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Results for “molecular biology”

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bioRxiv2025

Continuous Serial Electron Diffraction for High Quality Protein Structures

Determining macromolecular structures is crucial for understanding biological mechanisms and advancing drug discovery. Three-dimensional electron diffraction (3D ED), also known as microcrystal electron diffraction (MicroED) using continuous sample rotation has emerged as a powful method for solving structures from sub-micrometre-sized crystals. However, the resolution of MicroED data from protein crystals is often limited by radiation damage. Serial electron diffraction (SerialED) overcomes this limitation by merging single-shot diffraction patterns from thousands of crystals, but its widespread use has been hindered by the complexity and scarcity of equipment required for single shot data acquisition. Here, we introduce continuous SerialED (c-SerialED) - a simple, robust and widely accessible protocol. This approach collects diffraction data quickly and efficiently from all crystals within a given area, without prior crystal identification. We show that only using a standard cryo-EM instrument equipped with a simple widely available CMOS detector, c-SerialED greatly reduces radiation damage while improving the data quality. We demonstrate that c-SerialED enables determination of lysozyme structures at atomic resolution (0.83 [A]) and improves the data resolution of Dype Type Peroxidase Aa (DTPAa) crystals from 2.5 [A] (MicroED) to 1.3 [A]. Remarkably, the resulting structures are virtually free of radiation damage. The improved data quality and resolution allow visualization of radiation sensitive chemical features and protein-ligand interactions to state-of-the-art accuracy. By providing a convenient, fast, and damage-minimizing workflow on existing cryo-EM setups, c-SerialED significantly enhances the applicability of electron diffraction in structural biology. We anticipate our protocol will enable a wide range of studies requiring high-quality diffraction data from radiation-sensitive macromolecular crystals.

molecular biology↗

bioRxiv2025

IPSC-based modeling of resiliency in centenarians reveals longevity-specific signatures

Centenarians represent a human model of resilience to age-related decline, yet resiliency mechanisms remain elusive. Here, we establish an induced pluripotent stem cell (iPSC)-based platform to interrogate resilience signatures in centenarians. IPSC-derived neurons from centenarians exhibit transcriptional programs promoting synaptic integrity, calcium homeostasis, and cholesterol biosynthesis, while suppressing proteostatic stress pathways. Functionally, these neurons maintain stable calcium dynamics, reduced baseline mitochondrial activity, and energy-efficient homeostasis. Upon challenge, centenarian-derived neurons mount a robust stress response, in contrast to attenuated responses in non-centenarian controls. This resilience signature parallels adaptations in long-lived mammals and aligns with healthy brain aging, while showing erosion in Alzheimers disease and cancer. Our platform provides a scalable human model for dissecting resilience biology offering a framework to extend healthspan and mitigate age-related decline.

molecular biology↗

bioRxiv2026

SRC Knockdown Impairs Proliferation, Migration, and Invasion While Promoting Apoptosis in HTR8/SVneo Trophoblast Cells via Activation of the PI3K/Akt/Bcl-2 Signaling Pathway

SRC knockdown inhibits trophoblast cell proliferation, migration, and invasion while inducing apoptosis via activation of the PI3K/Akt/Bcl-2 signaling pathway. Trophoblast dysfunction is central to pregnancy disorders such as preeclampsia and miscarriage, yet the role of SRC, a non-receptor tyrosine kinase, in these cells remains poorly understood. This study aimed to elucidate the functional impact of SRC on trophoblast behavior and its underlying mechanism. Using siRNA-mediated knockdown in HTR8/SVneo cells, we confirmed efficient reduction of SRC mRNA and protein expression via RT-qPCR and Western blot. Functional assays demonstrated that SRC silencing significantly suppressed cell proliferation (CCK-8), migration (wound healing), and invasion (Transwell), while promoting apoptosis, evidenced by increased Annexin V-FITC/PI staining and upregulated Caspase-3 and Caspase-9 protein levels. Mechanistically, Western blot analysis revealed that SRC knockdown upregulated PI3K, Akt1, and Bcl-2 protein expression without altering IRS1 levels, indicating activation of the PI3K/Akt/Bcl-2 pro-survival pathway. This paradoxical activation appears to be a compensatory feedback insufficient to overcome SRC loss-induced dysfunction. Our findings identify SRC as a critical positive regulator of trophoblast proliferation, motility, and survival, acting through a non-canonical, IRS1-independent negative regulation of PI3K/Akt signaling. This study provides novel insights into trophoblast biology and suggests SRC as a potential therapeutic target for pregnancy complications; future in vivo studies are warranted to validate these mechanisms.

