Biology subjects

Jones, J. D.

Publications and source records attributed to Jones, J. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Integrating mass spectrometry with Nanopore direct RNA sequencing for de novo modification profiling of bacteriophage MS2

RNA modifications contribute to the regulatory and structural complexity of RNA molecules, influencing key biological processes. Nanopore direct RNA sequencing (DRS) enables the detection of these modifications at single-molecule resolution without chemical conversion. Oxford Nanopore Technologies recently introduced RNA004 sequencing chemistry and the Dorado basecaller, which improves accuracy and enables the identification of eight RNA modifications. However, the reliability of these predictions requires careful validation using orthogonal approaches. Here, we profiled RNA modifications in the MS2 bacteriophage genome using both LC-MS/MS and Nanopore DRS. LC-MS/MS analysis revealed that {Psi} is the predominant modification, present at approximately one site per RNA molecule, while all other modifications were found at low levels (< 0.04 modifications per RNA). In contrast, Dorado-based modification calling not only confirmed {Psi} but also predicted multiple m6A, m5C, 2OMe, and inosine sites not supported by LC-MS/MS data. To refine modification calling, we used in vitro transcribed (IVT) RNA as a negative control and subtracted IVT-derived false positive rates from native RNA predictions. This adjustment reduced overcalling and improved confidence in site-specific predictions. These data demonstrate that while Dorado can detect RNA modifications de novo, its predictions require careful filtering and validation. Our studies of provide a rare benchmark dataset for assessing the ability of transcriptome-wide methods to accurately identify RNA modifications and estimate modification stoichiometry. These findings support the use of orthogonal approaches, including LC-MS/MS and IVT controls, alongside Nanopore sequencing to provide a more reliable and interpretable strategy for studying RNA modification patterns. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/729303v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@637a55org.highwire.dtl.DTLVardef@f48272org.highwire.dtl.DTLVardef@a0465aorg.highwire.dtl.DTLVardef@6af045_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Canonical NLR immune receptor architecture enforces EDS1-dependency onto divergent TIR domains

Toll/interleukin-1 receptor (TIR) enzymes are prominent immune components in diverse organisms across the tree of life. In flowering plants, TIRs are often integrated into nucleotide binding leucine-rich repeat (NLR) receptors whose oligomerization-dependent biochemical activities create second messengers perceived by ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-family signaling complexes. TIR-NLRs and TIR proteins are present across the full spectrum of plant evolution, yet EDS1 signaling is a derived trait in seed plants. Here, we examined the functional dependency of diverse plant TIRs on the EDS1 pathway in the angiosperms Nicotiana benthamiana and Nicotiana tabacum. While the isolated TIR domains from non-seed plants generally required EDS1 for immune cell death activation, we also identified TIRs that functioned independent of EDS1. However, chimeric TIR-NLRs incorporating these diverse TIR domains onto the AtWRR4a receptor chassis showed a full reversion to EDS1-dependency. Extending this phenomenon further, we demonstrated that the AtWRR4a architecture enforces EDS1-dependence onto a bacterial TIR domain that is otherwise EDS1-independent. Collectively, our work demonstrates that NLR immune receptor architecture influences TIR-related immunity and provides further context to their ancient acquisition into plant immune systems.

plant biology↗

bioRxiv2026

Host specificity in cereal rust fungi is mediated by a conserved glycoside hydrolase family

Non-host resistance refers to the immunity of plant species to virtually all isolates of a potential pathogen and represents an underexplored avenue for breeding and engineering disease resistance. In domesticated and wild barley, cell surface-localized lectin receptor kinases (LecRKs) contribute to determining the host status to leaf rust fungi, which pose a major threat to global cereal production. Here, we identify a conserved family of leaf rust glycoside hydrolases as ligands for these barley LecRKs and show that direct ligand-receptor binding triggers immune responses. This mechanism of pathogen perception is conserved across multiple cereal species and can be functionally transferred between them. We also uncover previously uncharacterized recognition specificities among distinct LecRK variants, expanding the repertoire of LecRK-mediated rust pathogen detection. Our findings define a molecular mechanism underlying non-host resistance in cereals and provide a basis for harnessing non-host rust resistance across diverse crop-pathogen systems.

plant biology↗

bioRxiv2025

Diploid potato lines for the study and improvement of starch metabolism and structure

Diploid potato breeding enables faster genetic improvement via selection against deleterious alleles in inbred lines, unlike breeding by intercrossing tetraploid varieties. Starch is the major source of calories in potato tubers, but the starch properties of diploid lines have rarely been reported. In this study, we provide a comprehensive characterisation of tuber and starch properties in two diploid lines that are early isolates from the Solynta breeding program, B26 and B100, and their F1 hybrids. B100 produced fewer, but larger tubers compared to B26, and both diploid lines produced tubers that are smaller than the tetraploid variety, Clearwater Russet. The low tuber yield of B100 correlates with its high self-compatibility and fruit production. Pruning of fruits in B100 significantly increased total tuber yield per plant by stimulating more tuber initiations, but had no effect on average tuber weight, starch content or starch structure. Among the diploid, hybrid and tetraploid lines examined, there were no differences in the total starch content of tubers. Although amylopectin structure and amylose content were similar between the two diploid lines and the tetraploid comparison, B26 had elevated levels of resistant starch and a striking elongated granule morphology. Our results showcase the variation in source-sink relations and starch structure in diploid potato breeding material, demonstrating their potential for research into the genetics underpinning metabolic and quality traits.

plant biology↗

bioRxiv2025

Structural basis for heat tolerance in plant NLR immune receptors.

Nucleotide-binding leucine-rich repeat (NLR) immune receptors sense pathogen molecules and oligomerize, initiating defense signaling. Some NLRs function poorly at elevated temperatures for unknown reasons. We show that temperature-sensitive NLRs retain ligand binding at elevated temperatures but are impaired in oligomerization. We identify key residues involved in temperature resilience. Structural modeling reveals stabilizing intramolecular interactions of the NB-ARC domain with surface residues of the adjacent leucine-rich repeat (LRR) that preserve receptor integrity and functionality under heat stress. These insights enable in silico classification of NLRs as temperature-sensitive or -tolerant and underpin design of temperature tolerant variants of temperature sensitive NLRs. These findings provide a mechanistic basis for temperature sensitivity in plant immune receptors and enable engineering of temperature-tolerant disease resistance in crops.

plant biology↗

bioRxiv2025

Uncoupling hypersensitive cell death response and disease resistance activated by effector-triggered immunity

Effector-triggered immunity (ETI) is a major defence strategy in plants and is frequently associated with the hypersensitive response (HR), a localized form of programmed cell death long assumed to be essential for pathogen resistance. However, the causal relationship between HR and effective immunity remains unresolved. We show that the Arabidopsis cbp60g sard1 double mutant exhibits exaggerated ETI-associated HR but only partial resistance to bacterial and oomycete pathogens, thereby genetically uncoupling cell death from disease resistance without pleiotropic defects. Genome-wide transcriptome profiling reveals that the absence of CBP60g and SARD1 disrupts the balance between immune activators and suppressors, including reduced induction of the Nudix hydrolase NUDT7. Overexpression of NUDT7 diminishes but does not abolish the heightened HR phenotype in cbp60g sard1 mutant, indicating that multiple negative regulators act redundantly to restrain immune-associated cell death. These findings demonstrate that HR is not an obligatory determinant of effective resistance and provide mechanistic insight into how plants coordinate transcriptional networks to balance pathogen defence with the containment of host cell death. By refining the relationship between HR and immunity, this work challenges a long-standing paradigm in plant biology and advances our understanding of immune regulation.

