Biology subjects

Rodrigo, G.

Publications and source records attributed to Rodrigo, G..

7 recordsLinked to original sources

bioRxiv2026

Amplification-free CRISPR/Cas13a-based viroid detection in RNA extracts from infected plants

Viroids are plant infectious agents that threaten agricultural production. Current viroid detection methods rely on RT-PCR-based assays, which require specialized laboratory equipment and can sometimes produce false-negative results or non-specific amplification due to the high sequence conservation among closely related viroid species. CRISPR-based diagnostics, particularly Cas12-based systems for DNA detection (DETECTR) and Cas13a-based systems (SHERLOCK) for RNA detection, have emerged as powerful tools for nucleic acid diagnostics. However, most existing workflows still rely on target amplification and, in the case of Cas13a systems, require additional in vitro transcription steps, limiting their simplicity and direct applicability for plant diagnostics. Here, we developed a direct amplification-free Cas13a-based detection platform for viroids using potato spindle tuber viroid (PSTVd) as a model. We optimized CRISPR RNA (crRNA) design, identified inhibitory effects of plant total RNA on readout signal, and employed simplified viroid RNA enrichment workflows enabling robust detection in plant samples. The system further supported both PSTVd-specific and broad-spectrum pospiviroid (genus Pospiviroid) detection and was successfully extended to avocado sunblotch viroid (family Avsunviroidae), demonstrating its adaptability across distinct viroid families. Together, these results establish a practical and modular Cas13a-based platform, not only for viroid diagnostics, but also for broader applications in RNA-derived plant pathogen detection. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/736049v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1b62802org.highwire.dtl.DTLVardef@1fcd8b4org.highwire.dtl.DTLVardef@f73e8dorg.highwire.dtl.DTLVardef@2ba7c9_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance statementA simplified RNA enrichment workflow combined with CRISPR-Cas13a enables direct, amplification-free detection of plant viroids. The assay supports early and reliable diagnosis across different tomato varieties and provides a practical strategy for improving molecular detection of plant pathogens.

plant biology↗

bioRxiv2025

CRISPR-Cas9 trans-cleavage is hindered by a closed R-loop, an elongated spacer, and an inactive HNH domain

Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key structural determinants underlying this function. First, we show that open R-loops in the PAM-distal region favor trans-cleavage activity, which occur when targeting short double-stranded or single-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impairs this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Structural analyses of domain rearrangements provide mechanistic insight. Together, these findings illustrate a fine modulation of Cas9 function.

biochemistry↗

bioRxiv2025

Protection Against Pneumonia Induced By Vaccination With Fimbriae Subunits From Klebsiella pneumoniae

Klebsiella pneumoniae infections pose a great burden worldwide, causing high morbidity and mortality, which are worsened by the increase in multidrug resistant strains. New therapeutic/prophylactic strategies are urgently needed to overcome antibiotic resistance and reduce the health and economic impacts of diseases caused by this pathogen. Fimbriae are important virulence factors involved in biofilm formation and adhesion to host cells. Their exposed location, conservation among clinical isolates and adjuvant properties make them interesting candidates for inclusion in protein-based vaccines. Therefore, the present work investigated the immunological potential of type 1 and 3 fimbriae subunits in a murine model of K. pneumoniae lung infection. Subcutaneous immunization with recombinant FimA and MrkA induced high IgG1 production; the antibodies efficiently recognized the native proteins at the bacterial surface, promoted C3 deposition and reduced biofilm formation by K. pneumoniae in vitro. Mice vaccinated with the co-administered proteins reduced the bacterial loads in the lungs after intranasal challenge, less inflammation and tissue damage. The results suggest that both type 1 and type 3 fimbriae contribute to protection against K. pneumoniae lung infection, inducing antibodies that bind to the bacteria and favor complement deposition and clearance by the host, while inhibiting biofilm formation.

immunology↗

bioRxiv2023

Corynebacterium of the diphtheriae complex in companion animals: clinical and microbiological characterization of 64 cases from France

ObjectivesCorynebacteria of the diphtheriae complex (Cdc) can cause diphtheria in humans and have been reported from companion animals. We aimed to describe animal infection cases caused by Cdc isolates. Methods18 308 animals (dogs, cats, horses and small mammals) with rhinitis, dermatitis, non-healing wounds and otitis were sampled in metropolitan France (August 2019 to August 2021). Data on symptoms, age, breed, and the administrative region of origin were collected. Cultured bacteria were analyzed for tox gene presence, for production of the diphtheria toxin, for antimicrobial susceptibility, and genotyped by multilocus sequence typing. ResultsC. ulcerans was identified in 51 cases, 24 of which were toxigenic. Rhinitis was the most frequent presentation (18/51). Eleven cases (6 cats, 4 dogs, 1 rat) were mono-infections. Large breed dogs, especially German Shepherds (9 of 28 dogs; p < 0.00001) were overrepresented. C. ulcerans isolates were susceptible to all tested antibiotics. tox-positive C. diphtheriae was identified in 2 horses. Last, 11 infections cases (9 dogs, 2 cats; mostly chronic otitis, and 2 sores) had tox-negative C. rouxii, a recently defined species. C. rouxii and C. diphtheriae isolates were susceptible to most antibiotics tested, and almost all of these infections were polymicrobial. ConclusionsMonoinfections with C. ulcerans point towards a primary pathogenic potential to animals. C. ulcerans represents an important zoonotic risk, and C. rouxii may represent a novel zoonotic agent. This case series provides novel clinical and microbiological data on Cdc infections, and underlines the need for management of animals and their human contacts. ImportanceWe report on the occurrence, clinical and microbiological characteristics of infections caused by members of the Corynebacteria of the diphtheriae complex (Cdc) in companion animals. This is the first study based on the systematic analysis of a very large animal cohort (18 308 samples), which provides data on the frequency of Cdc isolates in various types of clinical samples from animals. Awareness of this zoonotic bacterial group remains low among veterinarians and veterinary laboratories, among which it is often considered a commensal bacteria of animals. We suggest that in case of Cdc detection in animals, the veterinary laboratories should be encouraged to send the samples to a reference laboratory for analysis of the presence of the tox gene. This work is relevant to the development of guidelines in case of Cdc infections in animals, and underlines their public health relevance given the zoonotic transmission risk.

microbiology↗

bioRxiv2022

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, to implement combinatorial regulation, and to reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

synthetic biology↗

bioRxiv2017

Viral fitness predicts the magnitude and direction of perturbations in the infected host transcriptome

Determining the fitness of viral genotypes has become a standard practice in virology as it is essential to evaluate their evolutionary potential. Darwinian fitness, defined as the advantage of a given genotype with respect to a reference one, is a mesoscopic property that captures into a single figure differences in performance at every stage of viral infection. But to which extent viral fitness results from particular molecular interactions with host factors and regulatory networks during infection? Can we identify host genes, and then functional classes, whose expression depends on viral fitness? Here, we compared the transcriptomes of tobacco plants infected with seven genotypes of tobacco etch potyvirus (TEV) that differ in fitness. We found that the larger the fitness differences among genotypes, the more dissimilar the transcriptomic profiles are. Consistently, two different mutations, one in the viral RNA polymerase and another in the viral suppressor of RNA silencing, that led to close fitness values, also resulted in significantly similar gene expression profiles. Moreover, we identified host genes whose expression showed a significant correlation, positive or negative, with TEV fitness. Over-expression of genes with positive correlation activates hormone-and RNA silencing-mediated pathways of plant defense. By contrast, under-expression of genes negatively correlated reduces metabolism, growth, and development. Overall, these results reveal the high information content of viral fitness, and suggest its potential use to predict differences in genomic profiles of infected hosts.

evolutionary biology↗

bioRxiv2017

Intrinsic adaptive value and early fate of gene duplication revealed by a bottom-up approach

Gene duplication is a major source of functional innovations and genome complexity, albeit this evolutionary process requires the preservation of duplicates in the genomes for long time. However, the population genetic mechanisms governing this preservation, especially in the critical very initial phase, have remained largely unknown. Here, we demonstrate that gene duplication confers per se a weak selective advantage in scenarios of fitness trade-offs. Through a precise quantitative description of a model system, we show that a second gene copy enhances the information transfer from the environmental signal to the phenotypic response by reducing gene expression inaccuracies derived from pervasive molecular noise and suboptimal gene regulation. We then reveal that such a phenotypic accuracy yields a selective advantage in the order of 0.1% on average, which would allow the positive selection of gene duplication in populations with moderate or large sizes. This advantage is greater at higher noise levels and intermediate concentrations of the environmental molecule, when fitness trade-offs become more evident. Moreover, we show that the genome rearrangement rates greatly condition the eventual fixation of duplicated genes, either by natural selection or by random genetic drift. Overall, our theoretical results highlight an original adaptive value for cells carrying new-born duplicates, broadly analyze the selective conditions that determine their early fates in different organisms, and reconcile population genetics with evolution by gene duplication.\n\nSIGNIFICANCEGene duplication is considered a major driver for the evolution of biological complexity. However, it is still enigmatic to what extent natural selection and genetic drift have governed this evolutionary process. This work uncovers a selective advantage for genotypes carrying duplicates, called phenotypic accuracy, widely characterized thanks to a multi-scale mathematical model coupling gene regulation with population genetics. Importantly, the integrative results presented here provide a detailed mechanistic description for the fixation of duplicates, which allows making predictions about the genome architectures, and which is relevant to understand the origins of complexity.

evolutionary biology↗