essential components of the host-pathogen interaction. Despite being important, most of the effector proteins remain unexplored due to the lack of conserved features and huge diversity in their primary sequence. In the present study, extensive secretome analysis was performed in sixteen major plant fungal pathogens to find the conserved features in the candidate secretory effector proteins (CSEPs) using homology and ab initio modeling approaches. Interestingly, a variable number of plant kiwellin proteins fold like secretory proteins were found in all the major rust fungal pathogens. Many of them are predicted as potential effector proteins. For instance, 26 out of 35 Kiwellin like proteins identified in Puccinia striiformis race 104E 137A were predicted as potential effector proteins. In addition, a kiwellin predicted effector gene, Pst_13960, from the Indian Puccinia striiformis race Yr9 was characterized using overexpression, localization, and deletion studies in Nicotiana benthamiana. The Pst_13960 suppressed the BAX-induced cell death and localized in the chloroplast. Furthermore, the expression of the kiwellin matching region (Pst_13960_kiwi) alone suppressed the BAX-induced cell death in N. benthamiana despite the change of location to the cytoplasm and nucleus, suggesting the novel function of the kiwellin fold in rust fungi. Further analysis of these proteins predicted these candidates to contain N-terminal Intrinsically disordered regions (IDRs) putatively associated with chloroplast translocation as deletion of region abolished the chloroplast localization of Pstr_13960. Overall, the current study reports the presence of kiwellin like proteins in rust fungi that act as a novel effector in plants. Author SummaryRust fungi are one of the most devastating plants infecting pathogens. These pathogens secrete several distinct proteins like effector proteins that help the pathogens in the establishment of infection by suppressing cell death induced by the plants. Despite being important, these effector proteins remain unexplored due to the lack of conserved features. Currently, different methods are being used to characterize them however, could not describe their specific function fully due to a lack of knowledge of the functional domain. Recent advancement in effector protein tertiary structure characterization using NMR (Nuclear magnetic resonance) and X-ray crystallography has been very helpful in identifying the conserved structural features defining functionality. However, these techniques are quite complicated and may take a lot of time and labor. On the other hand, the computational approaches for structural prediction of the effectors may help to identify known folds or domains with few efforts but at a significant level. Therefore, such computational approaches can be efficiently implemented in the preliminary screening of the candidates. In the present study using the computational structure prediction method, we were able to find several conserved novel kiwellin folds containing effectors, in different rust fungi. We characterized one of the candidates and it showed interference with artificially induced cell death in plants. This study highlights the novel function of the kiwellin like effector proteins of the rust fungi that are already identified to play a role in host defense against plant pathogens.

microbiology↗

bioRxiv2021

Horizontal transfer of a conserved npc-2 like effector gene in rust fungi that suppresses cell death in plants

ML/MD-2 is a conserved lipid/sterol-binding protein family having a role in sterol transfer and innate immunity in lower and higher eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 25 fungal and five oomycetes plant pathogen, having a different nutrition mode. All the fungal species were found to have varied numbers of family members, a distinctively substantial expansion of the ML gene family was observed in Rhizophagus irregularis (RI) with 33 genes. Our analysis also showed that NPC2 like proteins, a subfamily of ML domain superfamily, were not only restricted to animals and insect species but also present in plant fungal pathogens, including members of Clavicipitaceae, Pucciniacease, and Tremellaceae family. The phylogenetic analysis showed that these NPC2 like fungal proteins are more closely related to animals/insects than other fungal species. The molecular docking studies of these proteins with cholesterol and other derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. Further, the full length CDS of one of the npc2 like genes from Puccinia triticina (Pt5643) was PCR amplified and further characterized using various studies such as qRT-PCR, expression in onion epidermal cells, Nicotiana benthamiana for subcellular localization studies, yeast functional complementation, and expression studies. The mRNA abundance of Pt5643 was observed to be increased along with the infection progression and exhibits the highest expression at 5thday post-infection (dpi), suggesting its important role in the P. triticina infection cycle in wheat. The fluorescent confocal microscopy of transiently expressed YFP tagged Pt5643 in onion epidermal cells and N. benthamiana shows its location in cytoplasm and nucleus, indicating its involvement in the manipulation of host genes. The functional complementation of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic npc2 gene. Further, the overexpression of Pt5643 also suppressed the BAX and H2O2 induced program cell death in N. benthamiana and yeast, respectively thus proving to be a novel horizontally transferred effector in rust fungal pathogens. Altogether the present study reports the novel function of fungal NPC2 like proteins playing a crucial role in host defense manipulation possibly through lipid binding/transport similar to animals.

microbiology↗

bioRxiv2021

Unbuttoning the impact of N501Y mutant RBD on viral entry mechanism: A computational insight

The ongoing coronavirus disease 2019 (COVID-19) pandemic has become a serious global threat. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for this pandemic has imposed a severe burden on the medical settings. The spike (S) protein of SARS-CoV-2 is an important structural protein playing a key role in the viral entry. This protein is responsible for the receptor recognition and cell membrane fusion process. The recent reports of the appearance and spread of new SARS-CoV-2 strain has raised alarms. It was reported that this new variant containing the prominent active site mutation in the RBD (N501Y) was rapidly spreading within the population. The reported N501Y mutation within the spikes essential part, known as the receptor-binding domain has raised several questions. Here in this study we have tried to explore the effect of N501Y mutation within the spike protein using several in silico approaches

bioinformatics↗

bioRxiv2020

Wheat transcriptome profiling reveals abscisic and gibberellic acid treatments regulate early-stage phytohormone defense signaling, cell wall fortification, and metabolic switches following Fusarium graminearum-challenge

