Biology subjects

Cook, N.

Publications and source records attributed to Cook, N..

7 recordsLinked to original sources

bioRxiv2026

The role of reactive oxygen species and calcium signaling in antiviral defense in Arabidopsis

Plant viruses interfere with host signaling pathways, but it remains unclear how calcium (Ca2+) signaling, reactive oxygen species (ROS), and changes in the plasma membrane interact during viral infection. Here, we investigated how plantago asiatica mosaic virus (PlAMV) modulates host Ca2+ and ROS-associated signaling in Arabidopsis thaliana. Using live-cell imaging and the R-GECO1.2 Ca2+ sensor, we observed a rapid increase in cytoplasmic Ca2+ before the virus was detected, indicating that Ca2+ release occurs early in infection. Genetic analysis showed that GLR, CPK3, and CNGC, core components of Ca2+ signaling, limit PlAMV spread between cells, while the usual pattern-triggered immunity (PTI) co-receptors were not needed. This means that Ca2+-based antiviral restriction operates independently of PTI. With the plasma membrane-tethered and cytosolic HyPer7 biosensor, we found that ROS levels were lower inside infection foci in the inoculated leaves, but higher in nearby cells, respectively. The NADPH oxidases RBOHD and RBOHF, which produce ROS, slowed down the local viral propagation. The PM sphingolipid biosynthetic enzyme MOCA1 altered ROS patterns and reduced the viruss spread. Epistasis analysis revealed a functional interaction between RBOHD and MOCA1, suggesting that ROS signaling and plasma membrane sphingolipid homeostasis are interconnected in antiviral defense. Overall, our findings suggest that PlAMV triggers Ca2+ influx and ROS signaling at the plasma membrane, which induces sphingolipid reorganization and helps restrict the propagation of the virus. This study shows how Ca2+, ROS, and membrane sphingolipid signaling work together in plant antiviral immunity and points to possible ways to improve resistance to viruses.

plant biology↗

bioRxiv2024

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase, grapes have been thought to use a two-step pathway for methyl anthranilate biosynthesis. By mining available transcriptomics data, we identified two anthranilate methyltransferases in Vitis vinifera (wine grape), as well as one ortholog in Concord grape. Many angiosperms methylate the plant hormone salicylic acid to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) salicylic acid methyltransferase can methylate both anthranilate and salicylic acid, we used this enzyme to examine the molecular basis of anthranilate activity by introducing rational mutations, which identified several active site residues that increase activity with anthranilate. Reversing this approach, we introduced mutations that imparted activity with salicylic acid in the maize anthranilate methyltransferase, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis anthranilate methyltransferases shares an ancestor with jasmonic acid methyltransferases, similar to the anthranilate methyltransferase from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning anthranilate activity across methyltransferases and identified one-step enzymes by which grapes synthesize methyl anthranilate. Significance StatementWhile the two-step pathway responsible for the biosynthesis of the grape aroma molecule, methyl anthranilate, has remained incomplete in Vitis spp., we identified two one-step anthranilate methyltransferases in wine and one in Concord grapes that can methylate the tryptophan pathway intermediate anthranilate. Tracing the molecular basis of anthranilate activity in the maize and sweet orange methyltransferases uncovered distinct active site amino acids that impart substrate specificity.

