scholarly journals A High-Throughput, Seedling Screen for Plant Immunity

2020 ◽  
Vol 33 (3) ◽  
pp. 394-401
Author(s):  
Alexandre Martel ◽  
Timothy Lo ◽  
Darrell Desveaux ◽  
David S. Guttman
Keyword(s):  
Pseudomonas Syringae ◽  
High Throughput ◽  
Biological Diversity ◽  
Association Studies ◽  
Plant Immunity ◽  
Genome Wide Association Studies ◽  
Pathogen Effector ◽  
Immunity Genes ◽  
Microbe Interactions

An understanding of how biological diversity affects plant–microbe interactions is becoming increasingly important, particularly with respect to components of the pathogen effector arsenal and the plant immune system. Although technological improvements have greatly advanced our ability to examine molecular sequences and interactions, relatively few advances have been made that facilitate high-throughput, in vivo pathology screens. Here, we present a high-throughput, microplate-based, nondestructive seedling pathology assay, and apply it to identify Arabidopsis thaliana effector-triggered immunity (ETI) responses against Pseudomonas syringae type III secreted effectors. The assay was carried out in a 48-well microplate format with spray inoculation, and disease symptoms were quantitatively recorded in a semiautomated manner, thereby greatly reducing both time and costs. The assay requires only slight modifications of common labware and uses no proprietary software. We validated the assay by recapitulating known ETI responses induced by P. syringae in Arabidopsis. We also demonstrated that we can quantitatively differentiate responses from a diversity of plant genotypes grown in the same microplate. Finally, we showed that the results obtained from our assay can be used to perform genome-wide association studies to identify host immunity genes, recapitulating results that have been independently obtained with mature plants.

scholarly journals The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

2010 ◽  
Vol 107 (5) ◽  
pp. 2349-2354 ◽  
Author(s):  
Mike Wilton ◽  
Rajagopal Subramaniam ◽  
James Elmore ◽  
Corinna Felsensteiner ◽  
Gitta Coaker ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Immunity ◽  
Effector Proteins ◽  
Type Iii ◽  
Pathogen Effector ◽  
Effector Triggered Immunity ◽  
Type Iii Secretion Effector ◽  
Virulence Target

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as “non-self” features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as “non-self” features or induce a “modified-self” state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2Pto also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2Pto were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2Pto interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2Pto. In support of this hypothesis, HopF2Pto interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2Pto did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2Pto and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.

scholarly journals Genes in human obesity loci are causal obesity genes in C. elegans

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009736
Author(s):  
Wenfan Ke ◽  
Jordan N. Reed ◽  
Chenyu Yang ◽  
Noel Higgason ◽  
Leila Rayyan ◽  
...  
Keyword(s):  
High Throughput ◽  
Association Studies ◽  
The United States ◽  
Model Systems ◽  
Genome Wide Association Studies ◽  
Loss Of Function ◽  
Human Obesity ◽  
Fat Depots ◽  
Treatment And Prevention

Obesity and its associated metabolic syndrome are a leading cause of morbidity and mortality in the United States. Given the disease’s heavy burden on patients and the healthcare system, there has been increased interest in identifying pharmacological targets for the treatment and prevention of obesity. Towards this end, genome-wide association studies (GWAS) have identified hundreds of human genetic variants associated with obesity. The next challenge is to experimentally define which of these variants are causally linked to obesity, and could therefore become targets for the treatment or prevention of obesity. Here we employ high-throughput in vivo RNAi screening to test for causality 293 C. elegans orthologs of human obesity-candidate genes reported in GWAS. We RNAi screened these 293 genes in C. elegans subject to two different feeding regimens: (1) regular diet, and (2) high-fructose diet, which we developed and present here as an invertebrate model of diet-induced obesity (DIO). We report 14 genes that promote obesity and 3 genes that prevent DIO when silenced in C. elegans. Further, we show that knock-down of the 3 DIO genes not only prevents excessive fat accumulation in primary and ectopic fat depots but also improves the health and extends the lifespan of C. elegans overconsuming fructose. Importantly, the direction of the association between expression variants in these loci and obesity in mice and humans matches the phenotypic outcome of the loss-of-function of the C. elegans ortholog genes, supporting the notion that some of these genes would be causally linked to obesity across phylogeny. Therefore, in addition to defining causality for several genes so far merely correlated with obesity, this study demonstrates the value of model systems compatible with in vivo high-throughput genetic screening to causally link GWAS gene candidates to human diseases.

A Variant Noncoding Region Regulates Prrx1 and Predisposes to Atrial Arrhythmias

2021 ◽  
Author(s):  
Fernanda M Bosada ◽  
Mathilde R Rivaud ◽  
Jae-Sun Uhm ◽  
Sander Verheule ◽  
Karel van Duijvenboden ◽  
...  
Keyword(s):  
Sodium Current ◽  
Association Studies ◽  
Noncoding Region ◽  
Specific Gene ◽  
Genome Wide Association Studies ◽  
Enhancer Activity ◽  
Atrial Cardiomyocytes ◽  
The Impact ◽  
Lineage Specific Gene

Rationale: Atrial Fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1, encoding the Paired Related Homeobox 1 transcription factor. Objective: To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and Results: The mouse orthologue of the noncoding variant genomic region (R1A) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region (Kifap3, Prrx1, Fmo2, Prrc2c), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program (Mhrt, Myh6, Rbm20, Tnnt2, Ttn, Ckm) was upregulated in R1A-/- atrial cardiomyocytes, and that Mef2 binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A-/- mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared to controls. Conclusions: The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes.

scholarly journals Combined knockout of Lrrk2 and Rab29 does not result in behavioral abnormalities in vivo

2020 ◽  
Author(s):  
Melissa Conti Mazza ◽  
Victoria Nguyen ◽  
Alexandra Beilina ◽  
Jinhui Ding ◽  
Mark R. Cookson
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Association Studies ◽  
Dopamine Neurons ◽  
Substantia Nigra Pars Compacta ◽  
Genome Wide Association Studies ◽  
Double Knockout ◽  
Knockout Mouse Model

AbstractCoding mutations in the LRRK2 gene, encoding for a large protein kinase, have been shown to cause familial Parkinson’s disease (PD). The immediate biological consequence of LRRK2 mutations is to increase kinase activity, leading to the suggestion that inhibition of this enzyme might be useful therapeutically to slow disease progression. Genome-wide association studies have identified the chromosomal loci around LRRK2 and one of its proposed substrates, RAB29, as contributors towards the lifetime risk of sporadic PD. Considering the evidence for interactions between LRRK2 and RAB29 on the genetic and protein levels, here we generated a double knockout mouse model and determined whether there are any consequences on brain function with aging. From a battery of motor and non-motor behavioral tests, we noted only that 18-24 month Rab29-/- and double (Lrrk2-/-/Rab29-/-) knockout mice had diminished locomotor behavior in open field compared to wildtype mice. However, no genotype differences were seen in number of substantia nigra pars compacta (SNc) dopamine neurons or in tyrosine hydroxylase levels in the SNc and striatum, which might reflect a PD-like pathology. These results suggest that depletion of both Lrrk2 and Rab29 is tolerated, at least in mice, and support that this pathway might be able to be safely targeted for therapeutics in humans.Significance statementGenetic variation in LRRK2 that result in elevated kinase activity can cause Parkinson’s disease (PD), suggesting LRRK2 inhibition as a therapeutic strategy. RAB29, a substrate of LRRK2, has also been associated with increased PD risk. Evidence exists for an interactive relationship between LRRK2 and RAB29. Mouse models lacking either LRRK2 or RAB29 do not show brain pathologies. We hypothesized that the loss of both targets would result in additive effects across in vivo and post-mortem assessments in aging mice. We found that loss of both LRRK2 and RAB29 did not result in significant behavioral deficits or dopamine neuron loss. This evidence suggests that chronic inhibition of this pathway should be tolerated clinically.

scholarly journals Perspective of the GEMSTONE Consortium on Current and Future Approaches to Functional Validation for Skeletal Genetic Disease Using Cellular, Molecular and Animal-Modeling Techniques

2021 ◽  
Vol 12 ◽  
Author(s):  
Martina Rauner ◽  
Ines Foessl ◽  
Melissa M. Formosa ◽  
Erika Kague ◽  
Vid Prijatelj ◽  
...  
Keyword(s):  
Resource Sharing ◽  
Complex Traits ◽  
Cellular Localization ◽  
Target Genes ◽  
Mission Statement ◽  
Association Studies ◽  
Repetitive Sequences ◽  
Genome Wide Association Studies ◽  
Causal Genes

The availability of large human datasets for genome-wide association studies (GWAS) and the advancement of sequencing technologies have boosted the identification of genetic variants in complex and rare diseases in the skeletal field. Yet, interpreting results from human association studies remains a challenge. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary. Multiple unknowns exist for putative causal genes, including cellular localization of the molecular function. Intermediate traits (“endophenotypes”), e.g. molecular quantitative trait loci (molQTLs), are needed to identify mechanisms of underlying associations. Furthermore, index variants often reside in non-coding regions of the genome, therefore challenging for interpretation. Knowledge of non-coding variance (e.g. ncRNAs), repetitive sequences, and regulatory interactions between enhancers and their target genes is central for understanding causal genes in skeletal conditions. Animal models with deep skeletal phenotyping and cell culture models have already facilitated fine mapping of some association signals, elucidated gene mechanisms, and revealed disease-relevant biology. However, to accelerate research towards bridging the current gap between association and causality in skeletal diseases, alternative in vivo platforms need to be used and developed in parallel with the current -omics and traditional in vivo resources. Therefore, we argue that as a field we need to establish resource-sharing standards to collectively address complex research questions. These standards will promote data integration from various -omics technologies and functional dissection of human complex traits. In this mission statement, we review the current available resources and as a group propose a consensus to facilitate resource sharing using existing and future resources. Such coordination efforts will maximize the acquisition of knowledge from different approaches and thus reduce redundancy and duplication of resources. These measures will help to understand the pathogenesis of osteoporosis and other skeletal diseases towards defining new and more efficient therapeutic targets.