molecular biology↗

bioRxiv2026

Circulating miR-29a as a new biomarker of food anaphylaxis and endothelial glycocalyx regulation

To the editorO_ST_ABSBackgroundC_ST_ABSAnaphylaxis is an acute and potentially life-threatening hypersensitivity reaction often involving the cardiovascular system. Circulating microRNAs (miRNAs/miR), including those carried by extracellular vesicles (EVs), are emerging biomarkers that display regulatory functions in allergy. This study aims to investigate the role of miR-29a in anaphylaxis. MethodsMiR-29a (3p and 5p) levels were assessed by qPCR from acute and baseline samples of serum and EVs from 70 patients with food- and drug-mediated anaphylaxis. EVs purification was confirmed by Western blot, electron microscopy, and NanoSight. MiR-29a-3p target genes were studied in silico using systems biology analysis (SBA). Moreover, miR-29a levels were evaluated in vitro in endothelial cells (ECs) exposed to anaphylactic mediators. Additionally, a panel of endothelial glycocalyx (eGCX)-associated mRNA was analyzed after transfection with a miR-29a-3p inhibitor. ResultsPatients with food-induced anaphylaxis exhibited reduced miR-29a-3p levels in both serum and EVs during the acute reaction. In contrast, miR-29a-5p levels were decreased in serum but not in EVs. No significant modulation of either miRNA was observed in drug-induced anaphylaxis. SBA of miR-29a-3p identified molecular pathways, biological processes and functional networks associated with eGCX remodelling. Intracellular levels of miR-29a-3p were modulated in vitro in ECs following exposure to anaphylactic mediators. Inhibition of miR-29a-3p significantly reduced ESM1 expression. ConclusionsThe miR-29a-3p levels are decreased in serum and EVs from patients with acute food-induced anaphylaxis, suggesting its potential as a promising biomarker. Moreover, a role for miR-29a-3p in eGCX integrity under anaphylactic conditions was demonstrated, potentially regulating ESM1. Key MessageMiR-29a-3p is selectively reduced in serum and extracellular vesicles during acute food-induced anaphylaxis and may regulate endothelial glycocalyx-related pathways, which supports its potential as a novel biomarker and molecular mediator of vascular involvement in anaphylactic reactions.

immunology↗

bioRxiv2024

Heterodimerization of Endolysin Isoforms During Bacterial Infection by Staphylococcal Phage {varphi}2638A

AO_SCPLOWBSTRACTC_SCPLOWBacteriophage endolysins targeting Gram-positive bacteria typically feature a modular architecture of one or more enzymatically active domains (EADs) and cell wall binding domains (CBDs). Several endolysins also feature internal translational start sites (iTSSs) that produce short variant (SV) isoforms alongside the full-length (FL) endolysin. While the lytic activity of endolysins and their isoforms has been extensively studied as exogenous agents, the purpose behind producing the SV isoform during the phage infection cycle remains to be explored. In this study, we used staphylococcal phage {varphi}2638A as a model to determine the interplay between its full-length endolysin, Ply2638A, and its SV isoform during phage infection. X-ray crystallography structures and AlphaFold-generated models enabled elucidation of individual functions of the M23 endopeptidase, central amidase, and SH3b domains of Ply2638A. Production of the SV isoform (amidase and SH3b) was confirmed during phage infection and shown to form a heterodimer complex with Ply2638A via inter-amidase domain interactions. Using genetically engineered phage variants, we show that production of both isoforms provides an advantage during phage infection as phages producing only one isoform presented impaired lytic activity, which could be partly restored through recombinant protein complementation of the missing isoform. Importantly, when applied as an antimicrobial protein against Staphylococcus aureus in culture, the activity of Ply2638A remained constant regardless of SV isoform complementation. Drawing from our findings, we propose that SV isoform production provides its biological advantage upon endolysin entry to the periplasmic space to ensure optimal peptidoglycan degradation prior to cell wall lysis and progeny phage release.

