Prologue — Receptor Networks Supporting Plant Immunity

Plants boast a remarkably varied and adaptable collection of NLR immune receptors that share a common evolutionary lineage. Over time, many of these NLRs have undergone duplication and diversification, leading to their specialization into different receptor types, functioning either as 'sensors' or 'helpers.' This creates a range of interactions from straightforward pairs to intricate networks. In contrast to certain NLRs like ZAR1, which function as standalone entities, paired and networked NLRs display a much more complex interrelationship. ZAR1 has preserved its ancestral form and function, remaining largely unchanged for tens of millions of years.

To date, two principal NLR networks have been identified in plants, wherein numerous sensor NLRs—often recognized as disease resistance proteins—rely on a smaller set of helper NLRs to facilitate immune responses.

The first, termed the TIR-NLR network, consists of sensor NLRs that require downstream CCR-NLRs (also known as RPW8-NLRs or RNLs) to execute the immune response. This particular network is absent in monocots but can be significantly expanded in certain plant groups, particularly within rosid plants like the model organism Arabidopsis thaliana.

The second is the NRC network, which has developed within the asterid lineage. This network is distinct in its structure, where specific NLRs, referred to as NRCs, act as vital nodes downstream of various sensor NLRs that include well-studied disease resistance proteins in solanaceous plants such as potatoes and tomatoes. The NRC network is crucial for providing resistance to a wide range of pathogens and pests, thus playing a vital role in the dynamic immune system of asterid plants.

Both the TIR-NLR/CCR-NLR and NRC networks reveal unique phylogenetic frameworks, with their sensors and helpers classified into well-supported clades within the larger NLR phylogenetic tree. This suggests that these networks can trace their lineage back to a primitive pair of NLRs that have significantly expanded throughout plant evolution. Hence, incorporating evolutionary perspectives into mechanistic studies—such as those seen in the EVO-MPMI (Evolutionary Molecular Plant-Microbe Interactions) approach—is vital for a thorough understanding of NLR biology and network dynamics. This approach not only aids in unraveling the intricate evolutionary history of these networks but also offers essential insights into how NLRs have adapted over time in response to various pathogenic challenges.

Two Helpers for One Task

Within the NRC network, the helper NLRs have been found to form redundant nodes that can each contribute to disease resistance based on the upstream NLR sensor gene. A recent investigation led by Ning Zhang, under the mentorship of Greg Martin at the Boyce Thompson Institute in Cornell, challenged this paradigm by employing CRISPR gene editing on the tomato helper genes NRC2 and NRC3, which are critical for bacterial resistance linked to the Pto/Prf disease resistance genes.

Ning's research showed that both NRC2 and NRC3 must be knocked out to make the tomato as vulnerable to bacterial infection as plants lacking the disease resistance genes. This situation resembles a twin-engine aircraft; if one engine (either NRC2 or NRC3) fails, the other remains operational. Similarly, in tomatoes, the other gene continues to provide disease resistance. This redundancy enhances the system's reliability, ensuring that at least one backup is available.

Moreover, Ning observed that while NRC2 and NRC3 nodes are redundant, they can display additive effects against different bacterial strains. Notably, the NRC3 knockout mutant exhibited partial loss of Pto/Prf disease resistance. This raises interesting questions: Is this due to varying levels of protein accumulation, or does NRC3 possess a unique role in immunity, perhaps related to its involvement in immune responses activated by cell surface receptors? These are areas that require further investigation.

Click here for Ning’s X / Twitter thread about the paper.

Helper Zero

Researchers Toshiyuki Sakai and Hiroaki Adachi from the Crop Evolution Lab at Kyoto University, along with Chih-Hang Wu from the Institute of Plant and Microbial Biology (IPMB) at Academia Sinica in Taiwan, conducted a study on a distinct member of the NRC family, which they named NRC0 (NRC-zero). This so-called Helper Zero is conserved across a broader range of plant species than other NRCs. By utilizing phylogenomic methods, they traced the evolution of NRC0 and discovered its close relationship with other NLRs mapped to the NRC-sensor family in several plants, including tomatoes, wild sweet potatoes, coffee, and carrots. The findings suggest that this close genetic connection may reflect the ancestral layout of the NRC family prior to the development of the expanded network in plant evolution, illustrating how a previously cohesive gene cluster evolved into a widespread and diverse network over the past 125 million years.

Click here for Toshi’s X / Twitter thread about the paper.