BackgroundApplication of the wheat phytohormones abscisic acid (ABA) or gibberellic acid (GA) affect Fusarium head blight (FHB) disease severity; however, the molecular underpinnings of the elicited phenotypes remain unclear. Herein, the transcriptomic responses of an FHB-susceptible wheat cultivar Fielder were characterized upon treatment with ABA, an ABA receptor antagonist (AS6), or GA in the presence or absence of Fusarium graminearum (Fg) challenge. ResultsA total of 30,876 differentially expressed genes (DEGs) where identified in Fielder (26,004) and Fg (4,872). Fg challenge alone resulted in the most substantial wheat DEGs contributing to 57.2% of the total transcriptomic variation. Using a combination of topology overlap and correlation analyses, 9,689 Fg-related wheat DEGs were defined. Further enrichment analysis of the top 1% networked wheat DEGs identified critical expression changes within defense responses, cell structural metabolism, molecular transport, and membrane/lipid metabolism. Fg-challenged conditions also included the expression of a putative Fg ABA-biosynthetic cytochrome P450 and repression of wheat FUS3 for dysregulating ABA and GA crosstalk. ABA treatment alone elicited 4536 (32%) wheat DEGs common to those of the Fg-challenge, and Fg+ABA further enhanced 888 (12.5%) of them. These ABA elicited DEGs are involved in defense through both classical and non-classical phytohormone signaling and regulating cell wall structures including polyphenolic metabolism. Conversely, Fg+GA opposed 2239 (33%) Fg-elicited wheat DEGs, including modulating primary and secondary metabolism, defense responses, and flowering genes. ABA and jointly ABAFg[Fg+ABA] treatments repressed, while Fg+GA induced an over-representation of wheat DEGs mapping to chromosome 6BL. Finally, compared to Fg+ABA, co-application of Fg+AS6 did not antagonize ABA biosynthesis or signal but rather elicited antagonistic Fg (557) and wheat (11) DEGs responses directly tied to stress responses, phytohormone transport, and FHB. ConclusionsComparative transcriptomics highlight the effects of wheat phytohormones on individual pathway and global metabolism simultaneously. Application of ABA may reduce FHB severity through misregulating defense mechanisms and cell wall fortification pathways. GA application may alter primary and secondary metabolism, creating a metabolic shift to ultimately reduce FHB severity. By comparing these findings to those previously reported for four additional plant genotypes, an additive model of the wheat-Fg interaction is proposed.

genomics↗

bioRxiv2020

The Bromodomains of the mammalian SWI/SNF (mSWI/SNF) ATPases Brahma (BRM) and Brahma Related Gene 1 (BRG1) promote chromatin interaction and are critical for skeletal muscle differentiation

Skeletal muscle differentiation induces changes in the epigenome of myoblasts as they proceed towards a myogenic phenotype. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors and are key regulators of differentiation. Three mSWI/SNF proteins, the mutually exclusive ATPases, BRG1 and BRM, and the BAF180 protein (Polybromo1, PBRM1) contain bromodomains belonging to the same structural subfamily. Bromodomains bind to acetylated lysines on histone N-terminal tails and on other proteins. Pharmacological inhibition of mSWI/SNF bromodomain function using the selective inhibitor PFI-3 reduced differentiation, decreased expression of myogenic genes, and increased the expression of cell cycle-related genes, and the number of cells that remained in the cell cycle. Knockdown of BAF180 had no effect on differentiation, suggesting that only the BRG1 and BRM bromodomains contributed to differentiation. Comparison with existing gene expression data from myoblasts subjected to knockdown of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. ChIP analysis revealed decreased BRG1 and BRM binding to target gene promoters, indicating that the BRG1 and BRM bromodomains promote chromatin binding. Thus mSWI/SNF ATPase bromodomains contribute to cell cycle exit, to skeletal muscle-specific gene expression, and to stable promoter binding by the mSWI/SNF ATPases.

molecular biology↗

bioRxiv2020

Chromosome scale reference genome of Cluster bean (Cyamopsis tetragonoloba (L.) Taub.)

Clusterbean (Cyamopsis tetragonoloba (L.) Taub.), also known as Guar is a widely cultivated dryland legume of Western India and parts of Africa. Apart from being a vegetable crop, it is also an abundant source of a natural hetero-polysaccharide called guar gum or galactomannan which is widely used in cosmetics, pharmaceuticals, food processing, shale gas drilling etc. Here, for the first time we are reporting a chromosome-scale reference genome assembly of clusterbean, from a high galactomannan containing popular guar cultivar, RGC-936, by combining sequenced reads from Illumina, 10x Chromium and Oxford Nanopore technologies. The initial assembly of 1580 scaffolds with an N50 value of 7.12 Mbp was generated. Then, the final genome assembly was obtained by anchoring these scaffolds to a high density SNP map. Finally, a genome assembly of 550.31 Mbp was obtained in 7 pseudomolecules corresponding to 7 chromosomes with a very high N50 of 78.27 Mbp. We finally predicted 34,680 protein-coding genes in the guar genome. The high-quality chromosome-scale cluster bean genome assembly will facilitate understanding of the molecular basis of galactomannan biosynthesis and aid in genomics-assisted breeding of superior cultivars.

genomics↗