Modulation of multiple BAK1- dependent signaling pathways by two atypical receptor-like kinases

Transmembrane leucine-rich repeat (LRR) receptors are commonly used innate immune receptors in plants and animals but can also sense endogenous signals to regulate development. BAK1, a plant LRR-receptor-like kinase (RLK) with a small extracellular domain, interacts with several ligand-binding LRR-RLKs to positively regulate their functions. BAK1 is involved in brassinosteroiddependent growth and development, innate immunity, and cell-death control by interacting with the brassinosteroid receptor BRI1, immune receptors, such as FLS2 and EFR, and the small receptor kinase BIR1, respectively. In vivo study of BAK1 complex partners by LC/ESI-MS/MS led to the identification of two novel BAK1-interacting RLKs, BIR2 and BIR3. Functional analyses of bir2 mutants show differential impact on BAK1- regulated processes, such as hyperresponsiveness to microbe-associated molecular patterns (MAMP), enhanced cell death, and resistance to bacterial pathogens, but have no effect on brassinosteroid-regulated growth. BIR2 interacts constitutively with BAK1, thereby preventing interaction with the ligand-binding LRRRLK FLS2. MAMP perception leads to BIR2 release from the BAK1 complex and enables the recruitment of BAK1 into the FLS2 complex. These results provide evidence for a new regulatory mechanism for innate immune receptors with BIR2 acting as a negative regulator of MAMP-triggered immunity by limiting BAK1- receptor complex formation in the absence of ligands. In a second part, functional analysis of BIR3 overexpressing lines has been performed. These lines present a strong decrease in BL signaling with a morphology resembling bri1 mutants. MAMP signaling and cell death control are also downregulated pointing to a negative regulatory role of BIR3 on BAK1

A Genome-Wide Survey for Arabidopsis Leucine-Rich Repeat Receptor Kinases Implicated in Plant Immunity

Receptor-like kinases (RLK) are among the largest gene families encoded by plant genomes. Common structural features of plant RLKs are an extracellular ligand binding domain, a membrane spanning domain, and an intracellular protein kinase domain. The largest subfamily of plant RLKs is characterized by extracellular leucine-rich repeat (LRR-RLK) structures that are known biochemical modules for mediating ligand binding and protein–protein interactions. In the frame of the Arabidopsis Functional Genomics Network initiative of the German Research Foundation (DFG) we have conducted a comprehensive survey for and functional characterization of LRR-RLKs potentially implicated in Arabidopsis thaliana immunity to microbial infection. Arabidopsis gene expression patterns suggested an important role of this class of proteins in biotic stress adaptation. Detailed biochemical and physiological characterization of the brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) revealed brassinolide-independent roles of this protein in plant immunity, in addition to its well-established function in plant development. The LRR-RLK BAK1 has further been shown to form heteromeric complexes with various other LRR-RLKs in a ligand-dependent manner, suggesting a role as adapter or co-receptor in plant receptor complexes. Here, we review the current status of BAK1 and BAK1-interacting LRR-RLKs in plant immunity

Isolating strain and curvature effects in premixed flame/vortex interactions

This study focuses on the response of premixed flames to a transient hydrodynamic perturbation in an intermediate situation between laminar stretched flames and turbulent flames: an axisymmetric vortex interacting with a flame. The reasons motivating this choice are discussed in the framework of turbulent combustion models and flame response to the stretch rate. We experimentally quantify the dependence of the flame kinematic properties (displacement and consumption speeds) to geometrical scalars (stretch rate and curvature) in flames characterized by different effective Lewis numbers. Whilst the displacement speed can be readily measured using particle image velocimetry and tomographic diagnostics, providing a reliable estimate of the consumption speed from experiments remains particularly challenging. In the present work, a method based on a budget of fuel on a well chosen domain is proposed and validated both experimentally and numerically using two-dimensional direct numerical simulations of flame/vortex interactions. It is demonstrated that the Lewis number impact neither the geometrical nor the kinematic features of the flames, these quantities being much more influenced by the vortex intensity. While interacting with the vortex, the flame displacement (at an isotherm close to the leading edge) and consumption speeds are found to increase almost independently of the type of fuel. We show that the total stretch rate is not the only scalar quantity impacting the flame displacement and consumption speeds and that curvature has a significant influence. Experimental data are interpreted in the light of asymptotic theories revealing the existence of two distinct Markstein numbers, one characterizing the dependence of flame speed to curvature, the other to the total stretch rate. This theory appears to be well suited for representing the evolution of the displacement speed with respect to either the total stretch rate, curvature or strain rate. It also explains the limited dependence of the flame displacement speed to Lewis number and the strong correlation with curvature observed in the experiments. An explicit relationship between displacement and consumption speeds is also given, indicating that the fuel consumption rate is likely to be altered by both the total stretch rate and curvature

The Arabidopsis leucine-rich repeat receptor kinase BIR3 negatively regulates BAK1 receptor complex formation and stabilizes BAK1

BAK1 is a co-receptor and positive regulator of multiple ligand-binding leucine-rich-repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana. BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand-binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand-binding receptors to prevent BAK1 receptor complex formation

Meneco, a Topology-Based Gap-Filling Tool Applicable to Degraded Genome-Wide Metabolic Networks

