Genetic Assays Research Articles

PROthrombotic States in Transcatheter HEart Valve Leaflet thromboSIS (PROSTHESIS): Rationale and Early Results of the Observational Cohort Study

Subclinical leaflet thrombosis is an imaging phenomenon observed after transcatheter aortic valve implantation (TAVI) and characterized by hypoattenuating leaflet thickening (HALT) on computed tomography angiography. The clinical implications and underlying causes remain uncertain. Hypercoagulability, a component of Virchow’s triad, may contribute to thrombus formation on bioprosthetic leaflets, but data on hypercoagulable disorders in TAVI patients and their impact on HALT are limited. The PROSTHESIS study (PROthrombotic States in Transcatheter HEart valve Subclinical leaflet thrombosIS) is a single-center observational cohort study aiming to include 130 TAVI patients. This pilot study aimed to (i) assess the effect of hypercoagulable disorders on HALT prevalence and (ii) evaluate their impact on the natural history of HALT. Patients were screened for common hypercoagulable disorders using genetic and functional assays and underwent multimodal imaging one year after TAVI to detect HALT. In patients with HALT, post-implant imaging was repeated after three months to assess its progression. Early results comparing 52 TAVI patients with 52 matched controls undergoing coronary angiography showed similar thrombophilia prevalence between the groups (16% vs. 12%, p = 0.565). HALT occurred in 15% of TAVI patients, more extensively in those with thrombophilia (712 mm3 vs. 135 mm3, p = 0.036). However, thrombophilia was not an independent predictor of HALT. One-year follow-up CTA revealed spontaneous HALT resolution in 63%, stability in 25%, and progression in 12%. This study aims to provide insights into HALT and its mechanisms, which may help prevent complications and improve bioprosthesis durability.

Recolonisation or invasion? First insights into the origin of the newly established beaver populations in Bulgaria via eDNA

After a series of reintroductions (including unofficial releases) and natural recolonizations during the last decades, the Eurasian beaver (Castor fiber) is returning throughout Europe. However, it is hard to distinguish the species from its American relative, Castor canadensis, that has also been successfully introduced and is thriving in some parts of the continent as an invasive species. In this context, it is important to obtain genetic information for the newly recorded populations, in order to take appropriate management actions. The current paper presents results of the first genetic assay of five recently established beaver populations along the Danube River and two of its tributaries in Northern Bulgaria. The non-invasive method of eDNA sampling was applied. We found that all populations belong to Castor fiber and are of the haplotypes from the refugial populations in Norway and France. We hypothesise that they naturally spread from Romania, where reintroductions have been done using individuals from Bavaria, which were in turn translocated from Scandinavia, France, Russia and Poland.

Erlotinib therapy for Olmsted syndrome with p.L655P missense mutation in the TRPV3 gene: a case report

Olmsted syndrome (OS) is a rare disorder characterized by a mutilating palmoplantar keratoderma and periorificial keratotic plaques, but which shows considerable clinical heterogeneity. Recently, transient receptor potential vanilloid 3 (TRPV3) mutations associated with autosomal dominant or recessive OS have been reported. Here we describe a classically OS case with definitive diagnosis of OS based on clinical features and a genetic assay. Genetic analysis revealed heterozygous variants in the TRPV3 gene using whole-exome sequencing of case-parents’ trios. This mutation was not identified in his mother. Notably, a previously unreported heterozygous frameshift mutation, c.1964 T > C (p.L655P), was identified in exon 15 of the TRPV3 gene in this patient and his father. Additionally, the patient was effectively managed with oral erlotinib at a daily dose of 75 mg. After 3 months of treatment, most plantar lesions resolved, and the pain experienced was mildly alleviated. No significant adverse effects were observed in this case during treatment. In addition, we review the OS literature regarding TRPV3 gene mutations.

Liver transplantation for homozygous familial hypercholesterolemia: a retrospective analysis from Chinese experience

BackgroundHomozygous familial hypercholesterolaemia (HoFH) increases risk of premature cardiovascular events and cardiac death. In severe cases of HoFH, clinical signs and symptoms cannot be controlled well by non-surgical treatments, liver transplantation (LT) currently represents the viable option.MethodTo assess the clinical efficacy, prognosis, and optimal timing of LT for HoFH, a retrospective analysis was conducted on the preoperative, surgical conditions, and postoperative follow-up of children who received an LT for HoFH at the Beijing Friendship Hospital over the period from December 2014 to August 2022.ResultsXanthoma and decreased activity tolerance were the primary clinical manifestations in the 7 HoFH children initially assessed (one child died suddenly prior to surgery due to cardiac arrest). Accompanying these symptoms were increased blood total cholesterol (TC) and low density lipoprotein (LDL) levels, along with severe cardiovascular diseases. HoFH was confirmed in all cases by genetic and biochemical assays. Initial treatments administered to these patients consisted of low-fat diets and lipid-lowering drugs with poor outcomes. Accordingly, all 6 patients received orthotopic liver transplantations (OLT), with the result that significant postoperative reductions were observed in levels of TC and LDL. The median follow-up of these six cases was 37.41 months (range: 19.40–94.10 months). Regular postoperative follow-ups revealed that all survived and showed significant improvements in their clinical symptoms.ConclusionSo far, LT is the only way to heal HoFH. LT before the appearance of obvious cardiovascular atherosclerotic lesions can significantly improve the quality of life and prognosis of patients. At the same time, the blood cholesterol level of patients should be continuously monitored after LT to further control the progression of vascular complications.

The RAD6-like Ubiquitin Conjugase Gene OsUBC7 Has a Positive Role in the Early Cold Stress Tolerance Response of Rice.

Cold stress poses a significant threat to Asian rice cultivation, disrupting important physiological processes crucial for seedling establishment and overall plant growth. It is, thus, crucial to elucidate genetic pathways involved in cold stress tolerance response mechanisms. We mapped OsUBC7, a Radiation-sensitive 6 (RAD6)-type homolog of rice, to a low-temperature seedling survivability (LTSS) QTL and used genomics, molecular genetics, and physiological assays to assess its role in plant resilience against low-temperature stress. OsUBC7 is cold responsive and has higher expression levels in cold-tolerant japonica than cold-sensitive indica. Overexpression of OsUBC7 enhances LTSS of indica and freezing tolerance of Arabidopsis, increases levels of soluble sugars and chlorophyll A, boosts leaf development after cold exposure, and increases leaf cell numbers and plants size, but it does not affect membrane stability after cold stress exposure. Additionally, OsUBC7 has a positive role for germinability in the presence of salt and for flowering and yield-related traits. The OsUBC7 protein physically interacts with the developmental stage-specific and histone-modifying E3 ligases OsRFPH2-12 and OsHUB1/2, respectively, and potential target genes such as cell cycle dependent kinases were identified. OsUBC7 might contribute to cold resilience by regulating sugar metabolism to provide energy for promoting cellular homeostasis restoration after cold stress exposure via new cell growth, particularly in leaf cells crucial for photosynthesis and metabolic activity, possibly by interacting with cell cycle regulating proteins. Overall, the present study suggests that OsUBC7 may be involved in plant development, reproduction, and stress adaptation, and contributes to a deeper understanding of rice plant cold stress tolerance response mechanisms. OsUBC7 may be a promising candidate for improving crop productivity and resilience to stressful environments.

Unraveling the Genetic Landscape of Langerhans Cell Histiocytosis in Korean Patients: Comprehensive Insights from Mutational Profiles and Clinical Correlations.

This study aimed to conduct a comprehensive genetic analysis of patients with Langerhans cell histiocytosis (LCH), focusing on the frequency of MAPK pathway mutations, detailed mutation profiles of MAPK pathway genes, and their correlation with clinical features and prognosis in Korean LCH patients. We performed targeted next-generation sequencing, capable of capturing exons from 382 cancer-related genes, on genomic DNA extracted from formaldehyde-fixed and paraffin-embedded samples of 45 pathologically confirmed LCH patients. The majority of patients (91.1%) exhibited single-system disease, with bone being the most common location (84.4%). Initial treatments varied, and no patients died during a median follow-up of 6.8 years. Our genetic assays revealed that all patients had MAPK pathway alterations, including BRAF mutations in 51.2%, MAP2K1 mutations in 42.2%, RAF1 mutations in 4.4%, and a KRAS mutation in 2.2%. These mutations were mutually exclusive. Detailed mutation profiles indicated that among the BRAF mutations, there were 18 point mutations and 5 in-frame deletions, while most MAP2K1 mutations were in-frame deletions, with only one missense mutation. We detected previously unreported variations of point mutations in BRAF, MAP2K1, KRAS, and the first instance of a RAF1-KLC1 fusion in LCH. MAP2K1 mutations occurred more frequently in older patients, whereas BRAF V600 mutations were commonly associated with unifocal bone disease. Genetic mutations did not correlate with high-risk features or event-free survival. This study identified mutually exclusive MAPK pathway mutations in every LCH patient through comprehensive genetic analysis, highlighting the importance of inclusive testing in understanding the disease's genetics.

