Biology subjects

Thapa, M.

Publications and source records attributed to Thapa, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Species and strain diversity in Staphylococcus drive divergent host responses in human skin

The skin microbiome regulates key skin processes, yet the functional diversity of a dominant genus, Staphylococcus, remains poorly resolved at the strain level for multiple species across its pathogenic and commensal continuum. It is likely that Staphylococcus effects on skin are diverse at these finest taxonomic resolutions, but current skin models lack the physiological relevance and scalability needed to profile this diversity. Using an organotypic 3D human skin model (reconstructed human epidermis, RHE), we profiled skin responses to 187 Staphylococcus strains across seven dominant species. Canonically pathogenic species (e.g., S. aureus) induced broad inflammatory responses, whereas prototypical commensal species (e.g., S. hominis) elicited more nuanced effects on innate immune and skin barrier responses. Strikingly, S. epidermidis displayed pronounced strain-level heterogeneity, with subsets inducing either commensal or pathogen-like responses despite lacking canonical virulence factors, suggesting pleiotropic effects. Comparative genomics, dual-transcriptomics, untargeted metabolomics, and growth phenotyping revealed species- and strain-specific traits underlying these differential effects on RHE, including the presence of select cell surface proteins and differential arginine metabolism. Together, our study provides the first high-throughput, species- and strain-resolved analysis of skin-Staphylococcus interactions, offering mechanistic insights and a platform for microbiome-based strategies to modulate skin inflammation and diseases. One-line summaryHigh-throughput profiling of Staphylococcus in a human skin model shows that species- and strain-level diversity underlies a continuum of host barrier and immune responses.

microbiology↗

bioRxiv2026

Rapid Isolation and Genomic Characterization of Sewage-Derived Bacteriophages Targeting Multidrug-Resistant Avian Pathogenic Escherichia coli in Poultry

Antimicrobial resistance (AMR) in Escherichia coli is a major threat to global poultry production, particularly for controlling colibacillosis caused by avian pathogenic E. coli (APEC). Intensive antibiotic use has accelerated the emergence of multidrug-resistant (MDR) strains, undermining treatment efficacy and facilitating zoonotic transmission of resistance genes. This study evaluated bacteriophage therapy as a targeted, antibiotic-independent strategy to control MDR E. coli in Nepalese poultry systems. Seventeen E. coli isolates were obtained from commercial broiler and local-breed chicken farms. Using the double-layer agar method, 18 lytic phages were isolated from three urban sewage samples, infecting eight isolates. Real Time PCR screening revealed extensive AMR profiles in six isolates; one broiler-derived strain (EcI8) harbored 30 resistance genes, including carbapenemases, ESBLs, aminoglycoside, quinolone, and last-resort antibiotic determinants associated with class 1 and 3 integrons. Purified phages were whole-genome sequenced and all of them belonged to the genera Tequatrovirus, Phapecoctavirus, Vequintavirus, and Gamaleyavirus--lineages previously used in veterinary and human phage therapy. Several exhibited broad host ranges (up to seven isolates) and lacked detectable virulence or resistance genes; they also encoded potent lytic enzymes, including endolysins, holins, spanins, and transglycosylases. Notably, one Phapecoctavirus (PG7) showed close similarity to established APEC-infecting phages. These findings demonstrate that sewage can be a readily accessible source of safe, therapeutically relevant phages targeting highly resistant poultry E. coli. Phage cocktails and derived enzybiotics delivered through drinking water or feed offer a scalable alternative for preventing and treating colibacillosis, reducing antibiotic dependence, and mitigating zoonotic AMR risks. This study establishes a rapid, reproducible pipeline for local phage isolation and genomic validation, supporting field deployment of tailored phage-based interventions in resource-limited poultry systems.

microbiology↗

bioRxiv2025

Micropropagation and Assessment of Genetic Homogeneity in Dendrobium moniliforme (L.) Sw. using Molecular Markers

