A collection of novel Lotus japonicus LORE1 mutants perturbed in the nodulation program induced by the Agrobacterium pusense strain IRBG74.

The Lotus japonicus population carrying new Lotus retrotransposon 1 (LORE1) insertions represents a valuable biological resource for genetic research. New insertions were generated by activation of the endogenous retroelement LORE1a in the germline of the G329-3 plant line and arranged in a 2-D system for reverse genetics. LORE1 mutants identified in this collection contributes substantially to characterize candidate genes involved in symbiotic association of L. japonicus with its cognate symbiont, the nitrogen-fixing bacteria Mesorhizobium loti that infects root nodules intracellularly. In this study we aimed to identify novel players in the poorly explored intercellular infection induced by Agrobacterium pusense IRBG74 sp. For this purpose, a forward screen of > 200,000 LORE1 seedlings, obtained from bulk propagation of G329-3 plants, inoculated with IRBG74 was performed. Plants with perturbed nodulation were scored and the offspring were further tested on plates to confirm the symbiotic phenotype. A total of 110 Lotus mutants with impaired nodulation after inoculation with IRBG74 were obtained. A comparative analysis of nodulation kinetics in a subset of 20 mutants showed that most of the lines were predominantly affected in nodulation by IRBG74. Interestingly, additional defects in the main root growth were observed in some mutant lines. Sequencing of LORE1 flanking regions in 47 mutants revealed that 92 Lotus genes were disrupted by novel LORE1 insertions in these lines. In the IM-S34 mutant, one of the insertions was located in the 5´UTR of the LotjaGi5g1v0179800 gene, which encodes the AUTOPHAGY9 protein. Additional mutant alleles, named atg9-2 and atg9-3, were obtained in the reverse genetic collection. Nodule formation was significantly reduced in these mutant alleles after M. loti and IRBG74 inoculation, confirming the effectiveness of the mutant screening. This study describes an effective forward genetic approach to obtain novel mutants in Lotus with a phenotype of interest and to identify the causative gene(s).

The Lotus japonicus ROP3 Is Involved in the Establishment of the Nitrogen-Fixing Symbiosis but Not of the Arbuscular Mycorrhizal Symbiosis.

Legumes form root mutualistic symbioses with some soil microbes promoting their growth, rhizobia, and arbuscular mycorrhizal fungi (AMF). A conserved set of plant proteins rules the transduction of symbiotic signals from rhizobia and AMF in a so-called common symbiotic signaling pathway (CSSP). Despite considerable efforts and advances over the past 20 years, there are still key elements to be discovered about the establishment of these root symbioses. Rhizobia and AMF root colonization are possible after a deep cell reorganization. In the interaction between the model legume Lotus japonicus and Mesorhizobium loti, this reorganization has been shown to be dependent on a SCAR/Wave-like signaling module, including Rho-GTPase (ROP in plants). Here, we studied the potential role of ROP3 in the nitrogen-fixing symbiosis (NFS) as well as in the arbuscular mycorrhizal symbiosis (AMS). We performed a detailed phenotypic study on the effects of the loss of a single ROP on the establishment of both root symbioses. Moreover, we evaluated the expression of key genes related to CSSP and to the rhizobial-specific pathway. Under our experimental conditions, rop3 mutant showed less nodule formation at 7- and 21-days post inoculation as well as less microcolonies and a higher frequency of epidermal infection threads. However, AMF root colonization was not affected. These results suggest a role of ROP3 as a positive regulator of infection thread formation and nodulation in L. japonicus. In addition, CSSP gene expression was neither affected in NFS nor in AMS condition in rop3 mutant. whereas the expression level of some genes belonging to the rhizobial-specific pathway, like RACK1, decreased in the NFS. In conclusion, ROP3 appears to be involved in the NFS, but is neither required for intra-radical growth of AMF nor arbuscule formation.

Eco-Physiological Traits Related to Recovery from Complete Submergence in the Model Legume Lotus japonicus.

