www.nature.com/cdd REVIEW ARTICLE OPEN Dying in self-defence: a comparative overview of immunogenic cell death signalling in animals and plants 1,2 ✉ 3,4,5 ✉ 6,7 ✉ Takaki Maekawa , Hamid Kashkar and Núria S. Coll © The Author(s) 2022 Host organisms utilise a range of genetically encoded cell death programmes in response to pathogen challenge. Host cell death can restrict pathogen proliferation by depleting their replicative niche and at the same time dying cells can alert neighbouring cells to prepare environmental conditions favouring future pathogen attacks. As expected, many pathogenic microbes have strategies to subvert host cell death to promote their virulence. The structural and lifestyle differences between animals and plants have been anticipated to shape very different host defence mechanisms. However, an emerging body of evidence indicates that several components of the host–pathogen interaction machinery are shared between the two major branches of eukaryotic life. Many proteins involved in cell death execution or cell death-associated immunity in plants and animals exert direct effects on endomembrane and loss of membrane integrity has been proposed to explain the potential immunogenicity of dying cells. In this review we aim to provide a comparative view on how cell death processes are linked to anti-microbial defence mechanisms in 1234567890();,: plants and animals and how pathogens interfere with these cell death programmes. In comparison to the several well-defined cell death programmes in animals, immunogenic cell death in plant defence is broadly defined as the hypersensitive response. Our comparative overview may help discerning whether specific types of immunogenic cell death exist in plants, and correspondingly, it may provide new hints for previously undiscovered cell death mechanism in animals. Cell Death & Differentiation (2023) 30:258–268; https://doi.org/10.1038/s41418-022-01060-6 FACTS ● How does limited/controlled membrane disruption control cell death, neighbouring responses and pathogen progression in animals and plants? ● Cell death is a fundamental immune defence mechanism in ● Are there common molecular structures that are released from animals and plants. animal and plant cells and how do they impact immunity and ● Several components of cell death machinery are shared pathogen progression? between animals and plants. ● Does plant hypersensitive cell death resemble the distinct ● Alteration of endomembrane integrity represents a crucial step features of regulated necroptosis and/or pyroptosis in animals? in immunogenic cell death signalling in animals and plants. ● How does the increase of cation concentration in the cytosol ● Pathogenic microbes have evolved strategies to subvert host caused by the action of plant resistosomes translate into cell death in animals and plants to promote their virulence. immunogenic cell death in plants? OPEN QUESTIONS INTRODUCTION Cell death represents a common and fundamental process in ● Caspases are increasingly anticipated as a molecular switch in cell host-microbe interactions in both plants and animals [1, 2]. Not death and cellular signalling. What is the evolutionary conserved surprisingly, plant and animal pathogens have evolved various function of cell death proteases in animals and plants? means to specifically avoid or subvert host cell death as part ● Lytic cell death and membrane disintegration are viewed as an of their virulence strategies. Such co-evolutionary struggles initiator of neighbouring responses and systemic immunity in between hosts and pathogens have resulted in some of the animals. How does cell death in plants involve neighbouring cells most complex and interesting biological interactions [3]. The and provoke systemic immunity? structural and lifestyle differences between plants and animals 1 Department of Biology, Institute for Plant Sciences, University of Cologne, 50674 Cologne, Germany. 2CEPLAS Cluster of Excellence on Plant Sciences at the University of Cologne, Cologne, Germany. 3Faculty of Medicine and University Hospital of Cologne, Institute for Molecular Immunology, University of Cologne, 50931 Cologne, Germany. 4 Faculty of Medicine and University Hospital of Cologne, Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany. 5Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany. 6Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, 08193 Bellaterra, Spain. 7Consejo Superior de Investigaciones Científicas (CSIC), 08001 Barcelona, Spain. ✉email: tmaekawa@uni-koeln.de; h.kashkar@uni-koeln.de; nuria.sanchez-coll@cragenomica.es Edited by F Pentimalli Received: 14 May 2022 Revised: 29 August 2022 Accepted: 6 September 2022 Published online: 4 October 2022 Official journal of CDDpress T. Maekawa et al. 259 Fig. 1 Schematic comparison of molecular cell death features of animal and plant cell death types. Schematic display of cell death modalities of animals (apoptosis, necroptosis, pyroptosis and ferroptosis, in blue) and plants (hypersensitive response, green), highlighting the main regulators, formation of “deathosome” structures and impact on membrane integrity. Plant TNL resistosomes are not included, since the scheme mostly focuses on the impact of immune cell death on plasma membrane. and their respective pathogens have been expected to involve containing (NLR) receptor family [7–9]. The amplitude of HR can very different defence mechanisms and pathogenic strategies. be intensified upon recognition of conserved pathogen-associated However, accumulating evidence indicate that several compo- molecular patterns (PAMPs) by plasma membrane resident nents of host–pathogen interactions are shared between the receptors (PRRs) [10, 11]. Because NLR-triggered immunity also two phyla. intensifies PRR-triggered immunity [10, 11], this mutual potentia- The death of pathogen-infected cells can have beneficial or tion mechanism between NLR and PRR signalling acts as an detrimental consequences for the host. Host cell death can restrict amplification step in immune signalling and HR. Whether specific pathogen proliferation by destroying their replicative niche. types of immunogenic cell death exist in plants under the wide Alternatively, pathogens can induce host cell death to disseminate umbrella of HR remains to be clarified. and infect adjacent tissues. Beyond this categorical view of cell Despite the differences in nomenclature, both plant and animal death, the quality of cell death or how exactly cells die is emerging pathogens can be classified as killers or non-killers, as previously as a central determinant of the fate of the affected tissues. proposed [12]. This simplistic view is useful when comparing Alteration of membrane integrity represents one of the key steps host–pathogen interactions in animals and plants. Essentially, non- outlining a model that the immune system is more concerned killer pathogens feed on living tissue and need to maintain a living with entities that do damage rather than with those that are only host, whereas killer pathogens feed on dead or dying tissue. As a foreign [4]. The concept of damage-associated molecular patterns consequence, non-killer pathogens have evolved a variety of (DAMPs) has been originally proposed to explain the potential mechanisms to block host cell death, while in turn, killer immunogenicity of dying cells and immunogenic cell death pathogens can induce cell death in the host for their own benefit. describes cell death modalities that stimulate an immune Naturally, intermediate situations exist, whereby pathogens can response against dead cell-antigens [5, 6]. Accordingly, a number switch from non-killers to killers during their life cycle. of genetically controlled and molecularly defined cellular death In fact, suppression of cell death by certain pathogens as a processes that utilise distinct cellular death machinery and yield virulence strategy may have acted as an evolutionary driving force different tissue responses have been characterized in animal cells, for the host to evolve different cell death modalities [13, 14]. including apoptosis, necroptosis, pyroptosis and ferroptosis (Fig. 1 Furthermore, it is increasingly evident that the pathogenesis of and Table 1). Furthermore, accumulating evidence suggests that microbial infection does not only rely on the host cell death as a different cell death processes are deeply interlinked, serve as final event, but also depends on the mode of cell death and the backup mechanisms and thereby guarantee cellular death to quality of the intercellular communication within the infected mount immunity. tissue. These intercellular communications do not only activate In plants, immunogenic cell death has been broadly termed host defence mechanisms, but also boost tissue regeneration to hypersensitive response (HR). HR involves generation of reactive achieve full resolution of the infection. On the other hand, oxygen species (ROS) and elevation of intracellular Ca2+ levels successful pathogens are able to thwart this process and cause (Fig. 1) and is often caused by activation of members of overwhelming tissue damage that supports the propagation and the intracellular nucleotide-binding domain leucine-rich repeat spread of the pathogen. Cell Death & Differentiation (2023) 30:258 – 268 T. Maekawa et al. 260 Table 1. Comparison of the major cell death processes between animals (blue) and plants (green). “?” and “-” indicate “potentially, but not yet fully elucidated” and “not detected”, respectively. a Ferroptosis-like cell death has been described in plants (see text for more details), although molecular details remain limited. b The hypersensitive response (HR) can be considered “inflammatory”, as HR releases immunogenic molecules to the extracellular space. In the sections below we provide detailed descriptions of the particularly the case for viral pathogens, as they are obligate and death processes linked to microbial infection in animals and intracellular pathogens, therefore they have evolved multiple plants. strategies to block caspase activity, as shown with the cytokine response modifier A (CrmA) from cowpox virus [19–21], p35 Regulation of host–pathogen interaction by apoptosis from baculoviruses [22, 23] or FLICE/caspase-8-inhibitory proteins Apoptosis is the best studied form of animal cell death and is (v-FLIPs) from gamma-herpesviruses and the tumorigenic human morphologically characterized by the ordered disassembly of the molluscipoxvirus [24]. Other anti-apoptotic viral gene products dying cell within the boundaries of an intact plasma membrane. have been shown to inhibit apoptosis without inhibiting Apoptosis is controlled by cysteine-dependent aspartate-specific caspase activity, such as inhibitor of apoptosis protein (IAP) from proteases termed caspases. With the exception of caspase-1— baculoviruses [25]. A number of different viral pathogens enriched in monocytes/macrophages, and caspase-14—restricted to including Adenoviridae, Birnaviridae, Herpesviridae and Poxviridae keratinocytes, caspases are widely expressed as cytosolic/nucleoplas- express viral orthologues of Bcl-2, a key regulator of MOMP, mic zymogens [15]. Once activated by an upstream stimulus, caspases efficiently controlling intrinsic apoptosis during the course of viral perform limited proteolysis resulting in inactivation or activation of infection [26]. distinct downstream cascade of signalling events permitting the In contrast to viruses, only a minority of bacterial pathogens controlled demolition of cells. Within the family, apoptotic caspases are propagate within the cytosol and have direct access to caspases. distinguished as initiator or executioner caspases. The initiator Instead, they usually reside and multiply outside the cell or within caspases translate upstream death signals into proteolytic action intracellular vacuoles and deliver their effector proteins into their upon association with multicomponent signalling complexes which hosts’ cytosol via secretion systems [27]. Regardless, inhibition of generate an active protease [16]. Executioner caspases can be apoptosis plays an integral role in bacterial pathogenesis at early activated by initiator caspases and subsequently convert the stages of infection, and bacteria utilise a number of different proteolytic action into an apoptotic phenotype by processing distinct strategies for the inhibition of host cell apoptosis [28]. These include downstream substrates. The main pathways for caspase cascade caspase inhibition, as it has been shown for Rickettsia rickettsii, activation are the extrinsic and intrinsic pathways. In the extrinsic Shigella flexneri and Crassostrea gigas [29–32] and upon lipopoly- pathway, the activation of cell surface death receptor leads to the saccharide (LPS) direct binding to the executioner caspase-3 [31, 32]. formation of a protein complex including the initiator caspase-8 [17]. In Furthermore, Escherichia coli inhibits caspase activation by N-linked the intrinsic/mitochondrial pathway the initiator caspase-9 is activated glycosylation of the components of the death receptor signalling after mitochondrial outer membrane permeabilization (MOMP) and complex [33]. Intrinsic/mitocondrial apoptosis is also controlled by the release of cytochrome c. Once in the cytosol, cytochrome c many bacterial pathogens including Chlamydia [34, 35], Mycobacter- induces the formation of a cytosolic protein complex––the apopto- ium tuberculosis [36], and Coxiella burnetii [37] by interfering with some—consisting of the apoptotic protease activating factor 1 (Apaf- the expression or degradation of different members of the Bcl-2 1) [18] and caspase-9. Both caspase-8 and −9 proteolytically activate protein family. executioner caspase-3 and -7 which ultimately drive the characteristic Although initially and extensively studied in the context of morphology of apoptosis including membrane blebbing, chromoso- apoptosis, caspases are increasingly considered as versatile mal DNA fragmentation, packaging of cell constituents into apoptotic molecular switch controlling different cellular outcomes. In bodies and eventually cell death. particular, inhibition of caspase-8 not only fails to block cell Pathogens, particularly when growing inside a host cell, often death, but also causes inflammatory lytic cell death by involving activate a plethora of cellular stress responses that are normally necroptosis or pyroptosis (see below) [13, 14]. Lack of caspase-9 sensed by apoptotic machinery and translated into proteolytic activity indeed uncouples the apoptotic death from mitochondrial action involving caspases. Inhibition of apoptosis allows patho- damage, but it engages various inflammatory signalling and leads gens to optimize replication and progeny synthesis by prolonging to lytic cell death [38]. Accordingly, inhibition of caspase activity the infected cell life. Accordingly, caspases represent an important by pathogens may not solely increase the lifespan of the host cell, molecular target for pathogens to control apoptosis. This is it rather changes the nature of death and may cause beneficial or Cell Death & Differentiation (2023) 30:258 – 268 T. Maekawa et al. 261 Fig. 2 An amphipathic feature of cell death-inducing proteins compromises cell membrane integrity. A The pentameric ZAR1 resistosome of Arabidopsis thaliana that functions as a Ca2+ permeable channel. The solvent exposed funnel-shaped structure constituted by the N-terminal coiled-coil domain and the first helix (α1) of a ZAR1 protomer are highlighted in green and red, respectively. B Surface hydrophobicity of the pentameric structure of N-terminal coiled-coil domain of ZAR1. A, B The presumptive plasma membrane (PM)-inserted region of the ZAR1 resistosome is indicated in each right panel. b)The structures of the first N-terminal helices of selected plant cell death- inducing proteins. C The structures of the helices for ZAR1 (A. thaliana), MLA10 (barley), Sr33 (wheat), and AtMLKL2 (A. thaliana) are retrieved from the Protein Data Bank. D The hypothetical structure of the N-terminal part of NRC4 (Tomato) and the solved structure of PSM-alpha3 (Staphylococcus aureus). These two short peptides are able to cause lytic cell death or liposome rupture. C, D Wheel projections of the α‐helices were built using the server Heliquest (https://heliquest.ipmc.cnrs.fr/). The gray arrows point toward the hydrophobic face of the α‐helices. The hypothetical structure for NRC4 was generated by using the server RaptorX (http://raptorx.uchicago.edu/). B–D Surface hydrophobicity of helices are visualised by PyMOL using the script “Color h” https://pymolwiki.org/index.php/Color_h. E A hypothetical model for the amphipathic helical domain-induced membrane rupture. In