Suppression of LjBAK1-mediated immunity by SymRK promotes rhizobial infection in Lotus japonicus.

An important question in biology is how organisms can associate with different microbes that pose no threat (commensals), pose a severe threat (pathogens), and those that are beneficial (symbionts). The root nodule symbiosis serves as an important model system for addressing such questions in the context of plant-microbe interactions. It is now generally accepted that rhizobia can actively suppress host immune responses during the infection process, analogous to the way in which plant pathogens can evade immune recognition. However, much remains to be learned about the mechanisms by which the host recognizes the rhizobia as pathogens and how, subsequently, these pathways are suppressed to allow establishment of the nitrogen-fixing symbiosis. In this study, we found that SymRK (Symbiosis Receptor-like Kinase) is required for rhizobial suppression of plant innate immunity in Lotus japonicus. SymRK associates with LjBAK1 (BRASSINOSTEROID INSENSITIVE 1-Associated receptor Kinase 1), a well-characterized positive regulator of plant innate immunity, and directly inhibits LjBAK1 kinase activity. Rhizobial inoculation enhances the association between SymRK and LjBAK1 in planta. LjBAK1 is required for the regulation of plant innate immunity and plays a negative role in rhizobial infection in L. japonicus. The data indicate that the SymRK-LjBAK1 protein complex serves as an intersection point between rhizobial symbiotic signaling pathways and innate immunity pathways, and support that rhizobia may actively suppress the host's ability to mount a defense response during the legume-rhizobium symbiosis.

Anti-inflammatory and Antioxidant Effects of Lotus corniculatus on Paracetamol-induced Hepatitis in Rats.

BACKGROUND: Herbal medicines have been used in the treatment of liver diseases for a long time. The current study was elaborated to evaluate in vitro and in vivo antioxidant and anti-inflammatory effects of Lotus corniculatus (L. corniculatus) butanolic extract. METHODS: The in vitro antioxidant and anti-inflammatory properties of L. corniculatus were investigated by employing DPPH radical scavenging, H2O2 scavenging and BSA denaturation assays. In vivo antioxidant and anti-inflammatory effects of L. corniculatus were evaluated against paracetamol (APAP)-induced hepatitis in rats. L.corniculatus at doses of 100 and 200 mg/kg was administered orally once daily for seven days. Serum transaminases (AST and ALT) and lactate dehydrogenase (LDH), total bilirubin levels, liver malondialdehyde (MDA), reduced glutathione (GSH), glutathione S- transferase (GST) and superoxide dismutase (SOD) levels and inflammatory markers, such as serum Creactive protein (CRP), circulating and liver myeloperoxidase (MPO) levels were investigated. Further histopathological analysis of the liver sections was performed to support the effectiveness of L. corniculatus. RESULTS: L. corniculatus exhibited strong antioxidant and anti-inflammatory effects in vitro. In the in vivo study, our findings demonstrate that L. corniculatus (100 and 200 mg/kg) administration led to an amelioration of APAP effects on liver histology, liver functions parameters (AST, ALT, LDH, and total bilirubin levels) and liver oxidative stress markers (MDA, GSH, GST and SOD levels). Furthermore, serum CRP, circulating MPO and liver MPO levels were declined by both doses of L. corniculatus extract. The best benefits were observed with 200 mg/kg of L. corniculatus extract. CONCLUSION: Antioxidant and anti-inflammatory effects of L. corniculatus extract may be due to the presence of active components.

LYS12 LysM receptor decelerates Phytophthora palmivora disease progression in Lotus japonicus.

Phytophthora palmivora is a devastating oomycete plant pathogen. We found that P. palmivora induces disease in Lotus japonicus and used this interaction to identify cellular and molecular events in response to this oomycete, which has a broad host range. Transcript quantification revealed that Lys12 was highly and rapidly induced during P. palmivora infection. Mutants of Lys12 displayed accelerated disease progression, earlier plant death and a lower level of defence gene expression than the wild type, while the defence program after chitin, laminarin, oligogalacturonide or flg22 treatment and the root symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhiza were similar to the wild type. On the microbial side, we found that P. palmivora encodes an active chitin synthase-like protein, and mycelial growth is impaired after treatment with a chitin-synthase inhibitor. However, wheat germ agglutinin-detectable N-acetyl-glucosamine (GlcNAc) epitopes were not identified when the oomycete was grown in vitro or while infecting the roots. This indicates that conventional GlcNAc-mers are unlikely to be produced and/or accumulate in P. palmivora cell walls and that LYS12 might perceive an unknown carbohydrate. The impact of Lys12 on progression of root rot disease, together with the finding that similar genes are present in other P. palmivora hosts, suggests that LYS12 might mediate a common early response to this pathogen.

The Phenylalanine Ammonia Lyase Gene LjPAL1 Is Involved in Plant Defense Responses to Pathogens and Plays Diverse Roles in Lotus japonicus-Rhizobium Symbioses.