Comparison of targeted next generation sequencing assays in non-small cell lung cancer patients

Non-small cell lung cancer (NSCLC) is the most prevalent type of lung cancer the mutational spectrum of which has been extensively characterized. Treatment of patients with NSCLC based on their molecular profile is now part of the standard clinical care. The aim of this study was firstly to investigate two different NGS-based tumor profile genetic tests and secondly to assess the clinical actionability of the mutations and their association with survival and clinicopathological characteristics. Overall, 52 mutations were identified in 31 patients by either one or both assays. The most frequently mutated genes were TP53 (40.4%), KRAS (13.46%) and EGFR (9.62%). TP53 and KRAS mutations were associated with worst overall survival while KRAS was positively correlated with adenocarcinoma. The two methods showed a high concordance for the commonly covered genomic regions (97.14%). Ten mutations were identified in a genomic region exclusively covered by the MEDICOVER Genetics custom tumor profile assay. Likewise, one MET mutation was identified by the Ion Amliseq assay in a genomic region exclusively covered by Ion Amliseq. In conclusion both assays showed highly similar results in the commonly covered genomic areas, however, the MEDICOVER Genetics assay identified additional clinically actionable mutations that can be applied in clinical practice for personalized treatment decision making for patients with NSCLC.

A bacterial NLR-related protein recognizes multiple unrelated phage triggers to sense infection.

Immune systems must rapidly sense viral infections to initiate antiviral signaling and protect the host. Bacteria encode >100 distinct viral (phage) defense systems and each has evolved to sense crucial components or activities associated with the viral lifecycle. Here we used a high-throughput AlphaFold-multimer screen to discover that a bacterial NLR-related protein directly senses multiple phage proteins, thereby limiting immune evasion. Phages encoded as many as 5 unrelated activators that were predicted to bind the same interface of a C-terminal sensor domain. Genetic and biochemical assays confirmed activators bound to the bacterial NLR-related protein at high affinity, induced oligomerization, and initiated signaling. This work highlights how in silico strategies can identify complex protein interaction networks that regulate immune signaling across the tree of life.

A New Diagnostic Approach for Myelodysplastic Neoplasms Using a Combination of Scores Based on Flow Cytometry and Automated Hematology Sysmex XN Analyzers.

The first-step in diagnosis of myelodysplastic neoplasms (MDS) is essentially based on bone marrow cytomorphology. However, cytomorphology of MDS is often a difficult exercise, subject to inter-operator variability. Our study aims to evaluate whether the combination of two dysplasia scores, the extended Ogata score and the MDS-CBC score, could improve the screening of MDS patients among patients with chronic cytopenia. Extended Ogata score and MDS-CBC score have been measured on a retrospective cohort of 63 patients with a clinical suspicion of MDS based on the presence of cytopenia. Among these patients, 33 patients were diagnosed as MDS (MDS group) and 30 patients were diagnosed with another cause of cytopenia (non-MDS cytopenic control group). Our results show excellent performance of the combined scores in predicting MDS when the two scores are concordant: positive predictive value (PPV) = 96% and negative predictive value (NPV) = 92%. In comparison, in the same cohort, extended Ogata score alone showed a PPV = 90% and NPV = 79%, MDS-CBC score alone showed a PPV = 85% and NPV = 86%. For the first time, our results show that the combination of these two dysplasia scores constitutes a useful and rapid tool for the assessment of dysplasia associated with MDS. In the MDS diagnostic process, the use of combined scores could constitute a valuable tool to enable early strong prediction of MDS in cytopenic patients and to target patients who initially require additional genetic assays.

Chemogenomic Screening in a Patient-Derived 3D Fatty Liver Disease Model Reveals the CHRM1-TRPM8 Axis as a Novel Module for Targeted Intervention.

Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver modelfrom primary human hepatocytes and non-parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease-relevant endpoints, such as steatosis, inflammation and fibrosis, and multi-omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high-content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti-steatotic, anti-inflammatory, and anti-fibrotic effects are identified. Among these, activation of the muscarinic M1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti-fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate can be demonstrated. Combined, this study presents the first patient-derived 3D MASH model, identifies a novel signaling module with anti-fibrotic effects, and highlights the potential of organotypic culture systems for phenotype-based chemogenomic drug target identification at scale.

Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms

Telomeric DNA sequences are difficult to replicate. Replication forks frequently pause or stall at telomeres, which can lead to telomere truncation and dysfunction. In addition to being at chromosome ends, telomere repeats are also present at internal locations within chromosomes, known as interstitial telomeric sequences (ITSs). These sequences are unstable and prone to triggering gross chromosomal rearrangements (GCRs). In this study, we quantitatively examined the effect of ITSs on the GCR rate in Saccharomyces cerevisiae using a genetic assay. We find that the GCR rate increases exponentially with ITS length. This increase can be attributed to the telomere repeat binding protein Rap1 impeding DNA replication and a bias of repairing DNA breaks at or distal to the ITS via de novo telomere addition. Additionally, we performed a genome-wide screen for genes that modulate the rate of ITS-induced GCRs. We find that mutation of core components of the DNA replication machinery leads to an increase in GCRs, but many mutants known to increase the GCR rate in the absence of an ITS do not significantly affect the GCR rate when an ITS is present. We also identified genes that promote the formation of ITS-induced GCRs, including genes with roles in telomere maintenance, nucleotide excision repair, and transcription. Our work thus uncovers multiple mechanisms by which an ITS promotes GCR.

Plant PI4P is required for bacteria to translocate type-3 effectors.

Type-3 effectors (T3E) of phytopathogenic Gram-negative bacteria fulfill a virulent role, causing disease, or an avirulent role, inducing immunity, following their translocation into plant cells. This study aimed to validate the hypothesis that bacterial T3E translocation requires lipidic compounds in plant cell membranes. Based on genetic, molecular, and biochemical assays, we determined that phosphatidylinositol 4-phosphate (PI4P) associated with plant cell membranes is essential for the translocation of T3E by bacterial pathogens. Replicate experimental data revealed that PI4P cooperates with the type-3 translocase HrpF to facilitate the translocation of effectors TAL and Xop from Xanthomonas oryzae and Hop from Pseudomonas syringae into the cells of Oryza sativa and Nicotiana benthamiana, respectively. Genetic and molecular analyses confirmed that, once translocated into plant cells, the distinct effectors induce disease or immunity. Combined genetic and pharmacological analyses revealed that when PI4P content is suppressed via genetic or pharmacological measures, the T3 effector translocation is considerably suppressed, resulting in serious inhibition of bacterial infection. Overall, these findings demonstrate that cooperative functioning of HrpF-PI4P is conserved in bacterial effectors and plants.

High-frequency shoot regeneration, assessment of genetic fidelity, and histochemical analysis of forskolin production in Coleus forskohlii Briq.

Forskolin, a diterpenoid found in the roots of Coleus forskohlii, has generated significant interest in the medical field due to its various therapeutic uses. This study aimed to establish an effective system for regenerating C. forskohlii plants, ensuring a year-round supply of plant material and forskolin production. We tested different concentrations of cytokinins, either alone or combined with auxin, to see their impact on shoot multiplication and growth. We found that a medium supplemented with 1.5mg L-1 of meta-topolin (mT) resulted in the highest number of shoots (~ 12.66) and leaves (~ 20) within about 5days. When mT (1mg L-1) was combined with a low amount of auxin (0.05mg L-1 NAA), we obtained an even greater number of leaves (~ 23). The shoot regeneration capacity was consistent over five subculture passages, showing minimal variation in mean shoot length and number. During acclimatization, around 91% of the plantlets grown in vermiculite + sand survived. The photosynthetic pigment concentration in the plantlets modestly increased in the first 10days and reached its highest level after 30days. Genetic fidelity assays using inter simple sequence repeats (ISSRs) confirmed the similarity between the in vitro derived plantlets and the mother plant. Micro-morphological features of in vitro and ex-vitro acclimated plantlets also matched those of the mother plant, further confirming genetic accuracy. Histochemical staining with vanillin confirmed the presence of forskolin in the in vitro roots, indicated by the violet coloration in the cells. Forskolin quantification was also validated by HPLC where in vitro derived roots were documented to undergo an almost ~ 1.8-fold in comparison to that of the mother plant. This established protocol can effectively address resource scarcity for commercial-scale forskolin production and sustainable conservation techniques.

