Stuart Ronan Fisher

During disease and infestation, pathogens and pests deliver effectors inside their host to manipulate host responses, leading to immune suppression and altered nutrient availability. To be successful, pathogens and pests can produce and secrete large repertoires of effectors, consisting of up to hundreds of proteins. While effector virulence activities are usually studied in isolation, effectors can function together or cooperate to regulate transport and/or activity. An important example is the suppression of effector avirulence activity by plant pathogenic microbe effectors. Moreover, some effectors can physically associate with target host proteins and promote virulence. Here we explored the interaction of a conserved and co-regulated effector pair in aphids. Using a combination of computational modelling and crystallography combined with functional assays, we reveal a novel type of oligomeric assembly of the paired aphid effectors in a hexameric/octameric complex. Our data indicate that different oligomerization states exist among orthologs of this effector complex, suggesting that dynamic dissociation or subunit exchange may occur. Structure-based mutagenesis revealed that complex formation is essential for both effector protein stability and the in planta activity of one of these proteins. These findings emphasize the significance of effector hetero-oligomerization at the plant-insect interface, consistent with the model that this effector pair is an evolutionarily conserved module across aphid species.

Human calmodulin-1 (CaM) undergoes calcium-dependent conformational changes that are difficult to capture owing to substantial structural flexibility. This study aims to show that double electron-electron resonance (DEER) spectroscopy can measure ensembles of CaM conformations. The most probable distances are compared to structural models from experimental structures and deep-learning predictions. Four double-cysteine variants of a recombinant CaM were engineered for site-directed spin labeling. Spin-labeled CaM was measured with DEER spectroscopy in both the presence (holo CaM) and absence (apo CaM) of Ca(II). Distance distributions were compared with predictions derived from Protein Data Bank structures and models generated from AlphaFold2 and IntFOLD7. DEERefiner was used to generate conformers from these models using the distance-distributions as restraints. The distance distributions from DEER were consistent with conformational heterogeneity. Distance distributions differed between apo and holo conditions. The results with Ca(II) were consistent with an ensemble of conformations including the canonical crystallographic structure (1CLL). Apo state distance distributions were not reproduced well by the models. Upon refinement with the distance distributions the models aligned better with the most probable distances from the apo and holo results. Comparisons of the refined models indicated that Ca(II) binding causes subtle rearrangements between EF-hand domains. The DEER-derived distance distributions for the four doubly-labeled CaM variants demonstrate that apo and holo CaM populate broad but different conformational ensembles in frozen solution. Comparison of apo and holo distributions indicate that Ca(II) binding induces a subtle, yet significant, structural rearrangement. These results illustrate how DEER-guided modeling can provide deeper insight into flexible protein ensembles that are not captured by existing static structures.

Summary: Land plants secrete a protective outer cuticular layer with diverse functions. Barley ( Hordeum vulgare L.) develops two cuticular specialisations: the β‐diketone rich wax bloom on vegetative tissues and an adherent grain surface which sticks to the hulls, leading to barley's distinctive ‘covered’ grain phenotype. Two barley SHINE transcription factors, HvWIN1 and NUD, promote the wax bloom and covered grain phenotypes, respectively, yet we know little about other genes involved. We investigated a barley mutant showing defects in both cuticular specialisations to better understand the networks and processes underlying these traits. We identified the barley BODYGUARD1 ( HvBDG1 ) gene encoding an α/β hydrolase as crucial for leaf cuticular integrity and wax‐bloom deposition. Mining tetraploid wheat ( Triticum turgidum ssp. durum ) mutant populations demonstrated that BDG1 and WIN1 orthologues also control the wax bloom in wheat. We further reveal that NUD and HvWIN1 retain functional, independent overlap in leaf cuticle integrity and hull adhesion in barley, functions which involve upregulation of HvBDG1 and other direct or indirect targets, shared and distinct, depending on the stage and tissue. Our work greatly expands our knowledge of genetic and developmental mechanisms underlying shared and distinctive cuticular features in plants.

Pathogen and pest effectors play a crucial role in manipulating plant biological processes, facilitating infection and infestation. While pathogens and pests secrete repertoires of effectors into host plants, most effector function studies focus on characterizing individual proteins. Our previous work identified a genetically linked and co-regulated gene pair in the aphid pest Myzus persicae encoding effectors Mp1 and Mp58. Here, we explored the functional link between these two effectors. We revealed that effectors Mp1 and Mp58 interact in planta and in vitro and form an oligomeric complex. The putative orthologues of the Mp1–Mp58 pair in the aphid species Rhopalosiphum padi, Rp1 and Rp58, also interact, but members of the pair cannot interact across aphid species, suggesting that effector pairs have co-evolved within each aphid species but diversified across species. Both Mp1 and Mp58 associate with the host Vacuolar Protein Sorting associated Protein 52 (VPS52) to form an Mp1–Mp58–VPS52 complex, which localizes at vesicle-like structures. Our findings point to effector complex formation in plant–insect interactions and highlight a further layer of complexity in the molecular dialogue between insects and their host plants. Our work highlights the importance of considering the context in which effectors may function within a larger effector repertoire.

Lanthanide(III) complexes are invaluable tools for probing protein structure and dynamics, enabling precise distance measurements and site‐specific labeling across electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and luminescence‐based techniques. Designing an optimal lanthanide tag requires high thermodynamic and kinetic stability, minimal linker flexibility, and rapid, quantitative reactivity under mild conditions. Herein, a short series of Ln(III) complexes developed as efficient protein tags is reported, each showing high chemoselectivity for cysteine and enabling labeling in aqueous solution under mild conditions (pH 8, 37 °C). Subtle modifications to the reactive arm of the parent Ln.L1 complex has a pronounced effect on tagging kinetics, with Tb.QuNO achieving >95% labeling of the free amino acid cysteine within 10 min. The more compact and readily synthesized Gd.PyNO also labels efficiently and exhibits a narrow EPR linewidth (3.1 mT average at Q‐band across three protein‐labeled samples), making it an optimal choice for many EPR applications. These findings highlight the critical role of linker design in lanthanide‐based probes for EPR spectroscopy and biological imaging.

TonB-dependent uptake systems of Gram-negative bacterial pathogens constitute prominent virulence factors, allowing nutrient acquisition, primarily siderophore-bound iron, to cross the highly impermeable outer membrane (OM). Remarkably, the ferredoxin uptake system (Fus) of Pectobacteriaceae , a group of soft rot-inducing plant pathogens, imports an entire folded host protein into the periplasm and extracts its bound iron for growth. The inner membrane protein FusB, a TonB homologue, plays two roles in facilitating ferredoxin import. First, like other TonBs, it remodels the globular plug domain obstructing the lumen of the OM receptor FusA to allow ferredoxin passage. Unusually for a TonB protein, FusB then interacts directly with the FusA-bound ferredoxin substrate to facilitate its transport into the periplasm. In this work, we describe structures of FusB-ferredoxin and homodimeric FusB complexes and determine the key features of the binding interfaces formed by FusB with FusA and ferredoxin. We postulate that under resting conditions FusB exists a homodimer, stabilised by an intermolecular R241-D322 salt bridge. The homodimer dissociates when the “FusB-box” of FusA outcompetes one protomer, and FusA D53 displaces FusB D322. Upon ferredoxin binding, FusB undergoes a structural rearrangement, expanding its β-sheet from three to four strands. In agreement with the proposed sequence of events, ferredoxin binding displaces the receptor (FusA) from FusB with Arg241 forming an intramolecular salt bridge with Asp322 to stabilise the newly formed β-hairpin of FusB. We propose a mechanistic model for ferredoxin import in which FusB Arg241 acts as a molecular switch, and two distinct regions function as interaction hotspots.

The outer epidermis of land plants secretes a cuticular layer, a hydrophobic diffusion barrier which minimises water loss into the atmosphere and protects from pests, ultraviolet light and organ fusion. Cuticles typically comprise a polyester cutin matrix embedded and overlaid with cuticular waxes, but their exact chemical make-up, structure and functions can vary widely depending on the tissue and species. Barley shows two such cuticular specialisations: (1) deposition of a thick β-diketone-rich wax bloom on multiple organs at reproductive stage, common in other Poeceae species and linked to yield; and, (2) secretion of a sticky layer on the grain fruit (caryopsis) pericarp cuticle which adheres to inner floral hulls, leading to barley’s distinctive ‘covered’ grain used in animal feed and malting. Two SHINE/WAX-INDUCER transcription factors in barley, HvWIN1 and NUD, promote the wax bloom and hull to caryopsis adhesion, respectively, yet little is understood about other genes involved. Leveraging near-isogenic lines of wax-deficient mutants, we identify the barley BODYGUARD1 ( HvBDG1 ) gene encoding an α/β-hydrolase essential for leaf cuticular integrity and wax bloom deposition. Modelling of functional and defective alleles suggests that HvBDG1 N-terminal region control of protein flexibility is important for HvBDG1 function. In addition to their role in controlling barley epicuticular wax deposition, we show that both HvBDG1 and HvWIN1 are essential for strong hull to caryopsis adhesion. Along with NUD, these gene products differentially contribute to ultrastructural changes on the pericarp associated with a cuticular building programme driven by NUD and HvWIN1 regulation of cuticle metabolism and transport and cell wall-related genes, and correlate with shifts in pericarp surface chemistry. We also show that the previously asserted ‘grain-specific’ role of NUD should be revised, as our findings reveal that it is essential for maintaining leaf cuticle integrity. Our analyses in barley suggest that NUD and HvWIN1 control cuticular specialisations and cuticle integrity in part via promotion of HvBDG1 expression, while HvWIN1 and NUD likely act independently from each other. Lastly, mining tetraploid wheat mutant populations followed by crossing to combine mutated homoeologues demonstrated that BDG1 and WIN1 orthologues also control wax bloom in wheat. Taken together, our work greatly expands the genetic networks and molecular activities important for cuticle development in cereals and the underlying mechanisms for both shared and species-specific cuticular specialisations.