Dendrobium moniliforme (L.) Sw., is a valuable medicinal orchid widely used as tonic. This study aimed to develop an effective protocol for micropropagation of D. moniliforme and to compare the genetic similarities of the micro-propagated plants to the wild plants using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) molecular markers. Healthy seeds were cultured on Murashige-Skoog (MS) medium, supplemented with various concentrations of auxins, cytokinins and coconut water for shoot and root development. Genetic homogeneity was assessed using ten primers of each RAPD and ISSR marker. MS media fortified with 10% coconut water was efficient for seed germination and protocorm development. Maximum number of shoots were produced on half strength MS (HMS) medium supplemented with 0.25 mg/L NAA and 10% coconut water (CW) i.e 22 shoots. FMS supplemented with 0.5 mg/L indole butyric acid (IBA) was found to be best as plant hormone for root induction, as it induced ten roots/shoot. RAPD and ISSR amplified a total of 15 and 5 homogeneous DNA fragments. This protocol demonstrates a reliable and efficient method for large-scale propagation and conservation of D. moniliforme, supporting its commercial and medicinal value. HighlightsDendrobium moniliforme was micro-propagated from seeds using MS medium. RAPD and ISSR markers confirmed its genetic stability. The study supports large-scale true-to-type propagation and conservation of these valuable medicinal orchids.

molecular biology↗

bioRxiv2025

Fusion of Complement Fragment C3d Enhances Germinal Center Responses to HIV-1 Envelope Glycoproteins

Eliciting sustained germinal center (GC) responses is critical for the development of an effective HIV-1 vaccine, yet HIV-1 envelope glycoprotein (Env) immunogens often fail to elicit GC responses required for the maturation of cognate B cells that secrete broadly neutralizing antibodies (bNAbs). Effective antigen recognition is important for initial B cell priming, activation, and GC engagement. Since complement opsonization contributes to antigen recognition, we investigated whether C3d fusion could enhance the GC response of the stabilized HIV-1 Env immunogen based on a consensus sequence (ConM Env). Our results demonstrate that ConM Env-C3d induced potent HIV-specific B cell activation in vitro compared to ConM Env alone. We also observed that the C3d fusion enhanced antigen presentation by human tonsil-derived follicular dendritic cells (FDCs) to HIV-specific B cell lines. Moreover, mouse immunization studies combining ConM Env-C3d with the AddaS03 adjuvant revealed significantly enhanced early GC formation and prolonged antigen display and retention on FDCs for up to 56 days, highlighting improved antigen persistence within GCs. These immunological enhancements, including a more focused early antibody response, correlated with improved virus neutralization. Additionally, we observed sex-based differences in immune responses, with female mice showing stronger antibody responses and enhanced antigen retention compared to males. These findings suggest that C3d fusion can enhance GC engagement and improve the immunogenicity of HIV-1 vaccines.

immunology↗

bioRxiv2025

Constructing a consensus serum metabolome

Blood analysis is the most common in biomedical applications and a reference metabolome will be critical for effective annotation and for guiding scientific investigations. However, compiling such a reference is hindered by many technical challenges, despite the availability of large amount of metabolomics data today. Based on a new set of data structures and tools, we have assembled a consensus serum metabolome (CSM) from over 100,000 mass spectrometry acquisitions of more than 200 million spectra. This provides a comprehensive survey of human blood chemistry, revealing the frequency dependent nature of metabolome and exposome. Major gaps are found between CSM and the current databases. The CSM enables community-level data alignment and significantly improves annotation quality of LC-MS metabolomics. HighlightsO_LIA reference of human biochemistry linked to observation frequency C_LIO_LIMajor gaps revealed in current databases and experimental methods C_LIO_LIEnabling cross-laboratory, cross-platform data alignment C_LIO_LIAccelerated and cumulative metabolite annotation C_LI

