An official website of the United States government
Intracellular plant immune receptors, termed NLRs (Nucleotide-binding Leucine-rich repeat Receptors), confer effector-triggered immunity. Sensor NLRs are responsible for pathogen effector recognition. Helper NLRs function downstream of sensor NLRs to transduce signaling and induce cell death and immunity. Activation of sensor NLRs that contain TIR (Toll/interleukin-1receptor) domains generates small molecules that induce an association between a downstream heterodimer signalosome of EDS1 (EnhancedDisease Susceptibility 1)/SAG101 (Senescence-AssociatedGene 101) and the helper NLR of NRG1 (NRequired Gene 1). Autoactive NRG1s oligomerize and form calcium signaling channels largely localized at the plasma membrane (PM). The molecular mechanisms of helper NLR PM association and effector-induced NRG1 oligomerization are not well characterized. We demonstrate that helper NLRs require positively charged residues in their N-terminal domains for phospholipid binding and PM association before and after activation, despite oligomerization and conformational changes that accompany activation. We demonstrate that effector activation of a TIR-containing sensor NLR induces NRG1 oligomerization at the PM and that the cytoplasmic pool of EDS1/SAG101 is critical for cell death function. EDS1/SAG101 cannot be detected in the oligomerized NRG1 resistosome, suggesting that additional unknown triggers might be required to induce the dissociation of EDS1/SAG101 from the previously described NRG1/EDS1/SAG101 heterotrimer before subsequent NRG1 oligomerization. Alternatively, the conformational changes resulting from NRG1 oligomerization abrogate the interface for EDS1/SAG101 association. Our data provide observations regarding dynamic PM association during helper NLR activation and underpin an updated model for effector-induced NRG1 resistosome formation.
more »
« less
Plant “helper” immune receptors are Ca 2+ -permeable nonselective cation channels
Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. In Arabidopsis , a subfamily of “helper” NLRs is required by many “sensor” NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta, and conferred cytoplasmic calcium ion (Ca 2+ ) influx in plant and human cells. NRG1.1-dependent Ca 2+ influx and cell death were sensitive to Ca 2+ channel blockers and were suppressed by mutations affecting oligomerization or plasma membrane enrichment. Ca 2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another helper NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrated that Arabidopsis helper NLRs form Ca 2+ -permeable cation channels to directly regulate cytoplasmic Ca 2+ levels and consequent cell death. Thus, helper NLRs transduce cell death signals directly.
more »
« less
- Award ID(s):
- 1758400
- PAR ID:
- 10323914
- Date Published:
- Journal Name:
- Science
- Volume:
- 373
- Issue:
- 6553
- ISSN:
- 0036-8075
- Page Range / eLocation ID:
- 420 to 425
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Iron- and reactive oxygen species (ROS)-dependent ferroptosis occurs in plant cells. Ca2+acts as a conserved key mediator to control plant immune responses. Here, we report a novel role of cytoplasmic Ca2+influx regulating ferroptotic cell death in rice immunity using pharmacological approaches. High Ca2+influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). DuringMagnaporthe oryzaeinfection, 14 different Ca2+influx regulators altered Ca2+, ROS and Fe2+accumulation,glutathione reductase(GR) expression, glutathione (GSH) depletion and lipid peroxidation, leading to ferroptotic cell death in rice. High Ca2+levels inhibited the reduction of glutathione isulphide (GSSG) to GSHin vitro. Ca2+chelation by ethylene glycol-bis (2-aminoethylether)-N, N, N’, N’-tetra-acetic acid (EGTA) suppressed apoplastic Ca2+influx in rice leaf sheaths during infection. Blocking apoplastic Ca2+influx into the cytoplasm by Ca2+chelation effectively suppressed Ca2+-mediated iron-dependent ROS accumulation and ferroptotic cell death. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+influx, as well as ROS and iron accumulation to trigger ferroptotic cell death in rice. The cytoplasmic Ca2+influx through calcium-permeable cation channels, including the putative resistosomes, could mediate iron- and ROS-dependent ferroptotic cell death under reducedGRexpression levels in rice immune responses.more » « less
-
TIR domains are NAD-degrading enzymes that function during immune signaling in prokaryotes, plants, and animals. In plants, most TIR domains are incorporated into intracellular immune receptors termed TNLs. In Arabidopsis, TIR-derived small molecules bind and activate EDS1 heterodimers, which in turn activate RNLs, a class of cation channel–forming immune receptors. RNL activation drives cytoplasmic Ca 2+ influx, transcriptional reprogramming, pathogen resistance, and host cell death. We screened for mutants that suppress an RNL activation mimic allele and identified a TNL, SADR1. Despite being required for the function of an autoactivated RNL, SADR1 is not required for defense signaling triggered by other tested TNLs. SADR1 is required for defense signaling initiated by some transmembrane pattern recognition receptors and contributes to the unbridled spread of cell death in lesion simulating disease 1 . Together with RNLs, SADR1 regulates defense gene expression at infection site borders, likely in a non-cell autonomous manner. RNL mutants that cannot sustain this pattern of gene expression are unable to prevent disease spread beyond localized infection sites, suggesting that this pattern corresponds to a pathogen containment mechanism. SADR1 potentiates RNL-driven immune signaling not only through the activation of EDS1 but also partially independently of EDS1. We studied EDS1-independent TIR function using nicotinamide, an NADase inhibitor. Nicotinamide decreased defense induction from transmembrane pattern recognition receptors and decreased calcium influx, pathogen growth restriction, and host cell death following intracellular immune receptor activation. We demonstrate that TIR domains can potentiate calcium influx and defense and are thus broadly required for Arabidopsis immunity.more » « less
-
Abstract The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca 2+ -imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism.more » « less
-
The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a “plasma membrane on a chip,” also known as a supported lipid bilayer. Here, we create the “plant-membrane-on-a-chip,″ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein–protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein–protein and protein–lipid interactions in a convenient, cell-free platform.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1126/science.abg7917
