Project description:Intracellular NLR receptors with a central nucleotide-binding domain allow plants to detect specific pathogen-secreted proteins and initiate immune signaling. A family of conserved Enhanced disease susceptibility 1 proteins with a lipase-like and the EP domain transduces the signal from activated NLRs downstream. This family includes EDS1, PAD4 and SAG101 that form mutually exclusive heterodimers EDS1-PAD4 and EDS1-SAG101. Despite sequence similarities, PAD4 and SAG101 control different aspects of NLR immune signaling. More specifically, PAD4 and SAG101 regulate resistance and cell death outputs of the NLR receptor pair RRS1-RPS4, respectively. Also, PAD4 and SAG101 genetically cooperate with distinct downstream signaling NLRs - ADR1 and NRG1. The main aim of this IP-LC/MS project is to assess whether PAD4 and SAG101 form complexes with ADR1 and NRG1 and to gain insight into mechanisms of the genetically established functional links between EDS1 family proteins and downstream signaling NLRs.
Project description:Arabidopsis Enhanced Disease Susceptibility 1 (EDS1) and its direct partner Phytoalexin Deficient 4 (PAD4) are required for both basal resistance against virulent pathogens and innate immune responses mediated by all tested TIR (Toll-Interleukin-1 Receptors) type nucleotide-binding/leucine-rich-repeat (NLR) receptors. Using transgenic Arabidopsis plants in Col-0 eds1-2 pad4-1 background conditionally expressing PAD4 and constitutively expressing EDS1 (ED-P4E1), an EDS1/PAD4 immune response can be triggered by estradiol treatment and can induce expression of defense-related genes and lead to enhanced basal resistance. In order to capture primary transcriptional changes in response to EDS1/PAD4, we performed RNA-seq gene expression analysis in ED-P4E1 plants after estradiol or mock treatment at three time points: 6, 12 and 24 h.
Project description:Arabidopsis pathogen effector-triggered immunity (ETI) is controlled by a family of three lipase-like proteins EDS1, PAD4 and SAG101 and two sub-families of HET-S/LOB-B (HeLo)-domain “helper” NLRs, ADR1s and NRG1s. EDS1-PAD4 dimers cooperate with ADR1s, and EDS1-SAG101 dimers with NRG1s, in two separate defense-promoting modules. EDS1-PAD4-ADR1 and EDS1- SAG101-NRG1 complexes were detected in immune-activated leaf extracts but the molecular determinants for specific complex formation and function remain unknown. EDS1 signaling is mediated by a C-terminal EP domain (EPD) surface surrounding a cavity formed by the heterodimer. Here we investigated whether the EPDs of PAD4 and SAG101 contribute to EDS1 dimer functions. Using a structure-guided approach, we undertook a comprehensive mutational analysis of Arabidopsis PAD4. We identify two conserved residues (Arg314 and Lys380) lining the PAD4 EPD cavity that are essential for EDS1-PAD4 mediated pathogen resistance, but are dispensible for PAD4 mediated restriction of green peach aphid infestation. Positionally equivalent Met304 and Arg373 at the SAG101 EPD cavity are required for EDS1-SAG101 promotion of ETI-related cell death. In a PAD4 and SAG101 interactome analysis of ETI-activated tissues, PAD4R314A and SAG101M304R EPD variants maintain interaction with EDS1 but lose association, respectively, with helper NLRs ADR1-L1 and NRG1.1, and other immune-related proteins. Our data reveal a fundamental contribution of similar but non-identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity.