A Modular Genetic Approach to Newborn Screening from Spinal Muscular Atrophy to Sickle Cell Disease-Results from Six Years of Genetic Newborn Screening.

Genetic newborn screening (NBS) has already entered the phase of common practice in many countries. In Germany, spinal muscular atrophy (SMA), severe combined immunodeficiency (SCID) and sickle cell disease (SCD) are currently a mandatory part of NBS. Here, we describe the experience of six years of genetic NBS including the prevalence of those three diseases in Germany. Samples and nucleic acids were extracted from dried blood spot cards, commonly used for NBS. A qPCR assay was used to detect disease-causing variants for SMA and SCD, and the detection of T-cell receptor excision circles (TRECs) was performed for SCID screening. The results of the NBS of over 1 million newborns for SMA, approximately 770,000 for SCID and over 410,000 for SCD are discussed in detail. In these newborns, we have identified 121 cases of SMA, 15 cases of SCID and syndrome-based immunodeficiencies and 77 cases of SCD or β-thalassemia. The flexibility of multiplex qPCR is assessed as an effective tool for incorporating different molecular genetic markers for screening. The processing of dried blood spot (DBS) filter cards for molecular genetic assays and the assays are described in detail; turn-around times and cost estimations are included to give an insight into the processes and discuss further options for optimization. The identified cases are in the range expected for the total number of screened newborns, but present a more exact view on the actual prevalences for Germany.

Measuring Meiotic Recombination Frequency in Schizosaccharomyces pombe Using an Engineered Genetic Interval.

The fission yeast Schizosaccharomyces pombe has been used to elucidate meiotic recombination mechanisms for decades. Alongside the budding yeast Saccharomyces cerevisiae, research employing fission yeast has been instrumental in advancing our knowledge of double-stranded DNA break (DSB) formation and repair during meiosis. Genetic recombination assays are the workhorses of gene conversion and crossover frequency analysis; these have been employed to investigate cis and trans determinants of meiotic recombination. Here, I describe meiotic recombination assays engineered by the introduction of nutritional markers up- and downstream of the ade6 and ade7 genes. These particular setups enable a comprehensive assessment of reproductive success in a single assay because spore viability and the frequency of gene conversion, crossovers, and crossovers associated with gene conversion events are simultaneously measured.

TRIM32 inhibits Venezuelan equine encephalitis virus infection by targeting a late step in viral entry.

Alphaviruses are mosquito borne RNA viruses that are a reemerging public health threat. Alphaviruses have a broad host range, and can cause diverse disease outcomes like arthritis, and encephalitis. The host ubiquitin proteasome system (UPS) plays critical roles in regulating cellular processes to control the infections with various viruses, including alphaviruses. Previous studies suggest alphaviruses hijack UPS for virus infection, but the molecular mechanisms remain poorly characterized. In addition, whether certain E3 ubiquitin ligases or deubiquitinases act as alphavirus restriction factors remains poorly understood. Here, we employed a cDNA expression screen to identify E3 ubiquitin ligase TRIM32 as a novel intrinsic restriction factor against alphavirus infection, including VEEV-TC83, SINV, and ONNV. Ectopic expression of TRIM32 reduces alphavirus infection, whereas depletion of TRIM32 with CRISPR-Cas9 increases infection. We demonstrate that TRIM32 inhibits alphaviruses through a mechanism that is independent of the TRIM32-STING-IFN axis. Combining reverse genetics and biochemical assays, we found that TRIM32 interferes with genome translation after membrane fusion, prior to replication of the incoming viral genome. Furthermore, our data indicate that the monoubiquitination of TRIM32 is important for its antiviral activity. Notably, we also show two TRIM32 pathogenic mutants R394H and D487N, related to Limb-girdle muscular dystrophy (LGMD), have a loss of antiviral activity against VEEV-TC83. Collectively, these results reveal that TRIM32 acts as a novel intrinsic restriction factor suppressing alphavirus infection and provides insights into the interaction between alphaviruses and the host UPS.