this model, hydrophilic surfaces of helical domains are buried in the acyl chains of the phospholipid bilayer that compromises integrity of cell membrane. The cartoon representation is adapted from [119]. A partial membrane rupture could cause ion influxes from extracellular spaces, which might activate cell death program as seen during ferroptosis [108]. detrimental tissue responses which deserves further consideration death that involves activation of proteolytic enzymes [39]. As we during the course of host–pathogen interaction. discuss in this review, several exciting similarities between HR and In plants, there is no apoptosis. As outlined above, plant different forms of regulated lytic cell death (pyroptosis, necrop- pathogens can trigger HR, a plant-specific type of regulated cell tosis) are emerging, especially at the molecular level. However, Cell Death & Differentiation (2023) 30:258 – 268 T. Maekawa et al. 262 considering HR as a form of apoptotic-like cell death is a very may be important for the understanding of the evolution of limiting misnomer often used in the literature that leads to regulated necrosis in immunity. Currently, no GSDM-like proteins confusion and should be avoided. have been identified in plants. Further structural and functional Proteases are probably one of the most ancient families of analyses will be instrumental for identifying GSDM-like proteins in enzymes [40]. However, plants do not encode caspases in their plants, paving the way for a better understanding of cell death in genomes [41]. Although caspase-like activities have been shown these organisms. to be important for HR, these are carried out by proteases with little structural resemblance to caspases [39]. In turn, metacas- Resistosome-initiated cell death in plants. In the last few years, a pases, which belong to the same superfamily as caspases and major breakthrough in plant immunity has been the identification share certain structural similarities with them, do not cleave after of supramolecular complexes assembled upon NLR activation with a aspartate residues (positively charged), but rather lysine or key role in defence, which have been dubbed “resistosomes” [65]. In arginine (negatively charged) and therefore they cannot be plants, NLRs are broadly classified according to their N-terminal considered caspases [42]. Metacaspases have been shown to be domain as coiled-coil (CC) domain NLRs (CNLs) or TOLL/interleukin 1 important for HR regulation [43], although their mode of action receptor (TIR)-like domain NLRs (TNLs). Additionally, a subclass of in this process remains unclear. Interestingly, available crystal CNLs has been identified as RPW8-like CC domain containing NLRs structures suggest that functional metacaspases act as mono- (RNLs) [66–68]. The RPW8-like CC domain is alternatively called the mers [44, 45]. HeLo-domain due to a characteristic N-terminal four-helix bundle structure (also see below) [69, 70]. As TNL-mediated HR and “Deathosomes”, macromolecular apparatuses causing cell immunity require RNLs, RNLs are also known as “helper-NLR” [68]. death Structural and biochemical data on resistosomes have helped Inflammasome-initiated pyroptosis in animal cells. Inflammasomes understanding the differences of CNL- and TNL-RNL pathways and are multiprotein complexes that are formed upon activation of PRRs their mechanistic similarities to animal immune deathosomes. following the detection of PAMPs or DAMPs in the cytosol of host Structurally, CNL resistosomes resemble the wheel-like inflamma- cells [46]. Inflammasomes serve as platforms for the activation of some comprising animal NLRs [71, 72]. caspase-1 which promotes the proteolytic maturation of the In the context of the two-tiered plant immune system [73], it has cytokines interleukin-1β (IL-1β) and IL-18. Caspase-1 additionally been proposed that initial PAMP recognition by surface receptors cleaves gasdermin D (GSDMD) unleashing its pore-forming domain triggers a fast and transient elevation of cytosolic Ca2+ concentra- and inducing a lytic type of cell death that is known as pyroptosis. tion (i.e, Ca2+ influx), whereas NLR activation leads to a long-lasting Although initially described as caspase-1–dependent cell death, Ca2+ influx that would result in cell death, overcoming potential pyroptosis can be additionally induced by caspase-4 (or its pathogen-mediated immune suppression [74]. The spatiotemporal orthologues caspase-11 in mouse) and caspase-5 each of which pattern of Ca2+ spikes upon NLR activation at a single cell level cleaves GSDMD and induces membrane pores [47, 48] (Fig. 1). as well as at tissue-level might be distinctive to activate cell death- Activation of caspase-1 requires the formation of an inflammasome executing proteins. In addition, Ca2+ could directly act as an which is initiated upon pattern recognition by NLRs (Nlrp3, Nlrp1b activator of many proteolytic enzymes, which have been shown and Nlrc4), the cytosolic DNA sensor AIM2 (absent in melanoma 2) to play a role in HR [39, 75]. It has been recently shown and Pyrin, which subsequently recruits caspase-1 zymogen directly that GSDM pores induce calcium influx, which modulates pore or through the adaptor protein ASC (apoptosis-associated speck- opening/closing kinetics through a phospholipid-mediated feed- like protein containing a CARD) [49]. Caspase-4, −5 and −11 are back mechanism [76]. activated by the detection of cytosolic LPS and do not require the (a) CNL resistosome-initiated cell death formation of canonical inflammasomes [50]. Unlike caspase-1, −4, The first resistosome