plant biology↗

bioRxiv2025

An activated wheat CCG10-NLR immune receptor forms an octameric resistosome

Nucleotide-binding and leucine-rich repeat (LRR) receptors (NLRs) are widespread intracellular immune sensors across kingdoms. In plants, the G10-type coiled-coil (CCG10)-NLRs form a distinct phylogenetic clade that remains poorly characterized. Here, we identified a gain-of-function (GOF) mutant of Wheat Autoimmunity 3 (WAI3), designated WAI3GOF, which encodes a constitutively activated CCG10-NLR protein due to an amino acid substitution. Cryo-EM structural analysis revealed that activated WAI3 assembles into a distinctive octameric resistosome. Arabidopsis RPS2, another CCG10-NLR, also forms an octamer, indicating a conserved structural property across monocot and dicot plants. The WAI3 resistosome mediates a prolonged and sustained increase in cytosolic calcium influx, facilitated by a unique channel architecture arising from its divergent CC domain configuration. Notably, this domain arrangement may be shared by many plant NLRs that lack the conserved EDVID motif in their CC domains. Our findings uncover a previously uncharacterized resistosome structure and provide insights into plant immune receptor plasticity.

plant biology↗

bioRxiv2025

Cell-type-specific execution of effector-triggered immunity

Effector-triggered immunity (ETI) is a central component of host defense, but whether all cell types execute ETI similarly remains unknown. We combined chemically imposed immune activation with single-cell transcriptomics to profile ETI responses across all leaf cell types in Arabidopsis. Despite uniform ETI perception, we find striking divergence between transcriptional outputs: a core set of defense genes is broadly induced, while distinct cell types activate specialized immune modules. We infer that downstream immune execution is shaped not only by immune receptor activation, but also by cell identity and its associated transcriptional regulatory context, including local transcription factor availability and chromatin accessibility. We further demonstrate that transcriptional regulators preferentially induced in epidermal cells are required to restrict invasion by non-adapted pathogens. Their absence permits pathogen entry into deeper tissues despite intact recognition, revealing a spatial division of immune functions. Our findings uncover a layered immune architecture in plants, challenges the assumption of uniform immune execution, and provides a framework for exploring cell-type-specific resistance logic in multicellular hosts.

plant biology↗

bioRxiv2025

Defense-Suppressive Fragments of RIN4 generated by AvrRpt2 Participate in NDR1-dependent Activation of RPS2

Plant nucleotide-binding, leucine-rich-repeat (NLR) immune receptors recognize pathogen effectors and activate immunity. The NLR RPS2 recognizes AvrRpt2, a Pseudomonas effector that promotes virulence by proteolytically cleaving a membrane-tethered host protein, RIN4. RIN4 cleavage by AvrRpt2 generates fragments that activate RPS2. A model for RPS2 activation by RIN4 destruction is consistent with the ectopic activity of RPS2 in plants lacking RIN4 but does not explain the link between AvrRpt2s virulence activity and RPS2 activation. We found that non-membrane-tethered RIN4 derivatives are potent cytosolic activators of RPS2. Activation of RPS2 by these RIN4 derivatives, like AvrRpt2-induced activation, and unlike ectopic activation in the absence of RIN4, requires the defense signaling protein NDR1. Cleavage products of RIN4 produced by AvrRpt2 play contrasting roles in the activation of RPS2, with the membrane-tethered C-terminal fragment suppressing RPS2 and the non-membrane-tethered internal fragment, dependent on compatibility with the C-terminal fragment, overcoming its suppression of RPS2. HighlightsO_LINon-membrane tethered derivatives of RIN4 activate RPS2-induced cell death C_LIO_LIActivation of RPS2 by non-membrane-tethered derivatives of RIN4 requires NDR1 C_LIO_LIAvrRpt2-induced cleavage fragments of RIN4 play contrasting roles in RPS2 activation C_LI

plant biology↗

bioRxiv2025

TlyA is a 23S and 16S 2'-O-methylcytidine methyltransferase important for ribosome assembly in Bacillus subtilis

