Group 2 innate lymphoid cells are key in lipid transfer protein allergy pathogenesis.

Background: Immunopathology in food allergy is characterized by an uncontrolled type 2 immune response and specific-IgE production. Recent studies have determined that group 2 innate lymphoid cells (ILC2) participate in the food allergy pathogenic mechanism and their severity. Our objective was to investigate the role of ILC2 in peach-allergic patients due to non-specific lipid transfer protein (Pru p 3) sensitization. Methods: The immune response in peripheral blood mononuclear cells was characterized in lipid transfer protein-allergic patients and healthy controls. We have analyzed the Pru p 3 uptake on ILC2, the expression of costimulatory molecules, and their involvement on the T-cell proliferative response and cytokine production under different experimental conditions: cytokines involved in group 2 innate lymphoid cell activation (IL-33 and IL-25), Pru p 3 as main food allergen, and the combination of both components (IL-33/IL-25+Pru p 3) using cell sorting, EliSpot, flow cytometry, and confocal microscopy. Results: Our results show that Pru p 3 allergen is taken up by group 2 innate lymphoid cells, regulating their costimulatory molecule expression (CD83 and HLA-DR) depending on the presence of Pru p 3 and its combination with IL-33/IL-25. The Pru p 3-stimulated ILC2 induced specific GATA3+Th2 proliferation and cytokine (IL-4, IL-5, and IL-13) production in lipid transfer protein-allergic patients in a cell contact-dependent manner with no changes in Tbet+Th1- and FOXP3+Treg cell differentiation. Conclusions: The results indicate that in lipid transfer protein-allergic patients, the responsible allergen, Pru p 3, interacts with group 2 innate lymphoid cells, promoting a Th2 cell response. Our results might be of interest in vivo, as they show a role of group 2 innate lymphoid cells as antigen-presenting cells, contributing to the development of food allergy. Consequently, group 2 innate lymphoid cells may be considered as potential therapeutic targets.

Peanut oral immunotherapy using an extensively heated and baked novel composition of peanuts.

BACKGROUND: Oral immunotherapy (OIT) is an increasingly acceptable therapeutic option for peanut-allergic (PA) children, despite significant side effects. Major peanut allergenic proteins are heat-resistant and are not rendered hypoallergenic after baking or cooking. Lyophilized peanut protein-MH (LPP-MH) is a novel composition from developing peanuts, enabling cooking-induced reduction in allergenicity. We aimed to explore the safety and efficacy of OIT, with extensively heated and baked (EHEB) LPP-MH in PA children. METHODS: In a single-arm, single-center, pilot study, PA children with a single highest tolerated dose of <100 mg peanut protein were placed on a 40-week OIT protocol with 300 mg daily of heat-treated LPP-MH. A repeat open peanut food challenge was performed after 40 weeks of treatment and at a 6-12 months of follow-up visit. RESULTS: Thirty-three children with PA were enrolled, with a mean cumulative tolerated dose (MCTD) of 71.2 mg PP (95% CI 45-100 mg). After 40 weeks, 32/33 patients were able to consume more than 300 mg of natural PP, with MCTD of 1709 mg (CI 365-3675 mg). There were no severe allergic reactions requiring epinephrine, during any of the observed LPP-MH challenges or any treatment related doses at home. After 6-12 months on daily maintenance, the MCTD was 8821 mg (95% CI 1930-13,500 mg). This enabled most children age-appropriate dietary inclusion of peanuts. CONCLUSION: An OIT protocol with heat-treated LPP-MH, a novel composition from developing peanuts, seems a potentially safe and efficacious OIT modality for PA children, enabling the introduction of dietary levels of peanut proteins in highly allergic PA children. Validation in randomized controlled studies is mandated.

Effects of enzymatic hydrolysis combined with pressured heating on tree nut allergenicity.

