Overexpression of rice jacalin-related mannose-binding lectin (OsJAC1) enhances resistance to ionizing radiation in Arabidopsis.

BACKGROUND: Jacalin-related lectins in plants are important in defense signaling and regulate growth, development, and response to abiotic stress. We characterized the function of a rice mannose-binding jacalin-related lectin (OsJAC1) in the response to DNA damage from gamma radiation. RESULTS: Time- and dose-dependent changes of OsJAC1 expression in rice were detected in response to gamma radiation. To identify OsJAC1 function, OsJAC1-overexpressing transgenic Arabidopsis plants were generated. Interestingly, OsJAC1 overexpression conferred hyper-resistance to gamma radiation in these plants. Using comparative transcriptome analysis, genes related to pathogen defense were identified among 22 differentially expressed genes in OsJAC1-overexpressing Arabidopsis lines following gamma irradiation. Furthermore, expression profiles of genes associated with the plant response to DNA damage were determined in these transgenic lines, revealing expression changes of important DNA damage checkpoint and perception regulatory components, namely MCMs, RPA, ATM, and MRE11. CONCLUSIONS: OsJAC1 overexpression may confer hyper-resistance to gamma radiation via activation of DNA damage perception and DNA damage checkpoints in Arabidopsis, implicating OsJAC1 as a key player in DNA damage response in plants. This study is the first report of a role for mannose-binding jacalin-related lectin in DNA damage.

Populus euphratica JRL Mediates ABA Response, Ionic and ROS Homeostasis in Arabidopsis under Salt Stress.

Sodium chloride (NaCl) induced expression of a jacalin-related mannose-binding lectin (JRL) gene in leaves, roots, and callus cultures of Populus euphratica (salt-resistant poplar). To explore the mechanism of the PeJRL in salinity tolerance, the full length of PeJRL was cloned from P. euphratica and was transformed into Arabidopsis. PeJRL was localized to the cytoplasm in mesophyll cells. Overexpression of PeJRL in Arabidopsis significantly improved the salt tolerance of transgenic plants, in terms of seed germination, root growth, and electrolyte leakage during seedling establishment. Under NaCl stress, transgenic plants retained K⁺ and limited the accumulation of Na⁺. PeJRL-transgenic lines increased Na⁺ extrusion, which was associated with the upward regulation of SOS1, AHA1, and AHA2 genes encoding plasma membrane Na⁺/proton (H⁺) antiporter and H⁺-pumps. The activated H⁺-ATPases in PeJRL-overexpressed plants restricted the channel-mediated loss of K⁺ that was activated by NaCl-induced depolarization. Under salt stress, PeJRL⁻transgenic Arabidopsis maintained reactive oxygen species (ROS) homeostasis by activating the antioxidant enzymes and reducing the production of O2- through downregulation of NADPH oxidases. Of note, the PeJRL-transgenic Arabidopsis repressed abscisic acid (ABA) biosynthesis, thus reducing the ABA-elicited ROS production and the oxidative damage during the period of salt stress. A schematic model was proposed to show the mediation of PeJRL on ABA response, and ionic and ROS homeostasis under NaCl stress.

Distinct roles for each N-glycan branch interacting with mannose-binding type Jacalin-related lectins Orysata and Calsepa.

Mannose-binding type Jacalin-related lectins (mJRLs) bind to branched N-glycans via conserved sugar-binding sites. Despite, significant 3D structural similarities, each mJRL is known to have a unique binding preference toward various N-glycans. However, the molecular basis of varying binding preference is substantially unknown. Here, we report a detailed comparison of N-glycan-binding preference for two mJRLs, Orysata and Calsepa using frontal affinity chromatography (FAC), X-ray and molecular modeling. The FAC analysis using a panel of N-glycans shows difference in N-glycan-binding preference between the lectins. Orysata shows broader specificity toward most high-mannose-type glycans as well as biantennary complex-type glycans bearing an extension on the Manα1-6 branch. Whereas, Calsepa shows narrow specificity to complex-type glycans with bisecting GlcNAc. The X-ray crystallographic structure reveals that two Orysata lectins bind to one biantennary N-glycan (2:1 binding) where one lectin binds to mannose of the α1-3 branch, while the other interacts with an N-acetylglucosamine of the α1-6 branch. In contrast, Calsepa shows 1:1 binding where α1-3 branch and core chitobiose region N-glycan interacts with lectin, while α1-6 branch is flipped-back to the chitobiose core. Molecular dynamics study of Orysata bound to N-glycans substantiate possibility of two-binding modes for each N-glycan. Binding free energies calculated separately for α1-3 and α1-6 branches of each N-glycan suggest both branches can bind to Orysata. Overall these results suggest that each branch of N-glycan has a distinct role in binding to mJRLs and the nonbinding branch can contribute significantly to the binding affinity and hence to the specificity.

