In most legume-rhizobium symbioses, rhizobial colonization occurs through host-derived intracellular infection threads, which enable rhizobial recruitment while presumably modulating the host immune system to prevent rejection. To investigate post-translational regulation of immune responses during rhizobial infection, we focused on Cyclophilin A (CyPA), a peptidyl-prolyl cis/trans isomerase. The model legume Lotus japonicus encodes three canonical CyPA genes. Using CRISPR/Cas9 mutagenesis, structural modeling, and phylogenomics, we characterized LjCyPA1 as essential for normal intracellular infection by compatible rhizobia. A gain-of-function LjCyPA1 variant in a soybean cultivar promoted symbiosis with both compatible and incompatible rhizobia. Functional association between LjCyPA1 and the immune hub protein LjRIN4 is essential for symbiosis. The putative cis-LjRIN4 promoted intracellular rhizobial infection, while the putative trans-LjRIN4 suppressed it. LjCyPA1 and LjRIN4 acted in concert with the rhizobial type III secretion system (T3SS), highlighting a cooperative role between host and symbiont in facilitating infection. Our results contribute to the understanding of how legumes accept symbiotic partners while balancing immune responses.

Nitrogen-fixing nodule symbiosis is an ecologically and economically important trait in legumes and some related species. A critical step in the evolution of nodulation is the recruitment of NODULE INCEPTION (NIN); a homolog of the nitrate-sensing NIN-LIKE PROTEIN (NLP) transcription factors. However, whether adaptations have occurred in the NIN protein upon its recruitment in symbiosis remains elusive. Here we show that non-symbiotic NIN orthologs can function in intracellular infection and even nodule initiation, indicating that these properties of NIN predate the evolution of nodulation. Concurrent with the evolution of nodulation, symbiotic NIN proteins were optimized for their role in symbiosis by acquiring nitrate independent functionality, including constitutive nuclear localization. A single amino acid substitution in the non-symbiotic Arabidopsis AtNLP2 enhances its nuclear localization under low nitrate conditions, making it functionally comparable to the symbiotic Parasponia PanNIN. Our study provides insight in the evolutionary trajectory and molecular adaptation that allowed NIN to function as the central regulator of nitrogen-fixing nodule symbiosis.

Legume symbiosis with nitrogen-fixing bacteria is controlled by a cascade of signaling events leading to root nodule development. While plant cell-surface receptors initiate this process, the link between receptors and cytoplasmic signaling components remains unclear. Here we identify Early Phosphorylated Protein 1 (EPP1) as a central mediator of this pathway. EPP1 is recruited to the activated SYMRK receptor, where it is phosphorylated on a key serine residue, an event essential and sufficient to propagate symbiotic signaling. We provide structural and functional validation of the SYMRK-EPP1 signaling complex and demonstrate that EPP1 is required for root nodule formation. Synthetic engineering of the SYMRK-EPP1 interaction bypasses the need for symbiotic bacteria to initiate the pathway and triggers nodule organogenesis. These findings establish EPP1 as a crucial cytoplasmic component between receptor activation and intracellular signaling, advancing our understanding of nitrogen-fixing symbiosis. ### Competing Interest Statement The authors have declared no competing interest.

Nitrogen-fixing nodule symbiosis is an ecologically and economically important trait in legumes and some related species. A critical step in the evolution of nodulation is the recruitment of NODULE INCEPTION (NIN); a homolog of the nitrate-sensing NIN-LIKE PROTEIN (NLP) transcription factors. However, whether adaptations have occurred in the NIN protein upon its recruitment in symbiosis remains elusive. Here we show that non-symbiotic NIN orthologs can function in intracellular infection and even nodule initiation, demonstrating that these properties of NIN predate the evolution of nodulation. Concurrent with the evolution of nodulation, symbiotic NIN proteins were optimized for their role in symbiosis by acquiring nitrate independent functionality, including constitutive nuclear localization. A single amino acid substitution in Arabidopsis AtNLP2 enhances its nuclear localization under low nitrate conditions, making it functionally comparable to a symbiotic NIN. These findings highlight that NIN was predisposed to function in nodulation at the time of its recruitment into nitrogen-fixing symbiosis. Our study provides novel insight in how and why non-symbiotic NIN orthologues could be recruited to function in nitrogen-fixing root nodule symbiosis. ### Competing Interest Statement Some findings in this manuscript are considered for patent application. Enabling Nutrient Symbioses in Agriculture (ENSA) that is funded by Bill & Melinda Gates Agricultural Innovations, INV- 57461 China Scholarship Council, 201506300062, 201906170085, 202008150090 Nederlandse Organisatie voor Wetenschappelijk Onderzoek, VI.Veni.212.132