Hazelnut, pistachio and cashew are tree nuts with health benefits but also with allergenic properties being prevalent food allergens in Europe. The allergic characteristics of these tree nuts after processing combining heat, pressure and enzymatic digestion were analyzed through in vitro (Western blot and ELISA) and in vivo test (Prick-Prick). In the analyzed population, the patients sensitized to Cor a 8 (nsLTP) were predominant over those sensitized against hazelnut seed storage proteins (Sprot, Cor a 9 and 14), which displayed higher IgE reactivity. The protease E5 effectively hydrolyzed proteins from hazelnut and pistachio, while E7 was efficient for cashew protein hydrolysis. When combined with pressured heating (autoclave and Controlled Instantaneous Depressurization (DIC)), these proteases notably reduced the allergenic reactivity. The combination of DIC treatment before enzymatic digestion resulted in the most effective methodology to drastically reduce or indeed eliminate the allergenic capacity of tree nuts.

Release of a ubiquitin brake activates OsCERK1-triggered immunity in rice.

Plant pattern-recognition receptors perceive microorganism-associated molecular patterns to activate immune signalling1,2. Activation of the pattern-recognition receptor kinase CERK1 is essential for immunity, but tight inhibition of receptor kinases in the absence of pathogen is crucial to prevent autoimmunity3,4. Here we find that the U-box ubiquitin E3 ligase OsCIE1 acts as a molecular brake to inhibit OsCERK1 in rice. During homeostasis, OsCIE1 ubiquitinates OsCERK1, reducing its kinase activity. In the presence of the microorganism-associated molecular pattern chitin, active OsCERK1 phosphorylates OsCIE1 and blocks its E3 ligase activity, thus releasing the brake and promoting immunity. Phosphorylation of a serine within the U-box of OsCIE1 prevents its interaction with E2 ubiquitin-conjugating enzymes and serves as a phosphorylation switch. This phosphorylation site is conserved in E3 ligases from plants to animals. Our work identifies a ligand-released brake that enables dynamic immune regulation.

Insect Cell-Expressed Major Ragweed Allergen Amb a 1.01 Exhibits Similar Allergenic Properties to Its Natural Counterpart from Common Ragweed Pollen.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Fabrication of an Antifouling Surface Plasmon Resonance Sensor with Stratified Zwitterionic Peptides for Highly Efficient Detection of Peanut Allergens in Biscuits.

Peanut allergen monitoring is currently an effective strategy to avoid allergic diseases, while food matrix interference is a critical challenge during detection. Here, we developed an antifouling surface plasmon resonance sensor (SPR) with stratified zwitterionic peptides, which provides both excellent antifouling and sensing properties. The antifouling performance was measured by the SPR, which showed that stratified peptide coatings showed much better protein resistance, reaching ultralow adsorption levels (<5 ng/cm2). Atomic force microscopy was used to further analyze the antifouling mechanism from a mechanical perspective, which demonstrated lower adsorption forces on hybrid peptide coatings, confirming the better antifouling performance of stratified surfaces. Moreover, the recognition of peanut allergens in biscuits was performed using an SPR with high efficiency and appropriate recovery results (98.2-112%), which verified the feasibility of this assay. Therefore, the fabrication of antifouling sensors with stratified zwitterionic peptides provides an efficient strategy for food safety inspection.

Structural and Immunological Features of PR-10 Allergens: Focusing on the Major Alder Pollen Allergen Aln g 1.

Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can significantly increase their allergenicity potential. It has been recently shown that not only the birch pollen allergen Bet v 1 but also the alder pollen allergen Aln g 1, might act as a true sensitizer of the immune system. The current investigation is aimed at the further study of the allergenic and structural features of Aln g 1. By using qPCR, we showed that Aln g 1 was able to upregulate alarmins in epithelial cells, playing an important role in sensitization. With the use of CD-spectroscopy and ELISA assays with the sera of allergic patients, we demonstrated that Aln g 1 did not completely restore its structure after thermal denaturation, which led to a decrease in its IgE-binding capacity. Using site-directed mutagenesis, we revealed that the replacement of two residues (Asp27 and Leu30) in the structure of Aln g 1 led to a decrease in its ability to bind to both IgE from sera of allergic patients and lipid ligands. The obtained data open a prospect for the development of hypoallergenic variants of the major alder allergen Aln g 1 for allergen-specific immunotherapy.