described in plants was a pentameric −5 and −11 are not able to process IL-1β and IL-18. structure containing the activated form of the CNL ZAR1 (HOPZ- Pathogens manipulate various aspects of pyroptosis to escape ACTIVATED RESISTANCE1) in complex with the pseudokinase PBL2 the immediate and efficient killing of the host cells. Yersinia outer [72, 77]. Recognition of the cognate bacterial effector AvrAC causes protein K (YopK) and M (YopM) block the activation of the a post-translational modification of PBL2 that results in subsequent inflammasome [51, 52]. Pseudomonas aeruginosa effectors activation and oligomerization of ZAR1 into a pentameric wheel C120HSL, ExoU and ExoS inhibit NLRC4, NLRP3 and caspase-1 structure. In the activated ZAR1 resistosome, the first N-terminal [53, 54]. Bacterial pathogens also efficiently manipulate caspase-4, alpha-helixes of the CC domains form a solvent exposed funnel- −5 and −11-mediated pyroptosis. Francisella and Shigella reshape shaped structure, which constitutes a cation channel (Fig. 2A, B). their LPS and escape efficient cytosolic detection by caspase-4, −5 This structural configuration is required for plasma membrane and −11 [55, 56]. Shigella OspC3 effector can efficiently inhibit association and Ca2+ influx [72, 77]. The CNL Sr35 also assembles caspase-4 and −11 by direct binding or post-translational into a resistosome upon direct binding of the fungal effector modification [57, 58]. Caspase-1 can also be inhibited by viruses AvrSr35, forming a structure that is highly similar to the ZAR1 such as poxvirus serpins [19]. Poxviruses also express pyrin-only resistosome [71]. Similar to the CNL resistosomes of ZAR1 and Sr35, proteins that inhibit inflammasome activation by direct binding ASC members of the RNL subclass also exhibit a calcium-permeable and NLRs [59]. Papillomaviruses inhibit pyroptosis by inducing unselective cation channel activity [71, 78, 79], likely contributing to proteosomal degradation of the inflammasome [60]. Kaposi’s the aforementioned long-lasting Ca2+ influx. The elevation of sarcoma-associated herpesviruses express the NLRP1 homolog cytosolic Ca2+ is essential for HR execution, since chemically Orf63, which subverts the function of NLRs [61]. Measles virus and blocking calcium channels prevents HR cell death [80]. It has been influenza virus inhibit the NLRP3 inflammasome [62, 63]. Enterovirus shown that cations other than Ca2+ can be transported through 71 inhibits pyroptosis by cleaving and inactivating GSDMD [64]. resistosome channels [79], indicating that other cations could be GSDM-like proteins have been identified in taxa outside of important for HR. animals such as fungi and bacteria [62]. Fungal and bacterial GSDM- (b) TNL resistosome-initiated cell death like proteins share many features with mammalian GSDMs: they are Some TNLs assemble into a tetrameric resistosome upon ligand activated by proteases and they have the ability to form pores and binding, in which two pairs of TIR homodimers function as a permeabilize plasma membranes mediating cell death upon nicotinamide adenine dinucleotide (NAD+) cleaving enzyme allorecognition and upon phage infection, respectively [63, 64]. (NADase) [81, 82]. The plant genome encodes a number of TIR- These observations establish GSDMs as an extremely ancient only proteins, which lack some of the typical domains present in mechanism of cell death and membrane pore formation, which TNL proteins (e.g., NB-ARC, LRR domains) [67, 83]. These TIR-only Cell Death & Differentiation (2023) 30:258 – 268 T. Maekawa et al. 263 proteins and artificially truncated TIR domains of TNLs can be domains in RNLs (i.e., ADR1 and NRG1 family) are structurally assembled into filament-like structures that produce 2′,3′-cAMP/ similar to the HeLo domain [69, 78] and the N-terminal domains cGMP by hydrolysing DNA or RNA [84]. As HR caused by TNLs and alone of the ADR1 and NRG1 are sufficient to cause HR cell TIR-only proteins genetically requires RNLs [85, 68], some of death [104]. products of the TIR enzymes are anticipated to act as signalling A conserved protein family across seed plants that structurally molecule(s) to facilitate the formation of RNL-resistosomes. Recently resembles animal MLKL was recently discovered [70]. Experiments identified TIR products, 2’-(5”-phosphoribosyl)-5’-adenosine mono-/ using combinatorial mutants of the three Arabidopsis MLKLs di-phosphate (pRib-AMP/ADP) [86], and ADP-ribosylated ADPR (di- (AtMLKLs) indicate that they act redundantly in conferring disease ADPR) or (ADP-ribosylated ATP or ADPr-ATP) [87] are proposed to resistance mediated by TNLs but not CNLs [70]. Considering that be perceived by EDS1-PAD4 and EDS1-SAG101 receptor complexes, the HeLo domain-containing RNLs are also required for TNL- respectively, for the formation of RNL-resistosomes [86, 87]. Plant mediated HR and immunity [68], it may be surmised that two pathogens have evolved effectors to manipulate both precursors distinctive HeLo domain-containing protein families, namely plant and products of TIR-enzymes. For example, an effector of MLKL and RNL families, cooperatively participate in TNL-mediated Xanthomonas phosphorylates NAD+ [88] and effectors from immunity. Furthermore, the mobility of AtMLKLs on microtubules oomycete and bacterial pathogens hydrolyse 2′,3′-cAMP/cGMP is linked to their immune output [70]. This is interesting, but not its regioisomer 3′,5′-cAMP/cGMP [84, 89] emphasising the considering the fact that microtubules are attributed to the importance of the TIR-catalysed products in plant disease