Ribosomal RNA (rRNA) is methylated in organisms ranging from bacteria to metazoans. Despite the pervasiveness of rRNA methylation in biology, the function of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2'-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside antibiotics that target the 16S rRNA. We show that{Delta} tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach and based on sequence alignments with other rRNA methyltransferases we tested the importance of potential catalytic residues and S-adenosyl-L-methionine (SAM) cofactor binding sites. We show that TlyA shares the common rRNA methyltransferase catalytic triad KDK and a SAM binding motif GxSxG which differs from Mtb TlyA. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since E. coli lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between B. subtilis, Mtb and more divergent Gram-negative bacteria providing new insight into translation and antibiotic resistance mechanisms.

microbiology↗

bioRxiv2025

Recognition-dependent activation of the RRS1-R/RPS4 immune receptor complex

The Arabidopsis TIR-NLR immune receptors RPS4 and RRS1 function together to enable recognition of multiple effector proteins including AvrRps4 and PopP2. We show here that both in the presence and absence of effector, RPS4 and RRS1 form an oligomer that does not change in size upon effector provision. Oligomer formation involves interactions between the RPS4 and RRS1 TIR domains and requires nucleotide binding capacity in RPS4. RPS4 mutants that lose TIR domain NADase activity abrogate immune activation but retain oligomerization. A cysteine residue in the RPS4 LRR domain contributes to oligomer stabilization. We propose that upon effector recognition, conformational changes in the complex relieve inhibition of RPS4 TIR domains by RRS1 TIR domains, enabling proximity between RPS4 TIR domains to create NADase activity.

plant biology↗

bioRxiv2025

Corticotropin-Releasing Factor in the Nucleus Accumbens Does Not Drive High Levels of Cocaine Consumption

Uncovering the neurobiological processes underlying substance use disorder informs future therapeutic interventions. Prior research implicates the corticotropin releasing factor (CRF) system as a major player in a wide variety of substance use disorder-like phenotypes. However, the complexity of the CRF system in regard to brain region specific effects and experience-dependent changes in activity is poorly understood. Employing a cocaine self-administration paradigm that induces escalation of cocaine consumption in a subset of subjects, we investigated the role of CRF activity in the Nucleus Accumbens (NAc) in cocaine-taking patterns both before and after chronic cocaine experience. Our results showed that pharmacologically inhibiting CRF-R1 in the NAc did not reduce cocaine consumption following escalation and genetically deleting CRF-R1 from cells in the NAc did not prevent escalation. Overall, this suggests that any effect of CRF activity driving escalation or high levels of cocaine consumption is not through its actions on CRF-R1 in the NAc.

neuroscience↗

bioRxiv2024

Interfamily co-transfer of sensor and helper NLRs extends immune receptor functionality between angiosperms

Plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors recognize pathogen effectors and activate defense. NLR genes can be non-functional in distantly related plants (restricted taxonomic functionality, RTF). Here, we enable Solanaceae NLR gene function in rice, soybean and Arabidopsis by co-delivering sensor NLR genes with their cognate NRC-type helper NLRs. In soybean protoplasts and in Arabidopsis plants, Solanum americanum Rpi-amr1, Rpi-amr3 and pepper Bs2 sensor NLRs confer cognate effector responsiveness if co-expressed with NRC helper NLRs. Rice carrying pepper Bs2 and NRCs recognizes the conserved effector, AvrBs2, and resists an important pathogen, Xanthomonas oryzae pv. oryzicola for which no resistance gene is available in rice. Rice lines carrying sensor and helper NLR genes otherwise resemble wild-type, with unaltered basal resistance or field fitness. Thus, interfamily co-transfer of sensor and helper NLRs can broaden utility of sensor NLRs, extending the tools available to control diseases of rice, soybean, Brassicas and other crops.