scholarly journals Asthma-associated genetic variants induce IL33 differential expression through an enhancer-blocking regulatory region

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ivy Aneas ◽  
Donna C. Decker ◽  
Chanie L. Howard ◽  
Débora R. Sobreira ◽  
Noboru J. Sakabe ◽  
...  
Keyword(s):  
Association Studies ◽  
Regulatory Region ◽  
Airway Epithelial Cells ◽  
Genome Wide Association Studies ◽  
Airway Epithelial ◽  
Allele Specific ◽  
Enhancer Blocking ◽  
Underlying Mechanisms

AbstractGenome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

scholarly journals High-Throughput Approaches onto Uncover (Epi)Genomic Architecture of Type 2 Diabetes

Genes ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 374 ◽  
Author(s):  
Anna Dziewulska ◽  
Aneta Dobosz ◽  
Agnieszka Dobrzyn
Keyword(s):  
Type 2 Diabetes ◽  
Pancreatic Islets ◽  
High Throughput ◽  
Target Genes ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Genomic Landscape ◽  
A Genome ◽  
Next Generation Sequencing Ngs

Type 2 diabetes (T2D) is a complex disorder that is caused by a combination of genetic, epigenetic, and environmental factors. High-throughput approaches have opened a new avenue toward a better understanding of the molecular bases of T2D. A genome-wide association studies (GWASs) identified a group of the most common susceptibility genes for T2D (i.e., TCF7L2, PPARG, KCNJ1, HNF1A, PTPN1, and CDKAL1) and illuminated novel disease-causing pathways. Next-generation sequencing (NGS)-based techniques have shed light on rare-coding genetic variants that account for an appreciable fraction of T2D heritability (KCNQ1 and ADRA2A) and population risk of T2D (SLC16A11, TPCN2, PAM, and CCND2). Moreover, single-cell sequencing of human pancreatic islets identified gene signatures that are exclusive to α-cells (GCG, IRX2, and IGFBP2) and β-cells (INS, ADCYAP1, INS-IGF2, and MAFA). Ongoing epigenome-wide association studies (EWASs) have progressively defined links between epigenetic markers and the transcriptional activity of T2D target genes. Differentially methylated regions were found in TCF7L2, THADA, KCNQ1, TXNIP, SOCS3, SREBF1, and KLF14 loci that are related to T2D. Additionally, chromatin state maps in pancreatic islets were provided and several non-coding RNAs (ncRNA) that are key to T2D pathogenesis were identified (i.e., miR-375). The present review summarizes major progress that has been made in mapping the (epi)genomic landscape of T2D within the last few years.

Abstract 49: Adipose-Specific Knockout of Trib1 Reduces Plasma Lipids and Diet-Induced Insulin Resistance, and Increases Circulating Adiponectin

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Mikhaila A Smith ◽  
Jian Cui ◽  
Sumeet A Kheterpal ◽  
Daniel J Rader ◽  
Robert C Bauer
Keyword(s):  
Body Mass ◽  
Plasma Lipids ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Genetic Associations ◽  
Tissue Mass ◽  
Fed State ◽  
Plasma Adiponectin ◽  
Metabolic Role