Analysis of Methylesterase Gene Family in Fragaria vesca Unveils Novel Insights into the Role of FvMES2 in Methyl Salicylate-Mediated Resistance against Strawberry Gray Mold.

Methylesterases (MESs) hydrolyze carboxylic ester and are important for plant metabolism and defense. However, the understanding of MES' role in strawberries against pathogens remains limited. This study identified 15 FvMESs with a conserved catalytic triad from the Fragaria vesca genome. Spatiotemporal expression data demonstrated the upregulated expression of FvMESs in roots and developing fruits, suggesting growth involvement. The FvMES promoter regions harbored numerous stress-related cis-acting elements and transcription factors associated with plant defense mechanisms. Moreover, FvMES2 exhibited a significant response to Botrytis cinerea stress and showed a remarkable correlation with the salicylic acid (SA) signaling pathway. Molecular docking showed an efficient binding potential between FvMES2 and methyl salicylate (MeSA). The role of FvMES2 in MeSA demethylation to produce SA was further confirmed through in vitro and in vivo assays. After MeSA was applied, the transient overexpression of FvMES2 in strawberries enhanced their resistance to B. cinerea compared to wild-type plants.

Techno-Functions and Safety Concerns of Plant-Based Peptides in Food Matrices.

Plant-based peptides (PBPs) benefit functional food development and environmental sustainability. Proteolysis remains the primary method of peptide production because it is a mild and nontoxic technique. However, potential safety concerns still emanate from toxic or allergenic sequences, amino acid racemization, iso-peptide bond formation, Maillard reaction, dose usage, and frequency. The main aim of this review is to investigate the techno-functions of PBPs in food matrices, as well as their safety concerns. The distinctive characteristics of PBPs exhibit their techno-functions for improving food quality and functionality by contributing to several crucial food formulations and processing. The techno-functions of PBPs include solubility, hydrophobicity, bitterness, foaming, oil-binding, and water-holding capacities, which subsequently affect food matrices. The safety and quality of foodstuff containing PBPs depend on the proper source of plant proteins, the selection of processing approaches, and compliance with legal regulations for allergen labeling and safety evaluations. The safety concerns in allergenicity and toxicity were discussed. The conclusion is that food technologists must apply safe limits and consider potential allergenic components generated during the development of food products with PBPs. Therefore, functional food products containing PBPs can be a promising strategy to provide consumers with wholesome health benefits.

Pattern recognition receptors as potential therapeutic targets for developing immunological engineered plants.

There is an urgent need to combat pathogen infestations in crop plants to ensure food security worldwide. To counter this, plants have developed innate immunity mediated by Pattern Recognition Receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and damage- associated molecular patterns (DAMPs). PRRs activate Pattern-Triggered Immunity (PTI), a defence mechanism involving intricate cell-surface and intracellular receptors. The diverse ligand-binding ectodomains of PRRs, including leucine-rich repeats (LRRs) and lectin domains, facilitate the recognition of MAMPs and DAMPs. Pathogen resistance is mediated by a variety of PTI responses, including membrane depolarization, ROS production, and the induction of defence genes. An integral part of intracellular immunity is the Nucleotide-binding Oligomerization Domain, Leucine-rich Repeat proteins (NLRs) which recognize and respond to effectors in a potent manner. Enhanced understanding of PRRs, their ligands, and downstream signalling pathways has contributed to the identification of potential targets for genetically modified plants. By transferring PRRs across plant species, it is possible to create broad-spectrum resistance, potentially offering innovative solutions for plant protection and global food security. The purpose of this chapter is to provide an update on PRRs involved in disease resistance, clarify the mechanisms by which PRRs recognize ligands to form active receptor complexes and present various applications of PRRs and PTI in disease resistance management for plants.

CaWRKY01-10 and CaWRKY08-4 Confer Pepper's Resistance to Phytophthora capsici Infection by Directly Activating a Cluster of Defense-Related Genes.