plant biology↗

bioRxiv2020

Genomics of sex allocation in the parasitoid wasp Nasonia vitripennis

BackgroundWhilst adaptive facultative sex allocation has been widely studied at the phenotypic level across a broad range of organisms, we still know remarkably little about its genetic architecture. Here, we explore the genome-wide basis of sex ratio variation in the parasitoid wasp Nasonia vitripennis, perhaps the best studied organism in terms of sex allocation, and well known for its response to local mate competition (LMC). ResultsWe performed a genome-wide association study (GWAS) for single foundress sex ratios using iso-female lines derived from the recently developed outbred N. vitripennis laboratory strain HVRx. The iso-female lines capture a sample of the genetic variation in HVRx and we present them as the first iteration of the Nasonia vitripennis Genome Reference Panel (NVGRP 1.0). This panel provides an assessment of the standing genetic variation for sex ratio in the study population. Using the NVGRP, we discovered a cluster of 18 linked SNPs, encompassing 9 annotated loci associated with sex ratio variation. Furthermore, we found evidence that sex ratio has a shared genetic basis with clutch size on three different chromosomes. ConclusionsOur approach provides a thorough description of the quantitative genetic basis of sex ratio variation in Nasonia at the genome level and reveals a number of inter-related candidate loci underlying sex allocation regulation.

evolutionary biology↗

bioRxiv2018

Genome-wide disruption of DNA methylation by 5-aza-2’-deoxycytidine in the parasitioid wasp Nasonia vitripennis

DNA methylation of cytosine residues across the genome influences how genes and phenotypes are regulated in a wide range of organisms. As such, understanding the role of DNA methylation and other epigenetic mechanisms has become very much a part of mapping genotype to phenotype, a major question in evolutionary biology. Ideally, we would like to manipulate DNA methylation patterns on a genome-wide scale, to help us to elucidate the role that epigenetic modifications play in phenotypic expression. Recently, the demethylating agent 5-aza-2-deoxycytidine (5-aza-dC; commonly used in the epigenetic treatment of certain cancers), has been deployed to explore the epigenetic regulation of a number of traits of interest to evolutionary ecologists, including facultative sex allocation in the parasitoid wasp Nasonia vitripennis. In a recent study, we showed that treatment with 5-aza-dC did not ablate the facultative sex allocation response in Nasonia, but shifted the patterns of sex allocation in a way predicted by genomic conflict theory. This was the first (albeit indirect) experimental evidence for genomic conflict over sex allocation facilitated by DNA methylation. However, that work lacked direct evidence of the effects of 5-aza-dC on DNA methylation, and indeed the effect of the chemical has since been questioned in Nasonia. Here, using whole-genome bisulphite sequencing of more than 4 million CpGs, across more than 11,000 genes, we demonstrate unequivocally that 5-aza-dC disrupts methylation on a large scale across the Nasonia vitripennis genome. We show that the disruption can lead to both hypo- and hyper-methylation, may vary across tissues and time of sampling, and that the effects of 5-aza-dC are context- and sequence specific. We conclude that 5-aza-dC does indeed have the potential to be repurposed as a tool for studying the role of DNA methylation in evolutionary ecology, whilst many details of its action remain to be discovered. Author SummaryShedding light on the mechanistic basis of phenotypes is a major aim in the field of evolutionary biology. If we understand how phenotypes are controlled at the molecular level, we can begin to understand how evolution has shaped that phenotype and conversely, how genetic architecture may constrain trait evolution. Epigenetic markers (such as DNA methylation) also influence phenotypic expression by regulating how and when genes are expressed. Recently, 5-aza-2-deoxycytidine (5-aza-dC), a hypomethylating agent used in the treatment of certain cancers, has been used to explore the epigenetic regulation of traits of interest to evolutionary ecologists. Previously, we used 5-aza-dC to validate a role for DNA methylation in facultative sex allocation behaviour in the parasitoid wasp Nasonia vitripennis. However, the direct effects of the chemical were not examined at that point and its efficacy in insects was questioned. Here, we demonstrate that 5-aza-dC disrupts DNA methylation on a genome-wide scale in a context- and sequence-specific manner and results in both hypo- and hyper-methylation. Our work demonstrates that 5-aza-dC has the potential to be repurposed as a tool for studying the role of DNA methylation in phenotypic expression.