Lamiids Gone Wild

In a complementary study, Foong-Jing Goh and Ching-Yi Huang, working in Chih-Hang Wu's lab at IPMB and collaborating with Lida Derevnina from Cambridge University, employed phylogenomic methods to reconstruct the evolutionary history of the NRC network. Their work revealed that NRCs were somewhat restricted to a limited number of genes in asterid plants until they underwent substantial expansion in the lamiid lineage. This group includes a diverse range of species such as coffee, sweet potatoes, peppers, and tomatoes. In these plants, the phylogenetic superclade grouping NRC sensors and helpers constitutes a significant portion of all NLRs. For instance, in the parasitic plant Striga asiatica, an impressive 89% of all NLRs cluster within the NRC superclade.

This remarkable proliferation of the NRC network in lamiid plants serves as a striking example of NLR evolution across all life domains. From a mere pair of genes, these NLRs have diversified and expanded over the last 100 million years, forming intricate immune receptor networks.

Click here for Chih-Hang’s X / Twitter thread about the paper.

Returning to the Roots

Daniel Luedke from The Sainsbury Lab undertook a significant investigation into plant immunity, addressing a critical question: How organ-specific is NLR immunity? Collaborating with various scientists worldwide, including Toshi and Chih-Hang, Daniel discovered a unique branch of the NRC network. This branch encompasses two NLR genes, Hero and Mer1, which confer resistance specifically against cyst and root knot nematodes—parasites notorious for their devastating effects on crops and posing significant management challenges once they infest farmland.

Notably, genes within 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 as a response to particular parasites, a strategy to mitigate the risk of unintended activation in non-target tissues.

This study adds another layer to the intricate tapestry of the NRC network’s diversification in plants. The duplication of one of the NRC nodes and its sensor partners has given rise to yet another evolutionary innovation: the emergence of an organ-specific sub-network in roots.

Click here for Daniel’s X / Twitter thread about the paper.

It Takes Two to Tango

For an immune response to be effectively initiated, a disease resistance sensor NLR protein must be genetically and biochemically compatible with its NRC partner. Ching-Yi Huang, Yu-Seng Huang, and their colleagues from Chih-Hang Wu's lab at IPMB, in collaboration with Yu Sugihara at The Sainsbury Lab and Lida Derevnina at Cambridge University, explored the natural variants of NRC3, a crucial node in the network linking NLRs with cell surface receptors. Their research revealed that different NRC3 variants display varying genetic compatibilities with the disease resistance protein Rpi-blb2, which provides resistance to Phytophthora infestans, the pathogen responsible for the Irish potato famine.

Using ancestral sequence reconstruction, the team mapped the functional diversification of NRC3. Their findings indicate that NRC3 has undergone subfunctionalization throughout its evolutionary history, leading to the emergence of smaller, more specialized subnetworks, thereby increasing the complexity of the plant immune system.

Click here for Chih-Hang’s X / Twitter thread about the paper.

Minimal Unit

In a recent paper adapted from his thesis work, Mauricio (Mau) Contreras, in collaboration with Hsuan Pai at The Sainsbury Laboratory, addressed a fundamental aspect of NRC activation by a disease resistance sensor protein. This study also pays tribute to the pioneering efforts of Peter Moffett and other scientists from two decades ago. Mau and Pai's research demonstrated that the nucleotide-binding domain (NBD) of the virus resistance protein Rx, which spans approximately 150 amino acids, is both necessary and sufficient for activating its downstream helper NRC2 and triggering its oligomerization into a resistosome. This finding underscores the significance of the central NBD of Rx and related sensor NLRs as a signaling domain, challenging the conventional view that signaling activity in NLR proteins is exclusively mediated by the N-terminal domain.

These earlier observations by Peter Moffett and colleagues, which previously appeared as an unresolved anomaly, are now clarified by Mau’s study.

Mau, Pai, and their team propose a model in which sensor NLRs, upon activation by pathogens, 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. It is also consistent with an experiment conducted by Ching-Yi, Yu-Seng, and colleagues, who demonstrated transient interactions between the Rpi-blb2 sensor and its NRC3 helper—a result that has been challenging to achieve, given the tendency of NRC sensors and helpers to non-specifically bind each other in co-immunoprecipitation assays.

Mau and colleagues further illustrated that this system, which consists of a sensor NBD and its corresponding helper NRC2, forms a minimal functional unit that can be transferred across distantly related plant species. This capability extends from solanaceous plants (lamiids) to the Campanulid species lettuce (Lactuca sativa). This discovery has significant implications for bioengineering disease resistance, as it opens up the possibility of transferring NRC sensor-helper pairs across different crop species, potentially aiding in the management of plant diseases across various agricultural settings.

Click here for Mau’s X / Twitter thread about the paper.

Helper Structures: Part One

The three-dimensional structures of helper NLR proteins have not been well understood until recently. In one of two studies that reported on Cryo-EM structures of NRC family helper NLRs, M. Selavaraj and colleagues at The Sainsbury Laboratory found that NRC2 generally exists as a two-protein complex (homodimer) in its resting state. Upon activation by an upstream sensor NLR protein (in this case, the virus resistance protein Rx), the NRC2 homodimer transitions into a larger resistosome complex.

This research highlights the diverse activation mechanisms utilized by plant NLRs. Notably, NRC2 undergoes a unique activation process compared to other NLRs studied to date. For example, the well-known Arabidopsis NLR protein ZAR1 is typically found in an inactive, single-unit (monomeric) form, bound to its partner pseudokinase RKS1. This starkly contrasts with NRC2’s distinctive activation pathway, emphasizing the diversity of mechanisms involved in NLR immune receptor activation in plants.

Click here for Mau’s X / Twitter thread about the paper.

Click here for Selavaraj’s X / Twitter thread about the paper.

Helper Structures: Part Deux

At the University of California, Berkeley, Furong Liu, Zhenlin Yang, and colleagues, collaborating with Eva Nogales and Brian Staskawicz, elucidated the structure of an autoactive form of the helper NRC4 in plants. They discovered that NRC4 forms a hexameric (six-unit) configuration upon activation, associated with an influx of calcium ions (Ca2+) into the cell’s cytosol. This hexameric formation of NRC4 resistosomes differs from the previously reported pentameric (five-unit) resistosomes of ZAR1 and other coiled-coil class (CC-NLR) proteins. This study reinforces the concept of an 'activation-and-release' model for NRC sensors and helpers, as the hexameric resistosome is composed solely of NRC4 proteins.

It is important to note that prior investigations examined resistosome structures formed by CC-NLRs in vitro or in insect cells. Therefore, it remains uncertain whether the pentameric formation can occur naturally in plants.

Furong Liu, Zhenlin Yang, and their team have made another fascinating discovery through their Cryo-EM analyses. They found that the autoactive form of NRC4 can form a double hexameric complex, essentially a 12-unit assembly. In this large 12-mer state, the two hexamers are interconnected, and the functionally significant N-terminal CC domain becomes integrated within the overall structure.

Based on these findings, the authors propose that this specific configuration represents a previously unknown inhibited state of oligomerized NRC4, potentially playing a role in the regulation of these helper NLR proteins, thereby enriching our understanding of their functional dynamics.

Drifting Apart

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 adaptability of the plant immune system. However, the precise biochemical underpinnings of this redundancy remained elusive. M. Selavaraj, AmirAli Toghani, Hsuan Pai, Mau Contreras, and their colleagues have posited that mutations in the dimerization interfaces of NRC paralogs may be crucial in isolating these helpers from one another. Their research substantiates this theory, confirming that different NRC paralogs have indeed diverged at their dimerization interfaces. For instance, NRC2 does not associate with its paralogs NRC3 and NRC4, thereby establishing insulated pathways within the immune network.

One hypothesis arising from these findings is that having redundant nodes in immune receptor networks may enable plants to evade suppression by pathogen effectors, thereby enhancing the resilience of the overall immune system.

Epilogue — Monitoring the Guardians

It has been six years since Chih-Hang Wu and colleagues introduced the NRC network in 2017. During this relatively brief period, characterized by a surge of publications (eight in the last few weeks alone!), we have established a mechanistic model that elucidates how this network translates pathogen detection into immunity and disease resistance.

Traditionally, NLRs are recognized for their role in monitoring—guarding, in plant pathology terminology—host components, reacting swiftly to the slightest disturbances caused by pathogenic infections. Often, the host elements overseen by NLRs have evolved into ‘decoys,’ losing some of their original functions while retaining the ability to lure pathogens into initiating an immune response. However, recent research has expanded this concept: helper NLRs may also serve as guardians of sensor NLRs.

Sensors within the NRC network have lost the ability to perform many standard NLR functions independently, such as oligomerizing into resistosomes and instigating cell death. Their primary role is pathogen detection and undergoing conformational changes, which are then recognized by NRC helpers. We propose that they have evolved into "decoys" that are protected by NRCs.

Despite six years of remarkable collaboration and breakthroughs, the journey of discovery is far from over. One aspect remains abundantly clear: NLR biology is never a one-size-fits-all affair. From monomers versus homodimers, pentamers versus hexamers, to the varying natures of inflammasome heterocomplexes and the activation-and-release dynamics of NRC resistosomes, the deeper we delve into this subject, the more diversity and evolutionary innovation we unveil. The excitement is palpable as we continue to navigate the complexities of these captivating plant protein families, anticipating more groundbreaking discoveries in the coming years.

This article is available under 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*

Additional Resources and Suggested Readings

Wu, C.H., Derevnina, L., and Kamoun, S. 2018. Receptor networks underpin plant immunity. Science, 360:1300–1301. YouTube.

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.

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