Biology subjects

Toghani, A.

Publications and source records attributed to Toghani, A..

16 recordsLinked to original sources

bioRxiv2026

A conserved structural logic underlies sensor-helper NLR communication in the NRC immune receptor network

NLR immune receptor networks consist of expanded disease resistance proteins (sensor NLRs) that signal via core executors of immunity known as helper NLRs. Although some sensor NLRs are thought to activate their cognate helpers via an activation-and-release mechanism, the structural basis of sensor-helper communication remains poorly understood. Here, we identify and validate sensor-helper NLR interfaces that are critical for immune activation in the NRC network of coiled-coil NLR immune receptors. Using AlphaFold 3 we predicted a high confidence model between the virus resistance protein Rx and its helper NLR NRC2. We validated the interfaces by loss and gain-of-function mutagenesis, including reconstituting a critical salt bridge through reciprocal mutations. We showed that these interfaces are conserved across the NRC network of asterid plants despite over 120 million years of divergence and validated the sensor-NRC interfaces within the common lettuce network. Structure-guided bioengineering of a lettuce sensor NLR enabled expansion of its NRC helper compatibility profile. These results are consistent with the activation-and-release model and point to bioengineering sensor- helper specificity in economically important crop species.

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

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↗

bioRxiv2025

A conserved small RNA-generating gene cluster undergoes sequence diversification and contributes to plant immunity

Small RNA-mediated gene silencing contributes to plant immunity. The secondary small interfering RNA (siRNA) pathway promotes defense by silencing target genes in invading fungal and oomycete pathogens. Many secondary siRNAs derive from transcripts potentially encoding pentatricopeptide repeat (PPR) proteins. Here, we report that siRNA production is an ancient function of an evolutionarily conserved clade of PPR genes that undergo extensive within-species diversification. In Arabidopsis thaliana, siRNA-source PPRs are physically clustered in one locus on Chromosome 1. These sequences are diversified through gene duplication followed by sequence diversification as well as accumulation of high-impact variations including pseudogenization. This diversity leads to the accumulation of a diverse PPR-siRNA pool, consistent with an engagement in a co-evolutionary arms race with the pathogens. This study defines siRNA-producing PPRs as a family of defense genes and highlights the potential of PPR-siRNA-based engineering for enhancing broad-spectrum disease resistance.

plant biology↗

bioRxiv2025

An effector from the potato late blight pathogen bridges ENTH-domain protein TOL9a to an activated helper NLR to suppress immunity

Pathogens counteract central nodes of NLR immune receptor networks to suppress immunity. However, the mechanisms by which pathogens hijack helper NLR pathways are poorly understood. Here, we show that an effector from the potato late blight pathogen Phytophthora infestans bridges the host protein NbTOL9a, a putative member of the host ESCRT pathway, to a helper NLR to suppress immunity. In this work, we solved the crystal structure of the RXLR-LWY effector AVRcap1b in complex with the ENTH domain of NbTOL9a. The structure revealed that unlike other RXLR-LWY effectors, AVRcap1b has a novel L-shaped fold that defines a new structural family of effectors in the Phytophthora genus. Moreover, we defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that dont bind NbTOL9a, impairing immune suppression. This indicates that ENTH binding is required for full virulence activity of this effector. Lastly, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. This suggests that the effector functions as a bridge that interconnects NbNRC2 with the NbTOL9a pathway. These results illustrate an unprecedented pathogen mechanism to hijack helper NLR pathways and suppress immunity.

plant biology↗

bioRxiv2025

The autoactivity of tomato helper NLR immune proteins of the NRC clade is unaltered in prf mutants of Nicotiana benthamiana

Nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) can function in networks of sensors and helpers to induce hypersensitive cell death and immunity against pathogens. The tomato sensor NLR Prf guards the Pto kinase from AvrPto and AvrPtoB effector perturbation and activates the downstream helpers NRC2 and NRC3. Prf is conserved across the Solanaceae and its ortholog in the model species Nicotiana benthamiana is also required for detection of AvrPto/AvrPtoB function on Pto. A recent study reported that cell death induction after transient expression of an autoactive mutant of tomato NRC3 is abolished upon RNAi silencing of Prf in N. benthamiana. Here we generated loss-of-function prf mutants in N. benthamiana and demonstrate that autoactive mutants of eight canonical tomato NRCs (NRC0, NRC1, NRC2, NRC3, NRC4a, NRC4b, NRC6, and NRC7) still induce hypersensitive cell death when expressed transiently in the prf mutant background. Autoactive tomato NRCs also triggered cell death when expressed in lettuce (Lactuca sativa), an Asteraceae plant that does not have a Prf ortholog. These results confirm a unidirectional dependency of sensors and helpers in the NRC network and underscore the value of the N. benthamiana and lettuce model systems for studying functional relationships between paired and networked NLRs.

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

Can AI modelling of protein structures distinguish between sensor and helper NLR immune receptors?

NLR immune receptors can be functionally organized in genetically linked sensor-helper pairs. However, methods to categorize paired NLRs remain limited, primarily relying on the presence of non-canonical domains in some sensor NLRs. Here, we propose that the AI system AlphaFold 3 can classify paired NLR proteins into sensor or helper categories based on predicted structural characteristics. Helper NLRs showed higher AlphaFold 3 confidence scores than sensors when modelled in oligomeric configurations. Furthermore, funnel-shaped structures--essential for activating immune responses--were reliably predicted in helpers but not in sensors. Applying this method to uncharacterized NLR pairs from rice, we found that AlphaFold 3 can differentiate between putative sensors and helpers even when both proteins lack non-canonical domain annotations. These findings suggest that AlphaFold 3 offers a new approach to categorize NLRs and enhances our understanding of the functional configurations in plant immune systems, even in the absence of non-canonical domain annotations.

plant biology↗

bioRxiv2024

Semaphorin 4A maintains functional diversity of the hematopoietic stem cell pool

Somatic stem cell pools are comprised of diverse, highly specialized subsets whose individual contribution is critical for the overall regenerative function. In the bone marrow, myeloid-biased HSC (myHSC) are indispensable for replenishment of myeloid cells and platelets during inflammatory response but at the same time, become irreversibly damaged during inflammation and aging. Here, we identify an extrinsic factor, Semaphorin 4A (Sema4A), which non cell-autonomously confers myHSC resilience to inflammatory stress. We show that the absence of Sema4A, myHSC inflammatory hyper-responsiveness in young mice drives excessive myHSC expansion, myeloid bias and profound loss of regenerative function with age. Mechanistically, Sema4A is mainly produced by neutrophils, signals via a cell surface receptor Plexin D1 and safeguards myHSC epigenetic state. Our study shows that by selectively protecting a distinct stem cell subset, an extrinsic factor preserves functional diversity of somatic stem cell pool throughout organismal lifespan.

cell 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↗

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↗

bioRxiv2022

Resurrection of plant disease resistance proteins via helper NLR bioengineering

Parasites counteract host immunity by suppressing helper NLR proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. A single amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a novel strategy for resurrecting disease resistance in crop genomes. One sentence summaryA helper NLR is mutated to evade inhibition by a parasite effector.

plant biology↗