Phytophthora blight of pepper, which is caused by the notorious oomycete pathogen Phytophthora capsici, is a serious disease in global pepper production regions. Our previous study had identified two WRKY transcription factors (TFs), CaWRKY01-10 and CaWRKY08-4, which are prominent modulators in the resistant pepper line CM334 against P. capsici infection. However, their functional mechanisms and underlying signaling networks remain unknown. Herein, we determined that CaWRKY01-10 and CaWRKY08-4 are localized in plant nuclei. Transient overexpression assays indicated that both CaWRKY01-10 and CaWRKY08-4 act as positive regulators in pepper resistance to P. capsici. Besides, the stable overexpression of CaWRKY01-10 and CaWRKY08-4 in transgenic Nicotiana benthamiana plants also significantly enhanced the resistance to P. capsici. Using comprehensive approaches including RNA-seq, CUT&RUN-qPCR, and dual-luciferase reporter assays, we revealed that overexpression of CaWRKY01-10 and CaWRKY08-4 can activate the expressions of the same four Capsicum annuum defense-related genes (one PR1, two PR4, and one pathogen-related gene) by directly binding to their promoters. However, we did not observe protein-protein interactions and transcriptional amplification/inhibition effects of their shared target genes when coexpressing these two WRKY TFs. In conclusion, these data suggest that both of the resistant line specific upregulated WRKY TFs (CaWRKY01-10 and CaWRKY08-4) can confer pepper's resistance to P. capsici infection by directly activating a cluster of defense-related genes and are potentially useful for genetic improvement against Phytophthora blight of pepper and other crops.

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 µM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

[Analysis of the electrophoretic profiles of the protein extracts of the species Manihot esculenta (Crantz, 1975), Persea Americana (Mill., 1768) and Actinidia delicious (A. Chevalier., 1984), and their association with the latex-fruit syndrome for searching for potential allergens]. / Análisis de los perfiles electroforéticos de los extractos proteicos de las especies Manihot esculenta (Crantz,1975), Persea americana (Mill.,1768) y Actinidia deliciosa (A. Chevalier., 1984), y su asociación con el síndrome látex-fruta para la búsqueda de alérgenos potenciales.

OBJECTIVE: Determine the electrophoretic profiles of the extracts of Manihot esculenta, Actinidia Deliciosa and Persea Americana and their possible relationship with Latex-Fruit Syndrome. METHODS: Protein extracts of M. esculenta, P. Americana and A. Deliciosa were prepared through the processes of maceration and solvent extraction from plant samples. In the case of the avocado, a prior extraction by soxhlet was carried out to eliminate the fat. The extracts were vacuum filtered, dialyzed and finally lyophilized. Separation of proteins based on molecular weight was performed by SDS PAGE electrophoresis. The electrophoretic profiles obtained were compared with the allergenic proteins previously identified in the latex extract, in order to determine a possible relationship with Latex-Fruit Syndrome, depending on the molecular weight. RESULTS: The extracts of M. esculenta and P. Americana showed a wide range of protein fractions with molecular weights varying from 10 to 250 KD, finding that the region with the highest concentration of bands was between 20 and 89 KD, (60 and 65%), respectively. A 20-band profile was obtained for the M. esculenta extract (Figure 1), with seven bands sharing similar weights with the latex allergens (Hev b 1, Hev b 2, Hev b3, Hev b 4, Hev b 5, Hev b 6.03, Hev b 8 and Hev b 10) (3-5). For the P. Americana extract, 20 bands were also observed (Figure 2), seven of which presented approximate weights to the Latex allergens (Hev b 1, Hev b 2 Hev b 4 Hev b 6.01 Hev b 6.03 Hev b 8 , Hev b 10 Hev b 11 Hev b 14). The Kiwi extract showed two bands of 19.1 and 22.9 KD, with weights close to latex proteins (figure 3), (Hev b 3 and Hev b 6.01), and allergens (Act d 2 and Act d 6), reported in the literature for this fruit. CONCLUSIONS: When analyzing the relationship between the separated protein fractions and the latex allergens described in the literature, a possible association of 35% was found for the extracts of M. esculenta and P. Americana, and 10% for A. Delicious, with great relevance being the association found with the allergens Hev b 4, Hev b 2, Hev 8 and Hev b 11, which are involved in Latex-Fruit Syndrome. The electrophoretic profiles of the prepared extracts were determined and compared with the Latex allergens. This information generates a contribution for the development of new research and advances in the standardization of these extracts on a large scale and for their future use in diagnostic tests.