Evolutionary analysis of the jacalin-related lectin family genes in 11 fishes.

Jacalin-related lectins are a type of carbohydrate-binding proteins, which are distributed across a wide variety of organisms and involved in some important biological processes. The evolution of this gene family in fishes is unknown. Here, 47 putative jacalin genes in 11 fish species were identified and divided into 4 groups through phylogenetic analysis. Conserved gene organization and motif distribution existed in each group, suggesting their functional conservation. Some fishes have eleven jacalin genes, while others have only one or zero gene in their genomes, suggesting dynamic changes in the number of jacalin genes during the evolution of fishes. Intragenic recombination played a key role in the evolution of jacalin genes. Synteny analyses of jacalin genes in some fishes implied conserved and dynamic evolution characteristics of this gene family and related genome segments. Moreover, a few functional divergence sites were identified within each group pairs. Divergent expression profiles of the zebra fish jacalin genes were further investigated in different stresses. The results provided a foundation for exploring the characterization of the jacalin genes in fishes and will offer insights for additional functional studies.

Two jacalin-related lectins from seeds of the African breadfruit (Treculia africana L.).

Two jacalin-related lectins (JRLs) were purified by mannose-agarose and melibiose-agarose from seeds of Treculia africana. One is galactose-recognizing JRL (gJRL), named T. africana agglutinin-G (TAA-G), and another one is mannose-recognizing JRL (mJRL), TAA-M. The yields of the two lectins from the seed flour were approximately 7.0 mg/g for gJRL and 7.2 mg/g for mJRL. The primary structure of TAA-G was determined by protein sequencing of lysyl endopeptic peptides and chymotryptic peptides. The sequence identity of TAA-G to other gJRLs was around 70%. Two-residue insertion was found around the sugar-binding sites, compared with the sequences of other gJRLs. Crystallographic studies on other gJRLs have shown that the primary sugar-binding site of gJRLs can accommodate Gal, GalNAc, and GalNAc residue of T-antigen (Galß1-3GalNAcα-). However, hemagglutination inhibition and glycan array showed that TAA-G did not recognize GalNAc itself and T-antigen. TAA-G preferred melibiose and core 3 O-glycan.

Quantitative proteomics analysis identified new interacting proteins of JAL30 in Arabidopsis.

Jacalin-related lectins (JALs) are a unique group of plant lectins derived from the jacalin protein family, which play important roles in plant defense responses. JAL30/PBP1 (PYK10 binding protein 1) interacts with inactive PYK10, exerting negative regulatory control over the size of the PYK10 complex, which is formed and activated upon insect or pathogen invasion. However, the precise interplay between JAL30 and other components remains elusive. In this study, we found JAL30 as a nucleocytoplasmic protein, but no obvious phenotype was observed in jal30-1 single mutant. Through immunoprecipitation (IP) enrichment combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), dozens of new JAL30 interacting proteins were found in addition to several reported ones. Gene Ontology (GO) analysis revealed that these interacting proteins were highly related to the wounding and bacterial stimuli, suggesting their potential involvement in the jasmonate (JA) response. Importantly, the expression of JAL30 was induced by MeJA treatment, further highlighting its relevance in plant defense mechanisms. A novel JAL30 interacting protein, ESM1, was identified and its interaction with JAL30 was confirmed by Co-immunoprecipitation. Moreover, ESM1 was found as an O-GlcNAcylated protein, suggesting that JAL30 may possess glycosylated protein binding ability, particularly in O-GlcNAcylated protein and peptide recognition. Overall, our study provides valuable insights into the interacting protein network and biological function of JAL30, demonstrates the interaction between JAL30 and ESM1, and uncovers the potential significance of JAL30 in plant defense system, potentially through its association with PYK10 complex or JA response. SIGNIFICANCE: The biological functions of lectin proteins, including defense responses, immunity responses, signal transduction, have been well studied. Lectin proteins were also utilized to enrich glycosylated proteins for their specific carbohydrates binding capability. Jacalin-related lectins (JALs) were found to involve in plant defense mechanism. However, it is not yet clear whether JALs could use for enrichment of glycosylated proteins. In this study, we used label-free quantification method to identify interacting proteins of JAL30. A novel interacting protein, ESM1, as an O-GlcNAcylated protein was found. ESM1 has been reported to take part in defense against insect herbivory. Therefore, our findings provided experimental evidence to confirm that JALs have potential to be developed as the bio-tools to enrich glycosylated proteins. Finally, our data not only illustrated the vital biological role of JALs in plants, but also verified unique function of JAL30 in recognizing O-GlcNAcylated proteins.