biochemistry↗

bioRxiv2024

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design. Author summaryEnterotoxigenic E. coli (ETEC), a leading cause of diarrhea disproportionately affecting young children in low-income regions, are a priority for vaccine development. Individuals possessing A blood-type are more susceptible to severe cholera-like disease. EtpA, a secreted, immunogenic, blood group A binding protein, is a current vaccine target antigen. Here, we determined the atomic structure of EtpA in complex with protective as well as non-protective monoclonal antibodies targeting two different domains of the protein, allowing us to pinpoint key regions involved in blood-group A antigen recognition and uncover the mechanism of antibody-based protection. In addition, we show through mass-spectrometry that EtpA is extensively and heterogeneously glycosylated at surface-exposed asparagine residues by a promiscuous and low-fidelity glycosyltransferase, EtpC, and that this unique form of bacterial glycosylation is critical for to development of protective immune responses. Lastly, polyclonal antibodies from vaccinated mice as well as monoclonal antibodies obtained from ETEC-infected human volunteers revealed that the highly antigenic surface of EtpA exhibits both protective and non-protective epitopes. These results greatly expand our understanding of ETEC pathogenesis, and the immune responses elicited by these common infections, providing valuable information to aid in the rational design and testing of subunit vaccines.

microbiology↗

bioRxiv2024

Common data models to streamline metabolomics processing and annotation, and implementation in a Python pipeline

To standardize metabolomics data analysis and facilitate future computational developments, it is essential is have a set of well-defined templates for common data structures. Here we describe a collection of data structures involved in metabolomics data processing and illustrate how they are utilized in a full-featured Python-centric pipeline. We demonstrate the performance of the pipeline, and the details in annotation and quality control using large-scale LC-MS metabolomics and lipidomics data and LC-MS/MS data. Multiple previously published datasets are also reanalyzed to showcase its utility in biological data analysis. This pipeline allows users to streamline data processing, quality control, annotation, and standardization in an efficient and transparent manner. This work fills a major gap in the Python ecosystem for computational metabolomics. Author SummaryAll life processes involve the consumption, creation, and interconversion of metabolites. Metabolomics is the comprehensive study of these small molecules, often using mass spectrometry, to provide critical information of health and disease. Automated processing of such metabolomics data is desired, especially for the bioinformatics community with familiar tools and infrastructures. Despite of Pythons popularity in bioinformatics and machine learning, the Python ecosystem in computational metabolomics still misses a complete data pipeline. We have developed an end-to-end computational metabolomics data processing pipeline, based on the raw data preprocessor Asari [1]. Our pipeline takes experimental data in .mzML or .raw format and outputs annotated feature tables for subsequent biological interpretation. We demonstrate the application of this pipeline to multiple metabolomics and lipidomics datasets. Accompanying the pipeline, we have designed a set of reusable data structures, released as the MetDataModel package, which shall promote more consistent terminology and software interoperability in this area.

bioinformatics↗

bioRxiv2023

Translocation of gut commensal bacteria to the brain

The gut-brain axis, a bidirectional signaling network between the intestine and the central nervous system, is crucial to the regulation of host physiology and inflammation. Recent advances suggest a strong correlation between gut dysbiosis and neurological diseases, however, relatively little is known about how gut bacteria impact the brain. Here, we reveal that gut commensal bacteria can translocate directly to the brain when mice are fed an altered diet that causes dysbiosis and intestinal permeability, and that this also occurs without diet alteration in distinct murine models of neurological disease. The bacteria were not found in other systemic sites or the blood, but were detected in the vagus nerve. Unilateral cervical vagotomy significantly reduced the number of bacteria in the brain, implicating the vagus nerve as a conduit for translocation. The presence of bacteria in the brain correlated with microglial activation, a marker of neuroinflammation, and with neural protein aggregation, a hallmark of several neurodegenerative diseases. In at least one model, the presence of bacteria in the brain was reversible as a switch from high-fat to standard diet resulted in amelioration of intestinal permeability, led to a gradual loss of detectable bacteria in the brain, and reduced the number of neural protein aggregates. Further, in murine models of Alzheimers disease, Parkinsons disease, and autism spectrum disorder, we observed gut dysbiosis, gut leakiness, bacterial translocation to the brain, and microglial activation. These data reveal a commensal bacterial translocation axis to the brain in models of diverse neurological diseases.

neuroscience↗

bioRxiv2023

GSK3α/β restrains IFNγ-inducible costimulatory molecule expression in alveolar macrophages, limiting CD4+ T cell activation