resistance. translocation of animal MLKLs from cytoplasmic necrosomes to the plasma membrane [105].Taken together, an analogous MLKL-mediated necroptosis biochemical mode of action for plant and animal MLKL- In animals, necroptosis has been defined as a form of regulated mediated cell death and immunity is anticipated. necrotic cell death induced by receptor interacting protein kinase 3 (RIPK3) and its substrate mixed lineage kinase-like (MLKL) [90]. Iron-dependent lytic cell death RIPK3 is activated by upstream pathways via RIP homotypic Ferroptosis is an iron-dependent, oxidative form of necrotic cell interaction motif (RHIM)-dependent protein-protein interactions death that does not share genetic similarities with apoptosis, with the three additional proteins in the mammalian genome that pyroptosis or necroptosis. In animals, ferroptosis can be triggered by contain conserved RHIMs, namely RIPK1, TRIF and ZBP1/DAI depleting the cell of the amino acid cysteine, or by inhibiting [90, 91]. RIPK1 links RIPK3 to death receptor (DR) signalling: TRIF phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4) mediates RIPK3 activation downstream of TLR3 and TLR4, whilst [106]. In contrast to apoptosis, pyroptosis and necroptosis, ZBP1/DAI mediates RIPK3 activation in response to certain viruses ferroptotic cell death does not appear to involve molecularly [91] and endogenous Z-form nucleic acid [92]. Importantly, defined cascades of events, but represents a distinct cellular death caspase-8 inhibits RIPK3 activation and the induction of necrop- outcome involving iron-dependent peroxidation of lipids associated tosis by mechanisms that likely involve cleavage of RIPK1/RIPK3 with plasma membrane damage [107]. During ferroptosis, a partial complex components. Necroptosis execution requires the RIPK3- membrane rupture could cause ion influxes from extracellular dependent phosphorylation of MLKL. Phosphorylated MLKL spaces, which might activate further downstream cell death induces plasma membrane damage via incompletely character- programme [108]. ized mechanisms [91, 93]. Lipid peroxidation has been long known to be involved in the Pathogens utilize a plethora of mechanisms to inhibit necrop- pathogenesis of diverse infectious diseases, although knowledge tosis. Several bacterial peptidases expressed by EPEC and Shigella about the role of ferroptosis in previous data dealing with lipid degrade components of necroptotic machinery [94, 95]. However, peroxidation during the course of infection is lacking. In particular, interference of necroptosis seems to be a common pathogenic ROS are frequently formed after viral infections and an imbalance in strategy of viral pathogens. Viral inhibitor of RIP activation (vIRA) cellular redox responses has been viewed as one the of the drivers of proteins of MCMV (M45) can interact with TRIF, ZBP1, RIPK1, and virus-induced inflammatory destruction of a tissue [109]. Therefore, RIPK3 via RHIM homotypic interaction to impair necroptosis [96]. one can speculate that oxidative stress, lipid peroxidation and Unlike MCMV, human CMV (HCMV) ortholog of M45, UL45, does ferroptosis can participate in various pathological states of viral not contain a RHIM domain but efficiently inhibits necroptosis by infection. In line with this notion, T cell lipid peroxidation and acting downstream of RIPK3 and MLKL [97]. Cowpox virus ferroptosis were shown to prevent immunity against Choriomenin- expresses viral inducer of RIPK3 degradation (vIRD) triggering gitis virus [110]. Similar to viral infection, a few bacterial pathogens K48-linked ubiquitylation of RIPK3 and its proteasomal degrada- have been associated with ferroptosis of the host cell. M. tuberculosis tion [98]. Other poxviruses express viral MLKL-like proteins has been characterized as a trigger of pathological ferroptosis in (vMLKL) and inhibit necroptosis by sequestering RIPK3 [99]. macrophages [111]. P. aeruginosa utilizes host polyunsaturated In view of the fact that caspase-8 is the central inhibitor of phosphatidylethanolamines to trigger theft-ferroptosis in bronchial necroptosis, necroptosis has been frequently considered to have epithelium [112]. Whether and how ferroptosis interferes and evolved as a “back-up” cell death mechanism when death receptor impacts on the course of host–pathogen interaction in animals will activation fails to kill an infected cell via apoptosis. Intriguingly, constitute future experimental challenges. poxovirus crmA is able to preferentially inhibit caspase-8-mediated In plants, a ferroptosis-like process has been shown to take apoptosis without markedly altering caspase-8-mediated inhibition of place in rice upon NLR-mediated recognition of the fungal necroptosis [100]. Herpes simplex virus (HSV)-1 and HSV-2 inhibit pathogen Magnaporthe oryzae [113]. In this study, ferric ions apoptosis via direct binding of caspase-8 by the large subunit (R1) of (Fe3+) and ROS were shown to focally accumulate in cells ribonucleotide reductase (RR). HSV-1 and HSV-2 R1 proteins (ICP6 and undergoing HR cell death and neighbouring areas. Importantly, ICP10, respectively) also prevent necroptosis by inhibiting the the ferroptosis inhibitors deferoxamine and ferrostatin-1 attenu- interaction between RIPK1 and RIPK3 [101]. ated HR levels. The degree of conservation of ferroptosis between MLKLs contain a characteristic N-terminal four-helix bundle plants and animals and how extended is this type of cell death structure called the HeLo domain. This domain, named after within the green lineages remain to be determined. fungal Het and LopB