Regulation of magnesium ion transport in Escherichia coli: insights into the role of the 5’ upstream region in corA expression

ABSTRACT In Escherichia coli, transport of magnesium ions across the cellular membrane relies on MgtA and CorA transporters. While the expression of mgtA is controlled by the two-component system PhoQ/PhoP and 5’ upstream region elements, corA expression is considered to be constitutive and not to depend on cellular factors. Importantly, the 5’ upstream region of corA is predicted to fold into structures highly similar to the magnesium-sensing mgtA 5‘ upstream region. Here using biochemical and genetic assays, we show that the intracellular concentration of magnesium ions affects corA expression. Similarly to mgtA, we find that the effect of magnesium ions on corA expression is mediated by the 5’ upstream region. We demonstrate that the RNA structure is important for regulation and that the Rho transcription factor is involved in the modulation of transcription termination. Consistent with previous studies, we find that translation of corL, a short ORF located within the 5’ upstream region, is important for corA regulation. Our data indicate that the efficiency of corL translation is inversely proportional to corA expression, similar to what has been described for mgtA and corA in Salmonella enterica. Using a novel assay to control the import of magnesium ions, we show that while the expression of mgtA is regulated by both extra- and intracellular magnesium ions, corA is regulated by variations in intracellular magnesium ions. Our results support a model in which the expression of corA is regulated by the 5’ upstream region that senses variations of intracellular magnesium ions.

Proteome integral solubility alteration via label-free DIA approach (PISA-DIA), game changer in drug target deconvolution.

Drug protein-target identification in past decades required screening compound libraries against known proteins to determine drugs binding to specific protein. Protein targets used in drug-target screening were selected predominantly used laborious genetic manipulation assays. In 2013, a team led by Pär Nordlund from Karolinska Institutet(Stockholm, Sweden) developed Cellular Thermal Shift Assay (CETSA), a method which, for the first time, enabled the possibility of drug protein-target identification in the complex cellular proteome. High throughput, quantitative mass spectrometry (MS) proteomics appeared as a compatible analytical method of choice to complement CETSA, aka Thermal Protein Profiling assay (TPP). Since the seminal CETSA-MS/ TPP-MS publications, different protein-target deconvolution strategies emerged including Proteome Integral Solubility Alteration (PISA). The work of Emery-Corbin etal.(Proteomics2024,2300644), titled Proteome Integral Solubility Alteration via label-free DIA approach (PISA-DIA), introduces Data-Independent Acquisition (DIA) as a quantification method, opening new avenues in drug target-deconvolution field. Application of DIA for target deconvolution offers attractive alternative to widely used data dependent methodology.

Tomato roots exhibit distinct, development-specific responses to bacterial-derived peptides.

Plants possess cell-surface recognition receptors that detect molecular patterns from microbial invaders and initiate an immune response. Understanding the conservation of pattern-triggered immunity within different plant organs and across species is crucial to its sustainable and effective use in plant disease management but is currently unclear.We examined the activation and immune response patterns of three pattern recognition receptors (PRRs: SlFLS2, SlFLS3, and SlCORE) in different developmental regions of roots and in leaves of multiple accessions of domesticated and wild tomato (Solanum lycopersicum and S. pimpinellifolium) using biochemical and genetic assays.Roots from different tomato accessions differed in the amplitude and dynamics of their immune response, but all exhibited developmental-specific PTI responses in which the root early differentiation zone was the most sensitive to molecular patterns. PRR signaling pathways also showed distinct but occasionally overlapping responses downstream of each immune receptor in tomato roots.These results reveal that each PRR initiates a unique PTI pathway and suggest that the specificity and complexity of tomato root immunity are tightly linked to the developmental stage, emphasizing the importance of spatial and temporal regulation in PTI.

The Arabidopsis E3 ubiquitin ligase DOA10A promotes localization of abscisic acid (ABA) receptors to the membrane through mono-ubiquitination in ABA signaling.

The endoplasmic reticulum-associated degradation (ERAD) system eliminates misfolded and short-lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain-of-function mutant, doa10a-1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono-ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING-type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono-ubiquitination.