OBJETIVO: Determinar los perfiles electroforéticos de los extractos de Manihot esculenta, Actinidia deliciosa y Persea americana y su posible relación con el Síndrome de Látex ­ Fruta. MÉTODOS: Se prepararon extractos proteicos de M. esculenta, P. Americana y A. Deliciosa, a través de los procesos de macerado y extracción con solventes a partir muestras vegetales. En el caso del aguacate, se realizó una extracción previa por soxhlet, para eliminar la grasa. Los extractos se filtraron al vacío, se sometieron a diálisis y por último se liofilizaron. La separación de las proteínas en función del peso molecular se realizó mediante electroforesis SDS PAGE. Se compararon los perfiles electroforéticos obtenidos con las proteínas alergénicas previamente identificadas en el extracto de látex, con el fin de determinar una posible relación con el Síndrome de Látex-Fruta, en función del peso molecular. RESULTADOS: Los extractos de M. esculenta y P. americana mostraron una amplia gama de fracciones proteicas con pesos moleculares que varían desde 10 a 250 KD, encontrando que la región con mayor concentración de bandas se situó entre 20 y 89 KD, (60 y 65 %), respectivamente. Se obtuvo un perfil de 20 bandas para el extracto de M. esculenta (figura 1), con siete bandas que comparten pesos similares con los alérgenos del látex (Hev b 1, Hev b 2, Hev b3, Hev b 4, Hev b 5, Hev b 6.03, Hev b 8 y Hev b 10) (3-5). Para el extracto de P. americana, también se observaron 20 bandas (figura 2), siete de las cuales presentaron pesos aproximados a los alérgenos de Látex (Hev b 1, Hev b 2 Hev b 4 Hev b 6.01 Hev b 6.03 Hev b 8, Hev b 10 Hev b 11 Hev b 14). El extracto de Kiwi mostró dos bandas de 19,1 y 22,9 KD, con pesos cercanos a proteínas de látex (figura 3), (Hev b 3 y Hev b 6.01), y los alérgenos (Act d 2 y Act d 6), reportados en la literatura para esta fruta. CONCLUSIONES: Al analizar la relación existente entre las fracciones proteicas separadas y los alérgenos de los látex descritos en la literatura, se encontró una posible asociación del 35% para los extractos de M. esculenta y P. Americana, y del 10% para A. Deliciosa, siendo de gran relevancia la asociación encontrada con los alérgenos Hev b 4, Hev b 2, Hev 8 y Hev b 11, los cuales se encuentran implicados en el Síndrome de Látex-Fruto. Se lograron determinar los perfiles electroforéticos de los extractos elaborados y se compararon con los alérgenos del Látex. Está información genera un aporte para el desarrollo de nuevas investigaciones y avances en la estandarización de estos extractos a gran escala y para su uso futuro en pruebas diagnósticas.

[GmAGB1 plays a positive regulatory role in soybean defense responses].