proteins, is commonly present in a number of cell death-inducing proteins in animals, fungi and plants Loss of plasma membrane integrity in plant and animal cell [68–70, 102, 103]. In fungi, Het-S or Het-s (Heterokaryon death incompatibility protein S/s) trigger hyphal death in heterokaryon It is becoming increasingly clear that a transient or permanent loss incompatibility [69]. In plants, the N-terminal RPW8-like CC of endomembrane integrity is a key step of many forms of cell Cell Death & Differentiation (2023) 30:258 – 268 T. Maekawa et al. 264 Fig. 3 Communication between cells during immunogenic cell death. A In animals, activation of host defence systems can initiate a range of genetically encoded cell death, including gasdermin-induced pyroptosis and MLKL-induced necroptosis. Some pathogenic microbes secrete toxins to manipulate host immune systems. Ion- and water- fluxes are commonly seen during cell death progression. The water entry into cells results in cell swelling and blebbing and cell rupture is finally executed by Ninjurin-1 (NINJ1) [114]. Cell rupture facilitates the release of damage-associated molecular patterns (DAMPs), which stimulate inflammatory responses of neighbouring cells. B Like in animals, plant cell death plays fundamental roles in immunity. The hypersensitive response (HR) is a rapid localized cell death at pathogen invading sites mostly mediated by intracellular NLR immune receptors. Unlike animal cell death, rigid cell walls prevent cell swelling and blebbing. Cell rupture facilitates the release of damage-associated molecular patterns (DAMPs), which stimulate immune responses of neighbouring cells. death both in animals (pyroptosis, necroptosis, ferroptosis, proteins enchases or are required for their cell lytic activities apoptosis) and plants (HR). In the case of animal cell death, [114, 117, 118]. Partial membrane damage leading to formation of a plasma membrane rupture and catastrophic cell lysis are transient pore can cause ion fluxes, which might activate a cell facilitated by the transmembrane protein Ninjurin-1 (NINJ1), upon death programme as described in the case of ferroptosis [108] oligomerization of its amphiphatic extracellular α-helix [114]. An (Fig. 2E). This model suggests that cell death can be initiated by a amphiphatic α-helix carries both but segregated hydrophilic and range of proteins carrying an amphipathic helical domain, although hydrophobic surfaces, a common feature of cation channel- they may not form well-organised pores or channels and this model forming pores or pore-forming proteins/toxins [72, 79, 93] (Fig. 2). might apply to the MLKL-induced plasma membrane permeabiliza- In plants, an amphiphatic helical domain is located at the tion as previously proposed [93]. N-termini of many CNLs -including ZAR1, MLA10, Sr33 [72, 115, 116] and they share the “MADA” motif named after the consensus Signalling molecules released from dying cells MADAxVSFxVxKLxxLLxxEx [117] (Fig. 2). Although this motif could Conceptually, lytic cell death including pyroptosis, necroptosis and be attributed to their cation channel functions associated with ferroptosis in animals has been originally considered to cause an resistosomes, overexpression of the N-terminal part of NRC uncontrolled release of cellular contents and exposure of DAMPs, containing the “MADA” motif is sufficient to cause HR-like cell since the cell boundary is lost due to membrane rupture (Fig. 3A). It death [117]. This is reminiscent of lytic cell death caused by is however increasingly evident that different modes of regulated amphiphatic PSM-alpha3 (Staphylococcus aureus) [118]. Such an necrosis provoke the release of a distinct battery of DAMPs. amphipathic feature is also present in the HeLo-helices of mouse, Accordingly, HMGB1 released by pyroptosis is hyperacetylated, human, and plant MLKLs [70, 93] (Fig. 2C), although how MLKLs which is not the case when it is released from necrotic or apoptotic induce plasma membrane permeabilization is not well established cells [120]. Furthermore, in contrast to initial views, apoptotic cells [70, 91, 93]. can also release different factors such as nuclear DNA (below How does the amphipathic helical domain cause membrane 180 bp), ATP (early phase) and HMGB1 (late phase) [121]. disruption? Hydrophilic surfaces of the amphipathic helical domain The composition and signatures of apoptotic and necroptotic could be buried inside the acyl chains of the phospholipid bilayer of DAMPs might be qualitatively and quantitatively different. In the plasma membrane, which would partially compromise integrity apoptosis, caspases process different factors such as phospholi- of cell membrane like a surfactant [119] (Fig. 2E). Experimental pase A2 (PLA2) and inhibitor of caspase-activated DNase (called evidence suggests that oligomerization of amphipathic helical ICAD) to induce phagocytosis and to produce small and weak Cell Death & Differentiation (2023) 30:258 – 268 T. Maekawa et al. 265 immunostimulatory DNA fragments. In addition, full-length IL-33 is elevated cytoplasmic Ca2+ concentration was shown to cause released during necroptosis, but during apoptosis it can undergo irreversible formation of ceramide platforms within the plasma caspase-dependent proteolysis into a non-immunogenic form membrane, which is proposed to induce a proximity of FAS (CD95) [122]. DAMP release has been significantly less studied in the receptors to fully activate caspase-8 [136]. The generation of context of necroptosis. However, as membrane integrity is lost in ceramide-enriched membrane is