Molecular components of PAMP-triggered oxidative burst in plant immunity

Reactive oxygen species (ROS) are important molecules that are rapidly generated in response to abiotic and biotic stimuli and which regulate diverse physiological processes such as stomatal aperture and cell death. Plant immunity involves the detection of pathogen-associated molecular patterns (PAMPs) through cognate pattern recognition receptors (PRRs). Perception of PAMPs induces an extracellular oxidative burst, which requires the function of the NADPH oxidase AtRbohD. However, little is known about the regulation of PAMP-elicited ROS and its role in plant PAMP-triggered immunity. We investigated ROS production mediated by the FLS2 receptor kinase responsible for the detection of bacterial flagellin (flg22) in Arabidopsis to elucidate components of early flg22 signaling. Rboh proteins are assumed to predominantly mediate extracellular ROS production in plants. We observed AtRbohD to be rate limiting for flg22-elicited ROS production. Moreover, bacterial multiplication monitored at early stages of infection with a disarmed but pathogenic Pseudomonas syringae strain indicated ROS accumulation important for plant immunity. A forward genetic screen led to the isolation of rio1 to rio5 mutants, all of which were severely reduced in flg22-induced ROS production. Rio1, rio2 and rio3 carry mutations in FLS2 and its co-receptor BAK1, respectively. The responsible gene mutations of rio4 and rio5 remain to be identified in a subsequent mapping approach. A candidate gene approach revealed that flg22-stimulated oxidative burst was specifically inhibited in ethylene signaling mutants. Impaired ROS production in etr1 and ein2 mutants could be partially rescued by chemical interference with ethylene accumulation. Notably, wounding partially complemented the ROS reduced phenotype. Furthermore, accumulation of FLS2 but not BAK1 was in part dependent on ethylene signaling. Bacterial multiplication at early time points was significantly enhanced in ethylene signaling mutants indicating the importance of PAMP-triggered ROS production in plant defence responses

Stability of change point detection methods

Change point detection is a common technique to analyse time-indexed data and today's literature offers a wide variety of methods for change point detection. Assessing the performance of a change point detection method usually involves evaluating a method's ability to detect the true change point locations (if known). Additional potentially interesting properties, well-studied for other predictive algorithms, are left unstudied. Beyond conventional performance measures, we explore the consistency of estimated change point locations when a method is challenged with slightly perturbed input signals, a concept known as algorithmic stability in the machine learning literature. In particular, we conceptualise and formalise ideas relating to stability for change point detection, and present a simulation study that compares stabilities of popular change point detection methods including Binary Segmentation, Wild Binary Segmentation, and PELT (Pruned Exact Linear Time). We verify prior beliefs that Binary Segmentation's greedy search procedure is vulnerable to data perturbations, and introduce an extension to Binary Segmentation that stabilises its search procedure to some extent. We also find that PELT, as an exact method, is often more stable than non-optimal competitors, given the optimal parameter setting is known. Finally, we explore the potential for using stability as a means for setting control parameters in the absence of ground truth.Open Acces

Transcriptional regulation of the CRK/DUF26 group of Receptor-like protein kinases by ozone and plant hormones in Arabidopsis

Peer reviewe

Ethylene Signaling Regulates Accumulation of the FLS2 Receptor and Is Required for the Oxidative Burst Contributing to Plant Immunity

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response

A novel and robust method for counting components within bio-molecular complexes using fluorescence microscopy and statistical modelling.