Heterotrimeric GTP-binding protein (G-proteins) complex, which consists of Gα, Gß and Gγ subunits, plays critical roles in defense signaling. Arabidopsis genome contains only a single Gß-encoding gene, AGB1. Loss function of AGB1 in Arabidopsis results in enhanced susceptibility to a wide range of pathogens. However, the function of soybean AGB1 in immunity has not been previously interrogated. Bioinformatic analysis indicated that there are four GmAGB1 homologous genes in soybean genome, sharing homology of 86%-97%. To overcome the functional redundancy of these GmAGB1 homologs, virus-induced gene silencing (VIGS) mediated by the bean pod mottle virus (BPMV) was used to silence these four genes simultaneously. As expected, these four GmAGB1 homologous genes were indeed silenced by a single BPMV-VIGS vector carrying a conserved fragments among these four genes. A dwarfed phenotype was observed in GmAGB1s-silenced soybean plants, suggesting that GmAGB1s play a crucial role in growth and development. Disease resistance analysis indicated that silencing GmAGB1s significantly compromised the resistance of soybean plants against Xanthomonas campestris pv. glycinea (Xag). This reduced resistance was correlated with the decreased accumulation of pathogen-induced reactive oxygen species (ROS) and the reduced activation of GmMPK3 in response to flg22, a conserved N-terminal peptide of flagellin protein. These results indicate that GmAGB1 functions as a positive regulator in disease resistance and GmAGB1 is indispensable for the ROS production and GmMPK3 activation induced by pathogen infection. Yeast two hybrid assay showed that GmAGB1 interacted with GmAGG1, suggesting that an evolutionary conserved heterotrimeric G protein complex similarly functions in soybean.

[In silico analysis of Cit s 2: A highly conserved profilin]. / Análisis in silico de cit s 2: una profilina altamente conservada.

OBJECTIVE: Analyze phylogenetic relationships and molecular mimicry of Cit s 2 and other plant profilins. METHODS: Online bioinformatics tools including Basic Local Alignment Search Tool (BLASTP), PRALINE and MEGA were used for multiple alignments and phylogenetic analysis. A 3D-homology model of Cit s 2 was predicted. Models were calculated with MODELLER. The best model was selected with the model scoring option of MAESTRO. Conserved regions between Cit s 2 and other profilins were located on the 3D model and antigenic regions were predicted by ElliPro server (3-5). RESULTS: Cit s 2 amino acid sequence (Uniprot code:P84177) was compared with other 30 profilins from different allergenic sources. The identity between Cit s 2 and other profilins ranged between 82 and 99%. The highest identity was observed with Cucumis melo (99%) followed by Prunus persica (98%) and Malus domestica (92%). High conserved antigenic regions were observed on the 3D predicted model. Seven lineal and six discontinuous epitopes were found in Cit s 2. CONCLUSION: High conserved antigenic regions were observed on the 3D predicted model of Cit s 2, which might involve potential cross-reactivity between Cit s 2 and other profilins. Future studies are needed to further analyze these results.

OBJETIVO: Analizar las relaciones filogenéticas y el mimetismo molecular de Cit s 2 y otras profilinas vegetales. MÉTODOS: Se utilizaron herramientas bioinformáticas en línea, incluida la de búsqueda de alineación local básica (BLASTP), PRALINE y MEGA, para alineamientos múltiples y análisis filogenético. Se predijo un modelo de homología 3D de Cit s 2. Los modelos se calcularon con MODELLER. El mejor modelo fue seleccionado con la opción de puntuación de modelo de Maestro. Las regiones conservadas entre Cit s 2 y otras profilinas se ubicaron en el modelo 3D y las regiones antigénicas fueron predichas por el servidor ElliPro (3-5). RESULTADOS: La secuencia de aminoácidos de Cit s 2 (código Uniprot: P84177), se comparó con otras 30 profilinas de diferentes fuentes alergénicas. La mayor identidad se observó con Cucumis melo (99%) seguida de Prunus persica (98%) y Malus domestica (92%). Se observaron regiones antigénicas altamente conservadas en el modelo predicho en 3D. Se encontraron siete epítopes lineales, y seis epítopes discontinuos en Cit s 2. CONCLUSIÓN: Se observaron regiones antigénicas altamente conservadas en el modelo 3D predicho de Cit s 2, lo que podría implicar una posible reactividad cruzada entre Cit s 2 y otras profilinas. Se necesitan estudios futuros para analizar más a fondo estos resultados.

