Biology subjects

Kourelis, J.

Publications and source records attributed to Kourelis, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Conserved core and dynamic periphery NRC helper NLRs underpin immune receptor network evolution across Solanaceae

Plant nucleotide-binding leucine-rich repeat (NLR) proteins function as intracellular immune receptors that detect pathogen-derived signals and activate defense responses. The NRC (NLR required for cell death) receptor network plays central roles in immunity of solanaceous crops, yet its evolutionary diversification across Solanaceae remains poorly understood. Here, we combined comparative phylogenomics and comprehensive functional complementation assays to investigate the evolution and functional diversification of NRC helper NLRs across nine representative species from diverse genera within the Solanaceae. Phylogenetic analyses resolved 11 NRC helper subfamilies with distinct evolutionary trajectories, revealing a conserved core and dynamic periphery within the NLR receptor network. NRC2, NRC3, and NRC4 were broadly conserved across all examined species, whereas other NRC lineages exhibited degrees of presence-absence polymorphisms, lineage-specific expansion, and rapid diversification. Comparative genomic analyses revealed highly dynamic helper-sensor NLR cluster organization, indicating substantial genomic restructuring during Solanaceae evolution. Functional assays further showed that some NRC subfamilies retained broad compatibility with multiple sensor NLRs despite extensive sequence and genomic divergence, whereas other helpers displayed lineage-specific gains and losses of compatibility, revealing extensive rewiring of helper-sensor functional connections. Together, our study provides a cross-Solanaceae evolutionary and functional atlas of the NRC immune receptor network and demonstrates how a conserved core and dynamic periphery of NRC helper NLRs underpin the evolution of immune signaling specificity across Solanaceae.

plant biology↗

bioRxiv2026

AI-guided discovery of atypical protein assemblies

Artificial intelligence (AI) systems such as AlphaFold have transformed structural biology by enabling accurate prediction of protein structures. However, their capacity to uncover new classes of macromolecular assemblies remains largely untapped. We developed the Structural Novelty Index (SNI), a quantitative framework for identifying protein complexes that diverge from canonical architectures. As one implementation of SNI, we developed SNINRC-Hexa, to identify unconventional resistosomes formed by nucleotide-binding, leucine-rich repeat immune receptors (NLRs). We used it to analyze AlphaFold 3 models of 637 non-redundant NRC proteins from 346 genomes representing 85 plant species. This analysis identified candidates with predicted architectures distinct from the canonical hexameric resistosomes of NRC proteins. Biochemical purification and negative-stain transmission electron microscopy of NRC7 orthologs from multiple species supported the SNI prediction and revealed an unexpected undecameric (11-mer) assembly. Our results establish SNI as a scalable approach for discovering atypical protein complexes.

bioinformatics↗

bioRxiv2026

NbRD21 protease controls receptor kinase homeostasis in Nicotiana benthamiana

RD21-like proteases are papain-like cysteine proteases with a C-terminal granulin domain that are abundant and ubiquitous in angiosperms and have often been implicated in immunity. We previously found that the activity of RD21 in Nicotiana benthamiana (NbRD21) is suppressed during infection with Pseudomonas syringae. Here, we studied the role of NbRD21 in immunity and proteome processing. NbRD21 was disrupted by genome editing and rd21 mutants were subjected to disease assays and shot-gun proteomics. Dipeptide substrate zLR-AMC was used in protease assays and agroinfiltration was used to transiently express NbRD21 and candidate substrates. Genome edited lines lacking NbRD21 develop normally but have drastically reduced zLRase activity and are significantly more susceptible to P. syringae. Shot gun proteomics revealed an increased accumulation of [~]20 diverse receptor-like kinases (RLKs) in untreated rd21 knockout lines, but their transcript levels are unaltered when compared to wild-type plants. 35S-driven GFP-tagged RLKs accumulate more upon transient expression in rd21 plants than in wild- type plants. These data indicate that NbRD21 post-translationally controls RLK homeostasis, either by directly degrading RLKs, or indirectly by regulating endocytic RLK recycling.

plant biology↗

bioRxiv2026

A broad-host-range Rhizobium rhizogenes strain enables transient expression across diverse crops and establishes functional assays in faba bean