Macrophages play a crucial role in eliminating respiratory pathogens. Both pulmonary resident alveolar macrophages (AMs) and recruited macrophages contribute to detecting, responding to, and resolving infections in the lungs. Despite their distinct functions, it remains unclear how these macrophage subsets regulate their responses to infection, including how activation by the cytokine IFN{gamma} is regulated. This shortcoming prevents the development of therapeutics that effectively target distinct lung macrophage populations without exacerbating inflammation. We aimed to better understand the transcriptional regulation of resting and IFN{gamma}-activated cells using a new ex vivo model of AMs from mice, fetal liver-derived alveolar-like macrophages (FLAMs), and immortalized bone marrow-derived macrophages (iBMDMs). Our findings reveal that IFN{gamma} robustly activates both macrophage types; however, the profile of activated IFN{gamma}-stimulated genes varies greatly between these cell types. Notably, FLAMs show limited expression of costimulatory markers essential for T cell activation upon stimulation with only IFN{gamma}. To understand cell type-specific differences, we examined how the inhibition of the regulatory kinases GSK3/{beta} alters the IFN{gamma} response. GSK3/{beta} controlled distinct IFN{gamma} responses, and in AM-like cells, we found GSK3/{beta} restrained the induction of type I IFN and TNF, thus preventing the robust expression of costimulatory molecules and limiting CD4+ T cell activation. Together, these data suggest that the capacity of AMs to respond to IFN{gamma} is restricted in a GSK3/{beta}-dependent manner and that IFN{gamma} responses differ across distinct macrophage populations. These findings lay the groundwork to identify new therapeutic targets that activate protective pulmonary responses without driving deleterious inflammation.

immunology↗

bioRxiv2023

Population encoding of stimulus features along the visual hierarchy

The retina and primary visual cortex (V1) both exhibit diverse neural populations sensitive to diverse visual features. Yet it remains unclear how neural populations in each area partition stimulus space to span these features. One possibility is that neural populations are organized into discrete groups of neurons, with each group signaling a particular constellation of features. Alternatively, neurons could be continuously distributed across feature-encoding space. To distinguish these possibilities, we presented a battery of visual stimuli to mouse retina and V1 while measuring neural responses with multi-electrode arrays. Using machine learning approaches, we developed a manifold embedding technique that captures how neural populations partition feature space and how visual responses correlate with physiological and anatomical properties of individual neurons. We show that retinal populations discretely encode features, while V1 populations provide a more continuous representation. Applying the same analysis approach to convolutional neural networks that model visual processing, we demonstrate that they partition features much more similarly to the retina, indicating they are more like big retinas than little brains.

neuroscience↗

bioRxiv2023

Late gene therapy limits the restoration of retinal function in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is an inherited photoreceptor degeneration that begins with rod loss followed by cone loss and eventual blindness. Gene therapies are being developed, but it is unknown how retinal function depends on the time of intervention. To uncover this dependence, we utilized a mouse model of retinitis pigmentosa capable of artificial genetic rescue. This model enables a benchmark of best-case gene therapy by removing the variables that complicate the ability to answer this vital question. Complete genetic rescue was performed at 25%, 50%, and 70% rod loss (early, mid and late, respectively). Early and mid treatment restored retinal function to near wild-type levels, specifically the sensitivity and signal fidelity of retinal ganglion cells (RGCs), the output neurons of the retina. However, some anatomical defects persisted. Late treatment retinas exhibited continued, albeit slowed, loss of sensitivity and signal fidelity among RGCs, as well as persistent gliosis. We conclude that gene replacement therapies delivered after 50% rod loss are unlikely to restore visual function to normal. This is critical information for administering gene therapies to rescue vision.