molecular biology↗

bioRxiv2024

Synergy between cis-regulatory elements can render cohesin dispensable for distal enhancer function

Enhancers are critical genetic elements controlling transcription from promoters, but the mechanisms by which they convey regulatory information across large genomic distances remain elusive. Here, we engineered pluripotent stem cells in which cohesin loop extrusion can be inducibly disrupted without causing confounding cell cycle defects. While evident, transcriptional dysregulation was cell-type specific, and not all loci with distal enhancers depend equally on cohesin extrusion. Using comparative genome editing, we demonstrate that enhancer-promoter communication across as little as 20 kilobases can rely on cohesin. However, promoter-proximal regulatory elements can support long-range, cohesin-independent enhancer action - either upon disabling extrusion or across strong CTCF insulators. Finally, transcriptional dynamics and the emergence of new embryonic cell types in response to differentiation cues remained largely robust to disrupting cohesin extrusion. Beyond establishing novel experimental strategies to study cohesin functions in enhancer biology, our work provides mechanistic insight accounting for both cell type- and genomic context-specificity.

molecular biology↗

bioRxiv2019

Caveolae and scaffold detection from single molecule localization microscopy data using deep learning

Caveolae are plasma membrane invaginations whose formation requires caveolin-1 (Cav1), the adaptor protein polymerase I, and the transcript release factor (PTRF or CAVIN1). Caveolae have an important role in cell functioning, signaling, and disease. In the absence of CAVIN1/PTRF, Cav1 forms non-caveolar membrane domains called scaffolds. In this work, we train machine learning models to automatically distinguish between caveolae and scaffolds from single molecule localization microscopy (SMLM) data. We apply machine learning algorithms to discriminate biological structures from SMLM data. Our work is the first that is leveraging machine learning approaches (including deep learning models) to automatically identifying biological structures from SMLM data. In particular, we develop and compare three binary classification methods to identify whether or not a given 3D cluster of Cav1 proteins is a caveolae. The first uses a random forest classifier applied to 28 hand-crafted/designed features, the second uses a convolutional neural net (CNN) applied to a projection of the point clouds onto three planes, and the third uses a PointNet model, a recent development that can directly take point clouds as its input. We validate our methods on a dataset of super-resolution microscopy images of PC3 prostate cancer cells labeled for Cav1. Specifically, we have images from two cell populations: 10 PC3 and 10 CAVIN1/PTRF-transfected PC3 cells (PC3-PTRF cells) that form caveolae. We obtained a balanced set of 1714 different cellular structures. Our results show that both the random forest on hand-designed features and the deep learning approach achieve high accuracy in distinguishing the intrinsic features of the caveolae and non-caveolae biological structures. More specifically, both random forest and deep CNN classifiers achieve classification accuracy reaching 94% on our test set, while the PointNet model only reached 83% accuracy. We also discuss the pros and cons of the different approaches.

molecular biology↗

bioRxiv2026

Accurate quantification of canine mitochondrial DNA copy number and its evaluation as a biomarker of brain injury

Acute brain injury is challenging to manage in veterinary medicine, with limited validated means of prognostication currently available. The brain is rich in mitochondrial content and contains thousands of copies of mitochondrial DNA (mtDNA) per cell. We hypothesized that brain cell loss following acute brain injury may result in release of mtDNA into the systemic circulation. To investigate this, mtDNA-CN was measured in blood (n=4-6/group) and cerebral cortex (n=1/group) samples from dogs with and without brain injury using absolute quantification by real-time qPCR. By filtering out regions with homology to nuclear mitochondrial insertion sequences (NumtS) and repetitive regions, oligonucleotide primers were designed to the canine mitochondrial and nuclear genomes. In controls, blood mtDNA-CN ranged from 98 to 288 (mean 193{+/-}72), in cases of brain injury, there was a non-significant trend for higher mtDNA-CN, ranging from 163-453 (mean 244{+/-}106). As expected, cerebral cortex contained higher mtDNA-CN than the blood. In a single case with serial sampling, mtDNA-CN increased five days post-injury. We present for the first time an assay to accurately quantify mtDNA-CN in canine samples, with potential as a biomarker for acute brain injury in veterinary practice. Simple SummaryThis work describes a novel assay to accurately measure absolute levels of canine mitochondrial DNA copy number (mtDNA-CN) in biological samples. We describe the range of mtDNA-CN in canine blood and show pilot data suggesting that blood mtDNA-CN should be evaluated as a potential biomarker of acute brain injury.