Cellular biology occurs through myriad interactions between diverse molecular components, many of which assemble in to specific complexes. Various techniques can provide a qualitative survey of which components are found in a given complex. However, quantitative analysis of the absolute number of molecules within a complex (known as stoichiometry) remains challenging. Here we provide a novel method that combines fluorescence microscopy and statistical modelling to derive accurate molecular counts. We have devised a system in which batches of a given biomolecule are differentially labelled with spectrally distinct fluorescent dyes (label A or B), and mixed such that B-labelled molecules are vastly outnumbered by those with label A. Complexes, containing this component, are then simply scored as either being positive or negative for label B. The frequency of positive complexes is directly related to the stoichiometry of interaction and molecular counts can be inferred by statistical modelling. We demonstrate this method using complexes of Adenovirus particles and monoclonal antibodies, achieving counts that are in excellent agreement with previous estimates. Beyond virology, this approach is readily transferable to other experimental systems and, therefore, provides a powerful tool for quantitative molecular biology

PhyreRisk: A Dynamic Web Application to Bridge Genomics, Proteomics and 3D Structural Data to Guide Interpretation of Human Genetic Variants

This work is licensed under a Creative Commons Attribution 4.0 International License.PhyreRisk is an open-access, publicly accessible web application for interactively bridging genomic, proteomic and structural data facilitating the mapping of human variants onto protein structures. A major advance over other tools for sequence-structure variant mapping is that PhyreRisk provides information on 20,214 human canonical proteins and an additional 22,271 alternative protein sequences (isoforms). Specifically, PhyreRisk provides structural coverage (partial or complete) for 70% (14,035 of 20,214 canonical proteins) of the human proteome, by storing 18,874 experimental structures and 84,818 pre-built models of canonical proteins and their isoforms generated using our in house Phyre2. PhyreRisk reports 55,732 experimentally, multi-validated protein interactions from IntAct and 24,260 experimental structures of protein complexes. Another major feature of PhyreRisk is that, rather than presenting a limited set of precomputed variant-structure mapping of known genetic variants, it allows the user to explore novel variants using, as input, genomic coordinates formats (Ensembl, VCF, reference SNP ID and HGVS notations) and Human Build GRCh37 and GRCh38. PhyreRisk also supports mapping variants using amino acid coordinates and searching for genes or proteins of interest. PhyreRisk is designed to empower researchers to translate genetic data into protein structural information, thereby providing a more comprehensive appreciation of the functional impact of variants. PhyreRisk is freely available at http://phyrerisk.bc.ic.ac.ukWellcome Trust 104955/Z/14/ZWellcome Trust PhD studentship 108908/B/15/ZBBSRC BB/M011526/1BBSRC BB/P011705/1NSF DBI1565107NIH R01GM07425

Plant Pattern-Recognition Receptor FLS2 Is Directed for Degradation by the Bacterial Ubiquitin Ligase AvrPtoB

Background: An important layer of active defense in plant immunity is the detection of pathogen-associated molecular patterns (PAMPs) mediated by cell-surface receptors. For the establishment of disease, pathogens depend on the ability to overcome PAMP perception and disable plant signaling pathways activated in response to PAMPs. Pattern recognition receptors (PRRs) are therefore prime targets for pathogen effectors. FLS2, its coreceptor BAK1, and EFR encode receptor-like kinases that play a role in immunity against bacterial pathogens. Results: Here, we report that virulence of Pseudomonas syringae pv tomato DC3000 (PtoDC3000) in Arabidopsis is enhanced through the action of its effector AvrPtoB, which promotes degradation of FLS2. We show that AvrPtoB, through its N terminus, associates with FLS2 and BAK1, of which interaction with FLS2 is enhanced by flg22 activation. In vitro, AvrPtoB is active as an E3 ligase to catalyze polyubiquitination of the kinase domain of FLS2, a process confirmed in planta. Full enhancement of PtoDC3000 virulence appears to require the E3 ligase activity of AvrPtoB. Conclusions: AvrPtoB, initially identified through its activation of hypersensitive resistance in tomato cultivars expressing the Pto kinase, is composed of at least two functional domains: the N terminus is responsible for interaction with Pto, and the C terminus carries an E3 ligase activity. Based on our findings, we propose that both domains of AvrPtoB act together to support the virulence of PtoDC3000 in Arabidopsis through their ability to eliminate FLS2 from the cell periphery, and probably also other PAMP sensors that are constitutively expressed or induced after pathogen challenge

A roadmap to generate renewable protein binders to the human proteome

Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders