Why are we so excited about these receptor networks?
Plants possess an extraordinarily diverse and dynamic array of NLR immune receptors that share a common evolutionary origin. Over time, the majority of these NLRs have undergone duplication and diversification, leading to their subfunctionalization into distinct types of receptors. These can operate either as ‘sensors’ or ‘helpers,’ creating a spectrum of connections that range from simple pairs to complex networks. Paired and networked NLRs stand in contrast to certain NLRs like ZAR1, which are classified as functional singletons. ZAR1 has maintained its ancestral structure and function, remaining remarkably conserved over tens of millions of years.
So far, two major NLR networks have been described in plants, wherein numerous sensor NLRs, often first identified as disease resistance proteins, depend on a relatively smaller number of helper NLRs to carry out the immune response.
The first, known as the TIR-NLR network, involves sensor NLRs that necessitate downstream CCR-NLRs (also referred to as RPW8-NLRs or RNLs) for executing the immune response. This TIR-NLR/CCR-NLR network is absent in monocots but can be extensively expanded in certain plant taxa, notably in rosid plants such as the model Arabidopsis thaliana.
The second is the NRC network, which has evolved within the asterid lineage. This network is characterized by its unique configuration, where specific NLRs, identified as NRCs, function as crucial nodes downstream of a variety of sensor NLRs that include numerous well known disease resistance proteins of solanaceous plants, e.g. potato and tomato. The NRC network is pivotal in conferring resistance to a broad spectrum of pathogens and pests, thereby playing a central role in the dynamic immune system of asterid plants.
The TIR-NLR/CCR-NLR and NRC networks both showcase distinct phylogenetic structures, with their sensors and helpers falling into well-supported clades within the broader NLR phylogenetic tree. This pattern suggests that these networks trace their origins back to an ancestral pair of NLRs that have undergone extensive expansion throughout the course of plant evolution. Consequently, the integration of evolutionary perspectives into mechanistic studies — as exemplified in the EVO-MPMI (Evolutionary Molecular Plant-Microbe Interactions) approach — is essential for a comprehensive understanding of NLR biology and their network dynamics. Such an approach not only aids in deciphering the complex evolutionary history of these networks but also provides crucial insights into how NLRs have adapted and specialized in response to diverse pathogenic challenges over time.
Two helpers for one job
In the NRC network, the helpers have been shown to form redundant nodes that can equally contribute to disease resistance depending on the upstream NLR sensor gene. A recent study led by Ning Zhang, under the guidance of Greg Martin at the Cornell-based Boyce Thompson Institute, challenged this model using CRISPR gene editing of the tomato helper genes NRC2 and NRC3. These genes are essential for bacterial resistance governed by the Pto/Prf disease resistance genes.
Ning demonstrated that both NRC2 and NRC3 need to be knocked out to render the tomato as susceptible to bacterial infection as plants lacking the disease resistance genes. This situation is akin to a twin-engine airplane; if one engine (either NRC2 or NRC3) fails, the other can keep the plane airborne. Similarly, in tomato, the remaining gene continues to confer disease resistance. This redundancy adds a layer of robustness against system failure, ensuring that one backup is always in place.
Adding another layer of complexity, Ning observed that while NRC2 and NRC3 nodes are redundant, they can exhibit additive effects against different bacterial strains. Notably, the NRC3 knockout mutant was partially compromised in its Pto/Prf disease resistance. This raises intriguing questions: Is this due to differential accumulation of the two proteins, or does NRC3 have a unique role in immunity, perhaps linked to its capacity to contribute to immune responses triggered by cell surface receptors? These are areas yet to be explored.
Our colleagues Toshiyuki (Toshi) Sakai and Hiroaki (Aki) Adachi at the Crop Evolution Lab in Kyoto University along with Chih-Hang Wu at Institute of Plant and Microbial Biology (IPMB) in Academia Sinica in Taiwan led a study on a unique member of the NRC family, they named NRC0 (NRC-zero). This is Helper Zero, a member of the NRC family that is conserved across a wider spectrum of plant species than other NRCs. Using phylogenomic methods, they traced the evolution of NRC0 and discovered that in many plants, such as tomato, wild sweet potato, coffee, and carrot, it is closely linked and functionally connected to other NLRs that map to the NRC-sensor family. The study suggests that this close genetic clustering might reflect the ancestral configuration of the NRC family before the emergence of the expanded network in plant evolution. This work reveals how a once closely-knit gene cluster evolved into a widespread and diverse network over 125 million years, highlighting the dynamic evolution of plant immune systems.