evolutionary biology↗

bioRxiv2018

Ventral tegmental area astrocytes orchestrate avoidance and approach behavior

The ventral tegmental area (VTA) is a heterogeneous midbrain structure, containing neurons and astrocytes, that coordinates approach and avoidance behaviors by integrating activity from numerous afferents. Within neuron-astrocyte networks, astrocytes control signals from distinct afferents in a circuit-specific manner, but whether this capacity scales up to drive motivated behavior has been undetermined. Using genetic and optical dissection strategies in vitro and during behavior we report that VTA astrocytes tune glutamatergic signaling selectively on local inhibitory neurons to drive a functional circuit for learned avoidance. In this circuit, VTA astrocytes facilitate excitation of local GABA neurons to increase inhibition of dopamine neurons. The increased inhibition of dopamine neurons elicits real-time and learned avoidance behavior that is sufficient to impede expression of learned preference for reward. Despite the large number of functions performed by astrocytes, loss of one glutamate transporter (GLT-1) from VTA astrocytes selectively blocks these avoidance behaviors and spares preference for reward. Thus, VTA astrocytes selectively regulate excitation of local GABA neurons to drive a distinct learned avoidance circuit that opposes learned approach behavior.

neuroscience↗

bioRxiv2018

Signaling pathway screening platforms are an efficient approach toidentify therapeutic targets in precision-medicine oriented early phaseclinical trials

Precision medicine aims to tailor cancer therapies to target specific tumorpromoting aberrations. For tumors that lack actionable drivers, extensive molecular characterization and pre-clinical drug efficacy studies will be required to match patients with the appropriate targeted therapy. A cell line maintained at low passage and a patient-derived xenograft model (PDX) were generated using a fresh biopsy from a patient with a poorly-differentiated neuroendocrine tumor of unknown primary origin. Next-generation sequencing, high throughput signaling network analysis, and drug efficacy trials were then conducted to identify actionable targets for therapeutic intervention. No actionable mutations were identified after whole exome sequencing of the patients DNA; however, whole genome sequencing revealed amplification of the 3q and 5p chromosomal arms, that include the PIK3CA and RICTOR genes, respectively. Consistent with amplification of these genes, pathway analysis revealed activation of the AKT pathway. Based on this analysis, efficacy of PIK3CA and AKT inhibitors were evaluated in the tumor biopsy-derived cell culture and PDX, and response to the AKT inhibitor AZD5363 was observed both in vitro and in vivo indicating the patient would benefit from targeted therapies directed against the serine/threonine kinase AKT. In conclusion, our study demonstrates that high throughput signaling pathway analysis complements next-generation sequencing approaches for detection of actionable alterations and will aid in patient stratification into early-phase clinical trials.

clinical trials↗

bioRxiv2017

Integration of plasticity mechanisms within a single sensory neuron of C. elegans actuates a memory

Neural plasticity--the ability of a neuron to change its cellular properties in response to past experiences--underpins the nervous systems capacity to form memories and actuate behaviors. How different plasticity mechanisms act together in vivo and at a cellular level to transform sensory information into behavior is not well understood. Here we show that in the nematode C. elegans two plasticity mechanisms--sensory adaptation and presynaptic plasticity--act within a single cell to encode thermosensory information and actuate a temperature-preference memory. Sensory adaptation enables the primary thermosensory neuron, AFD, to adjust the temperature range of its sensitivity to the local environment, thereby optimizing its ability to detect temperature fluctuations associated with migration. Presynaptic plasticity transforms this thermosensory information into a behavioral preference by gating synaptic communication between sensory neuron AFD and its postsynaptic partner, AIY. The gating of synaptic communication is regulated at AFD presynaptic sites by the conserved kinase nPKC{varepsilon}. Bypassing or altering AFD presynaptic plasticity predictably changes the learned behavioral preferences without affecting sensory responses. Our findings indicate that two distinct and modular neuroplasticity mechanisms function together within a single sensory neuron to encode multiple components of information required to enact thermotactic behavior. The integration of these plasticity mechanisms result in a single-cell logic system that can both represent sensory stimuli and guide memory-based behavioral preference.

neuroscience↗