molecular biology↗

bioRxiv2020

Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology

Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible, which makes them unsuitable as structural building blocks. Here we show that the ER/K motif, a single -helical domain (SAH)1, can be seamlessly fused2 to terminal helices of proteins, forming an extended and partially free-standing rigid helix. Through the intrinsic stability of the SAH, two domains can be connected with a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano, and we show that a single SAH allows the connection of two separate structural domains with sufficient rigidity to form ordered crystals. The analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact in construct flexibility (for the study of protein dynamics), and to design novel biomaterials.

molecular biology↗

bioRxiv2025

Development of a Bacterial Colorimetric Reporter System for Functional Screening of SARS-Cov-2 Main Protease Inhibitors Using Plant Preparations (juices): A Proof-of-Concept Study

SARS-CoV-2 main protease (Mpro) is essential for viral polyprotein processing and represents a prime target for antiviral drug discovery. However, most available screening strategies rely on biochemical and computational approaches that lack the biological context of living cells, or costly mammalian-cell based models. Therefore, there remains a shortage of simple and biosafe cellular models enabling rapid, functional screening of potential Mpro inhibitors, particularly those derived from natural sources and in urgent situations such as the COVID-19 pandemic. In this study, a bacterial colorimetric reporter system was developed that directly links SARS-CoV-2 Mpro activity to {beta}-galactosidase function in Escherichia coli. To the best of our knowledge, the developed system represents the first bacterial colorimetric model for direct monitoring of SARS-CoV-2 Mpro inhibition in living cells. The system enables real-time visual detection of protease inhibition on X-gal-containing medium and provides a cost-effective, biologically relevant, biosafe alternative to existing screening assays. Functional validation was performed using pomegranate juice as a representative natural inhibitor source. The system provides a simple, scalable, and biosafe platform for the primary screening of antiviral candidates, including phytochemicals, under standard laboratory conditions.

molecular biology↗

bioRxiv2020

Resources for genome editing in livestock: Cas9-expressing chickens and pigs

Genetically modified animals continue to provide important insights in biomedical sciences. Research has focused mostly on genetically modified mice so far, but other species like pigs resemble more closely the human physiology. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic chickens and pigs that allow in vivo genome editing in these two important agricultural species. We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number of different target genes and for a variety of cell types. Taken together, these transgenic animal species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, and will facilitate organ specific reverse genetics as well as cross-species comparisons. Significance statementGenome engineering of animals is crucial for translational medicine and the study of genetic traits. Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, providing the basis for in vivo genome editing. We demonstrated the functionality of this system by successful genome editing in chicken and porcine cells and tissues. These animals facilitate organ specific in vivo genome editing in both species without laborious germ line modifications, which will reduce the number of animals needed for genetic studies. They also provide a new tool for functional genomics, developmental biology and numerous other applications in biomedical and agricultural science.

molecular biology↗

bioRxiv2020

Bacteriome diversity of blackflies gut and association with Onchocerca volvulus, the causative agent of onchocerciasis in Mbam valley (Center Region, Cameroon)