International audienceIncreasing amounts of sequence data are becoming available for a wide range of non-model organisms. Investigating and modelling the metabolic behaviour of those organisms is highly relevant to understand their biology and ecology. As sequences are often incomplete and poorly annotated, draft networks of their metabolism largely suffer from incompleteness. Appropriate gap-filling methods to identify and add missing reactions are therefore required to address this issue. However, current tools rely on phenotypic or taxonomic information, or are very sensitive to the stoichiometric balance of metabolic reactions, especially concerning the co-factors. This type of information is often not available or at least prone to errors for newly-explored organisms. Here we introduce Meneco, a tool dedicated to the topological gap-filling of genome-scale draft metabolic networks. Meneco reformulates gap-filling as a qualitative combinatorial optimization problem, omitting constraints raised by the stoichiometry of a metabolic network considered in other methods, and solves this problem using Answer Set Programming. Run on several artificial test sets gathering 10,800 degraded Escherichia coli networks Meneco was able to efficiently identify essential reactions missing in networks at high degradation rates, outperforming the stoichiometry-based tools in scalability. To demonstrate the utility of Meneco we applied it to two case studies. Its application to recent metabolic networks reconstructed for the brown algal model Ectocarpus siliculosus and an associated bacterium Candidatus Phaeomarinobacter ectocarpi revealed several candidate metabolic pathways for algal-bacterial interactions. Then Meneco was used to reconstruct, from transcriptomic and metabolomic data, the first metabolic network for the microalga Euglena mutabilis. These two case studies show that Meneco is a versatile tool to complete draft genome-scale metabolic networks produced from heterogeneous data, and to suggest relevant reactions that explain the metabolic capacity of a biological system

Flame front analysis of high-pressure turbulent lean premixed methane–air flames

International audienceAn experimental study on lean turbulent premixed methane–air flames at high pressure is conducted by using a turbulent Bunsen flame configuration. A single equivalence ratio flame at U = 0.6 is explored for pressures ranging from atmospheric pressure to 0.9 MPa. LDA measurements of the cold flow indicate that turbulence intensities and the integral length scale are not sensitive to pressure. Due to the decreased kinematic viscosity with increasing pressure, the turbulent Reynolds numbers increase, and isotropic turbulence scaling relations indicate a large decrease of the smallest turbulence scales. Available experimental results and PREMIX code computations indicate a decrease in laminar flame propagation velocities with increasing pressure, essentially between the atmospheric pressure and 0.5 MPa. The u0/SL ratio increases thereforeaccordingly. Instantaneous flame images are obtained by Mie scattering tomography. The images and their analysis show that pressure increase generates small scale flame structures. In an attempt to generalize these results, the variance of the flamelet curvatures, the standard deviation of the flamelet orientation angle, and the flamelet crossing lengths have been plotted against Re1=2 t which is proportional to the ratio between theintegral and Taylor length scales, and which increases with pressure. These three parameters vary linearly with the ratio between large and small turbulence scales and clearly indicate the strong effect of this parameter on premixed turbulent flame dynamics and structure. An obvious consequence is the increase in flame surface density and hence burning rate with pressure, as confirmed by its direct determination from 2D tomographic images

The Arabidopsis active demethylase ROS1 cis-regulates defense genes by erasing DNA methylation at promoter-regulatory regions

International audienceActive DNA demethylation has emerged as an important regulatory process of plant and mammalian immunity. However, very little is known about the mechanisms by which active demethylation controls transcriptional immune reprogramming and disease resistance. Here, we first show that the Arabidopsis active demethylase ROS1 promotes basal resistance towards Pseudomonas syringae by antagonizing RNA-directed DNA methylation (RdDM). Furthermore, we demonstrate that ROS1 facilitates the flagellin-triggered induction of the disease resistance gene RMG1 by limiting RdDM at the 3' boundary of a transposable element (TE)-derived repeat embedded in its promoter. We further identify flagellin-responsive ROS1 putative primary targets and show that at a subset of promoters, ROS1 erases methylation at discrete regions exhibiting WRKY transcription factors (TFs) binding. In particular, we demonstrate that ROS1 removes methylation at the orphan immune receptor RLP43 promoter, to ensure DNA binding of WRKY TFs. Finally, we show that ROS1-directed demethylation of RMG1 and RLP43 promoters is causal for both flagellin responsiveness of these genes and for basal resistance. Overall, these findings significantly advance our understanding of how active demethylases shape transcriptional immune reprogramming to enable antibacterial resistance

DNA methylation at a single cytosine embedded in the W-box cis -element repels binding of WRKY transcription factors through steric hindrance

ABSTRACT Previously, we showed that the Arabidopsis active demethylase ROS1 prunes DNA methylation at the promoters of a subset of immune-responsive genes to facilitate their transcriptional activation during antibacterial defence (Halter et al., 2021). In particular, ROS1 was shown to demethylate the RLP43 promoter in a region carrying a functional W-box cis -element, thereby ensuring a tight binding of WRKY transcriptional factors (TFs) onto DNA. Here, we first extend these findings by showing that DNA methylation at W-box elements decreases the binding of several Arabidopsis WRKY TFs in vitro . Furthermore, we provide evidence that DNA methylation at a single cytosine located in the W-box of the RLP43 promoter strongly repels DNA binding of an Arabidopsis WRKY TF in vitro . Using structural modelling, we demonstrate that this cytosine interacts through van der Waals contacts with the conserved tyrosine of WRKY DNA binding domains. Significantly, our model predicts steric hindrance when a 5-methyl group is present on this specific cytosine, thereby likely preventing tight binding of WRKY DNA binding domains. Finally, because the WRKY motif and the residues involved in DNA contacts are conserved, we propose that this methylation-dependent WRKY-DNA binding inhibitory mechanism must be widespread across plant species