plant biology↗

bioRxiv2024

A helper NLR targets organellar membranes to trigger immunity

Upon activation, plant NLR immune receptors are known to assemble into oligomeric resistosomes that insert into the plasma membrane, forming Ca2+-permeable channels and triggering immunity. Here, we show that the RPW8-like coiled-coil (CCR-) NLR NRG1 primarily targets organelles instead of the plasma membrane. Unlike canonical CC-NLRs, activated NRG1 accumulates at the chloroplast envelope and channels stromal Ca2+ into the cytosol. AlphaFold modeling of the NRG1 resistosome reveals an unusually long N-terminal membrane-insertion structure that would span the double chloroplast membranes. Nanobody-mediated relocalization shows functional membrane specificity: chloroplast trapping abolishes activity of the canonical helper CC-NLR NRC4 but not NRG1. NRG1 orthologs, from non-flowering lineages to angiosperms, target chloroplasts, suggesting that organelle-centered defense dates back to at least [~]360 million years. We propose that coiled-coil NLR diversification has enabled compartment-specific immune signaling to capture diverse Ca2+ stores.

plant biology↗

bioRxiv2024

Reversible ubiquitination of integrated domain controls paired NLR immune receptor complex homeostasis

AbstractPlant intracellular NLR immune receptors can function individually or in pairs to detect pathogen effectors and activate immune responses. NLR homeostasis has to be tightly regulated to ensure proper defense without triggering autoimmunity. However, in contrast to singleton NLRs, the mechanisms controlling the paired NLRs complex homeostasis are less understood. The paired Arabidopsis RRS1/RPS4 immune receptor complex confers disease resistance through effector recognition mediated by the integrated WRKY domain of RRS1. Here, through proximity labelling, we reveal a ubiquitination-deubiquitination cycle that controls the homeostasis of the RRS1/RPS4 complex. E3 ligase RARE directly binds and ubiquitinates RRS1s WRKY domain to promote its proteasomal degradation, thereby destabilizing RPS4 indirectly and compromising the stability and function of the RRS1/RPS4 complex. Conversely, the deubiquitinating enzymes UBP12/UBP13 deubiquitinate RRS1s WRKY domain, counteracting RAREs effects. Interestingly, the abundance of WRKY transcription factors WRKY70 and WRKY41 is also regulated by RARE and UBP12/UBP13. Phylogenetic analysis suggests this regulation likely transferred from WRKY70/WRKY41 to RRS1 upon WRKY domain integration. Our findings improve our understanding of homeostatic regulation of paired NLR complex and uncover a new paradigm whereby domain integration can co-opt preexisting post-translational modification to regulate novel protein functions.

plant biology↗

bioRxiv2024

Berberine Bridge enzyme-like oxidases orchestrate homeostatic control and signaling of oligogalacturonides in defense and wounding

Plant immunity is triggered by endogenous elicitors known as damage-associated molecular patterns (DAMPs). Oligogalacturonides (OGs) are DAMPs released from the cell wall (CW) demethylated homogalacturonan during microbial colonization, mechanical or pest-provoked tissue damage, and physiological CW remodeling. Berberine Bridge Enzyme-like (BBE-l) proteins named OG oxidases (OGOXs) have been proposed to govern OGs homeostasis, which is necessary to avoid deleterious growth-affecting hyper-immunity and possible cell death. Using OGOX1 over-expressing lines and ogox1/2 double mutants, we show that these enzymes determine the levels of active OGs vs. inactive oxidized products (oxOGs). The ogox1/2-deficient plants have elevated levels of OGs, while plants overexpressing OGOX1 accumulate oxOGs. The balance between OGs and oxOGs affect disease resistance against Pseudomonas syringae pv tomato, Pectobacterium carotovorum, and Botrytis cinerea depending on the microbial capacity to respond to OGs and metabolize oxOGs. Gene expression upon plant infiltration with OGs reveals that OGOXs orchestrate OG signaling in defense as well as upon tissue damage, pointing to these enzymes as apoplastic players in immunity and tissue repair. TeaserOxidases control the homeostasis of oligogalacturonides in the cell wall and play a pivotal role in the plant immunity.