Project description:LSD1, EDS1 and PAD4 Conditionally Regulate Cellular Signaling Homeostasis, Photosynthesis, Water Use Efficiency and Seed Yield in Arabidopsis
Project description:There is growing evidence that for a comprehensive insight into the function of plant genes it is crucial to assess their functionalities under a wide range of conditions. In this study we examined the role of LESION SIMULATING DISEASE1 (LSD1), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) in the regulation of photosynthesis, water use efficiency (WUE), ROS/hormonal homeostasis and seed yield in Arabidopsis thaliana grown in the laboratory and in the field. We demonstrate that the LSD1 null mutant (lsd1), which is known to exhibit a runaway cell death in non-permissive conditions, proves to be more tolerant to combined drought and high-light stress than the wild type. Moreover, depending on growing conditions, it shows variations in WUE, salicylic acid and hydrogen peroxide concentrations, photosystem II maximum efficiency and in transcription profiles. However, despite these changes, lsd1 demonstrates similar seed yield under all tested conditions. All of these traits depend on EDS1 and PAD4. The differences in the pathways prevailing in the lsd1 in various growing environments are manifested by the significantly smaller number of transcripts deregulated in the field compared to the laboratory, with only 43 commonly regulated genes. Our data indicate that LSD1, EDS1 and PAD4 participate in the regulation of various molecular and physiological processes that influence Arabidopsis fitness. On the basis of these results we emphasize that the function of such important regulators as LSD1, EDS1 and PAD4 should be studied not only under stable laboratory conditions, but also in the environment abounding in multiple stresses. Six genotypes x two conditions experiment including five-week-old WT (Ws), lsd1, pad4, eds1, eds1lsd1, and pad4lsd1 plants grown in laboratory or field conditions, hybridized in two loop-designs (lab and field). Two biological replicates. In total, 2 x 12 samples were hybridized on 24 Arabidopsis Gene Expression microarrays V4 (Agilent, two-color array) including color swap of replicate samples.
Project description:There is growing evidence that for a comprehensive insight into the function of plant genes it is crucial to assess their functionalities under a wide range of conditions. In this study we examined the role of LESION SIMULATING DISEASE1 (LSD1), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) in the regulation of photosynthesis, water use efficiency (WUE), ROS/hormonal homeostasis and seed yield in Arabidopsis thaliana grown in the laboratory and in the field. We demonstrate that the LSD1 null mutant (lsd1), which is known to exhibit a runaway cell death in non-permissive conditions, proves to be more tolerant to combined drought and high-light stress than the wild type. Moreover, depending on growing conditions, it shows variations in WUE, salicylic acid and hydrogen peroxide concentrations, photosystem II maximum efficiency and in transcription profiles. However, despite these changes, lsd1 demonstrates similar seed yield under all tested conditions. All of these traits depend on EDS1 and PAD4. The differences in the pathways prevailing in the lsd1 in various growing environments are manifested by the significantly smaller number of transcripts deregulated in the field compared to the laboratory, with only 43 commonly regulated genes. Our data indicate that LSD1, EDS1 and PAD4 participate in the regulation of various molecular and physiological processes that influence Arabidopsis fitness. On the basis of these results we emphasize that the function of such important regulators as LSD1, EDS1 and PAD4 should be studied not only under stable laboratory conditions, but also in the environment abounding in multiple stresses.
Project description:Nod-like receptors (NLRs) belong to AAA+ ATPases and act as ATP-dependent molecular switches, which detect activity of pathogens. Function of a large class of plant NLRs with Toll-like domain (TNL) is fully dependent on a class of EDS1-like proteins specific to seed plants. EDS1 (Enhanced Disease Susceptibility 1) – like proteins are defined as fusions of two domains: lipase-like a/b hydrolase domain and an a-helical bundle domain specific to the EDS1 family. In Arabidopsis, RRS1-RPS4 TNL signaling is dependent on the nuclear localization of EDS1 and formation of heterodimers between EDS1 and its sequence-related partners, PAD4 and SAG101. Here, we deposited results of affinity purification and LC-MS analyses are deposited for the EDS1 complexes after triggering NLR-dependent immune responses in Arabidopsis leaves. As a negative control, plants expressing GFP-tagged Telomere Repeat Binding 1 were used. The complexes were purified from nuclear enriched fractions of Arabidopsis complementation lines infected with the TNL-triggering bacteria.
Project description:Plants deploy cell surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens. LRR receptor kinases (LRR-RKs) and LRR receptor proteins (LRR-RPs) recognise microbe-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR (NLR) proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes, suggesting pathway convergence upstream of nuclear events. We report that PTI triggered by Arabidopsis LRR-RP (RLP23) requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and ADR1-family helper NLRs, which are all components of ETI. The cell surface LRR-RK SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting formation of constitutive supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane.