Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness, regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modulation of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases.

Concerted expansion and contraction of immune receptor gene repertoires in plant genomes

Recent reports suggest that cell-surface and intracellular immune receptors function synergistically to activate robust defence against pathogens, but whether or not they co-evolve is unclear. Here we determined the copy numbers of cell-surface and intracellular immune receptors in 208 species. Surprisingly, these receptor gene families contract and/or expand together in plant genomes, suggesting the mutual potentiation of immunity initiated by cell-surface and intracellular receptors is reflected in the concerted co-evolution of the size of their repertoires across plant species.

A lineage-specific Exo70 is required for receptor kinase-mediated immunity in barley

In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity, but lack causal association. Here, we show that Rps8-mediated resistance in barley to the fungal pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: LRR-RK and Exo70FX12, which are together necessary and sufficient. The Rps8 LRR-RK is the ortholog of rice extracellular immune receptor Xa21 and Exo70FX12 is a member of the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae, and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Rps8 LRR-RK immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling.

Seven Mysteries of LAG-3: A Multi-faceted Immune Receptor of Increasing Complexity

Despite three decades of research to its name and increasing interest in immunotherapies which target it, LAG-3 remains an elusive co-inhibitory receptor in comparison to the well-established PD-1 and CTLA-4. As such, LAG-3 targeting therapies have yet to achieve the clinical success of therapies targeting other checkpoints. This could, in part, be attributed to the many unanswered questions that remain regarding LAG-3 biology. Of these, we (i) the function of the many LAG-3:ligand interactions; (ii) the hurdles that remain to acquire a high resolution structure of LAG-3; (iii) the under-studied LAG-3 signal transduction mechanism; (iv) the elusive soluble form of LAG-3; (v) the implications of the lack of (significant) phenotype of LAG-3 knockout mice; (vi) the reports of LAG-3 expression on epithelium and (vii) the conflicting reports of LAG-3 expression (and potential contributions to pathology) in the brain. These mysteries which surround LAG-3 highlight how the ever-evolving study of its biology continues to reveal ever-increasing complexity in its role as an immune receptor. Importantly, answering the questions which shroud LAG-3 in mystery will allow maximum therapeutic benefit of LAG-3 targeting immunotherapies in cancer, autoimmunity and beyond.

DALI (Diversity AnaLysis Interface): a novel tool for the integrated analysis of multimodal single cell RNAseq data and immune receptor profiling.

Single-cell RNA sequencing is instrumental to unravel the cellular and transcriptomic heterogeneity of T and B cells in health and disease. Recent technological advances add additional layers of information allowing researchers to simultaneously explore the transcriptomic, surface protein and immune receptor diversity during adaptive immune responses. The increasing data complexicity poses a burden on the workload for bioinformaticians, who are often not familiar with the specificities and biology of immune receptor profiling. The wet-lab modalities and sequencing capabilities currently have outpaced bioinformatics solutions, which forms an ever-increasing barrier for many biologists to analyze their datasets. Here, we present DALI (Diversity AnaLysis Interface), a software package to identify and analyze T cell and B cell receptor diversity in high-throughput single-cell sequencing data. DALI aims to support bioinformaticians with a functional toolbox, allowing seamless integration of multimodel scRNAseq and immune receptor profiling data. The R-based package builds further on workflows using the Seurat package and other existing tools for BCR/TCR analyses. In addition, DALI is designed to engage immunologists having limited coding experience with their data, using a browser-based interactive graphical user interface. The implementation of DALI can effectively lead to a two-way communication between wet-lab scientists and bioinformaticians to advance the analysis of complex datasets.

Seven chain adaptive immune receptor repertoire analysis in rheumatoid arthritis: association to disease and clinically relevant phenotypes

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease characterized by a defective adaptive immune receptor repertoire (AIRR) that fails to distinguish self from non-self antigens. The AIRR is vast, encompassing four T cell receptor (TCR) and three B cell receptor (BCR) chains, each of which displays an extraordinary amino acid sequence variability in the antigen-binding site. How the concerted action of T and B cell clones is associated with the development and clinical evolution of immune-mediated diseases is still not known. Using a new immunosequencing technology that allows the unbiased amplification of the seven receptor chains, we conducted an in-depth quantitative analysis of the seven-receptor chain variability in RA. Compared to healthy controls, the AIRR in RA was found to be characterized by a lower BCR diversity, the depletion of highly similar BCR clones, an isotype-specific signature as well as a skewed IGL chain and gene segment usage. A predictor based on quantitative multi-chain AIRR information was able to accurately predict disease, including the elusive seronegative subset of RA patients. AIRR features of the seven immune receptor chains were also different between patients with distinct clinically relevant phenotypes. Incorporating HLA variation data, we were able to identify the TCR clones that are specifically associated with disease risk variants. The longitudinal analysis of the AIRR revealed that treatment with Tumor Necrosis Factor (TNF) inhibitors selectively restores the diversity of B cell clones in RA patients by reducing the frequency of clones with a similar biochemical profile. The biochemical properties of the TNFi-modulated clones were also found to differ between responders and non-responders, supporting a different antigenic reactivity in the B cell compartment of these two groups of RA patients. Our comprehensive analysis of the TCR and BCR repertoire reveals a complex T and B cell architecture in RA, and provides the basis for precision medicine strategies based on the highly informative features of the adaptive immune response.

An atypical NLR protein modulates the NRC immune receptor network

The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical, but essential member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, transient RNA interference of NRCX in mature N. benthamiana leaves does not result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.