Biology subjects

Scheer, H.

Publications and source records attributed to Scheer, H..

4 recordsLinked to original sources

bioRxiv2026

BIRD-Seq: B2 Protein Integrated End-to-End Pipeline for dsRNA Detection and Nanopore Sequencing for Virus Monitoring

Double-stranded RNA (dsRNA) is a near-universal hallmark of active viral infection. Despite its role as a pan-viral replication intermediate, dsRNA-centred technologies for virus monitoring remain scarce and largely rely on monoclonal antibody-based approaches that, while highly sensitive, are costly and difficult to engineer, scale, or integrate with downstream assays. Here, we present a modular and antibody-free pipeline for quantitative and qualitative dsRNA analysis built around an engineered B2 protein from Flock House virus with nanomolar-range binding affinity. The pipeline is compatible with absorbance- or luminescence-based measurement formats. In a sandwich assay configuration (Sand-BIRD), sub-ng mL-1 quantification of dsRNA is achieved, comparable to the gold standard J2 monoclonal antibody, directly from crude biological samples without RNA extraction. Sand-BIRD reliably detects viral infection in both plant samples (Tomato bushy stunt virus and Grapevine fanleaf virus) and mosquitoes (West Nile virus and Dengue virus) with commercial-grade reliability. dsRNA eluted from positive samples were further processed directly by Oxford Nanopore direct sequencing, enabling identification of virus species without prior sequence knowledge or total RNA extraction. Together, this work establishes an end-to-end, sequence-agnostic workflow for direct RNA-duplex quantification and sequencing (BIRD-Seq), which has compelling potential for emerging infectious disease surveillance and next-generation point-of-care (PoC) diagnostics. Technology ReadinessBIRD-Seq is an integrated pipeline for agnostic dsRNA detection and sequencing designed for broad-spectrum virus monitoring. A Technology Readiness Level (TRL) 5 under NASAs classification framework has been reached as BIRD-Seq has been validated in laboratory-relevant environments using real-world samples, including virus-infected plants and mosquitoes. The ELISA-based sensing platform employs engineered variants of the B2 protein (from Flock House virus) in a sandwich assay format for dsRNA capture and detection, achieving sensitivity comparable to the gold-standard J2 monoclonal antibody. Unlike traditional antibody-based methods, the B2 protein offers key practical advantages: straightforward production in bacterial expression systems, high versatility, and reduced manufacturing costs, as well as direct compatibility with crude extract monitoring, eliminating the need for RNA extraction. Captured B2/RNA duplexes can then be directly eluted from the ELISA microplates and subjected to downstream nanopore direct RNA sequencing, providing both quantitative and qualitative information on the underlying virus infection, a capacity enabled by the near-universal nature of dsRNA as a pathogen-associated molecular pattern. That said, further validation on large-scale field-collected and clinical samples will be essential before widespread deployment can be envisioned. While the B2 sandwich assay offers favorable cost-efficiency over antibody-based alternatives, the relatively high cost of Oxford Nanopore direct RNA sequencing remains an important economic constraint. Nevertheless, the growing importance of dsRNA detection across virus sensing, mRNA vaccine development, innate immunity research, and human disease diagnostics, combined with the increasing role of portable long-read sequencing in emerging infectious disease (EIDs) surveillance, positions BIRD-Seq as an innovative and competitive diagnostic platform. Highlights- Double-stranded RNA (dsRNA) is one of the critical pathogen-associated molecular patterns for viral invasion in the host. A protein-based sandwich assay for dsRNA detection in crude biological samples with a sub-ng mL-1 order detection limit was developed, achieving similar sensing efficiency in comparison to expensive and proprietary monoclonal antibody-based ELISA methods. - The quantitative detection of RNA duplex is coupled with an Oxford nanopore direct dsRNA sequencing method for virus species identification and qualitative analysis. - This is one of the very first dsRNA-centered end-to-end workflows for virus monitoring and sequencing, validated for both infected plant and animal samples.

bioengineering↗

bioRxiv2022

The Hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

plant biology↗

bioRxiv2020

Molecular connection between the TUTase URT1 and decapping activators

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3 terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

molecular biology↗

bioRxiv2019

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes

Plant RNA viruses form highly organized membrane-bound virus replication complexes (VRCs) to replicate their genome and multiply. This process requires both virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) intermediates of replication that trigger potent antiviral defenses in all eukaryotes. In this work, we describe the use of A. thaliana constitutively expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down viral replicating RNA and associated proteins in planta upon infection with tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from TRV-infected plants revealed the presence of (i) viral proteins such as the replicase, which attested to the successful isolation of VRCs, and (ii) a number of host proteins, some of which have previously been involved in virus infection. Among a set of nine selected such host candidate proteins, eight showed dramatic re-localization upon infection, and seven of these co-localized with B2-labeled TRV replication complexes, providing ample validation for the immunoprecipitation results. Infection of A. thaliana T-DNA mutant lines for eight of these factors revealed that genetic knock-out of the Double-stranded RNA-Binding protein 2 (DRB2) leads to increased TRV accumulation. In addition, over-expression of this protein caused a dramatic decrease in the accumulation of four unrelated plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We therefore propose B2:GFP-mediated pull down of dsRNA to be a novel and robust method to explore the proteome of VRCs in planta, allowing the discovery of key players in the viral life cycle. AUTHOR SUMMARYViruses are an important class of pathogens that represent a major problem for human, animal and plant health. They hijack the molecular machinery of host cells to complete their replication cycle, a process frequently associated with the production of double-stranded RNA (dsRNA) that is regarded as a universal hallmark of infection by RNA viruses. Here we exploited the capacity of a GFP-tagged dsRNA-binding protein stably expressed in transgenic Arabidopsis to pull down dsRNA and associated proteins upon virus infection. In this manner we specifically captured short and long dsRNA from tobacco rattle virus (TRV) infected plants, and successfully isolated viral proteins such as the replicase, which attested to the successful isolation of virus replication complexes (VRCs). More excitingly, a number of host proteins, some of which have previously been involved in virus infection, were also captured. Remarkably, among a set of nine host candidates that were analyzed, eight showed dramatic re-localization to viral factories upon infection, and seven of these co-localized dsRNA-labeled VRCs. Genetic knock-out and over-expression experiments revealed that one of these proteins, A. thaliana DRB2, has a remarkable antiviral effect on four plant RNA viruses belonging to different families, providing ample validation of the potential of this experimental approach in the discovery of novel defense pathways and potential biotech tools to combat virus infections in the field. Being compatible with any plant virus as long as it infects Arabidopsis, we propose our dsRNA-centered strategy to be a novel and robust method to explore the proteome of VRCs in planta.

microbiology↗