Plants coordinate systemic immunity through mobile signals that prime distal tissues for enhanced defense. Two recent studies show that an upstream ORF-encoded peptide induces systemic stomatal closure, whereas apoplastic alkalinization enhances immune responsiveness, revealing complementary, systemic acquired resistance-independent mechanisms that integrate peptide signaling and extracellular pH dynamics.

Abstract Plant plasma membrane proteins are organized into distinct domains; however, the principles that govern this spatial organization are not fully understood. HYPERSENSITIVE-INDUCED REACTION (HIR) proteins are plant-specific members of the stomatin/prohibitin/ flotillin/HflK/C (SPFH) family implicated in membrane organization. Arabidopsis thaliana HIR2 interacts with multiple plasma membrane proteins, including receptor kinases such as BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE (BAK1)-INTERACTING RECEPTORS 2 and 3 (BIR2 and 3) and BAK1. These interactions connect HIR2 to BAK1-mediated signaling pathways, as evidenced by impaired growth and immunity phenotypes in hir2 mutants. HIR2 is anchored to the inner leaflet of the plasma membrane through a hydrophobic domain and S-acylation. Single-particle-tracking photoactivated localization microscopy (sptPALM) revealed that HIR2 affects BAK1 mobility and clustering, exemplifying a role in regulating plasma membrane dynamics. Structural modeling with AlphaFold 3 predicts a multimeric cup-like assembly for HIR2, consistent with high molecular weight complexes identified through blue-native PAGE. These findings indicate that HIR2 contributes to the formation of membrane sub-compartments, providing a potential structural framework for spatial membrane organization that influences the dynamics and function of membrane-resident receptors.

The distribution of proteins across the plasma membrane is not uniform; however, the principles governing their organization remain not fully understood. Hypersensitive-induced reaction (HIR) proteins are plant-specific members of the stomatin/prohibitin/flotillin/HflK/C (SPFH) family that have been shown to influence membrane organization. Arabidopsis thaliana HIR2 interacts with multiple plasma membrane proteins, including receptor kinases such as BAK1-INTERACTING RECEPTORS 2 and 3 (BIR2 and 3), BRI1-ASSOCIATED KINASE 1 (BAK1), FLAGELLIN SENSING 2 (FLS2), and BRASSINOSTEROID INSENSITIVE 1 (BRI1). These interactions connect HIR2 to BAK1-mediated signaling pathways, as evidenced by impaired growth and immunity phenotypes in hir2 mutants. HIR2 is anchored to the inner leaflet of the plasma membrane through a hydrophobic interaction domain and S-acylation. Using single-particle tracking photoactivated localization microscopy (sptPALM), we showed that HIR2 affects receptor kinase dynamics and clustering, suggesting a role in spatially coordinating receptor complex activities. Structural modeling with AlphaFold 3 predicts a multimeric circular cup-like assembly for HIR2, consistent with high molecular weight complexes identified through blue native polyacrylamide gel electrophoresis. These findings indicate that HIR2 forms a discrete membrane compartment, providing a novel structural framework for spatial membrane organization and thereby modulating the function of membrane-resident receptors. ### Competing Interest Statement The authors have declared no competing interest.

Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-by-step manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44).

Arabidopsis BAK1/SERK3, a co-receptor of leucine-rich repeat pattern recognition receptors (PRRs), mediates pattern-triggered immunity (PTI). Genetic inactivation of BAK1 or BAK1-interacting receptor-like kinases (BIRs) causes cell death, but the direct mechanisms leading to such deregulation remains unclear. Here, we found that the TIR-NBS-LRR protein CONSTITUTIVE SHADE AVOIDANCE 1 (CSA1) physically interacts with BIR3, but not with BAK1. CSA1 mediates cell death in bak1-4 and bak1-4 bir3-2 mutants via components of effector-triggered immunity-(ETI) pathways. Effector HopB1-mediated perturbation of BAK1 also results in CSA1-dependent cell death. Likewise, microbial pattern pg23-induced cell death, but not PTI responses, requires CSA1. Thus, we show that CSA1 guards BIR3 BAK1 homeostasis and integrates pattern- and effector-mediated cell death pathways downstream of BAK1. De-repression of CSA1 in the absence of intact BAK1 and BIR3 triggers ETI cell death. This suggests that PTI and ETI pathways are activated downstream of BAK1 for efficient plant immunity.