neuroscience↗

bioRxiv2022

Thousandfold Cell-Specific Pharmacology of Neurotransmission

Cell-specific pharmaceutical technologies promise mechanistic insight into clinical drugs[-]those that treat, and often define, human disease. In particular, DART (drug acutely restricted by tethering) achieves genetically programmable control of drug concentration over cellular dimensions. The method is compatible with clinical pharmaceuticals and amenable to studies in behaving animals. Here, we describe DART.2, comprising three advances. First, we improve the efficiency of chemical capture, enabling cell-specific accumulation of drug to [~]3,000-times the ambient concentration in 15 min. Second, we develop tracer reagents, providing a behavior-independent measure of cellular target engagement in each animal. Third, we extend the method to positive allosteric modulators and outline design principles for this clinically significant class. We showcase the platform with four pharmaceuticals[-]two that weaken excitatory (AMPAR) or inhibitory (GABAAR) chemical neurotransmission, and two that strengthen these forms of synaptic communication. Across four labs, we tested reagents in the mouse cerebellum, basal ganglia, visual cortex, and retina. Collectively, we demonstrate robust, bidirectional editing of chemical neurotransmission. We provide for distribution of validated reagents, community design principles, and synthetic building blocks for application to diverse pharmaceuticals.

neuroscience↗

bioRxiv2022

Large scale interrogation of retinal cell functions by 1-photon light-sheet microscopy

Visual processing in the retina depends on the collective activity of large ensembles of neurons organized in different layers. Current techniques for measuring activity of layer-specific neural ensembles rely on expensive pulsed infrared lasers to drive 2-photon activation of calcium-dependent fluorescent reporters. Here, we present a 1-photon light-sheet imaging system that can measure the activity in hundreds of ex vivo retinal neurons over a large field of view while simultaneously presenting visual stimuli. This allowed for a reliable functional classification of different retinal ganglion cell types. We also demonstrate that the system has sufficient resolution to image calcium entry at individual synaptic release sites across the axon terminals of dozens of simultaneously imaged bipolar cells. The simple design, a large field of view, and fast image acquisition, make this a powerful system for high-throughput and high-resolution measurements of retinal processing at a fraction of the cost of alternative approaches.

neuroscience↗

bioRxiv2022

Cones and Cone Pathways Remain Functional in Advanced Retinal Degeneration

Most defects causing retinal degeneration in retinitis pigmentosa (RP) are rod-specific mutations, but the subsequent degeneration of cones, which produces loss of daylight vision and high-acuity perception, is the most debilitating feature of the disease. To understand better why cones degenerate and how cone vision might be restored, we have made the first single-cell recordings of light responses from degenerating cones and retinal interneurons after most rods have died and cones have lost their outer-segment disk membranes and synaptic pedicles. We show that degenerating cones have functional cyclic-nucleotide-gated channels and can continue to give light responses, apparently produced by opsin localized either to small areas of organized membrane near the ciliary axoneme or distributed throughout the inner segment. Light responses of second-order horizontal and bipolar cells are smaller and less sensitive but surprisingly similar to those of normal retina. Furthermore, retinal output as reflected in responses of ganglion cells is largely intact at cone-mediated light levels. Together, these findings show that cones and their retinal pathways can remain functional even as degeneration is progressing, an encouraging result for future research aimed at enhancing the light sensitivity of residual cones to restore vision in patients with genetically inherited retinal degeneration.

neuroscience↗

bioRxiv2022

Trackable and scalable LC-MS metabolomics data processing using asari

Significant challenges still exist in the computational processing of data from LC-MS metabolomic experiments into metabolite features. In this study, we examine the issues of provenance and reproducibility in the current software tools. The inconsistency among these tools is attributed to the deficiencies of mass alignment and controls of feature quality. To address these issues, we have developed a new open-source software tool, asari, for LC-MS metabolomics data processing. Asari is designed with a set of new algorithmic framework and data structures, and all steps are explicitly trackable. Asari compares favorably to other tools in feature detection and quantification. It offers substantial improvement of computational performance over current tools, and is highly scalable.

biochemistry↗

bioRxiv2022

Robust cone-mediated signaling persists late into rod photoreceptor degeneration

Rod photoreceptor degeneration causes deterioration in the morphology and physiology of cone photoreceptors along with changes in retinal circuits. These changes could diminish visual signaling at cone-mediated light levels, thereby limiting the efficacy of treatments such as gene therapy for rescuing normal, cone-mediated vision. However, the impact of progressive rod death on cone-mediated signaling remains unclear. A mouse model of rod degeneration was used to investigate the fidelity of retinal ganglion cell (RGC) signaling throughout disease progression. Despite clear deterioration of cone morphology with rod death, cone-mediated signaling among RGCs remained surprisingly robust: spatiotemporal receptive fields changed little and the mutual information between stimuli and spiking responses was relatively constant. This relative stability held until nearly all rods had died and cones had completely lost well-formed outer segments. Interestingly, RGC information rates were higher and more stable for natural movies than checkerboard noise as degeneration progressed. The main change in RGC responses with photoreceptor degeneration was a decrease in response gain. These results suggest that gene therapies for rod degenerative diseases are likely to successfully prolong cone-mediated vision even if there are changes to cone morphology and density.

neuroscience↗

bioRxiv2021

Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination

Germinal centres (GC) are lymphoid structures where vaccine-responding B cells acquire affinity-enhancing somatic hypermutations (SHM), with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells (BMPCs)1-4. Induction of the latter is a hallmark of durable immunity after vaccination5. SARS-CoV-2 mRNA vaccination induces a robust GC response in humans6-8, but the maturation dynamics of GC B cells and propagation of their progeny throughout the B cell diaspora have not been elucidated. Here we show that anti-SARS-CoV-2 spike (S)-binding GC B cells were detectable in draining lymph nodes for at least six months in 10 out of 15 individuals who had received two doses of BNT162b2, a SARS-CoV-2 mRNA vaccine. Six months after vaccination, circulating S-binding MBCs were detected in all participants (n=42) and S-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of single-cell RNA sequencing of responding blood and lymph node B cells from eight participants and expression of the corresponding monoclonal antibodies, we tracked the evolution of 1540 S-specific B cell clones. SHM accumulated along the B cell differentiation trajectory, with early blood plasmablasts showing the lowest frequencies, followed by MBCs and lymph node plasma cells whose SHM largely overlapped with GC B cells. By three months after vaccination, the frequency of SHM within GC B cells had doubled. Strikingly, S+ BMPCs detected six months after vaccination accumulated the highest level of SHM, corresponding with significantly enhanced anti-S polyclonal antibody avidity in blood at that time point. This study documents the induction of affinity-matured BMPCs after two doses of SARS-CoV-2 mRNA vaccination in humans, providing a foundation for the sustained high efficacy observed with these vaccines.

immunology↗

bioRxiv2021

PcGCE is a potent elicitor of defense responses in aspen

Using microbial enzymes in transgenesis is a powerful means to introduce new functionalities in plants. Glucuronoyl esterase (GCE) is a microbial enzyme hydrolyzing the ester bond between lignin and 4-O-methyl--D-glucuronic acid present as a side chain of glucuronoxylan. This bond mediates lignin-carbohydrate complex (LCC) formation, considered as crucial factor of lignocellulose recalcitrance to saccharification. Previous studies showed that hybrid aspen (Populus tremula L. x tremuloides Michx.) constitutively expressing Phanerochaete carnosa Burt GCE (PcGCE) had better efficiency of cellulose-to-glucose conversion but were stunned and had lower cellulose content indicating that more studies are needed to design strategy for deployment of this enzyme in planta. Here we report that the transgenic plants exhibit premature leaf senescence, increased accumulation of calcium oxalate crystals, tyloses and necrotic lesions and have strongly activated immune defense reactions as revealed by their altered profiles of transcriptomes, metabolomes and hormones in the leaves. To elucidate if these effects are triggered by damage-associated molecular patterns (DAMPs) or by PcGCE protein perceived as a pathogen-associated molecular pattern (PAMP), we ectopically expressed in aspen an enzymatically inactive PcGCES217A. The mutated PcGCE induced similar growth retardation, leaf necrosis and premature senescence as the active one, providing evidence that PcGCE protein is recognized as PAMP. Transcriptomics analysis of young expanding leaves of 35S:PcGCE plants identified several candidates for receptors of PcGCE, which were not expressed in developing wood tissues. Grafting experiments showed that PcGCE transcripts are not cell-to-cell mobile and that PcGCE expressing leaves augment systemic responses. In agreement, expressing PcGCE in developing wood by using the wood-specific promoter (WP), avoided all off-target effects. Moreover, WP:PcGCE lines grew better than control plants providing evidence that this strategy can be used in transgenic crops dedicated for biorefinery.

bioengineering↗