Agrobacterium-mediated transient expression has revolutionized plant research, enabling numerous landmark discoveries across diverse areas of plant biology. Yet this powerful approach remains largely confined to solanaceous species, leaving most economically important crop families without a comparable rapid assay platform. Here, we show that an engineered Rhizobium rhizogenes strain, AS109, mediates efficient transient expression across diverse dicot species spanning multiple taxonomic families, consistently outperforming commonly used laboratory agrobacterial strains. Leveraging the broad host range of AS109, we establish a suite of functional assays in faba bean (Vicia faba), including protein localisation, RNA interference-mediated gene silencing, cell-surface elicitor recognition screens, nucleotide-binding leucine-rich repeat receptor (NLR) activation, and infection cell biology at the host-pathogen interface. We further demonstrate that both singleton NLRs and sensor-helper NLR pairs from Solanaceae retain effector recognition and cell death activity when transferred into faba bean, establishing a rapid platform for evaluating cross-family transferability of disease-resistance genes. AS109 thus provides an accessible and versatile chassis for functional genomics in non-model crops, bridging the widening gap between hypothesis generation and experimental validation across diverse plant species.

plant biology↗

bioRxiv2026

NLR immune receptors can exhibit tissue-specific expression patterns across legume species

Pathogen pressure threatens legume crop productivity worldwide. Nucleotide-binding leucine-rich repeat (NLR) immune receptors serve as crucial plant resistance genes, recognizing pathogens and triggering immunity. However, the extent and patterns of NLR expression in different tissues and organs, notably across evolutionary time, remain largely uncharacterized. To investigate tissue-specificity of NLR expression in the Fabaceae (legumes), we conducted comparative analyses integrating phylogenomics and transcriptomics in root and shoot tissues across different legume species. The NLR repertoires of 28 legumes were grouped into five monophyletic clades: coiled-coil NLR (CC-NLR), Toll/interleukin-1 receptor NLR (TIR-NLR), G10-subclade CC NLR (CCG10-NLR), RESISTANCE TO POWDERY MILDEW 8-like CC NLR (CCR-NLR), and TIR-NB-ARC-like {beta}-propeller WD40/tetratricopeptide repeats (TNPs). Most legume NLRs belonged to CC-NLR and TIR-NLR clades, followed by CCG10-NLR, CCR-NLR, and TNP clades. In seven of these species, comparative analysis of NLR expression in leaves versus roots revealed that over half ([~]57%) of expressed NLR genes showed predominant expression in one tissue: 34% in roots (451/1336), and 23% in leaves (311/1336). We identified 324 root-specific NLRs, 171 leaf-specific NLRs, and 841 non-specific NLRs, with an average tissue specificity per species of 32%. The closely related species grass pea (Lathyrus sativus) and pea (Pisum sativum) were an exception, showing higher levels of leaf-specific rather than root-specific NLR expression. We also identified conserved tissue expression patterns across legume species, resulting in a comprehensive resource describing tissue expression bias, enrichment, and specificity for 113 phylogenetic NLR subclasses. These legume NLR repertoires will support comparative studies between species and inform precision-breeding programs considering tissue expression patterns.

plant biology↗

bioRxiv2025

The Pik NLR pair accumulates at the plasma membrane as a hetero-oligomeric sensor-helper immune protein complex prior to activation

Following the perception of pathogen virulence proteins in plants, nucleotide-binding and leucine-rich repeat immune receptors (NLRs) are activated via a wide range of mechanisms. Singleton NLRs can both perceive effectors and trigger an immune response, whereas other NLRs specialise in either pathogen recognition (sensor NLRs) or activation of the immune response (helper NLRs). Sensor and helper NLRs can function as genetically linked pairs or in unlinked receptor networks. Although growing evidence suggests that NLRs conditionally oligomerise upon activation, our understanding of the resting state of NLRs prior to effector perception remains limited. Here, we investigated the oligomeric state of the genetically linked rice (Oryza sativa) sensor Pik-1 and helper Pik-2 NLR pair prior to effector activation when transiently expressed in Nicotiana benthamiana leaves. We show that both wild-type Pikm-1 and engineered Pikm-1Enhancer sensors associate with Pikm-2 and form [~]1 MDa hetero-complexes in the resting state that accumulate at the plasma membrane. Our findings contribute to the growing evidence that pre-activation mechanisms vary widely across NLRs. This knowledge could be leveraged for disease resistance engineering strategies complementary to approaches focussing solely on effector binding. One sentence summaryThe Pik sensor-helper pair of rice immune receptor proteins forms a hetero-oligomer that accumulates at the plasma membrane in its resting state. SynopsisNucleotide-binding and leucine-rich repeat receptors (NLRs) are key players in plant immune systems that recognise and respond to harmful pathogen effector proteins. NLRs can play specialised roles in either detecting effectors (sensor NLRs) or triggering the immune response (helper NLRs) and might function together as pairs or within larger networks. Although many NLRs are known to oligomerise upon the perception of effectors, much less is understood about their resting state prior to pathogen recognition. In this study, we investigated the resting state of a pair of rice NLRs, Pik-1 and Pik-2, transiently expressed in Nicotiana benthamiana leaves. These two proteins formed a sensor/helper complex at the cell membrane even in the absence of the effector protein. This discovery is significant because it reveals a new aspect of how these immune proteins are organised before activation, contributing to our understanding of the diverse ways plants prepare for immune responses. This finding adds to the growing body of evidence that, although many NLR proteins form complexes to respond to pathogens, their resting states and pre-activation mechanisms can vary widely.

plant biology↗

bioRxiv2025

A hierarchical immune receptor network in lettuce reveals contrasting patterns of evolution in sensor and helper NLRs

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) are known for their rapid evolution, even at the intraspecific level, yet the rates of evolution differ significantly across various NLR classes. Within the NRC (NLR Required for Cell Death) network, NLRs operate in complex sensor-helper configurations to confer immunity against a diverse array of pathogens, particularly in Asterids. While helper NLRs are typically conserved and evolve slowly, sensor NLRs tend to evolve more rapidly. However, the functional connections between slow and fast-evolving NLRs remain poorly understood, notably in important crop species. We conducted a comparative analysis of NLRs across 40 Solanales and 29 Asterales genomes to explore NRC network expansion and diversification within the less-studied Asterales order. Our findings reveal that the NRC network has expanded less in Asterales compared to Solanales. We functionally validated a minimal Asterales NRC network with 2 helpers and 9 sensors in common lettuce (Lactuca sativa). Through selection and diversification analysis and structural modeling of NRC helper and sensor subclades in the Lactuca genus, we found varying evolutionary diversification rates between NRC helpers and sensors. We found a correlation between sensor diversification rates and helper dependency, with sensors reliant on a phylogenetically conserved helpers experiencing limited diversification pressure. Our results highlight the lineage- and function-specific evolution of the NRC network, offering insights into the evolutionary pressures shaping plant immune receptor networks.

plant biology↗

bioRxiv2024

A helper NLR targets organellar membranes to trigger immunity

Upon activation, plant NLR immune receptors are known to assemble into oligomeric resistosomes that insert into the plasma membrane, forming Ca2+-permeable channels and triggering immunity. Here, we show that the RPW8-like coiled-coil (CCR-) NLR NRG1 primarily targets organelles instead of the plasma membrane. Unlike canonical CC-NLRs, activated NRG1 accumulates at the chloroplast envelope and channels stromal Ca2+ into the cytosol. AlphaFold modeling of the NRG1 resistosome reveals an unusually long N-terminal membrane-insertion structure that would span the double chloroplast membranes. Nanobody-mediated relocalization shows functional membrane specificity: chloroplast trapping abolishes activity of the canonical helper CC-NLR NRC4 but not NRG1. NRG1 orthologs, from non-flowering lineages to angiosperms, target chloroplasts, suggesting that organelle-centered defense dates back to at least [~]360 million years. We propose that coiled-coil NLR diversification has enabled compartment-specific immune signaling to capture diverse Ca2+ stores.

plant biology↗

bioRxiv2024

A disease resistance protein triggers oligomerization of its NLR helper into a hexameric resistosome to mediate innate immunity

NRCs are essential helper NLR (nucleotide-binding domain and leucine-rich repeat) proteins that execute the immune response triggered by disease resistance proteins, also known as sensor NLRs. The structure of the resting state of NbNRC2 was recently revealed to be a homodimer. However, the sensor-activated state has not yet been elucidated. In this study, we used cryo-EM to determine the structure of sensor-activated NbNRC2, which forms a hexameric inflammasome-like structure known as resistosome. To confirm the functional significance of the hexamer, we mutagenized the interfaces involved in oligomerization and found that mutations in three nucleotide-binding domain interface residues abolish oligomerization and immune signalling. Comparative structural analyses between the resting state NbNRC2 homodimer and the sensor-activated homohexamer revealed significant structural rearrangements before and after activation, providing insights into NLR activation mechanisms. Furthermore, structural comparisons between the NbNRC2 hexamer and previously reported CC-NLR pentameric assemblies revealed features in NbNRC2 that allow for the integration of an additional protomer. We also used the NbNRC2 hexamer structure to assess the recently released AlphaFold 3 for the prediction of activated CC-NLR oligomers. This revealed that AlphaFold 3 allows for high-confidence modelling of the N-terminal 1-helices of NbNRC2 and other CC-NLRs, a region that has proven difficult to fully resolve using structural approaches. Overall, our work sheds light on the structural and biochemical mechanisms underpinning NLR activation and expands our understanding of NLR structural diversity.

plant biology↗

bioRxiv2024

Helper NLR immune protein NRC3 evolved to evade inhibition by a cyst nematode virulence effector

Parasites can counteract host immunity by suppressing nucleotide binding and leucine-rich repeat (NLR) proteins that function as immune receptors. We previously showed that a cyst nematode virulence effector SPRYSEC15 (SS15) binds and inhibits oligomerisation of helper NLR proteins in the expanded NRC1/2/3 clade by preventing intramolecular rearrangements required for NRC oligomerisation into an activated resistosome. Here we examined the degree to which NRC proteins from multiple Solanaceae species are sensitive to suppression by SS15 and tested hypotheses about adaptive evolution of the binding interface between the SS15 inhibitor and NRC proteins. Whereas all tested orthologs of NRC2 were inhibited by SS15, some natural variants of NRC1 and NRC3 are insensitive to SS15 suppression. Ancestral sequence reconstruction combined with functional assays revealed that NRC3 transitioned from an ancestral suppressed form to an insensitive one over 19 million years ago. Our analyses revealed the evolutionary trajectory of coevolution between a parasite inhibitor and its NLR immune receptor target, identifying key evolutionary transitions in helper NLRs that counteract this inhibition. This work reveals a distinct type of gene-for-gene interaction between parasite or pathogen immunosuppressors and host immune receptors that contrasts with the coevolution between AVR effectors and immune receptors.

plant biology↗

bioRxiv2024

Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors

Secreted immune proteases Rcr3 and Pip1 of tomato are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvum but only Rcr3 act as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues from tomato Rcr3 required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2 dependent immunity. Despite substantial divergences in Rcr3 orthologs from eggplant and tobacco, only minimal alterations were sufficient to trigger Avr2/Cf-2-triggered immune signalling. Tomato Pip1, by contrast, was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side next to the substrate binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

plant biology↗

bioRxiv2024

The blast effector Pwl2 is a virulence factor that modifies the cellular localisation of host protein HIPP43 to suppress immunity.

The rice blast fungus Magnaporthe oryzae secretes a battery of effector proteins to facilitate host infection. Among these effectors, Pwl2 was first identified as a host specificity determinant for infection of weeping lovegrass (Eragrostis curvula) and is also recognised by the barley Mla3 resistance gene. However, its biological activity is not known. Here we show that PWL2 expression is regulated by the Pmk1 MAP kinase during cell-to-cell movement by M. oryzae at plasmodesmata (PD)-containing pit field sites. Consistent with its regulation, we provide evidence that Pwl2 binds to a barley heavy metal-binding isoprenylated protein HIPP43, which results in its displacement from plasmodesmata. Transgenic barley lines overexpressing either PWL2 or HIPP43 exhibit attenuated immune responses and increased disease susceptibility. By contrast, a Pwl2SNDEYWY mutant that does not interact with HIPP43, fails to alter the PD localisation of HIPP43. Targeted deletion of three copies of PWL2 in M. oryzae results in a{Delta} pwl2 mutant showing gain-of-virulence to weeping lovegrass and barley Mla3 lines, but also a reduction in severity of blast disease on susceptible host plants. Taken together, our results provide evidence that Pwl2 is a virulence factor that acts by suppressing host immunity through perturbing the plasmodesmatal deployment of HIPP43.

plant biology↗

bioRxiv2023

Activation of plant immunity through conversion of a helper NLR homodimer into a resistosome

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins can engage in complex interactions to detect pathogens and execute a robust immune response via downstream helper NLRs. However, the biochemical mechanisms of helper NLR activation by upstream sensor NLRs remain poorly understood. Here, we show that the coiled-coil helper NLR NRC2 accumulates in vivo as a homodimer that converts into a higher order oligomer upon activation by its upstream virus disease resistance protein Rx. The Cryo-EM structure of NRC2 in its resting state revealed intermolecular interactions that mediate homodimer formation. These dimerization interfaces have diverged between paralogous NRC proteins to insulate critical network nodes and enable redundant immune pathways. Our results expand the molecular mechanisms of NLR activation pointing to transition from homodimers to higher-order oligomeric resistosomes.

plant biology↗

bioRxiv2023

A root-specific NLR network confers resistance to plant parasitic nematodes

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) confer disease resistance to a multitude of foliar and root parasites of plants. However, the extent to which NLR immunity is expressed differentially between plant organs is poorly known. Here, we show that a large cluster of tomato genes, which encodes the cyst and root-knot nematode disease resistance proteins Hero and MeR1 as well as the NLR-helper NRC6, exhibits nearly exclusive expression in the roots. This root-specific gene cluster emerged in Solanum species about 21 million years ago through gene duplication from the ancient NRC network of asterid plants. NLR-sensors in this gene cluster exclusively signal through NRC6 helpers to trigger the hypersensitive cell death immune response. These findings indicate that the NRC6 gene cluster has sub-functionalized from the larger NRC network to specialize for resistance against root pathogens, including cyst and root-knot nematodes. We propose that NLR gene clusters and networks have evolved organ-specific gene expression as an adaptation to particular parasites and to reduce the risk of autoimmunity.

plant biology↗

bioRxiv2023

Functional divergence shaped the network architecture of plant immune receptors

In solanaceous plants, several sensor NLRs and their helper NLRs, known as NRC, form a complex network to confer immunity against pathogens. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, the evolution and molecular basis of the complex network structure remained elusive. Here we demonstrated that functional divergence of NRC3 variants has shaped the genetic architecture of the NLR network. Natural NRC3 variants form three allelic groups displaying distinct compatibilities with sensor NLRs. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility, with two residues critical for subfunctionalization. Co-functioning Rpi-blb2 and NRC3 variants showed stronger transient interactions upon effector detection, with NRC3 membrane-associated complexes forming subsequently. Our findings reveal how mutations in helper NLRs, particularly NRC3, have driven the evolution of their transient interactions with sensor NLRs, leading to subfunctionalization and contributing significantly to the complexity of the NRC network in plant immunity. TeaserHelper NLR subfunctionalization alters transient interactions with sensor NLRs, enhancing plant immune system complexity.

plant biology↗

bioRxiv2023

Enhanced late blight resistance by engineering an EpiC2B-insensitive immune protease

Crop protection strategies relying on the improvement of the natural plant immune system via genetic engineering are sustainable solutions against the pathogen thread on food security. Here we describe a novel way to improve the plant immune system by immune protease engineering. As proof of concept, we increased resistance against the late blight pathogen Phytopththora infestans by rendering the tomato secreted immune protease Pip1 insensitive to the P. infestans-secreted inhibitor Epic2B. This concept can be applied to secreted immune proteases in crops by precision breeding.

plant biology↗

bioRxiv2022

The N-terminal executioner domains of NLR immune receptors are functionally conserved across major plant lineages

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a prominent class of intracellular immune receptors that are present across diverse plant lineages. However, our understanding of plant NLR structure and function is limited to the evolutionarily young flowering plant clade (angiosperms). Here, we describe an extended spectrum of NLR diversity across major plant lineages and demonstrate functional conservation of N-terminal executioner domains that trigger immune responses. We show that broadly distributed CC (coiled-coil) and TIR (toll/interleukin-1 receptor) domains retain executioner function through trans-lineage activation of immune-related cell death in the model angiosperm Nicotiana benthamiana. Further examination of a CC subfamily specific to non-flowering lineages uncovered an essential N-terminal MAEPL motif with functional similarity to resistosome-forming CC-NLRs. Ectopic activation of the MAEPL-type CC in the divergent liverwort Marchantia polymorpha led to profound growth inhibition, defense gene activation, and signatures of cell death resembling CC activity in flowering plants. Moreover, comparative macroevolutionary transcriptomics in Marchantia and Nicotiana identified conserved CC responsive genes, providing further insight into the core aspects of CC function shared between flowering and non-flowering plants. Our findings highlight the need to understand NLR structure and function across the full spectrum of plant diversity.

plant biology↗

bioRxiv2022

Allelic compatibility in plant immune receptors facilitates engineering of new effector recognition specificities.

Engineering expanded effector recognition in plant immune receptors is a promising prospect for generating new disease resistant crop varieties. However, modification of plant NLR receptors has proven challenging due to the lack of understanding of their context as part of complex immune systems. Here, we demonstrate a new avenue for NLR-mediated engineering that exploits the allelic diversity in the Pik NLR pair to allow for the generation of receptors with expanded recognition specificities, which would otherwise result in constitutive cell death. This work lays the foundation for the incorporation of new effector recognition motifs into the Pik system and advances the development of designer NLRs that can be tailored to specific secreted pathogen signatures. AbstractEngineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine rich repeat (NLRs) superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering novel disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defence responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signalling networks that co-evolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. We have previously shown how alleles of the rice NLR pair Pik have differentially co-evolved, and how sensor/helper mismatching between non-co-evolved alleles triggers constitutive activation and cell death (De la Concepcion et al., 2021b). Here, we dissect incompatibility determinants in the Pik pair and found that HMA domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely co-evolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic HMA domains with novel and enhanced recognition of an effector from the rice blast fungus. Taken together, our results reveal a new strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.

plant biology↗