Lamiids gone wild
A complementary study by Foong-Jing Goh and Ching-Yi Huang working in the lab of Chih-Hang Wu at IPMB and in collaboration with Lida Derevnina at Cambridge University used phylogenomic approaches to reconstruct the evolutionary history of the NRC network. They showed that NRCs have remained somewhat limited to a few genes in asterid plants until they massively expanded in the lamiid lineage. This is the group of plants that includes species as diverse as coffee, sweet potato, pepper and tomato. In these species, the phylogenetic superclade (family) that groups NRC sensors and helpers can comprise a significant fraction of all NLRs. In the parasitic plant Striga asiatica, a whopping 89% of all NLRs cluster with the NRC superclade.
This explosion of the NRC network in lamiid plants stands as a remarkable case in the evolution of NLRs across all domains of life. From a pair of genes, these NLRs have expanded and diversified in the last 100 million years or so to form intricate immune receptor networks.
Going back to the roots
Daniel Luedke from our group at The Sainsbury Lab embarked on a veritable detective story in plant immunity, that ended up addressing a significant question: How organ-specific is NLR immunity? Collaborating with several scientists around the world, notably Toshi and Chih-Hang, Daniel uncovered a distinct branch of the NRC network. This branch includes two NLR genes, Hero and Mer1, which specifically confer resistance against cyst and root knot nematodes. These parasites are notorious for their destructive impact on crops and pose a significant challenge in management once they infest farmland.
Remarkably, genes in this sub-network, including the NRC6 helper NLRs, display almost exclusive expression in roots, not in leaves. This specificity aligns perfectly with the targeted organ of the parasitic nematodes. The team concluded that NLRs can evolve organ-specific gene expression. This adaptation could be a response to specific parasites and a strategy to mitigate the risk of misactivation in non-target tissues.
This study adds another layer to the already intricate tapestry of the NRC network’s diversification in plants. Duplication of one of the NRC nodes and its sensor partners resulted in yet another evolutionary innovation: the emergence of an organ-specific sub-network in roots.
It takes two to tango
For an immune response to be effectively activated, a disease resistance sensor NLR protein and its NRC partner must be genetically and biochemically compatible. Ching-Yi Huang, Yu-Seng Huang, and colleagues from the lab of Chih-Hang Wu at IPMB in Taiwan and in collaboration with Yu Sugihara at The Sainsbury Lab and Lida Derevnina at Cambridge University, investigated the natural variants of NRC3, a pivotal node in the network linking NLRs with cell surface receptors. Their research uncovered that various NRC3 variants exhibit different genetic compatibilities with the disease resistance protein Rpi-blb2, which confers resistance to Phytophthora infestans, the pathogen that triggered the Irish potato famine.
By employing ancestral sequence reconstruction, the team mapped the functional diversification of NRC3. Their findings illustrate that NRC3 has undergone subfunctionalization throughout its evolutionary journey. This has led to the emergence of smaller, more specialized subnetworks, adding layers to the complexity of the plant immune system.
In a paper, adapted from one of his thesis chapters, Mauricio (Mau) Contreras, working closely with Hsuan Pai also at The Sainsbury Laboratory, described a key aspect of NRC activation by a disease resistance sensor protein. This study also serves as a homage to the pioneering work of Peter Moffett and other scientists from two decades ago. Mau and Pai’s research revealed that the nucleotide-binding domain (NBD) of the virus resistance protein Rx, roughly 150 amino acids in size, is both necessary and sufficient for activating its downstream helper NRC2 and triggering its oligomerization into a resistosome. This finding highlights the role of the central NBD of Rx and related sensor NLRs as a signalling domain and challenges the general assumption that the signaling activity in NLR proteins is always mediated by the N-terminal domain.
These old observations of Peter Moffett and colleagues, which remained a puzzling anomaly, finally find resolution in Mau’s study.
Mau, Pai, and their team have proposed a model where sensor NLRs, upon pathogen activation, undergo conformational changes that expose the NBD. This exposed NBD then transiently associates with the NRC helper to activate it. This ‘activate-and-release’ model aligns with the absence of a sensor unit within NRC resistosomes. The model is also consistent with an experiment by Ching-Yi and Yu-Seng and colleagues who showed transient interactions between the Rpi-blb2 sensor and its NRC3 helper, a result that has been challenging to achieve given the propensity of NRC sensors and helpers to non-specifically bind each other in co-immunoprecipitation assays (see response to preprint review here).
Mau and colleagues further demonstrated that this system, comprising a sensor NBD and its corresponding helper NRC2, constitutes a minimal unit capable of being transferred across distantly related plant species. This transferability extends from solanaceous plants (lamiids) to the Campanulid species lettuce (Lactuca sativa). This discovery holds substantial implications for bioengineering disease resistance, as it raises the potential for transferring NRC sensor-helper pairs across different crop species. This could ultimately contribute to managing plant diseases in a diverse range of agricultural contexts.
Helper structures: Part One
The 3D structures of helper NLR proteins have not been well understood until now. In one of two studies that reported on Cryo-EM structures of helper NLRs of the NRC family, M. Selavaraj and his colleagues at The Sainsbury Laboratory showed that NRC2 typically exists as a two-protein complex (homodimer) in its resting state within the plant. When activated by an upstream sensor NLR protein (the virus resistance protein Rx in this case), the NRC2 homodimer converts into a larger resistosome complex.
This work underscores the diverse activation mechanisms across plant NLRs. Notably, NRC2 undertakes a unique activation process compared to other NLRs studied so far. For instance, the well-known Arabidopsis NLR protein ZAR1 is typically found in an inactive, single-unit (monomeric) state, bound to its partner pseudokinase RKS1. This stands in stark contrast to NRC2’s distinct activation pathway, highlighting the diversity of mechanisms in NLR immune receptor activation in plants.
Helper structures: Part Deux
At the University of California Berkeley, Furong Liu, Zhenlin Yang and colleagues, working with Eva Nogales and Brian Staskawicz, resolved the structure of an autoactive form of the helper NRC4 in plants. They showed that NRC4 forms a hexameric (six-unit) configuration upon activation, which is associated with the influx of calcium ions (Ca2+) into the cell’s cytosol. This hexameric formation of NRC4 resistosomes contrasts to the previously reported pentameric (five-unit) resistosomes of ZAR1 and other NLR proteins of the coiled-coil class (CC-NLRs). This study reinforces the concept of an ‘activation-and-release’ model for NRC sensors and helpers, as the hexameric resistosome comprises only NRC4 proteins.
It’s important to note that previous studies looked into resistosome structures formed by CC-NLRs in vitro or in insect cells. Therefore, it remains to be seen whether the pentameric formation can naturally occur in plants.
Furong Liu, Zhenlin Yang, and their colleagues have made another intriguing discovery from their CryoEM analyses. They found that the autoactive form of NRC4 is capable of forming a double hexameric complex, essentially a 12-unit assembly. In this large 12-mer state, the two hexamers are linked, and the functionally significant N-terminal CC domain becomes embedded within the overall structure.
Based on these findings, the authors propose that this particular configuration represents a previously unknown inhibited state of the oligomerized NRC4. This state may play a role in the regulation of these helper NLR proteins, adding a new layer to our understanding of their functional dynamics.
The dodecameric (12-mer) state of NRC4. F. Liu, Z. Yang et al. 2023.
In the approximately 100-million-year-old immune receptor network, paralogous NRC helpers evolved to form genetically redundant network nodes, enhancing both the robustness and the adaptability of the plant immune system. Yet, the precise biochemical basis for this redundancy remained unknown. M. Selvaraj, AmirAli Toghani, Hsuan Pai, Mau Contreras, and their colleagues have postulated that mutations in the dimerization interfaces of NRC paralogs could be key in insulating these helpers from one another. Their research confirms this theory: different NRC paralogs have indeed diverged at their dimerization interfaces. For example, NRC2 does not associate with its paralogs NRC3 and NRC4, thereby creating insulated pathways within the immune network.
One hypothesis arising from these findings is that having redundant nodes in immune receptor networks may allow plants to circumvent suppression by pathogen effectors, thereby bolstering the overall resilience of the immune system.
