Genetically encoded pigments are powerful visual reporters and creative tools for biology, yet in plants the palette of pigment biosynthesis genes has remained largely limited to red betacyanins encoded by RUBY. Here we develop and characterize three new polycistronic constructs AMBER\_v1, AMBER\_v2, and GOLD that contain betalain biosynthesis enzymes to produce yellow, fluorescent betaxanthins in plant tissues. These tools expand the palette of publicly available pigmentation genes for use in plant research, education, and floral design. ### Competing Interest Statement The authors have declared no competing interest. Gatsby Charitable Foundation, https://ror.org/0290hax27 Swiss National Science Foundation, https://ror.org/00yjd3n13 O'Shaughnessy Ventures

Plant nucleotide–binding leucine–rich repeat (NLR) immune receptors typically confer resistance through recognition of specific pathogen effectors. The Arabidopsis NLR WRR4A defies this paradigm by recognizing multiple sequence-divergent effectors from Albugo candida, conferring resistance to multiple pathogen races. Despite minimal sequence similarity, these effectors share a conserved N–terminal ferredoxin–like fold. Through cryo–EM structure determination of two WRR4A resistosomes bound to sequence–distinct effectors, combined with AlphaFold modelling, we reveal a shape–based recognition mechanism: WRR4A engages structurally conserved backbone features of the effectors in a mostly side chain-independent manner, enabling recognition of diverse effectors with similar three–dimensional architectures. These insights guided successful engineering of WRR4A to acquire novel recognition specificity. In addition, analysis of the monomeric WRR4A resting state reveals a distinct domain architecture characteristic of C–JID—containing TIR–NLRs and informs their activation mechanism. This work provides insights into NLR–mediated broad-spectrum recognition and the potential for structure–informed engineering of improved crop resistance. ### Competing Interest Statement The authors have declared no competing interest. BBSRC, BB/P021646/1, BB/S018832/1, BB/W017423/1 Gatsby Charitable Foundation UKRI Biotechnology and Biological Sciences Research Council Norwich Research Park Biosciences Doctoral Training Partnership, BB/T008717/1 Wellcome Trust, 202679/Z/16/Z, 206166/Z/17/Z EMBO Postdoctoral Fellowship, EMBO ALTF88-2021

Helper NLRs function as central nodes in plant immune networks. Upon activation, they oligomerize into inflammasome-like resistosomes to initiate immune signaling, yet the dynamics of resistosome assembly remain poorly understood. Here, we show that the virulence effector AVRcap1b from the Irish potato famine pathogen Phytophthora infestans suppresses immune activation by directly engaging oligomerization intermediates of the tomato helper NLR SlNRC3. Cryo-EM structures of SlNRC3 in AVRcap1b-bound and unbound states reveal that AVRcap1b bridges multiple protomers, stabilizing a stalled intermediate and preventing formation of a functional resistosome. Leveraging AVRcap1b as a molecular tool, we also capture an additional SlNRC3 resistosome intermediate showing that assembly proceeds in a stepwise manner from dissociated monomers. These findings uncover a previously unrecognized vulnerability in NLR activation and reveal a pathogen strategy that disrupts immune complex assembly. This work advances mechanistic understanding of resistosome formation and uncovers a previously unrecognized facet of pathogen-plant coevolution.

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 NLRs. However, the degree to which evolutionary patterns reflect functional divergence remains poorly understood, notably in important crop species. Within the NRC (NLR Required for Cell Death) network in Asterids, sensor NLRs detect pathogen presence but require NRC helpers for signaling and to confer immunity. 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 an Asterales NRC network with 2 helpers and 9 sensors in common lettuce (Lactuca sativa). Through selection analysis and structural modeling of NRC gene family in the Lactuca genus, we found distinct evolutionary trajectories between NRC helpers and sensors. Sensors reliant on the phylogenetically conserved helper NRC0 experience limited diversification, whereas sensors dependent on other NRC helpers show higher rates of positive selection and gene duplication. Our results highlight the lineage- and function-specific evolution of the NRC network, offering insights into the evolutionary pressures shaping plant immune receptor networks.

Abstract 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.

Protein evolution is influenced by historical contingencies and functional constraints, but their combined impact on rapidly diversifying pathogen virulence effectors remains poorly understood. Here, we combined ancestral state reconstructions and functional assays to recapitulate the evolution of the MAX-fold effector protein APikL2 of the plant pathogenic blast fungus Magnaporthe (syn. Pyricularia) oryzae, focusing on the ancestral and functionally critical amino acid residue D66 (Asp, codon: GAT). 'Rewinding the tape' experiments based on ancestral sequence resurrection revealed that, out of the seven potential amino acid substitutions derived from single nucleotide polymorphisms, only the naturally occurring D66N (Asp to Asn, GAT to AAT) expanded the binding spectrum to host plant proteins of the heavy metal associated (HMA) family. In contrast, three of the non-synonymous substitutions were deleterious resulting in loss of binding to HMA proteins. Additionally, we identified three cases of homoplasy in the APikL effector family, involving HMA-binding interfaces, indicating recurrent convergent evolution. Our findings suggest an experimental framework for predicting evolutionary outcomes of pathogen effector-host target interactions with implications for plant disease resistance breeding.

