Organelle-centered resistosome signaling in plant immunity

The hidden contribution of intracellular calcium stores

Calcium (Ca²⁺) signaling is one of the earliest and most conserved hallmarks of plant immune activation (Wang et al., 2024). During pattern-triggered immunity, pathogen perception induces rapid and transient increases in cytosolic Ca²⁺, whereas effector-triggered immunity (ETI) is typically associated with stronger and more sustained Ca²⁺ signatures that are tightly linked to hypersensitive cell death (Zhang et al., 2024). The recent discovery that activated nucleotide-binding leucine-rich repeat (NLR) resistosomes function as Ca²⁺-permeable channels at the plasma membrane (PM) has established a prevailing model in which extracellular Ca²⁺ influx serves as a central driver of immune activation (Wang et al., 2019; Bi et al., 2021).

However, this PM-centered framework leaves an important question unresolved: how are the unusually sustained and spatially complex Ca²⁺ signatures associated with ETI generated and maintained? Plant cells contain extensive intracellular Ca²⁺ stores, including the vacuole, the endoplasmic reticulum (ER), chloroplasts, and mitochondria, all of which maintain Ca²⁺ concentrations substantially higher than the Ca²⁺ concentration in the cytosol (Wang et al., 2024). Previous studies have suggested that these organelles actively shape immune-associated Ca²⁺ dynamics and downstream defense outputs (Wang et al., 2024). Yet, despite long-standing speculation about the contribution of intracellular Ca²⁺ stores to plant immunity, direct mechanistic links between organellar Ca²⁺ mobilization and NLR activation have remained largely elusive.

A recent study now provides compelling evidence that helper NLR resistosomes can directly mobilize intracellular Ca²⁺ pools (Ibrahim et al., 2026). Upon recognition of the Xanthomonas effector XopQ by the Nicotiana benthamiana Toll/interleukin-1 receptor (TIR)-type NLR (TIR-NLR) RECOGNITION OF XOPQ 1 (ROQ1), the N. benthamiana helper NLR N REQUIREMENT GENE 1 (NbNRG1) relocalizes to punctate structures associated with intracellular membranes, including the chloroplast envelope, in an ENHANCED DISEASE SUSCEPTIBILITY 1–SENESCENCE-ASSOCIATED GENE 101-dependent manner and mediates chloroplast Ca²⁺ efflux into the cytosol (Ibrahim et al., 2026). These findings establish a direct mechanistic link between organellar Ca²⁺ stores and resistosome signaling during ETI. More broadly, this work challenges the traditional view of resistosomes as immune pores that function exclusively at the PM, supporting instead an emerging model in which compartment-specialized resistosomes mobilize Ca²⁺ from distinct intracellular stores to coordinate immune signaling (Figure 1).

Figure 1.

Compartmentalized Ca²⁺ signaling mediated by resistosomes during ETI.

Recognition of pathogen effectors by sensor NLRs activates distinct helper NLR signaling modules that mobilize Ca²⁺ from extracellular and intracellular sources, thereby driving a sustained elevation in cytosolic Ca²⁺ and HR-associated cell death. Canonical CC-NLR helper resistosomes, exemplified by NRC4, assemble at the PM and function as Ca²⁺-permeable channels that mediate Ca²⁺ influx from the extracellular space. In parallel, activated TIR-NLRs engage the EDS1–SAG101–NRG1 and EDS1–PAD4–ADR1 pathways, culminating in the formation of CC_R-NLR helper resistosomes. Activated NRG1 accumulates at the chloroplast envelope, where it mediates Ca²⁺ efflux from chloroplast stores into the cytosol. NRG1 has also been observed at mitochondria and the ER, although whether it mobilizes Ca²⁺ from these organelles remains unknown. In addition, PM-localized CNGCs contribute to immune-associated Ca²⁺ influx. Together, these findings support a model in which distinct helper resistosomes access spatially separated Ca²⁺ sources by localizing to specific membranes, thereby shaping compartmentalized ETI signaling and HR-associated cell death. ETI, effector-triggered immunity; NLR, nucleotide-binding leucine-rich repeat; TIR-NLR (TNL), Toll/interleukin-1 receptor (TIR)-type NLR; CC-NLR (CNL), coiled-coil (CC)-type NLR; CC_R-NLR (RNL), RPW8-like coiled-coil (CC_R)-type NLR; NRC4, NLR-REQUIRED FOR CELL DEATH 4; ADR1, ACTIVATED DISEASE RESISTANCE 1; NRG1, N REQUIREMENT GENE 1; EDS1, ENHANCED DISEASE SUSCEPTIBILITY 1; PAD4, PHYTOALEXIN DEFICIENT 4; SAG101, SENESCENCE-ASSOCIATED GENE 101; CNGCs, cyclic nucleotide-gated channels; PM, plasma membrane; ER, endoplasmic reticulum; HR, hypersensitive response. Solid lines indicate direct relationships; dashed lines indicate indirect relationships.

Structural diversification enables membrane specialization of helper NLRs

A central conceptual advance of this study is the demonstration that structural diversification of helper NLRs underlies their membrane specialization and compartment-specific immune functions (Figure 1; Ibrahim et al., 2026). In the prevailing sensor–helper model of ETI, upstream sensor NLRs perceive pathogen effectors, whereas downstream helper NLRs execute immune signaling through resistosome formation (He et al., 2025). Previous studies have shown that canonical coiled-coil (CC)-type NLR (CC-NLR) helpers such as NLR-REQUIRED FOR CELL DEATH (NRC) proteins and the RPW8-like coiled-coil (CC_R)-type NLR (CC_R-NLR) helper ACTIVATED DISEASE RESISTANCE 1 (ADR1) form PM-associated resistosomes that function as Ca²⁺-permeable channels. By contrast, NRG1 has now been shown to employ a distinct organelle-centered strategy (Jacob et al., 2021; Liu et al., 2024; Ibrahim et al., 2026).

Using AlphaFold 3-based structural modeling, the authors found that CC_R-NLR resistosomes contain an unusually extended N-terminal funnel architecture compared with that of canonical CC-NLRs such as NRC4 (Ibrahim et al., 2026). In particular, the elongated α4 helix of NbNRG1 was identified as a key determinant of organellar membrane targeting, especially its targeting of the chloroplast envelope. Whereas single-point mutants within this extended helix showed partial relocalization to the PM, the K126E/L129E double mutant localized predominantly to the PM and exhibited strongly reduced immune activity, highlighting the importance of correct membrane targeting for helper NLR function (Ibrahim et al., 2026).

Importantly, this membrane specialization is supported not only by structural modeling but also by functional relocalization experiments. Nanobody-mediated trapping of the canonical helper CC-NLR NRC4 at the chloroplast envelope abolished the hypersensitive response and failed to deplete stromal Ca²⁺, whereas chloroplast-trapped NRG1 retained the ability to trigger both the hypersensitive response and chloroplast Ca²⁺ efflux (Ibrahim et al., 2026). These findings demonstrate that resistosomes are functionally adapted to specific membrane environments rather than functioning as universally interchangeable channels across different membranes.

Remarkably, chloroplast targeting is conserved among NRG1-like CC_R-NLRs spanning approximately 360 million years of land plant evolution, including an NRG1-like CC_R-NLR from a monilophyte lineage that predates the divergence of the NRG1 and ADR1 subfamilies (Ibrahim et al., 2026). Although these localization analyses relied on heterologous expression in N. benthamiana, the conserved targeting pattern suggests that organelle-centered immune signaling is an ancient feature of helper NLR evolution rather than a species-specific phenomenon (Ibrahim et al., 2026).

These findings raise the intriguing possibility that helper NLRs have evolved structurally specialized membrane-targeting modules that enable immune signaling across distinct cellular compartments. Rather than functioning solely as generic PM-localized immune pores, different classes of resistosomes may localize to specific membrane systems to mobilize Ca²⁺ from distinct extracellular or intracellular stores. Such membrane specialization could provide an elegant mechanism for diversifying immune outputs and expanding the spatial organization of ETI signaling within plant cells (Figure 1).

Compartmentalized resistosome signaling expands immune execution strategies

The discovery of NbNRG1-mediated chloroplast Ca²⁺ signaling expands the current view of resistosomes from PM-associated immune pores to compartment-specialized signaling modules (Ibrahim et al., 2026). Previous studies established that canonical CC-NLR resistosomes, including the singleton receptor HOPZ-ACTIVATED RESISTANCE 1 and helper NRC proteins, assemble at the PM to mediate extracellular Ca²⁺ influx during immune activation (Bi et al., 2021; Liu et al., 2024). By contrast, activated NbNRG1 associates with the chloroplast envelope and promotes the release of Ca²⁺ from chloroplast stores into the cytosol (Ibrahim et al., 2026). Activated NbNRG1 also forms punctate structures associated with multiple intracellular membrane systems, including the chloroplast envelope, the ER, and mitochondria, although chloroplast-associated Ca²⁺ signaling is currently the best-characterized example (Ibrahim et al., 2026). Together, these findings support a multicompartment model of resistosome signaling, in which distinct NLR resistosomes mobilize Ca²⁺ from different stores to shape immune-associated Ca²⁺ signatures in plant cells (Figure 1).

Such compartmentalized immune organization may provide several important advantages to plant defense. First, the use of multiple Ca²⁺ sources could enhance the robustness and persistence of ETI-associated Ca²⁺ signaling, particularly when pathogen effectors suppress PM Ca²⁺ influx pathways by targeting immune-associated Ca²⁺-permeable channels such as cyclic nucleotide-gated channels (Lee et al., 2024). Sustained elevations of cytosolic Ca²⁺ are a hallmark of ETI (Wang et al., 2024), yet such long-lasting Ca²⁺ signatures may not rely exclusively on extracellular Ca²⁺ influx. Instead, intracellular Ca²⁺ stores within the vacuole, the ER, chloroplasts, and mitochondria may collectively contribute to the amplitude and duration of immune Ca²⁺ signaling (Wang et al., 2024).

Second, organelle-localized resistosomes may directly couple Ca²⁺ release to organelle-specific immune functions. Chloroplasts are central hubs for salicylic acid biosynthesis, reactive oxygen species (ROS) production, stromule formation, and retrograde signaling during plant immunity (Rui et al., 2025). Consequently, chloroplast-derived Ca²⁺ signals may not only amplify cytosolic immune signaling but also coordinate local immune responses within chloroplasts. This spatial coupling between organelle-derived Ca²⁺ signals and organellar functions adds another layer of regulation to immune signaling.

Finally, compartmentalized resistosome signaling may increase the evolutionary flexibility of plant immune systems. Pathogen effectors frequently target PM-associated immune receptors and ion channels to suppress defense signaling (Lee et al., 2024). The emergence of organelle-associated resistosomes may therefore provide alternative immune signaling routes that are potentially less vulnerable to pathogen interference. Such diversification of immune execution strategies could enhance the resilience of plant innate immunity during host–pathogen coevolution.

Outstanding questions in compartmentalized Ca²⁺ immunity

The discovery that helper NLR resistosomes can mobilize Ca²⁺ from intracellular stores opens an important new avenue for understanding the spatial organization of plant immune signaling. Nevertheless, several fundamental questions remain unresolved.

First, how does NbNRG1 mediate chloroplast Ca²⁺ efflux across the double-membrane chloroplast envelope? Current evidence demonstrates that activated NbNRG1 associates with the chloroplast envelope and promotes stromal Ca²⁺ release into the cytosol (Ibrahim et al., 2026). However, it remains unclear whether NbNRG1 forms a functional trans-envelope pore spanning both chloroplast membranes or acts in concert with additional host components. Resolving the structural basis of chloroplast Ca²⁺ export will be essential for understanding how organellar resistosomes mediate ion transport.

Second, how are chloroplast-derived Ca²⁺ signals integrated with other chloroplast immune outputs? Chloroplasts not only serve as intracellular Ca²⁺ stores but also function as central hubs for ROS production, salicylic acid biosynthesis, stromule formation, and retrograde signaling during immunity (Rui et al., 2025). Whether NbNRG1-triggered Ca²⁺ efflux directly coordinates these pathways remains unknown. Of particular interest is how chloroplast Ca²⁺ signals interact with ROS signaling and how organelle-derived Ca²⁺ signals are transmitted to the nucleus to drive transcriptional reprogramming during ETI.

Third, is organellar specialization a general property of helper NLRs? In addition to NRG1 proteins, the ADR1 family constitutes the second major branch of CC_R-NLR helper proteins (Kourelis and Adachi, 2022). Whether ADR1 resistosomes also localize to specific intracellular membranes and mobilize Ca²⁺ from distinct organellar stores remains unknown. Notably, a recent study showed that ADR1-L1 can assemble with the ENHANCED DISEASE SUSCEPTIBILITY 1–PHYTOALEXIN DEFICIENT 4 complex into highly organized, ring-like resistosome clusters at the PM, forming a cell death-associated signaling hub (Ge et al., 2026). Whether organelle-associated NRG1 puncta similarly form higher-order supramolecular immune assemblies remains unexplored.

Fourth, have pathogens evolved effectors that specifically target organellar resistosome signaling? Numerous pathogen effectors suppress PM-associated immune receptors and Ca²⁺-permeable channels (Lee et al., 2024). The discovery of organelle-associated resistosomes raises the possibility that chloroplasts and other organelles may also serve as important battlegrounds during host–pathogen coevolution. Identifying effectors that interfere with organellar resistosome assembly, membrane targeting, or channel activity could reveal new mechanisms of immune suppression.

Finally, how do intracellular Ca²⁺ sources collectively shape ETI-associated Ca²⁺ signatures? Sustained cytosolic Ca²⁺ elevation during ETI may not arise from a single source (Wang et al., 2024). Chloroplasts, mitochondria, the vacuole, the ER, and extracellular Ca²⁺ pools may each contribute distinct temporal and spatial components to immune Ca²⁺ signaling. Understanding how these Ca²⁺ sources are spatially and temporally coordinated will be essential for establishing a systems-level framework for compartmentalized Ca²⁺ signaling in plant immunity.

Funding

This work was supported by the High-Level Talent Training Support Project of Guizhou Province (Scientific and Technological Innovation Field, KJLYRC-[2026] 010 to L.Z.), the National Natural Science Foundation of China Excellent Young Scientists Fund (Overseas; to L.Z.), the Start-up Fund for Advanced Talents of Nanjing Agricultural University (680-804020 to M.C.), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX25_1046 to Q.W.).

Acknowledgments

We thank BioRender (https://www.biorender.com) for providing the materials used in this work. No conflict of interest declared.

Author contributions

M.C. and L.Z. conceived and revised the manuscript. Q.W. and Y.Y. drafted the manuscript.

Declaration of generative AI and AI-assisted technologies in the writing process

In preparing this work, the authors cautiously used ChatGPT-5 to check syntax and improve readability. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of this article.

Published: July 24, 2026