Most phytophthora root rot (PRR) outbreaks in symptomatic commercially cultivated raspberry varieties are associated with the prevalence of Phytophthora rubi . Reduced availability of chemical actives and the persistent presence of Phytophthora oospores in the soil contribute to its devastating impact on raspberry-growing regions. In this study, we examined the variation in root morphology in two contrasting raspberry cultivars, Latham (PRR resistant) and Glen Moy (PRR susceptible). We performed RNA-sequencing on Latham roots challenged with P. rubi, to study the transcriptomic response and uncover mechanisms underpinning resistance. We established a new raspberry reference transcript dataset that allowed quantification of raspberry root gene expression. Transcripts significantly upregulated in Latham challenged with P. rubi , included many with characterised roles in resistance, such as Pathogenesis-related proteins and a Germin-like protein, designated RiABP19. The homologous Glen Moy RiABP19 gene showed no differential transcriptional response to PRR infection, indicating a resistance cultivar-specific induction signature following PRR challenge. Three-dimensional structural modelling predicts that RiABP19 contains conserved active sites implicated in auxin-binding and superoxide dismutase activity and can form a homo-hexamer like true germins. Co-immunoprecipitation assays confirmed that RiABP19 can form both homo- and heterodimers in planta . Virus-induced gene silencing of RiABP19 orthologs of in the model plant Nicotiana benthamiana strongly impacts immune signalling, enhancing Phytophthora infestans colonization and attenuating resistance and cell death triggered by the tomato Cf4/Avr4 interaction. These findings suggest that RiABP19 functions as a positive regulator of immunity and may represent a target for future crop improvement in raspberries.

Host–pathogen co‐evolution shapes resistance (R) proteins and their recognition of pathogen avirulence factors. However, little attention has been paid to naturally occurring genetic diversity in R genes. In this study, 12 Solanum bulbocastanum accessions from the Commonwealth Potato Collection were screened for resistance to Phytophthora infestans, identifying 11 resistant and one susceptible accession. Targeted enrichment sequencing of nucleotide‐binding leucine‐rich repeat (NLR) genes using RenSeq, followed by diagnostic RenSeq (dRenSeq) analysis, revealed that all accessions except 7650 contained Rpi‐blb1/RB variants. Variants in accessions 7641 and 7648 were non‐functional, while three novel functional variants were identified. Cloning and functional analysis of Rpi‐blb1/RB variants assessed their recognition of the avirulence factor IPI‐O1. Three variants were functional, conferring resistance to P. infestans. Variants in accessions 7644 and 7647 also recognized IPI‐O4, confirmed in transgenic potatoes. Analysis of a non‐functional variant in S. bulbocastanum accession 7648 identified amino acid Ser347 in the nucleotide‐binding (NB‐ARC) domain as critical for cell‐death initiation following IPI‐O1 recognition. Predictions from the FunFOLD2 protein–ligand interaction model suggested that Ser347 is essential for ATP binding, suggesting potential inhibition on pentameric resistosome assembly. Western blot analysis revealed that the mutation of Ser347 to Asn markedly compromises the Rpi‐blb1/RB protein stability, and co‐immunoprecipitation assay further confirmed that this mutation severely disrupts the self‐association of CCNB, thereby preventing Rpi‐blb1/RB activation. Consistently, substituting Asn347 with serine restored function, underscoring its key role in Rpi‐blb1/RB activity. Cell biology experiments demonstrated that Rpi‐blb1/RB relocalizes to the plasma membrane in response to IPI‐O1. This relocalization depends on Ser347, further supporting the idea that its mutation affects resistosome formation, impairing resistance. This study provides an in‐depth functional analysis of natural Rpi‐blb1/RB diversity, offering insights into NLR protein evolution and resistance mechanisms in potatoes.