Receptor signalling determines cellular responses and is crucial for defining specific biological outcomes. In legume root cells, highly similar and structurally conserved chitin and Nod factor receptor kinases activate immune or symbiotic pathways, respectively, when chitinous ligands are perceived1. Here we show that specific amino acid residues in the intracellular part of the Nod factor receptor NFR1 control signalling specificity and enable the distinction of immune and symbiotic responses. Functional investigation of CERK6, NFR1 and receptor variants thereof revealed a conserved motif that we term Symbiosis Determinant 1 in the juxtamembrane region of the kinase domain, which is key for symbiotic signalling. We show that two residues in Symbiosis Determinant 1 are indispensable hallmarks of NFR1-type receptors and are sufficient to convert Lotus CERK6 and barley RLK4 kinase outputs to enable symbiotic signalling in Lotus japonicus.

Plants perceive and respond to chitin derived from fungal cell walls through lysine motif (LysM) receptor kinases. In the model legume Medicago truncatula, CERK1 and LYR4 represent the LysM receptor pair important for chitin-triggered immunity signaling. Here, we show that both the active kinase receptor CERK1 and the pseudokinase receptor LYR4 contribute to immunity signaling, leading to the production of reactive oxygen species (ROS). We determine the crystal structure of the LYR4 core intracellular domain with a bound nucleotide analog in the active site. Biochemical characterization shows that LYR4 binds ATP and has both autophosphorylation as well as transphosphorylation activity towards CERK1. However, in planta experiments demonstrate that the phosphorylation ability is not necessary for the function of LYR4 in chitin-triggered ROS production, but that the presence of its intracellular domain is indispensable. Together, we show that in chitin-triggered immunity the intracellular domain of LYR4 serves as a signaling scaffold independent of its catalytic activity. ### Competing Interest Statement The authors have declared no competing interest.

Chronic thromboembolic pulmonary hypertension (CTEPH) can develop subsequent to acute pulmonary embolism, with exertional dyspnea being the predominant symptom. Initial evaluation of CTEPH patients should prioritize surgical intervention via pulmonary thromboendarterectomy (PEA). For cases where surgical intervention is not feasible, balloon pulmonary angioplasty (BPA) emerges as a potential treatment. Post-treatment, patients are expected to demonstrate both subjective and objective amelioration. The technique often requires multiple sessions. The safety profile of BPA is comparable to that of open surgical PEA. Further research is imperative to comprehensively evaluate the long-term efficacy and safety of this therapeutic modality.

Plants detect microbes through pattern recognition receptors that perceive conserved microbial surface motifs known as microbe-associated molecular patterns (MAMPs). LysM receptors recognize and mediate downstream responses to chitinous MAMPs. Here, we elucidate the mechanism for the specific recognition of long-chain chitin oligomers and identify a hallmark bridge domain characteristic for the CHIP receptor class. Structural analysis of receptor-ligand complexes, biochemistry, and in planta functional studies using inhibitory nanobodies reveal the mechanism of size-selective, high affinity chitin perception in Lotus japonicus and Medicago truncatula . Additionally, we identify CERKs as low-affinity, yet essential co-receptors and propose a mechanistic model for a ligand-induced core signaling heterocomplex. Our findings provide mechanistic insights into plant chitin perception and the formation of receptor complexes critical for immune signaling. ### Competing Interest Statement The authors have declared no competing interest. Novo Nordisk Foundation, https://ror.org/04txyc737, NNF18OC0052855 Det Frie Forskningsråd, 3103-00137B Carlsberg Foundation, CF21-0139 Danish National Research Foundation, DNRF79 LABoratoires d’EXcellence ARCANE, ANR-17-EURE-0003

Receptor signaling shapes cellular functions and specificity in the signaling output is central for delineating biological processes. In legume root cells, very similar and structurally conserved chitin and Nod factor receptor kinases activate immunity, or symbiosis, respectively after perception of chitinous ligands. Here we show that individual residues in the intracellular part of the Nod factor receptor NFR1 determine signal specificity and efficiency distinguishing immunity and symbiotic signaling. Functional investigation of CERK6, NFR1 and receptor variants hereof revealed a conserved motif that we term Symbiosis Determinant 1 in the juxtamembrane region of the kinase domain that is key for symbiotic signaling. We demonstrate that two residues in Symbiosis Determinant 1 are indispensable hallmarks for NFR1 receptors and are sufficient to convert Lotus CERK6 and barley RLK4 functioning in immunity, to initiate symbiotic signaling. ### Competing Interest Statement A provisional patent application authored by SR, KRA, MT, BWS, CS, CK, MML, DL, SBH, KG, on using these findings for engineering LysM receptor kinases has been filed. The other authors declare no competing interests.