Phenylalanine ammonia lyase (PAL) is important in the biosynthesis of plant secondary metabolites that regulate growth responses. Although its function is well-established in various plants, the functional significance of PAL genes in nodulation is poorly understood. Here, we demonstrate that the Lotus japonicus PAL (LjPAL1) gene is induced by Mesorhizobium loti infection and methyl-jasmonate (Me-JA) treatment in roots. LjPAL1 altered PAL activity, leading to changes in lignin contents and thicknesses of cell walls in roots and nodules of transgenic plants and, hence, to structural changes in roots and nodules. LjPAL1-knockdown plants (LjPAL1i) exhibited increased infection thread and nodule numbers and the induced upregulation of nodulin gene expression after M. loti infection. Conversely, LjPAL1 overexpression delayed the infection process and reduced infection thread and nodule numbers after M. loti inoculation. LjPAL1i plants also exhibited reduced endogenous salicylic acid (SA) accumulation and expression of the SA-dependent marker gene. Their infection phenotype could be partially restored by exogenous SA or Me-JA application. Our data demonstrate that LjPAL1 plays diverse roles in L. japonicus-rhizobium symbiosis, affecting rhizobial infection progress and nodule structure, likely by inducing lignin modification, regulating endogenous SA biosynthesis, and modulating SA signaling.

The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus.

Bacterial accommodation inside living plant cells is restricted to the nitrogen-fixing root nodule symbiosis. In many legumes, bacterial uptake is mediated via tubular structures called infection threads (ITs). To identify plant genes required for successful symbiotic infection, we screened an ethyl methanesulfonate mutagenized population of Lotus japonicus for mutants defective in IT formation and cloned the responsible gene, ERN1, encoding an AP2/ERF transcription factor. We performed phenotypic analysis of two independent L. japonicus mutant alleles and investigated the regulation of ERN1 via transactivation and DNA-protein interaction assays. In ern1 mutant roots, nodule primordia formed, but most remained uninfected and bacterial entry via ITs into the root epidermis was abolished. Infected cortical nodule cells contained bacteroids, but transcellular ITs were rarely observed. A subset exhibited localized cell wall degradation and loss of cell integrity associated with bacteroid spread into neighbouring cells and the apoplast. Functional promoter studies revealed that CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter and in combination with CCaMK positively regulates ERN1 transcription. We conclude that the activation of ERN1 by CCaMK/CYCLOPS complex is an important step controlling IT-mediated bacterial progression into plant cells.

Functional analysis of chimeric lysin motif domain receptors mediating Nod factor-induced defense signaling in Arabidopsis thaliana and chitin-induced nodulation signaling in Lotus japonicus.

The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1-AtCERK1 and LjNFR5-AtCERK1, were expressed in cerk1-2, an A. thaliana CERK1 mutant lacking chitin-induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1-2 expressing both chimeras accumulated reactive oxygen species, expressed chitin-responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor-binding protein), respectively. The chimeras, named OsCERK1-LjNFR1 and OsCEBiP-LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1-1; nfr5-2) carrying a GUS (ß-glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF-induced defense signaling in non-legumes and chitin-induced nodulation signaling in legumes.

Defense responses in two ecotypes of Lotus japonicus against non-pathogenic Pseudomonas syringae.

Lotus japonicus is a model legume broadly used to study many important processes as nitrogen fixing nodule formation and adaptation to salt stress. However, no studies on the defense responses occurring in this species against invading microorganisms have been carried out at the present. Understanding how this model plant protects itself against pathogens will certainly help to develop more tolerant cultivars in economically important Lotus species as well as in other legumes. In order to uncover the most important defense mechanisms activated upon bacterial attack, we explored in this work the main responses occurring in the phenotypically contrasting ecotypes MG-20 and Gifu B-129 of L. japonicus after inoculation with Pseudomonas syringae DC3000 pv. tomato. Our analysis demonstrated that this bacterial strain is unable to cause disease in these accessions, even though the defense mechanisms triggered in these ecotypes might differ. Thus, disease tolerance in MG-20 was characterized by bacterial multiplication, chlorosis and desiccation at the infiltrated tissues. In turn, Gifu B-129 plants did not show any symptom at all and were completely successful in restricting bacterial growth. We performed a microarray based analysis of these responses and determined the regulation of several genes that could play important roles in plant defense. Interestingly, we were also able to identify a set of defense genes with a relative high expression in Gifu B-129 plants under non-stress conditions, what could explain its higher tolerance. The participation of these genes in plant defense is discussed. Our results position the L. japonicus-P. syringae interaction as a interesting model to study defense mechanisms in legume species.

Distinct binding patterns of fucose-specific lectins from Biomphalaria alexandrina and Lotus tetragonolobus to murine lymphocyte subsets.

The putative expression of distinct terminally fucosylated glycoconjugates among murine lymphocyte subpopulations was sought using a Biomphalaria alexandrina-derived lectin (BaSI), of proven specificity to a fucosyllactose determinant, and the fucose-binding lectin from Lotus tetragonolobus seeds. Direct labeling of isolated lymphocyte subsets in suspension as well as immuno-histochemical and two-dimensional Western blotting assays demonstrated the exclusive expression of the BaSI-reactive ligand among multiple isoforms of two major 95 and 92 kDa and a minor 82 kDa acidic glycoproteins, selectively localized to the splenic marginal zone B lymphocytes of adult mice. The expression of the L. tetragonolobus lectin-reactive ligand was, on the other hand, restricted primarily to a single homogeneous 50 kDa acidic glycoprotein associated with a subset of the mature (PNA-) medullary thymocytes of adult mice as well as a minority of the immature (PNA +) thymocytes within the deep cortical region in newly born mice. The significance of these findings is discussed in relation to mechanisms that govern lymphocyte development and homing.