Publications

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. CryoEM 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 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.

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. CryoEM 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.

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.

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant-derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TOPGAP, to remodel the host-pathogen interface. The effector, PiE354, hijacks TOPGAP as a susceptibility factor to usurp its GAP activity on Rab8a, a key Rab GTPase crucial for defense-related secretion. By hijacking TOPGAP, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co-opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

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.

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.

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.

NRCs are essential helper NLR (nucleotide-binding domain and leucine-rich repeat) proteins that execute immune responses triggered by sensor NLRs. The resting state of NbNRC2 was recently shown to be a homodimer, but the sensor-activated state remains unclear. Using cryo-EM, we determined the structure of sensor-activated NbNRC2, which forms a hexameric inflammasome-like resistosome. Mutagenesis of the oligomerization interface abolished immune signaling, confirming the functional significance of the NbNRC2 resistosome. Comparative structural analyses between the resting state homodimer and sensor-activated homohexamer revealed substantial rearrangements, providing insights into NLR activation mechanisms. Furthermore, structural comparisons between NbNRC2 hexamer and previously reported CC-NLR pentameric assemblies revealed features allowing an additional protomer integration. Using the NbNRC2 hexamer structure, we assessed the recently released AlphaFold 3 for predicting activated CC-NLR oligomers, revealing high-confidence modeling of NbNRC2 and other CC-NLR amino-terminal α1 helices, a region proven difficult to resolve structurally. Overall, our work sheds light on NLR activation mechanisms and expands understanding of NLR structural diversity.