Substrate-induced condensation activates plant TIR domain proteins.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain mediate recognition of strain-specific pathogen effectors, typically via their C-terminal ligand-sensing domains1. Effector binding enables TIR-encoded enzymatic activities that are required for TIR-NLR (TNL)-mediated immunity2,3. Many truncated TNL proteins lack effector-sensing domains but retain similar enzymatic and immune activities4,5. The mechanism underlying the activation of these TIR domain proteins remain unclear. Here we show that binding of the TIR substrates NAD+ and ATP induces phase separation of TIR domain proteins in vitro. A similar condensation occurs with a TIR domain protein expressed via its native promoter in response to pathogen inoculation in planta. The formation of TIR condensates is mediated by conserved self-association interfaces and a predicted intrinsically disordered loop region of TIRs. Mutations that disrupt TIR condensates impair the cell death activity of TIR domain proteins. Our data reveal phase separation as a mechanism for the activation of TIR domain proteins and provide insight into substrate-induced autonomous activation of TIR signalling to confer plant immunity.

Two different viral proteins suppress NUCLEAR FACTOR-YC-mediated antiviral immunity during infection in rice.

Plant viruses have multiple strategies to counter and evade the host's antiviral immune response. However, limited research has been conducted on the antiviral defense mechanisms commonly targeted by distinct types of plant viruses. In this study, we discovered that NUCLEAR FACTOR-YC (NF-YC) and NUCLEAR FACTOR-YA (NF-YA), 2 essential components of the NF-Y complex, were commonly targeted by viral proteins encoded by 2 different rice (Oryza sativa L.) viruses, rice stripe virus (RSV, Tenuivirus) and southern rice black streaked dwarf virus (SRBSDV, Fijivirus). In vitro and in vivo experiments showed that OsNF-YCs associate with OsNF-YAs and inhibit their transcriptional activation activity, resulting in the suppression of OsNF-YA-mediated plant susceptibility to rice viruses. Different viral proteins RSV P2 and SRBSDV SP8 directly disrupted the association of OsNF-YCs with OsNF-YAs, thereby suppressing the antiviral defense mediated by OsNF-YCs. These findings suggest an approach for conferring broad-spectrum disease resistance in rice and reveal a common mechanism employed by viral proteins to evade the host's antiviral defense by hindering the antiviral capabilities of OsNF-YCs.

NLR surveillance of pathogen interference with hormone receptors induces immunity.

Phytohormone signalling pathways have an important role in defence against pathogens mediated by cell-surface pattern recognition receptors and intracellular nucleotide-binding leucine-rich repeat class immune receptors1,2 (NLR). Pathogens have evolved counter-defence strategies to manipulate phytohormone signalling pathways to dampen immunity and promote virulence3. However, little is known about the surveillance of pathogen interference of phytohormone signalling by the plant innate immune system. The pepper (Capsicum chinense) NLR Tsw, which recognizes the effector nonstructural protein NSs encoded by tomato spotted wilt orthotospovirus (TSWV), contains an unusually large leucine-rich repeat (LRR) domain. Structural modelling predicts similarity between the LRR domain of Tsw and those of the jasmonic acid receptor COI1, the auxin receptor TIR1 and the strigolactone receptor partner MAX2. This suggested that NSs could directly target hormone receptor signalling to promote infection, and that Tsw has evolved a LRR resembling those of phytohormone receptors LRR to induce immunity. Here we show that NSs associates with COI1, TIR1 and MAX2 through a common repressor-TCP21-which interacts directly with these phytohormone receptors. NSs enhances the interaction of COI1, TIR1 or MAX2 with TCP21 and blocks the degradation of corresponding transcriptional repressors to disable phytohormone-mediated host immunity to the virus. Tsw also interacts directly with TCP21 and this interaction is enhanced by viral NSs. Downregulation of TCP21 compromised Tsw-mediated defence against TSWV. Together, our findings reveal that a pathogen effector targets TCP21 to inhibit phytohormone receptor function, promoting virulence, and a plant NLR protein has evolved to recognize this interference as a counter-virulence strategy, thereby activating immunity.

A wheat resistosome defines common principles of immune receptor channels.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence.

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.