ZAR1 resistosome and helper NLRs: Bringing in calcium and inducing cell death

Abstract

To protect themselves from infectious diseases, plants have deployed a two-tiered surveillance system to recognize invading pathogens (Fu and Dong, 2013Fu Z.Q. Dong X. Systemic acquired resistance: turning local infection into global defense.Annu. Rev. Plant Biol. 2013; 64: 839-863https://doi.org/10.1146/annurev-arplant-042811-105606Crossref PubMed Scopus (841) Google Scholar). In the first layer of the immune system, plants evolved extracellular pattern recognition receptors to detect conserved pathogen-associated molecular patterns (PAMPs) and activate PAMP-triggered immunity (PTI) (Boller and Felix, 2009Boller T. Felix G. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors.Annu. Rev. Plant Biol. 2009; 60: 379-406https://doi.org/10.1146/annurev.arplant.57.032905.105346Crossref PubMed Scopus (2048) Google Scholar). The second layer of the immune system, which is termed effector-triggered immunity (ETI), relies on an array of nucleotide-binding leucine-rich repeat receptor (NLR) proteins to detect pathogen effectors, either directly or the activities of these effectors indirectly (Figure 1A) (Sun et al., 2020Sun Y. Zhu Y.X. Balint-Kurti P.J. Wang G.F. Fine-tuning immunity: players and regulators for plant NLRs.Trends Plant Sci. 2020; 25: 695-713https://doi.org/10.1016/j.tplants.2020.02.008Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). NLR activation often triggers a rapid localized cell death called hypersensitive response (HR). In general, NLRs are divided into two functional groups: sensor NLRs that are involved in the recognition of pathogen effectors and helper NLRs that are required by sensor NLRs for ETI activation (Jubic et al., 2019Jubic L.M. Saile S. Furzer O.J. El Kasmi F. Dangl J.L. Help wanted: helper NLRs and plant immune responses.Curr. Opin. Plant Biol. 2019; 50: 82-94https://doi.org/10.1016/j.pbi.2019.03.013Crossref PubMed Scopus (94) Google Scholar; Sun et al., 2020Sun Y. Zhu Y.X. Balint-Kurti P.J. Wang G.F. Fine-tuning immunity: players and regulators for plant NLRs.Trends Plant Sci. 2020; 25: 695-713https://doi.org/10.1016/j.tplants.2020.02.008Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Two major types of sensor NLRs are defined as TIR (Toll/interleukin-1 receptor)-NLR (TNL) and CC (coiled-coil)-NLR (CNL) based on their N-terminal TIR or CC domain. HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) is a sensor NLR containing a canonical CC domain that acts as a sensor for a number of pathogen effectors, such as HopZ1a, HopF1, HopX1, HopO1, AvrAC, etc. (Laflamme et al., 2020Laflamme B. Dillon M.M. Martel A. Almeida R.N.D. Desveaux D. Guttman D.S. The pan-genome effector-triggered immunity landscape of a host-pathogen interaction.Science. 2020; 367: 763-768https://doi.org/10.1126/science.aax4079Crossref PubMed Scopus (65) Google Scholar). In many flowering plants, N REQUIRED GENE1 (NRG1) and ACTIVATED DISEASE RESISTANCE 1 (ADR1), two sequence-related protein groups, function as helper NLRs that are required for the full function of several TNL and CNL receptors (Sun et al., 2020Sun Y. Zhu Y.X. Balint-Kurti P.J. Wang G.F. Fine-tuning immunity: players and regulators for plant NLRs.Trends Plant Sci. 2020; 25: 695-713https://doi.org/10.1016/j.tplants.2020.02.008Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Some NLRs, such as ZAR1, function as both sensors and executors, and largely do not require helper NLRs to activate ETI (Adachi et al., 2019Adachi H. Derevnina L. Kamoun S. NLR singletons, pairs, and networks: evolution, assembly, and regulation of the intracellular immunoreceptor circuitry of plants.Curr. Opin. Plant Biol. 2019; 50: 121-131https://doi.org/10.1016/j.pbi.2019.04.007Crossref PubMed Scopus (79) Google Scholar). Despite the importance of ZAR1 resistosome, NRG1, and ADR1 in plant immunity, the precise biochemical functions of these proteins have remained elusive until the recent studies published by Bi et al. (2021) and Wan et al. (2021). The funnel-shaped structure formed by the ZAR1 resistosome makes sense that it functions as a channel pore (Figure 1B). Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar tested this hypothesis by expressing wild-type ZAR1 along with AvrAC, RKS1, and PBL2 in Xenopus oocytes and performing two-electrode voltage clamp recordings. They observed strong current traces upon application of voltage, demonstrating that the activated ZAR1 resistosome indeed possesses a channel activity (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Furthermore, they found that the channel activity is dependent on Glu11, a conserved acidic residue in all ZAR1 proteins from different plant species (Wang et al., 2019Wang J. Hu M. Wang J. Qi J. Han Z. Wang G. Qi Y. Wang H.-W. Zhou J.-M. Chai J. Reconstitution and structure of a plant NLR resistosome conferring immunity.Science. 2019; 364: eaav5870https://doi.org/10.1126/science.aav5870Crossref PubMed Scopus (259) Google Scholar; Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). To further determine the channel activity of the ZAR1 resistosome, the authors implemented planar lipid bilayer-based electrophysiology studies. Pre-assembled ZAR1 resistosomes were reconstituted into planar lipid bilayers, and single-channel measurements were conducted. They found that the ZAR1 channel was permeable to Na+, K+, Cs+, Mg2+, and Ca2+ with distinctive current amplitudes (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The next question is whether ZAR1 activation in the plant cell can lead to increased Ca2+ in the cytosol. To answer this question, the authors tested the Ca2+ influx in the protoplast of a zar1 aequorin line expressing ZAR1 along with RKS1, PBL2, and AvrAC (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Aequorin is a calcium-sensitive photoprotein, which is used to measure calcium concentration. They found that the ZAR1 activation by AvrAC is sufficient to induce Ca2+ influx in the protoplast, and that Glu11 is required for the ZAR1-mediated Ca2+ influx (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Through observation of Ca2+ influx in the zar1 aequorin line complemented with ZAR1 after infiltration with Pseudomonas syringae pv. tomato DC3000 carrying HopZ1a, they also confirmed that activation of ZAR1 can trigger Ca2+ influx in leaves during infection (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). To further reveal how ZAR1 oligomerizes in the plant cell, the authors implemented state-of-the-art technology, single-molecule imaging, to visualize ZAR1 resistosome subcellular localization. Through this technology, they provided direct evidence that the AvrAC-induced ZAR1 complex indeed forms pentamers in living protoplasts (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Furthermore, they observed that the ZAR1-mEGFP signal in the presence of AvrAC had a similar distribution as the plasma membrane (PM) marker PIP2;1-mCherry, demonstrating that the ZAR1 resistosome can be localized in the PM (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Cell staining results showed that propidium iodide, which stains and enters into the dead cell after activation of ZAR1, indicating that the PM-localized ZAR1 resistosome causes a loss of PM integrity once activated (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). HR-associated cell death is often accompanied by oxidative burst. Thus, the authors examined reactive oxygen species (ROS) production and PM integrity and tracked the process by live-cell imaging. They found that protoplasts expressing ZAR1, RKS1, PBL2, and AvrAC showed a continuous increase of ROS production before cell death (Bi et al., 2021Bi G. Su M. Li N. Liang Y. Dang S. Xu J. Hu M. Wang J. Zou M. Deng Y. et al.The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.Cell. 2021; https://doi.org/10.1016/j.cell.2021.05.003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), indicating that activation of ZAR1 induces ROS production followed by a loss of PM integrity (Figure 1B). To summarize, Bi et al. demonstrated that the activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, production of reactive oxygen species, perturbation of PM integrity, and eventually cell death. The milestone discovery that ZAR1 resistosome functions as a calcium-permeable channel provided new insights into the functions of NLRs in plant immunity and changed our view of ETI-triggered cell death. More recently, Jacob et al., 2021Jacob P. Kim N.H. Wu F. El-Kasmi F. Chi Y. Walton W.G. Furzer O.J. Lietzan A.D. Sunil S. Kempthorn K. et al.Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels.Science. 2021; : eabg7917https://doi.org/10.1126/science.abg7917Crossref PubMed Scopus (46) Google Scholar discovered that two helper NLRs have similar biochemical activities as the ZAR1 resistosome. They obtained X-ray crystal structures of two mutant NRG1.1 CC-R domains (residues 1–124), K94E/K96E/R99E/K100E/R103E/K106E/K110E (7K(R)/E) and K94E/K96E (2K/E). The structures of these two mutants superimposed well with the N-terminal four-helical bundle of the resting-state CC domain of ZAR1 and the cation channel-forming domain of mammalian mixed-linage kinase domain-like. Because the wild-type NRG1.1 is inactive without NLR activation, the authors introduced the auto-active NRG1.1 D485V (DV) allele and demonstrated that NRG1.1 DV is sufficient to trigger cell death in planta (Jacob et al., 2021Jacob P. Kim N.H. Wu F. El-Kasmi F. Chi Y. Walton W.G. Furzer O.J. Lietzan A.D. Sunil S. Kempthorn K. et al.Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels.Science. 2021; : eabg7917https://doi.org/10.1126/science.abg7917Crossref PubMed Scopus (46) Google Scholar). NRG1.1 DV oligomerized and was enriched in PM (Figure 1A). These interesting observations prompted them to investigate whether NRG1.1 forms pores and functions as a channel. The authors found that NRG1.1 DV induced cell death in HeLa cells. Using scanning electron microscopy, they observed significantly increased numbers of PM pores in dying HeLa cells. Using Ca2+ reporter GCaMP3 transgenic Nicotiana benthamiana plants, Jacob et al., 2021Jacob P. Kim N.H. Wu F. El-Kasmi F. Chi Y. Walton W.G. Furzer O.J. Lietzan A.D. Sunil S. Kempthorn K. et al.Plant “helper” immune receptors are Ca2+-permeable nonselective cation channels.Science. 2021; : eabg7917https://doi.org/10.1126/science.abg7917Crossref PubMed Scopus (46) Google Scholar showed that both NRG1.1 DV and ADR1 triggered Ca2+ influx in planta. Therefore, the authors drew the conclusion that the active NRG1.1 forms Ca2+ permeable channels (Figure 1A). These breakthrough discoveries uncovered the mysterious biological functions of the helper NLRs NRG1.1 and ADR1. The fact that ZAR1 functions as a calcium channel does not mean that all CNLs function as calcium channels. Some CNLs in Arabidopsis require NDR1 for their functions, and NLR-REQUIRED FOR CELL DEATH (NRC) is required for cell death of some CNLs in solanaceous plants. It remains to be determined whether these CNLs, NDR1 or NRC, function as calcium channels. If they do not form calcium channels, what are the biochemical functions of these proteins? Future studies may focus on how calcium influx induces cell death. Elevated cytoplasmic Ca2+ levels can certainly cause Ca2+ influx into mitochondria and these higher levels of Ca2+ could disrupt mitochondria metabolism, leading to cell death (Ermak and Davies, 2002Ermak G. Davies K.J. Calcium and oxidative stress: from cell signaling to cell death.Mol. Immunol. 2002; 38: 713-721https://doi.org/10.1016/s0161-5890(01)00108-0Crossref PubMed Scopus (0) Google Scholar). Ca2+ could also bind to calmodulin to modulate the activity of many cytosolic and nuclear proteins as well as gene transcription to promote cell death. In addition, we still do not know how effector recognition triggers the activation of helper NLR. TIR domains in TNL receptors have been shown to function as NAD+ cleavage enzymes, whose activity is indispensable for the TNL protein-dependent cell death response (Horsefield et al., 2019Horsefield S. Burdett H. Zhang X. Manik M.K. Shi Y. Chen J. Qi T. Gilley J. Lai J.S. Rank M.X. et al.NAD(+) cleavage activity by animal and plant TIR domains in cell death pathways.Science. 2019; 365: 793-799https://doi.org/10.1126/science.aax1911Crossref PubMed Scopus (170) Google Scholar; Wan et al., 2019Wan L. Essuman K. Anderson R.G. Sasaki Y. Monteiro F. Chung E.H. Osborne Nishimura E. DiAntonio A. Milbrandt J. Dangl J.L. et al.TIR domains of plant immune receptors are NAD(+)-cleaving enzymes that promote cell death.Science. 2019; 365: 799-803https://doi.org/10.1126/science.aax1771Crossref PubMed Scopus (151) Google Scholar). It may be worth of exploring the possible connection between TIR domain’s NAD+ cleavage activity and helper NLR’s calcium channel formation.