ABSTRACT Super-resolution microscopy techniques have revolutionized cell biology by providing insights into the dynamics of single molecules and nanoscale organization within living cells. However, the application of dynamic live-cell methods in plants has been limited by the lack of suitable fluorophores for simultaneous visualization of multiple proteins. To address this challenge, we implemented a two-color sptPALM approach using codon-optimized photoactivatable fluorescent proteins PA-GFP and PATagRFP. Recently, we showed their individual usability in single-color experiments in Nicotiana benthamiana and Arabidopsis thaliana cells. Here, we now demonstrate the suitability of these fluorophores and their combined use for dual-color sptPALM for the simultaneous observation of two different protein fusions in the same plant cell.

BRI1-ASSOCIATED KINASE 1 (BAK1/SERK3) and its closest homolog BAK1-LIKE 1 (BKK1/SERK4) are leucine-rich repeat receptor kinases (LRR-RKs) belonging to the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family. They act as co-receptors of various other LRR-RKs and participate in multiple signaling events by complexing and transphosphorylating ligand-binding receptors. Initially identified as the brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) co-receptor, BAK1 also functions in plant immunity by interacting with pattern recognition receptors. Mutations in BAK1 and BKK1 cause severely stunted growth and cell death, characterized as autoimmune cell death. Several factors play a role in this type of cell death, including RKs and components of effector-triggered immunity (ETI) signaling pathways, glycosylation factors, ER quality control components, nuclear trafficking components, ion channels, and Nod-like receptors (NLRs). The Shan lab has recently discovered a novel RK BAK-TO-LIFE 2 (BTL2) that interacts with BAK1 and triggers cell death in the absence of BAK1 and BKK1. This RK compensates for the loss of BAK1-mediated pattern-triggered immunity (PTI) by activating phytocytokine-mediated immune and cell death responses.

Interaction of engineered nanomaterials (ENMs) with the immune system mainly occurs with cells and molecules of innate immunity, which are present in interface tissues of living organisms. Immuno-nanotoxicological studies aim at understanding if and when such interaction is inconsequential or may cause irreparable damage. Since innate immunity is the first line of immune reactivity towards exogenous agents and is highly conserved throughout evolution, this review focuses on the major effector cells of innate immunity, the phagocytes, and their major sensing receptors, Toll-like receptors (TLRs), for assessing the modes of successful versus pathological interaction between ENMs and host defences. By comparing the phagocyte- and TLR-dependent responses to ENMs in plants, molluscs, annelids, crustaceans, echinoderms and mammals, we aim to highlight common recognition and elimination mechanisms and the general sufficiency of innate immunity for maintaining tissue integrity and homeostasis.

Plants are indispensable on earth and their improvement in terms of food security is a need of time. The current study has been designed to investigate how biogenic zinc nanoparticles (Zn NPs) can improve the growth and development of Brassica napus L. In this study, Zn NPs were synthesized utilizing Mentha arvensis aqueous extracts, and their morphological and optical properties were assessed using UV-Visible spectrophotometry, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). The synthesized Zn NPs were irregular in shape, indicating aggregation in pattern, with an average particle size of 30 nm, while XRD analysis revealed the crystalline structure of nanoparticles. The growth and development of B. napus varieties (Faisal canola and Shiralee) were assessed after foliar treatments with different concentrations of biogenic Zn NPs. In B. napus varieties, exposure to 15 mg/L Zn NPs dramatically increased chlorophyll, carotenoid content, and biomass accumulation. Similarly, proteomic analyses, on the other hand, revealed that proteins associated with photosynthesis, transport, glycolysis, and stress response in both Brassica varieties were substantially altered. Such exposure to Zn NPs, differential expression of genes associated with photosynthesis, ribosome structural constituents, and oxidative stress response were considerably upregulated in B. napus var. (Faisal and Shiralee canola). The results of this study revealed that foliar applications of biogenic Zn NPs influence the transcriptome and protein profiling positively, therefore stimulating plant growth and development.

AbstractThe BRI1-associated kinase BAK1/SERK3 is a positive regulator of multiple leucine rich receptor kinase-mediated signaling pathways including pattern triggered immunity (PTI). Absence or overexpression of BAK1 leads to spontaneous cell death formation. BAK1-interacting receptors (BIR) constitutively interact with BAK1, and plants lacking or overexpressing BIR proteins phenocopy the cell death symptoms observed inbak1knock outs or overexpressors. In the interactome of BIR3, the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) was identified by mass spectrometry. CSA1 physically interacts with BIR proteins and can be detected in complexes with BAK1. Direct interaction was shown only for CSA1 with BIR proteins but not BAK1. Double mutantbak1 bir3genotypes develop strong dwarfism and cell death symptoms that are dependent on EDS1 and salicylic acid. Loss of CSA1 blocksbak1andbak1 bir3-mediated cell death formation thus demonstrating that CSA1 is causal for this type of cell death. We propose that CSA1 guards BIR proteins and initiates autoimmune cell death that is observed when BAK1 BIR complexes are impaired. Our findings reveal how cell death in the absence of BAK1 and BIR3 is executed and links BAK1, a common co-receptor of many pattern recognition receptors, to NLR proteins typically implicated in effector-triggered immunity.

The BRI1-associated kinase BAK1/SERK3 is a positive regulator of multiple leucine rich receptor kinase-mediated signaling pathways including pattern triggered immunity (PTI). Absence or overexpression of BAK1 leads to spontaneous cell death formation. BAK1-interacting receptors (BIR) constitutively interact with BAK1, and plants lacking or overexpressing BIR proteins phenocopy the cell death symptoms observed in bak1 knock outs or overexpressors. In the interactome of BIR3, the TIR-NBS-LRR protein CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1) was identified by mass spectrometry. CSA1 physically interacts with BIR proteins and can be detected in complexes with BAK1. Direct interaction was shown only for CSA1 with BIR proteins but not BAK1. Double mutant bak1 bir3 genotypes develop strong dwarfism and cell death symptoms that are dependent on EDS1 and salicylic acid. Loss of CSA1 blocks bak1 and bak1 bir3 -mediated cell death formation thus demonstrating that CSA1 is causal for this type of cell death. We propose that CSA1 guards BIR proteins and initiates autoimmune cell death that is observed when BAK1 BIR complexes are impaired. Our findings reveal how cell death in the absence of BAK1 and BIR3 is executed and links BAK1, a common co-receptor of many pattern recognition receptors, to NLR proteins typically implicated in effector-triggered immunity.### Competing Interest StatementThe authors have declared no competing interest.

Plant growth and development are modulated by both biotic and abiotic stress. Increasing evidence suggests that cellular integration of the corresponding signals occurs within preformed hubs at the plasma membrane called nanodomains. These membrane sub-compartments are organized by multivalent molecular scaffold proteins, such as remorins. Here, we demonstrate that Group 1 remorins form a hetero-oligomeric complex at the plasma membrane. While these remorins are functionally redundant for some pathways their multivalency also allows the recruitment of specific interaction partners. One of them, the receptor-like kinase REMORIN-INTERACTING RECEPTOR 1 (RIR1), that acts redundantly with the closely related receptor NILR2, is specifically recruited by REM1.2 in a phosphorylation-dependent manner. Overlapping developmental phenotypes suggest that the REM/RIR complex regulates key developmental pathways.