Submergence is a severe form of stress for most plants. Lotus japonicus is a model legume with potential use in assisting breeding programs of closely related forage Lotus species. Twelve L. japonicus genotypes (10 recombinant inbred lines (RILs) and 2 parental accessions) with different constitutive shoot to root dry mass ratios (S:R) were subjected to 7 days of submergence in clear water and allowed to recover for two weeks post-submergence; a set of non-submerged plants served as controls. Relative growth rate (RGR) was used to indicate the recovery ability of the plants. Leaf relative water content (RWC), stomatal conductance (gs), greenness of basal and apical leaves, and chlorophyll fluorescence (Fv/Fm, as a measure of photoinhibition) were monitored during recovery, and relationships among these variables and RGR were explored across genotypes. The main results showed (i) variation in recovery ability (RGR) from short-term complete submergence among genotypes, (ii) a trade-off between growth during vs. after the stress indicated by a negative correlation between RGR during submergence and RGR post-submergence, (iii) an inverse relationship between RGR during recovery and S:R upon de-submergence, (iv) positive relationships between RGR at early recovery and RWC and gs, which were negatively related to S:R, suggesting this parameter as a good estimator of plant water balance post-submergence, (v) chlorophyll retention allowed fast recovery as revealed by the positive relationship between greenness of basal and apical leaves and RGR during the first recovery week, and (vi) full repair of the submergence-damaged photosynthetic apparatus occurred more slowly (second recovery week) than full recovery of plant water relations. The inclusion of these traits contributing to submergence recovery in L. japonicus should be considered to speed up the breeding process of the closely related forage Lotus spp. used in current agriculture.

The fungal endophyte Fusarium solani provokes differential effects on the fitness of two Lotus species.

The interactions established between plants and endophytic fungi span a continuum from beneficial to pathogenic associations. The aim of this work was to isolate potentially beneficial fungal endophytes in the legume Lotus tenuis and explore the mechanisms underlying their effects. One of the nine fungal strains isolated was identified as Fusarium solani and shows the highest phosphate-solubilisation activity, and also grows endophytically in roots of L. japonicus and L. tenuis. Interestingly, fungal invasion enhances plant growth in L. japonicus but provokes a contrasting effect in L. tenuis. These differences were also evidenced when the rate of photosynthesis as well as sugars and K contents were assessed. Our results indicate that the differential responses observed are due to distinct mechanisms deployed during the establishment of the interactions that involve the regulation of photosynthesis, potassium homeostasis, and carbohydrate metabolism. These responses are employed by these plant species to maintain fitness during the endophytic interaction.

Lotus japonicus plants of the Gifu B-129 ecotype subjected to alkaline stress improve their Fe(2+) bio-availability through inoculation with Pantoea eucalypti M91.

Inoculation assays with Pantoea eucalypti M91 were performed on Lotus japonicus ecotype Gifu. Under alkaline conditions, this ecotype is characterized by the development of interveinal chlorosis of the apical leaves due to low mobilization of Fe(2+). Inoculation with P. eucalypti M91, a plant growth-promoting bacterial strain capable of producing pyoverdine-like and pyochelin-like siderophores under alkaline growth conditions, alters the root, resulting in a herringbone pattern of root branching. Additional features include improvement in Fe(2+) transport to the shoots, acidification of the hydroponic solution of the plant cultures, and an accompanying increase in the efficiency of the PSII parameters. In addition, there was an increase in the expression of the FRO1 and IRT1 genes, accompanied by a significant increase in FRO activity. Results showed that P. eucalypti M91 has a beneficial effect on the Fe acquisition machinery of Strategy I, as described for non-graminaceous monocots and dicots, suggesting its potential as an inoculant for legume crops cultivated in alkaline soils.

Understanding the roles of Lys33 and Arg45 in the binding-site stability of LjLTP10, an LTP related to drought stress in Lotus japonicus.

In Lotus japonicus, as in most plants, long-chain fatty acids are important components of cuticular wax, one of the principal functions of which is to act as a barrier to water loss in response to drought stress. It is thought that lipid transfer proteins (LTPs) are involved in the process of cuticle formation. We previously described LjLTP10 as an LTP involved in cuticle formation during acclimation response to drought stress in L. japonicus. The structural model of LjLTP10 had two residues (K33 and R45) in the hydrophobic cavity, although the role of these residues was unclear. In the present work, we investigated the molecular mechanism involved in the transport of lipid precursors in L. japonicus and clarified the importance of the residues K33 and R45. First, in silico site-directed mutagenesis studies were carried out on the LjLTP10 structure. Structural analysis showed that LjLTP10 mutants possess similar structures but their hydrophobic cavities are somewhat different. Unfavorable energies for the interactions of the mutant proteins with different ligands were found by molecular docking and molecular dynamics simulations. We also examined the contributions of energetic parameters to the free energy of the protein-ligand complex using the MM-GBSA method. Results showed that the different complexes present similar, favorable van der Waals interactions, whereas electrostatic interactions were not favored in the mutant structures. Our study indicates that the residues K33 and R45 play a crucial role in maintaining the binding pocket structure required for lipid transport.