irreversible, thereby defining the necroptosis in a fashion similar to necrosis, theoretically, ‘Point-of-no-return” of dying cells [137]. Mutations in the gene necroptosis also results in the release of DAMPs and other cellular encoding NLRP3 cause a spectrum of autoinflammatory diseases components including, but not limited to, HMGB1, ATP, histones, known as cryopyrin-associated periodic syndromes (CAPS). HSPs, exRNAs, and cfDNA. Pyroptosis is typically known for the Increased extracellular Ca2+ concentrations trigger activation of release of IL-1ß and IL-18, though some other DAMPs such as the NLRP3 inflammasome in monocytes through Ca2+-sensing HMGB1, ATP and cfDNA can be released by cells undergoing receptor (CaSR) which causes inflammation in human cryopyrin- pyroptosis. Although it is a relatively new concept and less has associated periodic syndrome (CAPS) [138, 139]. been elucidated yet, HMGB1 and cfDNA have been regarded to be In plants, the prolonged and elevated cytoplasmic Ca2+ released by ferroptosis [123]. concentration is tightly associated with HR [140]. Whether plants Plant cells also release a large range of DAMPs or phytocy- have genetically encoded-machinery to sabotage membrane repair tokines into the surrounding environment upon pathogen machinery or to detect a sustained Ca2+ influx for final cell death perception, to alert neighbouring cells and activate immunity, executioner activation during HR remains to be determined. including peptides, nucleotides, sugars and amino acids [124–126] (Fig. 3B). However, it remains to be established how Concluding remarks dying and dead cells contribute to the release of signalling Traditional studies focusing on the role of cell death and its molecules during plant-pathogen interactions. A hint for such a regulation in plants and animals mainly considered caspase- mechanism could be inferred from plant responses to wound- induced apoptosis as the only regulated cell death process and ing, oxidative, and biotic stresses [127, 128]. Upon mechanical viewed death as the ultimate end point. Those initial studies failed damage, the Ca2+-activated metacaspase AtMC4 cleaves a 23- to identify shared molecular features of cell death among the two residue peptide called PEP1 from its precursor PROPEP1. As kingdoms. In contrast to apoptosis, plant cell death is usually PROPEP1 and PEP1 do not contain canonical signal sequences accompanied by membrane disintegration and the characteristic for extracellular secretion [127], plasma membrane damage features of apoptosis are not induced in plants, including caspase during cell death can promote the release of the peptide activation. The recent discovery of molecularly controlled path- (Fig. 3B). The released PEP1 is recognized by its cognate plasma ways of lytic cell death in animals has redefined necrosis as a membrane localised receptor [129]. Other extracellularly regulated cell death process. Accumulating evidence in the past released DAMPs upon membrane damage may include extra- decades identified regulated necrosis as a powerful trigger of cellular ATP [130] and oligogalacturonides, a constituent of the inflammation, indicating that the mediators of cell death plant cell wall [131]. Collectively, these examples provide a additionally control inflammatory signalling. In particular caspases, mechanistic clue for how DAMPs that lack the signal sequences originally described as engines of apoptosis can, however, for secretion could be released into extracellular spaces upon independently control lytic cell death and membrane rupture. membrane disintegration. However, this field of research is still Accordingly, the lack of caspase-8 causes lethality in mice because in its infancy and these molecules could as well be released by of its failure to inhibit necroptosis, but not because of its failure to alternative secretion mechanisms that are so far unknown. induce apoptosis. Membrane pore formation has indeed emerged as a major Calcium entry as a danger signal driving membrane repair to shared characteristic between plant and animal immunogenic cell counteract cell death machinery death. This has become particularly intriguing with i) the discovery While a long-lasting Ca2+ influx would facilitate HR cell death, of plant resistosomes comprising activated NLRs that can directly Ca2+ entry or leakage (Fig. 2) caused by plasma membrane form pores in the membrane that allow Ca2+ influx and ii) the damage or pore forming proteins can activate membrane repair characterization of functional/structural homologs of animal machinery [132, 133]. Therefore, the point where membrane MLKLs in plants [70], revealing necroptosis as a relevant lytic cell damage exceeds the capacity for membrane repair would define death option in plants. Besides, the discovery of GSDM-like pore- “Point-of-no-return” of dying cells. In animals, the repair machin- forming proteins outside of mammals and vertebrates indicates ery removing lesions from the plasma membrane includes that the GSDM family is of extremely old evolutionary origin and endocytosis, ESCRT-complex mediated shedding, exocytosis- likely a core-component of immunity in divergent eukaryotic and mediated patching and annexin-mediated sealing [132, 133]. prokaryotic taxa [141]. It is important to note that some recently Although these processes can seal off lesions of Ca2+ entry, failure described GSDM-like proteins in fungi show remarkable structural to prevent Ca2+ entry can result in a prolonged Ca2+ influx. similarity to animal proteins [142]. 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