Copper is an essential micronutrient and yet is highly toxic to cells at elevated concentrations. P1B-ATPase proteins are critical for this regulation, providing active extrusion across cellular membranes. One unique molecular adaptation of P1B-ATPases compared to other P-type ATPases is the presence of metal-binding domains (MBDs) at the cytosolic termini, which however are poorly characterized with an elusive mechanistic role. Here we present the MBD architecture in metal-free and metal-bound forms of the archetype Cu+-specific P1B-ATPase LpCopA, determined using NMR. The MBD is composed of a flexible tail and a structured core with a metal ion binding site defined by three sulfur atoms, one of which is pertinent to the so-called CXXC motif. Furthermore, we demonstrate that the MBD rather than being involved in ion delivery likely serves a regulatory role, which is dependent on the classical P-type ATPase E1-E2 transport mechanism. Specifically, the flexible tail appears responsible for autoinhibition while the metal-binding core is used for copper sensing. This model is validated by a conformation-sensitive and MBD-targeting nanobody that can structurally and functionally replace the flexible tail. We propose that autoinhibition of Cu+-ATPases occurs at low copper conditions via MBD-mediated interference with the soluble domains of the ATPase core and that metal transport is enabled when copper levels rise, through metal-induced dissociation of the MBD. This allows P1B-ATPase 'vacuum cleaners' to tune their own activity, balancing the levels of critical micronutrients in the cells.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Establishment of root nodule symbiosis is initiated by the perception of bacterial Nod factor ligands by the plant LysM receptor kinases NFR1 and NFR5. Receptor signaling initiating the symbiotic pathway depends on the kinase activity of NFR1, while the signaling mechanism of the catalytically inactive NFR5 pseudokinase is unknown. Here, we present the crystal structure of the signaling-competent Lotus japonicus NFR5 intracellular domain, comprising the juxtamembrane region and pseudokinase domain. The juxtamembrane region is structurally well defined and forms two α-helices, αA and αA', which contain an exposed hydrophobic motif. We demonstrate that this "juxtamembrane motif" promotes NFR5-NFR5 and NFR1-NFR5 interactions and is essential for symbiotic signaling. Conservation analysis reveals that the juxtamembrane motif is present throughout NFR5-type receptors and is required for symbiosis signaling from barley RLK10, suggesting a conserved and broader function for this motif in plant-microbe symbioses.

Background: The participation of patients in clinical trials is crucial for the development of healthcare. There are several challenges in the recruitment of trial participants with acute medical conditions. The registry-based randomized DAPA-MI clinical trial recruited patients during hospitalization for myocardial infarction and provided study drugs in bottles with smart caps that used wireless technology to transmit monitoring data. This interview study aimed to investigate patients' experience of participation in a clinical trial and their attitude to the new bottle cap technology. Methods: A subset of patients participating in the DAPA-MI trial were recruited from four hospitals in Sweden. Semi-structured interviews were conducted and analysed using manifest content analysis. Results: Video interviews were performed including 21 patients (four women and 17 men). The median age was 59 years (range 44-80). Four categories of patients' experiences were identified. A willingness to contribute consisted of patients' positive attitudes to participation and to be a part of development and research. The perception of information emphasized the value of the oral information as well as the importance of time for reflection. Be in a vulnerable condition highlighted the impaired ability to perceive and remember in the acute medical condition. Adaptation to a new technology described the overall positive experiences of the smart bottle cap to evaluate adherence. Conclusions: Patients' experiences of trial participation were in general positive but some challenges in the acute setting of a myocardial infarction were revealed. The smart bottle cap was well accepted, despite some handling difficulties.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.

To better align with the clinical setting, the European Society of Cardiology has published new guidelines covering the whole spectrum of the acute coronary syndrome, including ST-elevation myocardial infarction, non-ST-elevation myocardial infarction and unstable angina.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined β-1,3/β-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Understanding the composition and activation of multicomponent receptor complexes is a challenge in biology. To address this, we developed a synthetic approach based on nanobodies to drive assembly and activation of cell surface receptors and apply the concept by manipulating receptors that govern plant symbiosis with nitrogen-fixing bacteria. We show that the Lotus japonicus Nod factor receptors NFR1 and NFR5 constitute the core receptor complex initiating the cortical root nodule organogenesis program as well as the epidermal program controlling infection. We find that organogenesis signaling is mediated by the intracellular kinase domains whereas infection requires functional ectodomains. Finally, we identify evolutionarily distant barley receptors that activate root nodule organogenesis, which could enable engineering of biological nitrogen-fixation into cereals.

For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.