BackgroundVector control using larvicides is the main alternative strategy to address limits of preventive chemotherapy using ivermectin to fight onchocerciasis. However, it remains substantially limited by implementation difficulties, ecological concerns and resistance of vector populations. Therefore, efficient and environmentally safe alternative control strategies are still needed. This study explores the role of blackfly bacterial communities both on vector competence and refractoriness to O. volvulus infection in order to determine their potential as a novel vector control-based approach to fight onchocerciasis. Principal findingsA total of 1,270 blackflies were dissected and the infection rate was 10.1%, indicative of ongoing transmission of onchocerciasis in the surveyed communities. Sequencing process revealed 19 phyla and 210 genera, highlighting the diversity of gut blackflies bacterial communities. Wolbachia was the predominant genus with 70% of relative abundance of blackflies gut bacterial communities. Serratia sp and Acidomonas genera were significantly abundant among infected blackflies (p=0.043 and p=0.027, respectively), whereas other genera as Brevibacterium were associated with the absence of infection (p=0.008). Conclusion/SignificanceThis study revealed that blackfly native bacteria are potentially involved in infection by O. volvulus, either by facilitating or preventing the parasite infestation of the vector. These bacteria represent an interesting potential as a biological target for a novel approach of vector control to fight onchocerciasis. Author summaryStudies of arthropods involved in vector-borne diseases (tsetse flies, mosquitoes, and drosophila) demonstrated the importance of their native bacteria either to ease infection and transmission of human pathogenic microorganisms including parasites or on the contrary to induce host protective effects against these parasites. Indeed, some native bacteria of arthropod vectors are now recognized to be associated either with the resistance of their hosts to parasitic infections, or the reduction of their hosts viability in case of the parasite infestation, thus highlighting the potential of such bacteria to be used as biological tool for vector control strategies. However, such bacteria have never been described on blackfly, an arthropod transmitting Onchocerca volvulus, which is the parasite responsible of onchocerciasis commonly known as river blindness. This study aimed to fill this gap by investigating the bacterial diversity of blackfly bacteriome and describing the possible role of bacteria communities in susceptibility/resistance features of the blackflies to O. volvulus infection, and therefore their potential as biological targets or tool for vector control. The screening of these blackflies native bacteria during this study, highlighted some bacteria genera of interest with significant association either with the absence of O. volvulus in blackfly or with vector infection.

molecular biology↗

bioRxiv2024

Map based cloning of CT2 and the pilot functional exploration in abiotic stress

Heterotrimeric G-proteins are multifunctional modulators that participate in a wide range of growth and developmental processes in eukaryotic species, from yeast to plants and animals. Component detection and the study of G protein signaling in most plants, including maize, are in the initial stages. In this study, we characterized a maize mutant, ct2, that showed a compact architecture and reproductive organ-related phenotypic variation. The target gene CT2 was cloned using bulked segregant analysis and map-based cloning. Gene structure prediction and phylogenetic analysis indicated that CT2 is a canonical G protein belonging to the monocotyledonous group. Promoter analysis of CT2 and RNA sequencing revealed cis-acting regulatory elements and differentially expressed genes involved in JA signaling and stress tolerance. The transcription of CT2 was repressed by NaCl and PEG treatments, and ct2 mutation in the ct2 line compromised stress tolerance in maize. On the basis of our results, we proposed a schema diagram of CT2-regulated biological process and their feedback on CT2 transcription. This research provides clues for further studies of CT2 function in hormone signaling and stress tolerance, which is beneficial for maize breeding through the screening and application of beneficial alleles.

molecular biology↗

bioRxiv2018

A synthetic non-histone substrate provides insight into substrate targeting by the Gcn5 HAT and sirtuin HDACs

Gcn5 and sirtuins are highly conserved HAT and HDAC enzymes that were first characterised as regulators of gene expression. Although histone tails are important substrates of these enzymes, these proteins also target many non-histone substrates that participate in diverse biological processes. The mechanisms used by these enzymes to choose their non-histone substrates is unclear. In this work, we use a unique synthetic biology approach in S. cerevisiae to demonstrate that a shared target sequence can act as a determinant of substrate selection for Gcn5 and sirtuins. We also exploit this system to define specific subunits of the Gcn5-containing ADA complex as regulators of non-histone acetylations proteome-wide.

molecular biology↗

bioRxiv2020

A multifaceted microRNA turnover complex from Caenorhabditis elegans

microRNAs are known to regulate expression of more than two third of all the eukaryotic genes by post-transcriptional means, and regulation of these tiny regulators play an important role in determining their activities. Here, we report a macromolecular microRNA turnover complex, whose components are crucial to microRNA homeostasis and development in Caenorhabditis elegans. Biochemical investigations with the purified complex in an isolated system not only unfolded the roles of the individual subunits critical for the functionality of the complex, but also unraveled the different modes of operations and regulatability of this biological machine. Our results reveal that this complex is highly receptive and capable of switching between an ATP-dependent and ATP-independent mode of operation depending on the availability of ATP in its environment, which might allow the complex to function dynamically during different physiological conditions.