A jacalin-like lectin domain-containing protein of Sclerospora graminicola acts as an apoplastic virulence effector in plant-oomycete interactions.

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant-pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from Sg (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs by protein mass spectrometry analysis of extracellular fluid from Sg-inoculated foxtail millet leaves. SgJRLs consist of a jacalin-like lectin domain and an N-terminal putative secretion signal; SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with green fluorescent protein (GFP) localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defence-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defence responses.

Pathogen Resistance Depending on Jacalin-Dirigent Chimeric Proteins Is Common among Poaceae but Absent in the Dicot Arabidopsis as Evidenced by Analysis of Homologous Single-Domain Proteins.

MonocotJRLs are Poaceae-specific two-domain proteins that consist of a jacalin-related lectin (JRL) and a dirigent (DIR) domain which participate in multiple developmental processes, including disease resistance. For OsJAC1, a monocotJRL from rice, it has been confirmed that constitutive expression in transgenic rice or barley plants facilitates broad-spectrum disease resistance. In this process, both domains of OsJAC1 act cooperatively, as evidenced from experiments with artificially separated JRL- or DIR-domain-containing proteins. Interestingly, these chimeric proteins did not evolve in dicotyledonous plants. Instead, proteins with a single JRL domain, multiple JRL domains or JRL domains fused to domains other than DIR domains are present. In this study, we wanted to test if the cooperative function of JRL and DIR proteins leading to pathogen resistance was conserved in the dicotyledonous plant Arabidopsis thaliana. In Arabidopsis, we identified 50 JRL and 24 DIR proteins, respectively, from which seven single-domain JRL and two single-domain DIR candidates were selected. A single-cell transient gene expression assay in barley revealed that specific combinations of the Arabidopsis JRL and DIR candidates reduced the penetration success of barley powdery mildew. Strikingly, one of these pairs, AtJAX1 and AtDIR19, is encoded by genes located next to each other on chromosome one. However, when using natural variation and analyzing Arabidopsis ecotypes that express full-length or truncated versions of AtJAX1, the presence/absence of the full-length AtJAX1 protein could not be correlated with resistance to the powdery mildew fungus Golovinomyces orontii. Furthermore, an analysis of the additional JRL and DIR candidates in a bi-fluorescence complementation assay in Nicotiana benthamiana revealed no direct interaction of these JRL/DIR pairs. Since transgenic Arabidopsis plants expressing OsJAC1-GFP also did not show increased resistance to G. orontii, it was concluded that the resistance mediated by the synergistic activities of DIR and JRL proteins is specific for members of the Poaceae, at least regarding the resistance against powdery mildew. Arabidopsis lacks the essential components of the DIR-JRL-dependent resistance pathway.

A jacalin-related lectin domain-containing lipase from chestnut (Castanea crenata): Purification, characterization, and protein identification.

A novel lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was discovered from Korean chestnut (Castanea crenata). The lipase was isolated and purified by ammonium sulfate precipitation and a fast protein liquid chromatography system equipped with HiTrap DEAE-Sepharose Fast Flow, HiTrap Q-Sepharose Fast Flow, and HiPrep Sephacryl S-100 Hi-Resolution columns. The purified C. crenata lipase showed a 15.8% yield, purification fold number of 465.8, and specific activity against triolein of 88.5 mU/mg. The enzyme exhibited hydrolytic activity toward tributyrin, trilaurin, and triolein, and was maximally active at pH 8.0 and 35 °C, with triolein used as the substrate. The activation energy (Ea) and deactivation energy (Ed) of triolein hydrolysis were 38.41 and 83.35 kJ/mol, respectively. In the enzyme kinetic study, Vmax, Km, and k cat were 110.58 mU/mg, 0.11 mM, and 0.221 min-1, respectively. The relatively low Km value indicated that the lipase has high affinity for its substrate. Moreover, Mg2+ and Ca2+ increased the lipase activity to 115.4% and 108.3%, respectively. The results of peptide fingerprinting revealed that the C. crenata lipase with a molecular weight of 33.3 kDa was structurally similar to the mannose-binding lectin of the jacalin-related lectin domain superfamily, implying that it has potential as a therapeutic agent for use in the biomedical industry.

