Lida Derevnina

Agrobacterium -mediated transient expression has revolutionised 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.

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.

Plant-parasitic nematodes are a diverse, polyphyletic group of plant pathogens which can infect most plant tissues and all major crops. Amongst the most damaging clades are the cyst nematodes, which can remain dormant in the soil for decades as infection-competent, developmentally arrested, second-stage juveniles in eggs. Hatching is stimulated by a variety of factors. However, the impact of hatching factor responsiveness on nematode morphology, physiology, gene expression, and infection biology has not been explored. We examined the impact of hatching time on the beet cyst nematode, Heterodera schachtii . We found that late hatchers invaded host roots and established feeding sites in greater numbers than early hatchers. We demonstrate variation in baseline parasitism gene expression and in responsiveness of genes to effectostimulins, small, plant-derived molecules which upregulate parasitism genes. Three quarters of effectostimulin-induced transcriptional changes were also modulated, either positively or negatively, by hatching time. While there were no observable morphological differences between early and late hatching nematodes on the day of their emergence from the egg, the late hatchers displayed signs of faster utilisation of internal energy reserves after 7 days at 4°C, as evidenced by less body area attributed to fat, than early hatchers. Finally, we found no evidence of substantive genetic differences between early and late hatchers, they were representative of a single population, despite the observed differences in infection, gene expression, and physiology. Taken together, non-genetic differences likely drive late hatchers to more rapidly utilise their internal energy reserves, to be more responsive to host-derived signals, and to be ultimately more infective than their early hatching counterparts.

Summary: Plant‐parasitic nematodes and their hosts engage in a continuous exchange of signals cross‐kingdom. On the one hand, parasites exploit host‐derived metabolites, proteins, and RNAs to sense host identity, proximity, and condition – and as a basis for manipulating host processes to their benefit. On the other hand, hosts respond to nematode‐derived pheromones, proteins, and metabolites to either subvert or accommodate parasitism. Our recent understanding is that microbes play a role mediating, at least in part, a communication, which ultimately shapes the developmental trajectories of both host and parasite, in concert. In this Tansley insight, we review the current understanding of communication between plants and parasitic nematodes, framed by the developmental timeline. We focus on notable, reciprocal signals between plant and parasite that gate key life cycle transitions in the parasite, while acknowledging that these processes are embedded within, and inseparable from, a plethora of other signals – and indeed communication with other organisms. The frequency, reciprocity, and gravity of the exchange leads us to argue that it is best described as a nuanced communication, in which parasitism succeeds or fails based on interpretation and response.

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.

Roots of quince (Cydonia oblonga Mill.) exhibiting lesions/darkening were collected from private property in Sheffield, England (53°23'23.1"N 1°30'53.4"W), along with rhizospheric soil. Nematode extraction revealed the presence of Pratylenchus specimens (60 nematodes per g of root; 100 nematodes/cm3 of soil). No above-ground symptoms were evident. Females were slender with the labial region slightly offset from the body and comprising three annuli (Figure 1A and 1B), measuring 505.6 µm (427.0 - 580.5), a = 22.5 (19.1 - 25.1), b= 6.85 (5.3 - 10.0), c= 17.3 (9.4 - 27.0), and V% = 81.6 (78.9 - 83.0) (n = 10). Basal knobs were rounded but frequently found as slightly indented/cupped in some individuals (1B and 1C). The tail was rounded and smooth (Figure 1E). Males were common and slightly smaller than females (Figure 1D), measuring 482 µm (477.81 - 489.22), a = 25.9 (24.6 - 27.3), b= 7.3 (6.2 - 9.5), c= 15.2 (14.0 - 15.0) (n = 4). These characteristics are consistent with Pratylenchus penetrans (Loof, 1991; Castillo and Vovlas, 2007). To validate morphological observations, molecular identification was carried out using 28S rDNA D2A/D3B primers (Gamboa-Cortés et al., 2023). DNA was extracted from hand-picked individuals according to Sonawala et al. (2024). PCR was carried out using 5 µL of the digested sample with KOD Xtreme polymerase (Sigma Aldrich) using an initial denaturation at 94˚C for 2 min; 40 cycles of 98˚C for 10 s, 55˚C for 30 s and 68˚C for 1 min). Purified PCR products (Monarch Gel Extraction Kit) were sequenced, compared to those publicly available for Pratylenchus spp. and Xiphinema index, and aligned and trimmed using Clustal Omega (Madeira et al., 2024) on Geneious (2025.0.3) using default settings. Model selection (TPM3+G4) and phylogenetic tree inference (SH-aLRT, 1000 bootstraps (Minh et al., 2020)) were performed on the aligned sequences using IQ-TREE 2 version 2.2.0 (Figure 2A). Phylogenetic analysis corroborated the morphological identification of P. penetrans (Figure 2A). Nematodes were multiplied and maintained on maize (Zea mays L.) 'B73' and tomato (Solanum lycopersicum L.) 'Moneymaker' in growth chambers (light/dark cycle of 16h/8h at 30°C/21°C, respectively). To assess the host status of quince, five plants of C. oblonga (acquired commercially), and an equal number of Maize 'B73', were inoculated with 1,200 individuals. The inoculum was administered into two holes (2 cm depth) close to the stem and partially filled with vermiculite to avoid heat stress. Plants were assessed 90 days post-inoculation, as previously reported for sufficient reproduction of Pratylenchus brachyurus on Eucalyptus spp. (Souza and Inomoto, 2021). For quince and maize, similar reproductive values (2.3 and 2.2) and nematodes per gram of root (410 and 426) were found, respectively. Quince-infested roots were lesioned and decaying (Figure 2B), and reduced in weight by 31% compared to non-inoculated controls (7.8 g and 11.3 g, respectively, p<0.05, Student's t-test). Taken together, these data demonstrate that P. penetrans successfully reproduces on quince, and induces significant damage to host roots, indicating both host status and potential economic impact. The presence of P. penetrans should be considered when establishing quince orchards, as trees can become a source of inoculum over-season and/or to fast-growing crops (e.g., wheat or maize) grown near them. To our knowledge, this is the first report of P. penetrans on quince in England.

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.

Plants’ complex immune systems include nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, which help recognize invading pathogens. In solanaceous plants, the NRC (NLR required for cell death) family includes helper NLRs that form a complex genetic network with multiple sensor NLRs to provide resistance against pathogens. However, the evolution and function of NRC networks outside solanaceous plants are currently unclear. Here, we conducted phylogenomic and macroevolutionary analyses comparing NLRs identified from different asterid lineages and found that NRC networks expanded significantly in most lamiids but not in Ericales and campanulids. Using transient expression assays in Nicotiana benthamiana, we showed that NRC networks are simple in Ericales and campanulids, but have high complexity in lamiids. Phylogenetic analyses grouped the NRC helper NLRs into three NRC0 subclades that are conserved, and several family-specific NRC subclades of lamiids that show signatures of diversifying selection. Functional analyses revealed that members of the NRC0 subclades are partially interchangeable, whereas family-specific NRC members in lamiids lack interchangeability. Our findings highlight the distinctive evolutionary patterns of the NRC networks in asterids and provide potential insights into transferring disease resistance across plant lineages.

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.

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. Teaser Helper NLR subfunctionalization alters transient interactions with sensor NLRs, enhancing plant immune system complexity.