Tribbles-1 (TRIB1) was recently identified through genome-wide association studies as a novel mediator of plasma lipids and coronary artery disease in humans. While subsequent in vivo mouse work confirmed a role for hepatic TRIB1 in these associations, little is known about metabolic roles for extra-hepatic Trib1. Interestingly, SNPs near the TRIB1 gene are significantly associated with circulating adiponectin levels in humans, suggesting a metabolic role for adipose TRIB1 . To further investigate this, we generated adipose-specific Trib1 KO mice (Trib1_ASKO) by crossing Trib1 cKO mice to transgenic Adiponectin-Cre mice. Chow-fed Trib1_ASKO mice exhibited no differences in adipose tissue mass and overall body mass as compared to control littermates (N=8/group). However, Trib1_ASKO mice had reduced total (-16.9%, p <0.01), HDL (-16.7%, p <0.01), and non-HDL cholesterol (-17.3%, p =0.068), as well as plasma triglycerides (-28.6%, p <0.001) as compared to WT mice. Trib1_ASKO mice also had increased plasma adiponectin levels, a finding more pronounced in female mice (+33.3%, p <0.001) than in males (+16.4%, p =0.072). Despite this increase, transcript levels of adipoQ were moderately decreased in Trib1_ASKO mice, suggesting a post-transcriptional mode of regulation. Transcript and protein levels of C/EBPα, the best described target of Trib1 and a key regulator of adipogenesis, remained unchanged. To further investigate the metabolic consequences of adipose-specific KO of Trib1 , WT and Trib1_ASKO mice were fed high-fat diet (HFD, 45% kCal fat) for 12 weeks to induce obesity. HFD-fed Trib1_ASKO mice had reduced fasting plasma glucose (-22.3%, p <0.05), insulin (-38.2%, p <0.05), and glucose tolerance (-19.8% AUC, p <0.05) compared to control mice. Body mass and fat mass of HFD-fed Trib1_ASKO mice remained unchanged from WT, and the reductions in plasma lipids and increase in plasma adiponectin persisted in the HFD-fed state. In summary, we present here the first in vivo validation of the human genetic association between TRIB1 and plasma adiponectin, and provide evidence suggesting that adipose TRIB1 contributes to the genetic associations observed in humans between TRIB1 and multiple metabolic parameters.

scholarly journals The VNTR of the AS3MT gene is associated with brain activations during a memory span task and their training-induced plasticity

2020 ◽  
pp. 1-6 ◽  
Author(s):  
Wan Zhao ◽  
Qiumei Zhang ◽  
Xiongying Chen ◽  
Yang Li ◽  
Xiaohong Li ◽  
...  
Keyword(s):  
Neural Plasticity ◽  
Large Scale ◽  
Memory Span ◽  
Association Studies ◽  
Variable Number Tandem Repeat ◽  
Brain Activation ◽  
Head Movements ◽  
Span Task ◽  
Genome Wide Association Studies

Abstract Background The Arsenic (+3 oxidation state) methyltransferase (AS3MT) gene has been identified as a top risk gene for schizophrenia in several large-scale genome-wide association studies. A variable number tandem repeat (VNTR) of this gene is the most significant expression quantitative trait locus, but its role in brain activity in vivo is still unknown. Methods We first performed a functional magnetic resonance imaging (fMRI) scan of 101 healthy subjects during a memory span task, trained all subjects on an adaptive memory span task for 1 month, and finally performed another fMRI scan after the training. After excluding subjects with excessive head movements for one or more scanning sessions, data from 93 subjects were included in the final analyses. Results The VNTR was significantly associated with both baseline brain activation and training-induced changes in multiple regions including the prefrontal cortex and the anterior and posterior cingulate cortex. Additionally, it was associated with baseline brain activation in the striatum and the parietal cortex. All these results were corrected based on the family-wise error rate method across the whole brain at the peak level. Conclusions This study sheds light on the role of AS3MT gene variants in neural plasticity related to memory span training.

scholarly journals Cysteine protease RD21A regulated by E3 ligase SINAT4 is required for drought-induced resistance to Pseudomonas syringae in Arabidopsis

2020 ◽  
Vol 71 (18) ◽  
pp. 5562-5576
Author(s):  
Yi Liu ◽  
Kunru Wang ◽  
Qiang Cheng ◽  
Danyu Kong ◽  
Xunzhong Zhang ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Cysteine Protease ◽  
Plant Immunity ◽  
Stomatal Closure ◽  
Bacterial Pathogen ◽  
E3 Ligase ◽  
Biotic Stresses ◽  
Ubiquitin E3 Ligase ◽  
Induced Immunity

Abstract Plants can be simultaneously exposed to multiple stresses. The interplay of abiotic and biotic stresses may result in synergistic or antagonistic effects on plant development and health. Temporary drought stress can stimulate plant immunity; however, the molecular mechanism of drought-induced immunity is largely unknown. In this study, we demonstrate that cysteine protease RD21A is required for drought-induced immunity. Temporarily drought-treated wild-type Arabidopsis plants became more sensitive to the bacterial pathogen-associated molecular pattern flg22, triggering stomatal closure, which resulted in increased resistance to Pseudomonas syringae pv. tomato DC3000 (Pst-DC3000). Knocking out rd21a inhibited flg22-triggered stomatal closure and compromised the drought-induced immunity. Ubiquitin E3 ligase SINAT4 interacted with RD21A and promoted its degradation in vivo. The overexpression of SINAT4 also consistently compromised the drought-induced immunity to Pst-DC3000. A bacterial type III effector, AvrRxo1, interacted with both SINAT4 and RD21A, enhancing SINAT4 activity and promoting the degradation of RD21A in vivo. Therefore, RD21A could be a positive regulator of drought-induced immunity, which could be targeted by pathogen virulence effectors during pathogenesis.

Sign in / Sign up

Export Citation Format

Share Document