plant biology↗

bioRxiv2024

The dorsal fan-shaped body is a neurochemically heterogeneous sleep-regulating center in Drosophila

Sleep is a behavior that is conserved throughout the animal kingdom. Yet, despite extensive studies in humans and animal models, the exact function or functions of sleep remain(s) unknown. A complicating factor in trying to elucidate the function of sleep is the complexity and multiplicity of neuronal circuits that are involved in sleep regulation. It is conceivable that distinct sleep-regulating circuits are only involved in specific aspects of sleep and may underlie different sleep functions. Thus, it would be beneficial to assess the contribution of individual circuits in sleeps putative functions. The intricacy of the mammalian brain makes this task extremely difficult. However, the fruit fly Drosophila melanogaster, with its simpler brain organization, available connectomics, and unparalleled genetics offers the opportunity to interrogate individual sleep-regulating centers. In Drosophila, neurons projecting to the dorsal Fan-Shaped Body (dFB) have been proposed to be key regulators of sleep, particularly sleep homeostasis. We recently demonstrated that the most widely used genetic tool to manipulate dFB neurons, the 23E10-GAL4 driver, expresses in two sleep-regulating neurons (VNC-SP neurons) located in the Ventral Nerve Cord (VNC), the fly analog of the vertebrate spinal cord. Since most data supporting a role for the dFB in sleep regulation have been obtained using 23E10-GAL4, it is unclear whether the sleep phenotypes reported in these studies are caused by dFB neurons or VNC-SP cells. A recent publication replicated our finding that 23E10-GAL4 contains sleep-promoting neurons in the VNC. However, it also proposed that the dFB is not involved in sleep regulation at all, but this suggestion was made using genetic tools that are not dFB-specific and a very mild sleep deprivation protocol. In this study, using a newly created dFB-specific genetic driver line, we demonstrate that the majority of 23E10-GAL4 dFB neurons can promote sleep when activated and that these neurons are involved in sleep homeostasis. We also show that dFB neurons require stronger stimulation than VNC-SP cells to promote sleep. In addition, we demonstrate that dFB-induced sleep can consolidate Short-Term Memory (STM) into Long-Term Memory (LTM), suggesting that the benefit of sleep on memory is not circuit-specific. Finally, we show that dFB neurons are neurochemically heterogeneous and can be divided in 3 populations. Most dFB neurons express both glutamate and acetylcholine, while a minority of cells express only one of these two neurotransmitters. Importantly, dFB neurons do not express GABA, as previously suggested. Using neurotransmitter-specific dFB tools, our data also points at cholinergic dFB neurons as particularly potent at regulating sleep and sleep homeostasis.

neuroscience↗

bioRxiv2024

Modular mechanisms of immune priming and growth inhibition mediated by plant effector-triggered immunity

Excessive activation of effector-triggered immunity (ETI) in plants inhibits plant growth and activates cell death. ETI mediated by intracellular Toll/Interleukin-1 receptor/Resistance protein (TIR) nucleotide-binding leucine-rich-repeat receptors (NLRs) involves two partially redundant signalling nodes in Arabidopsis, EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1. Genetic and transcriptomic analyses show that EDS1-PAD4-ADR1 primarily enhances the immune component abundance and is critical for limiting pathogen growth, whereas EDS1-SAG101-NRG1 mainly activates the hypersensitive cell death response (HR) but is dispensable for immune priming. This study enhances our understanding of the distinct contributions of these two signalling modules to ETI and suggests potential strategies for improving disease resistance in crops without compromising yield.

plant biology↗