Internalization of a sunflower mannose-binding lectin into phytopathogenic fungal cells induces cytotoxicity.

Lectins are carbohydrate-affinity proteins with the ability to recognize and reversibly bind specific glycoconjugates. We have previously isolated a bioactive sunflower mannose-binding lectin belonging to the jacalin-related family called Helja. Despite of the significant number of plant lectins described in the literature, only a small group exhibits antifungal activity and the mechanism by which they kill fungi is still not understood. The aim of this work was to explore Helja activity on plant pathogenic fungi, and provide insights into its mechanism of action. Through cellular and biochemical experimental approaches, here we show that Helja exerts an antifungal effect on Sclerotinia sclerotiorum, a sunflower pathogen. The lectin interacts with the fungal spore surface, permeabilizes its plasma membrane, can be internalized into the cell and induces oxidative stress, finally leading to the cell death. On the other hand, Helja is inactive towards Fusarium solani, a non-pathogen of sunflower, showing the selective action of the lectin. The mechanistic basis for the antifungal activity of an extracellular jacalin lectin is presented, suggesting its initial interaction with fungal cell wall carbohydrates and further internalization. The implication of our findings for plant defense is discussed.

Two carbohydrate recognizing domains from Cycas revoluta leaf lectin show the distinct sugar-binding specificity-A unique mannooligosaccharide recognition by N-terminal domain.

Cycas revoluta leaf lectin (CRLL) of mannose-recognizing jacalin-related lectin (mJRL) has two tandem repeated carbohydrate recognition domains, and shows the characteristic sugar-binding specificity toward high mannose-glycans, compared with other mJRLs. We expressed the N-terminal domain and C-terminal domain (CRLL-N and CRLL-C) separately, to determine the fine sugar-binding specificity of each domain, using frontal affinity chromatography, glycan array and equilibrium dialysis. The specificity of CRLL toward high mannose was basically derived from CRLL-N, whereas CRLL-C had affinity for α1-6 extended mono-antennary complex-type glycans. Notably, the affinity of CRLL-N was most potent to one of three Man 8 glycans and Man 9 glycan, whereas the affinity of CRLL-C decreased with the increase in the number of extended α1-2 linked mannose residue. The recognition of the Man 8 glycans by CRLL-N has not been found for other mannose recognizing lectins. Glycan array reflected these specificities of the two domains. Furthermore, it was revealed by equilibrium dialysis method that the each domain had two sugar-binding sites, similar with Banlec, banana mannose-binding Jacalin-related lectin.

Polarized Defense Against Fungal Pathogens Is Mediated by the Jacalin-Related Lectin Domain of Modular Poaceae-Specific Proteins.

Modular proteins are an evolutionary answer to optimize performance of proteins that physically interact with each other for functionality. Using a combination of genetic and biochemical experiments, we characterized the rice protein OsJAC1, which consists of a jacalin-related lectin (JRL) domain predicted to bind mannose-containing oligosaccharides, and a dirigent domain which might function in stereoselective coupling of monolignols. Transgenic overexpression of OsJAC1 in rice resulted in quantitative broad-spectrum resistance against different pathogens including bacteria, oomycetes, and fungi. Overexpression of this gene or its wheat ortholog TAJA1 in barley enhanced resistance against the powdery mildew fungus. Both protein domains of OsJAC1 are required to establish resistance as indicated by single or combined transient expression of individual domains. Expression of artificially separated and fluorescence-tagged protein domains showed that the JRL domain is sufficient for targeting the powdery mildew penetration site. Nevertheless, co-localization of the lectin and the dirigent domain occurred. Phylogenetic analyses revealed orthologs of OsJAC1 exclusively within the Poaceae plant family. Dicots, by contrast, only contain proteins with either JRL or dirigent domain(s). Altogether, our results identify OsJAC1 as a representative of a novel type of resistance protein derived from a plant lineage-specific gene fusion event for better function in local pathogen defense.