Abstract 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 SNI NRC-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.

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 β-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. ### Competing Interest Statement S.K. receives funding from industry to study NLR biology and is a co-founder of start-up companies that focus on plant disease resistance. J.K. and S.K. have filed patents on NLR biology. The other authors declare that they have no competing interests. Fundação para a Ciência e Tecnologia (FCT), UI/BD/151214/2021, CEECIND/00198/2017, UID/04551/2025, UID/PRR/04551/2025, LA/P/0087/2020 Gatsby Charitable Foundation, https://ror.org/0290hax27 Biotechnology and Biological Sciences Research Council (BBSRC), BB/P012574, BBS/E/J/000PR9795, BBS/E/J/000PR9796, BBS/E/J/000PR9797, BBS/E/J/000PR9798 European Research Council, 743165 Engineering and Physical Sciences Research Council, EP/Y032187/1

Nucleotide-binding, leucine-rich repeat (NLR) receptors are widespread intracellular immune sensors across kingdoms. Plant G10-type coiled-coil (CCG10)-NLRs constitute a distinct phylogenetic clade that remains poorly characterized. Here, we identified a gain-of-function mutant of wheat autoimmunity 3 (WAI3GOF), which encodes a constitutively active CCG10-NLR resulting from a residue substitution in the leucine-rich repeat (LRR) domain. Cryo-electron microscopy (cryo-EM) analysis reveals that activated WAI3 assembles into a distinctive octameric resistosome. Arabidopsis RPS2, another CCG10-NLR, also forms an octamer, indicating a conserved structural property across monocot and dicot plants. The WAI3 resistosome induces a prolonged and sustained increase in cytosolic calcium, likely facilitated by a unique channel architecture arising from its divergent coiled-coil (CC) domain configuration. Notably, this domain arrangement may be shared by plant NLRs that lack the conserved EDVID (Glu-Asp-Val-Ile-Asp) motif in their CC domains. Together, our findings uncover a conserved yet previously uncharacterized NLR resistosome structure and provide insights into the plant immune receptor plasticity.

NLR annotation files, data, and related analysis outputs for preprint "Legume NLR immune receptors exhibit tissue-specific expression patterns across species"

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. We show that an effector from the late blight pathogen Phytophthora infestans interacts with the host protein NbTOL9a and a helper NLR to suppress immunity. 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 previously unidentified L-shaped fold that defines a distinct structural family of effectors in the genus Phytophthora. We defined the AVRcap1b/NbTOL9a binding interface and designed effector mutants that do not bind NbTOL9a, impairing immune suppression. This suggests that ENTH binding is required for full virulence activity. Last, we show that AVRcap1b associates specifically with activated NbNRC2 independently of NbTOL9a binding. We propose a model in which the effector interconnects NbNRC2 with the NbTOL9a pathway. Our results illustrate a previously uncharacterized pathogen mechanism to hijack NLR pathways and suppress immunity.

Upon activation, plant nucleotide-binding leucine-rich repeat (NLR) immune receptors are known to assemble into oligomeric resistosomes that insert into the plasma membrane, forming calcium (Ca2+)-permeable channels and triggering immunity. Here, we found that the RPW8-like coiled-coil NLR (CCR-NLR) N requirement gene 1 (NRG1) primarily targets organelles instead of the plasma membrane. Unlike canonical CC-NLRs, activated NRG1 accumulated at the chloroplast envelope and channeled stromal Ca2+ into the cytosol. AlphaFold modeling of the NRG1 resistosome revealed an unusually long amino-terminal membrane-insertion structure that could span the double membrane of the chloroplast. Nanobody-mediated relocalization showed functional membrane specificity: Chloroplast trapping abolished activity of the canonical helper CC-NLR NRC4 but not NRG1. NRG1 orthologs, from nonflowering lineages to angiosperms, targeted chloroplasts, suggesting that organelle-centered defense dates back at least ~360 million years. We propose that CC-NLR diversification has enabled compartment-specific immune signaling to capture diverse Ca2+ stores.

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 an NLR immune receptor against a parasite inhibitor, 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.

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 don’t 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. ### Competing Interest Statement T.O.B. and S.K. receive funding from industry on NLR biology and cofounded a start-up company (Resurrect Bio Ltd.) on resurrecting disease resistance. M.P.C., L.D., and S.K. have filed patents on NLR biology. M.P.C. and L.D. have received fees from Resurrect Bio Ltd. The other authors declare that they have no competing interests. European Research Council, https://ror.org/0472cxd90, 743165 British Society for Plant Pathology, https://ror.org/04zj17j36 Biotechnology and Biological Sciences Research Council, BB/P012574, BBS/E/J/000PR9795, BBS/E/J/000PR9796, BBS/E/J/000PR9797, BBS/E/J/000PR9798, BB/V002937/1

Phytophthora is a long-established, well-known, and globally important genus of plant pathogens. Phylogenetic evidence has shown that the biologically distinct, obligate biotrophic downy mildews evolved from Phytophthora at least twice. Because, cladistically, this renders Phytophthora "paraphyletic," it has been proposed that Phytophthora evolutionary clades be split into multiple genera (Crous et al. 2021; Runge et al. 2011; Thines 2023, 2024). In this letter, we review arguments for the retention of the generic name Phytophthora with a broad circumscription made by Brasier et al. (2022) and by many delegates at an open workshop organized by The American Phytopathological Society. We present our well-considered responses to the genus splitting proposals, both in general terms and in terms of the specific proposals for new genera, alongside new information regarding the biological properties and mode of origin of the Phytophthora clades. We consider that the proposals are mostly non-rigorous and not supported by the scientific evidence. Further, given (i) the apparent lack of any distinguishing biological characteristics (synapomorphies) between the Phytophthora clades; (ii) the fundamental monophyly of Phytophthora in the original Haeckelian sense (Haeckel 1877); (iii) the fact that paraphyly is not a justification for taxonomic splitting; and (iv) the considerable likely damage to effective scientific communication and disease management from an unnecessary breakup of the genus, we report that workshop delegates voted unanimously in favor of preserving the current generic concept and for seeking endorsement of this view by a working group of the International Commission on the Taxonomy of Fungi. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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. ### Competing Interest Statement J.K, C.M. and M.P.C. received funding from industry to study NLR biology at the time of the study. S.K. receives funding from industry to study NLR biology and co-founded a start-up company (Resurrect Bio Ltd.). C.M., M.P.C., J.K., and S.K. have filed patents on NLR biology. M.P.C. received fees from Resurrect Bio Ltd. Gatsby Charitable Foundation, https://ror.org/0290hax27 Biotechnology and Biological Sciences Research Council, BB/P012574 European Research Council, 743165 BASF Plant Science DFG Walter Benjamin Programme, 464864389

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. ### Competing Interest Statement S.K. receives funding from the industry for NLR biology and has co-founded a start-up company (Resurrect Bio Ltd.) related to NLR biology. S.K., J.K., and M.P.C. have filed patents on NLR biology. M.P.C. has received fees from Resurrect Bio Ltd.

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. ### Competing Interest Statement S.K. has filed patents on NLR biology, receives funding from industry on NLR biology, and is a co-founder of start-up companies that focus on plant disease resistance.

Plant nucleotide-binding domain and leucine-rich repeat immune receptors (NLRs) confer disease resistance to many foliar and root parasites. However, the extent to which NLR-mediated immunity is differentially regulated between plant organs is poorly known. Here, we show that a large cluster of tomato (Solanum lycopersicum) genes, encoding the cyst and root-knot nematode disease resistance proteins Hero and MeR1 as well as the NLR helper NLR required for cell death 6 (NRC6), is nearly exclusively expressed in the roots. This root-specific gene cluster emerged in Solanum species about 21 million years ago through gene duplication of the ancient asterid NRC network. NLR sensors in this gene cluster function exclusively through NRC6 helpers to trigger hypersensitive cell death. These findings indicate that the NRC6 gene cluster has sub-functionalized from the larger NRC network to specialize in mediating resistance against root pathogens, including cyst and root-knot nematodes. We propose that some NLR gene clusters and networks may have evolved organ-specific gene expression as an adaptation to particular parasites and to reduce the risk of autoimmunity.

The rice blast fungus Magnaporthe oryzae secretes a battery of effector proteins to facilitate host infection. Among these effectors, pathogenicity toward weeping lovegrass 2 (Pwl2) was originally identified as a host specificity determinant for the infection of weeping lovegrass (Eragrostis curvula) and is also recognized by the barley (Hordeum vulgare) Mla3 resistance protein. However, the biological activity of Pwl2 remains unknown. Here, we showed that the Pmk1 MAP kinase regulates PWL2 expression during the cell-to-cell movement of M. oryzae at plasmodesmata-containing pit fields. Consistent with this finding, we provided evidence that Pwl2 binds to the barley heavy metal-binding isoprenylated protein HIPP43, which results in HIPP43 displacement from plasmodesmata. Transgenic barley lines overexpressing PWL2 or HIPP43 exhibit attenuated immune responses and increased disease susceptibility. In contrast, a Pwl2SNDEYWY variant that does not interact with HIPP43 fails to alter the plasmodesmata localization of HIPP43. Targeted deletion of 3 PWL2 copies in M. oryzae resulted in a Δpwl2 mutant showing gain of virulence toward weeping lovegrass and barley Mla3 lines, but reduced blast disease severity on susceptible host plants. Taken together, our results provide evidence that Pwl2 is a virulence factor that suppresses host immunity by perturbing the plasmodesmatal deployment of HIPP43.