The global economic success of man-made nanoscale materials has led to a higher production rate and diversification of emission sources in the environment. For these reasons, novel nanosafety approaches to assess the environmental impact of engineered nanomaterials are required. While studying the potential toxicity of metal nanoparticles (NPs), we realized that gold nanoparticles (AuNPs) have a growth-promoting rather than a stress-inducing effect. In this study we established stable short- and long-term exposition systems for testing plant responses to NPs. Exposure of plants to moderate concentrations of AuNPs resulted in enhanced growth of the plants with longer primary roots, more and longer lateral roots and increased rosette diameter, and reduced oxidative stress responses elicited by the immune-stimulatory PAMP flg22. Our data did not reveal any detrimental effects of AuNPs on plants but clearly showed positive effects on growth, presumably by their protective influence on oxidative stress responses. Differential transcriptomics and proteomics analyses revealed that oxidative stress responses are downregulated whereas growth-promoting genes/proteins are upregulated. These omics datasets after AuNP exposure can now be exploited to study the underlying molecular mechanisms of AuNP-induced growth-promotion.

The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified.

Plants utilize a two-tiered immune system consisting of pattern recognition receptor (PRR)-triggered immunity (PTI) and effector-triggered immunity (ETI) to defend themselves against pathogenic microbes. The receptor protein kinase BAK1 plays a central role in multiple PTI signaling pathways in Arabidopsis. However, double mutants made by BAK1 and its closest paralog BKK1 exhibit autoimmune phenotypes, including cell death resembling a typical nucleotide-binding leucine-rich repeat protein (NLR)-mediated ETI response. The molecular mechanisms of the cell death caused by the depletion of BAK1 and BKK1 are poorly understood. Here, we show that the cell-death phenotype of bak1 bkk1 is suppressed when a group of NLRs, ADR1s, are mutated, indicating the cell-death of bak1 bkk1 is the consequence of NLR activation. Furthermore, introduction of a Pseudomonas syringae effector HopB1, which proteolytically cleaves activated BAK1 and its paralogs via either gene transformation or bacterium-delivery, results in a cell-death phenotype in an ADR1s-dependent manner. Our study thus pinpoints that BAK1 and its paralogs are likely guarded by NLRs.

The use of nanomaterials in agriculture is a current need and could be helpful in overcoming food security risks. Brassica napus L. is the third most important crop for edible oil, having double low unsaturated fatty acids. In the present study, we investigated the effects of green synthesized Zn NPs on biochemical effects, antioxidant enzymes, nutritional quality parameters and on the fatty acid profile of rapeseed (B. napus). Plant-mediated synthesis of zinc nanoparticles (Zn NPs) was carried out using Mentha arvensis L. leaf extract followed by characterization through ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-Ray (EDX), and X-Ray diffraction (XRD). NPs exhibited irregular shapes ranging in size from 30-70 nm and EDX analysis confirmed 96.08% of Zn in the sample. The investigated biochemical characterization (protein content, proline content, total soluble sugar (TSS), total flavonoid content (TFC), and total phenolic content (TPC) showed a substantial change on exposure to Zn NPs. A dose-dependent gradual increase was observed in the antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Oil and moisture contents dropped significantly from the control level in the rapeseed (B. napus) varieties. However, different trends in nutritional (Zn, Na+, K+) and fatty acid profiling of B. napus have been noted. This study demonstrates that Zn NPs have the potential to improve the biochemical, nutritional, antioxidant enzymes, and fatty acid profile of B. napus varieties.

Here we discuss the potential of engineered nanoparticles to act as agents affecting the immune status in different environmentally relevant species, with a focus on the emerging experimental models launched by the EU Horizon 2020 project PANDORA.

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1-associated kinase 1 (BAK1)-interacting receptor-like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.