Epilogue — watching the watchmen
Six years have elapsed since Chih-Hang Wu and colleagues unveiled the NRC network in 2017. In this relatively short time, marked by a surge of publications (eight just in the past few weeks!), we reached a mechanistic model that describes how this network translates pathogen detection into immunity and disease resistance, as detailed below.
Traditionally, NLRs are renowned for their role in monitoring — or 'guarding' in plant pathology jargon — host components, swiftly responding to the subtlest disturbances triggered by pathogenic infections. Often, host elements guarded by NLRs have evolved into ‘decoys,’ losing some of their native activities but retaining the capacity to lure pathogens into triggering an immune response. However, an extension of this concept has emerged from the recent outpouring of research: helper NLRs might actually be functioning as guardians of sensor NLRs.
Sensors in the NRC network have lost the capacity to execute many standard NLR functions independently, such as oligomerizing into resistosomes and initiating cell death. Their primary function is pathogen detection and undergoing conformational changes, which are then recognized by NRC helpers. We propose that they have evolved into “decoys’ that are safeguarded by NRCs.
Despite six years of remarkable teamwork and breakthroughs, the journey of discovery is far from complete. One aspect remains unequivocally clear: NLR biology is never a one-size-fits-all matter. From monomers versus homodimers, pentamers versus hexamers, to the contrasting natures of inflammasome heterocomplexes and the activation-and-release dynamics of NRC resistosomes, the more we pull on this thread, the more diversity and evolutionary innovation we uncover. The excitement is palpable as we continue to explore the complexities of these fascinating plant protein families, with the anticipation of uncovering more groundbreaking biology in the years to come.
This article is available on a CC-BY license via Zenodo. Cite as: Contreras, M.P., and Kamoun, S. (2023) Papers galore: A year-end update on immune receptor networks. Zenodo. https://doi.org/10.5281/zenodo.10439409
Contreras, M.P., Luedke, D., Pai, H., Toghani, A., and Kamoun, S. 2023. NLR receptors in plant immunity: making sense of the alphabet soup. EMBO Reports, e57495.
Selvaraj, M., Toghani, A., Pai, H., Sugihara, Y., Kourelis, J., Yuen, E.L.H., Ibrahim, T., Zhao, H., Xie, R., Maqbool, A., De la Concepcion, J.C., Banfield, M.J., Derevnina, L., Petre, B., Lawson, D.M., Bozkurt, T.O., Wu, C.-H. Kamoun, S., and Contreras, M.P. 2023. Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome. bioRxiv, doi: https://doi.org/10.1101/2023.12.17.572070.
Contreras, M.P., Pai, H., Thompson, R., Claeys, J., Adachi, H., and Kamoun, S. 2023. The nucleotide binding domain of NRC-dependent disease resistance proteins is sufficient to activate downstream helper NLR oligomerization and immune signaling. bioRxiv, doi: https://doi.org/10.1101/2023.11.30.569466.
Luedke, D., Sakai, T., Kourelis, J., Toghani, A., Adachi, H., Posbeyikian, A., Frijters, R., Pai, H., Harant, A., Ernst, K., Ganal, M., Verhage, A.,Wu, C.-H., and Kamoun, S. 2023. A root-specific NLR network confers resistance to plant parasitic nematodes. bioRxiv, doi: https://doi.org/10.1101/2023.12.14.571630.
Huang, C.-Y., Huang, Y.-S., Sugihara, Y., Wang, H.-Y., Huang, L.T., Lopez-Agudelo, J.C., Chen, Y.-F., Lin, K.-Y., Chiang, B.-J., Toghani, A., Kourelis, J., Derevnina, L., Wu. C.-H. 2023. Functional divergence shaped the network architecture of plant immune receptors. bioRxiv, doi: https://doi.org/10.1101/2023.12.12.571219.
Sakai, T., Martinez-Anaya, C., Contreras, M.P., Kamoun, S., Wu, C.-H., and Adachi, H. 2023. The NRC0 gene cluster of sensor and helper NLR immune receptors is functionally conserved across asterid plants. bioRxiv, doi: https://doi.org/10.1101/2023.10.23.563533.
Schornack, S., and Kamoun, S. 2023. EVO-MPMI: From fundamental science to practical applications. Current Opinion in Plant Biology, 76:102469.