molecular biology↗

bioRxiv2021

Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCEMicrobial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals. We validate the approach by synthesizing and testing an array of bioreporters, including arsenic and mercury biosensors, that produce signal outputs in environments ranging from aerobic to highly reduced anaerobic growth conditions.

molecular biology↗

bioRxiv2023

Exploring G-quadruplex structure in PRCC-TFE3 fusion Oncogene: Plausible use as anti cancer therapy for translocation Renal cell carcinoma (tRCC)

The TFE3 fusion gene, byproduct of Xp11.2 translocation, is the diagnostic marker for translocation renal cell carcinoma (tRCC). Absence of any clinically recognized therapy for tRCC, pressing a need to create novel and efficient therapeutic approaches. Previous studies shown that stabilization of the G-quadruplex structure in oncogenes suppresses their expression machinery. To combat the oncogenesis caused by fusion genes, our objective is to locate and stabilize the G-quadruplex structure within the PRCC-TFE3 fusion gene. Using the Quadruplex- forming G Rich Sequences (QGRS) mapper and the Non-B DNA motif search tool (nBMST) online server, we found putative G-quadruplex forming sequences (PQS) in the PRCC-TFE3 fusion gene. Circular dichroism demonstrating a parallel G-quadruplex in the targeted sequence. Fluorescence and UV-vis spectroscopy results suggest that pyridostatin binds to this newly discovered G-quadruplex. The PCR stop assay, as well as transcriptional or translational inhibition by PQS, revealed that stable G-quadruplex formation affects biological processes. Confocal microscopy of HEK293T cells transfected with the fusion transcript confirmed G- quadruplexes formation in cell. This investigation may shed light on G-quadruplexs functions in fusion genes and may help in the development of therapies specifically targeted against fusion oncogenes, which would enhance the capability of current tRCC therapy approach.

molecular biology↗

bioRxiv2024

Multi omics aided small RNA profiling of wheat rhizosphere and their potential targets in contrasting soils for Rhizoctonia solani-AG8 suppression

Next-generation sequencing helps describe microbial communities in rhizosphere environments, but understanding rhizosphere-plant interactions synergistic effects on plant traits and health outcomes remains challenging. This study analyses rhizosphere sRNAs ability to manipulate host gene targets in plants grown in suppressive (SP) and non-suppressive (NSP) soils with an integrated multi omics dataset. The results showed that rhizosphere sRNAs exhibited specific compositional features that may be important for rhizosphere-plant interaction. Small RNAs, less than 30 nt in size, were predominant in both samples, with a 5-prime bias towards cytosine enrichment, suggesting potential association with wheat specific argonauts. Mapping of sRNA reads to microbial metagenomes assembled draft genomes from SP and NSP soils showed sRNA loci were differentially expressed (DE) between the soils with contrasting disease suppressive capacities. In total, 96 and 132 non redundant rhizosphere sRNAs were abundant in SP and NSP rhizosphere communities, respectively. While 55 known bacterial sRNA loci were predicted from both SP and NSP metagenomes, 127 sRNAs originated from these loci were differentially expressed. Global wheat target prediction and functional analysis from DE rhizosphere sRNAs showed both soil type specific and common pathways. Upregulated NSP sRNAs target metabolic pathways, secondary metabolite biosynthesis, MAPK signalling, while SP sRNAs target glycerophospholipid metabolism, pathways such as polycomb repressive complex, starch/sucrose metabolism, and plant-pathogen interactions were targeted by both sets of sRNAs. This is the first study showing evidence for rhizosphere sRNAs and their corresponding plant transcripts in the context of biological disease suppression in agricultural soils. ImportanceSmall RNAs (sRNAs) have gained attention in host-microbe interactions due to their diverse roles in controlling biological processes. Studies have identified numerous sRNAs with novel functions across various organisms. Echoing growing evidence of sRNAs in different plant-microbe interaction, we show an evidence of rhizosphere sRNAs regulating wheat genes in soil disease suppression context. This understanding could significantly enhance our comprehension of gene regulation in biological functions, potentially paving the way for the development of microbiome-based methods to influence host traits. Understanding the microbiome communitys mechanisms in different environments offers opportunities to modify them for agriculture, including modifying farming practices, host genetics/immunity, and synthetic communities for disease suppression.

molecular biology↗