Lotus japonicus is a well-characterized model legume widely used in the study of plant-microbe interactions. However, datasets from various Lotus studies are poorly integrated and lack interoperability. We recognize the need for a comprehensive repository that allows comprehensive and dynamic exploration of Lotus genomic and transcriptomic data. Equally important are user-friendly in-browser tools designed for data visualization and interpretation. Here, we present Lotus Base, which opens to the research community a large, established LORE1 insertion mutant population containing an excess of 120,000 lines, and serves the end-user tightly integrated data from Lotus, such as the reference genome, annotated proteins, and expression profiling data. We report the integration of expression data from the L. japonicus gene expression atlas project, and the development of tools to cluster and export such data, allowing users to construct, visualize, and annotate co-expression gene networks. Lotus Base takes advantage of modern advances in browser technology to deliver powerful data interpretation for biologists. Its modular construction and publicly available application programming interface enable developers to tap into the wealth of integrated Lotus data. Lotus Base is freely accessible at: https://lotus.au.dk.

SUMMARYLong terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

Establishment of symbiotic nitrogen-fixation in legumes is regulated by the plant hormone ethylene, but it has remained unclear whether and how its biosynthesis is regulated by the symbiotic pathway. We established a sensitive ethylene detection system for Lotus japonicus and found that ethylene production increased as early as 6 hours after inoculation with Mesorhizobium loti. This ethylene response was dependent on Nod factor production by compatible rhizobia. Analyses of nodulation mutants showed that perception of Nod factor was required for ethylene emission, while downstream transcription factors including CYCLOPS, NIN, and ERN1 were not required for this response. Activation of the nodulation signaling pathway in spontaneously nodulating mutants was also sufficient to elevate ethylene production. Ethylene signaling is controlled by EIN2, which is duplicated in L. japonicus. We obtained a L. japonicus Ljein2a Ljein2b double mutant that exhibits complete ethylene insensitivity and confirms that these two genes act redundantly in ethylene signaling. Consistent with this redundancy, both LjEin2a and LjEin2b are required for negative regulation of nodulation and Ljein2a Ljein2b double mutants are hypernodulating and hyperinfected. We also identified an unexpected role for ethylene in the onset of nitrogen fixation, with the Ljein2a Ljein2b double mutant showing severely reduced nitrogen fixation. These results demonstrate that ethylene production is an early and sustained nodulation response that acts at multiple stages to regulate infection, nodule organogenesis, and nitrogen fixation in L. japonicus.

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here, we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with colonization of Japan by Lotus japonicus over the past~20,000 years. Based on patterns of genomic variation, we infer the details of the colonization process where L. japonicus gradually spread from subtropical conditions to much colder climates in northern Japan. We identify genomic regions with extreme genetic differentiation between northern and southern subpopulations and perform population structure-corrected association mapping of phenotypic traits measured in a common garden. Comparing the results of these analyses, we find that signatures of extreme subpopulation differentiation overlap strongly with phenotype association signals for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection during colonization and point to associated candidate genes.

Lotus japonicus is a well-studied nodulating legume and a model organism for the investigation of plant-microbe interactions. The majority of legume transcriptome studies have focused on interactions with compatible symbionts, whereas responses to non-adapted rhizobia and pathogenic bacteria have not been well-characterized. In this study, we first characterized the transcriptomic response of L. japonicus to its compatible symbiont, Mesorhizobium loti R7A, through RNA-seq analysis of various plant tissues. Early symbiotic signaling was largely Nod factor-dependent and enhanced within root hairs, and we observed large-scale transcriptional reprogramming in nodule primordia and mature nitrogen-fixing nodules. We then characterized root transcriptional responses to a spectrum of L. japonicus interacting bacteria ranging from semi-compatible symbionts to pathogens. M. loti R7A and the semi-compatible strain Sinorhizobium fredii HH103 showed remarkably similar responses, allowing us to identify a small number of genes potentially involved in differentiating between fully and semi-compatible symbionts. The incompatible symbiont Bradyrhizobium elkanii USDA61 induced a more attenuated response, but the weakest response was observed for the foliar pathogen Pseudomonas syringae pv. tomato DC3000, where the affected genes also responded to other tested bacteria, pointing to a small set of common bacterial response genes. In contrast, the root pathogen Ralstonia solanacearum JS763 induced a pronounced and distinct transcriptomic pathogen response, which we compared to the results of the other treatments. This comparative analysis did not support the concept that an early defense-like response is generally evoked by compatible rhizobia during establishment of symbiosis.