Biology subjects

Mackey, D.

Publications and source records attributed to Mackey, D..

6 recordsLinked to original sources

bioRxiv2026

Effector-triggered stomatal immunity prevents leaf invasion by bacterial pathogens

The phytopathogen Pseudomonas syringae must enter the leaf interior through natural openings such as stomata to cause disease. Plants restrict pathogen entry into the leaf upon non-self recognition-driven closure of their stomata. P. syringae counters this stomatal immunity by deploying a range of toxins and effectors. Here, we show that stomatal immunity in Arabidopsis thaliana is reinforced upon recognition of the ubiquitous P. syringae type III effector AvrE1 by the resistance protein CAR1. CAR1 expression is enriched in guard cells, and its activation prolongs the effective duration of stomatal immunity. We also show that CAR1 prevents the invasion of leaf tissue by natural isolates of P. syringae, demonstrating that CAR1-mediated stomatal immunity against the conserved effector AvrE1 provides a broadly significant barrier to host accessibility by P. syringae.

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↗

bioRxiv2023

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma

INTRODUCTIONPrimary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterised by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. This study sought to investigate the morphological and transcriptional consequences of perturbation of key genes at IOP loci in trabecular meshwork cell (TMC); the cellular regulators of IOP. We aimed to identify genetic and morphological variation that can be attributed to TMC dysfunction and raised IOP in POAG. METHODS62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescent staining of key organelles, was coupled with high-throughput microscopy for single-cell morphological analysis using CellProfiler image analysis. RESULTSAcross many of the individual gene knockouts scRNA-seq highlighted genes relating to matrix metalloproteinases and interferon-induced proteins. Our work has prioritised genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified (ABO / CAV1 / MYOC, ANGPT2 / PKHD1 / TNS1 / TXNRD2, and CAPZA1 / KALRN / LMO7 / PLEKHA7 / GNB1L / TEX41), suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, whilst knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSIONHigh throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

genetics↗

bioRxiv2023

Deep Learning-based Identification of Intraocular Pressure-Associated Genes Influencing Trabecular Meshwork Cell and Organelle Morphology

PURPOSEThe exact pathogenesis of primary open-angle glaucoma (POAG) is poorly understood. Genome-wide association studies (GWAS) have recently uncovered many loci associated with variation in intraocular pressure (IOP); a crucial risk factor for POAG. Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs), the regulatory cells of IOP. METHODSSixty-two genes at fifty-five loci associated with IOP variation were knocked out in primary TMC lines. All cells underwent high-throughput microscopy imaging after being stained with a five-channel fluorescent cell staining protocol. A convolutional neural network (CNN) was trained to distinguish between gene knockout and normal control cell images. The area under the receiver operator curve (AUC) metric was used to quantify morphological variation in gene knockouts to identify potential pathological perturbations. RESULTSCells where RALGPS1 had been perturbed demonstrated the greatest morphological variation from normal TMCs (AUC 0.851, SD 0.030), followed by LTBP2 (AUC 0.846, SD 0.029) and BCAS3 (AUC 0.845, SD 0.020). Of seven multi-gene loci, five had statistically significant differences in AUC (p<0.05) between genes, allowing for pathological gene prioritisation. The mitochondrial channel most frequently showed the greatest degree of morphological variation (33.9% of cell lines). CONCLUSIONSWe demonstrate a robust method for functionally interrogating genome-wide association signals using high-throughput microscopy and AI. Genetic variations inducing marked morphological variation can be readily identified, allowing for the gene-based dissection of loci associated with complex traits.

genetics↗

bioRxiv2021

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration.

Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy individuals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 individuals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.

neuroscience↗

bioRxiv2018

Congenital glaucoma with anterior segment dysgenesis in individuals with biallelic CPAMD8 variants

PurposeCongenital glaucoma is a significant cause of irreversible blindness. In some instances glaucoma is associated with developmental abnormalities of the ocular anterior segment, which can impair drainage of aqueous humor, leading to an increase in intraocular pressure.\n\nMethodsGenome sequencing was performed on a parent-proband congenital glaucoma trio, with exome sequencing of 79 additional individuals with suspected primary congenital glaucoma.\n\nResultsWe describe a unique ocular anterior segment dysgenesis associated with congenital glaucoma in four individuals from three unrelated families. In each case, disease was associated with compound heterozygous variants in CPAMD8, a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. CPAMD8 expression was highest in neural crest-derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation of key drainage structures and the development of high intraocular pressure and glaucoma.\n\nConclusionsThis study reveals a unique genetic cause of childhood glaucoma, and expands the phenotypic spectrum of CPAMD8-associated ocular disease.

genetics↗