The Lotus japonicus-IRBG74 symbiosis was established as a novel working model to study the conditional intercellular infection in legumes and the main features of the Lotus-IRBG74 symbiosis was characterized. RNAseq analysis and the symbiotic performance of Lotus mutants allowed the elucidation of the transcriptome and genetic requirements for intercellular colonization in this symbiotic association. A transcriptome analysis has provided a list of Lotus genes mainly induced by IRBG74. The list was assembled by comparing the transcriptomic response of Lotus roots during an intra- and intercellular infection process. Homozygous Lotus LORE1 mutants were obtained for some of these genes and their symbiotic performance assessed. Complementing these approaches a Lotus LORE1 mutant population was screened for mutants impaired in intercellular infection. Screening a population of > 200,000 mutants induced by the LORE1 retrotransposon allowed isolation of > 100 mutants with a symbiotic phenotype. A valuable biological resource for future research. Additionally, a list of genes potentially responsible for the deficient symbiotic ability in this mutant population was obtained. Besides the symbiotic phenotype, at least one selected mutant exhibits an interesting and unexpected root phenotype, whose characterization would impact other plant science fields. To explore the biodiversity among Lotus japonicus ecotypes a GWAS approach was performed using more than 100 Lotus ecotypes from which genome sequences was available. Genomic regions associated to intercellular infection ability were identified by analyzing the nodulation performance with IRBG74 in > 100 Lotus japonicus ecotypes.
Taking a completely different approach to identify Lotus genes required for or involved in governing the rhizobial infection, a single cell sequencing project was initiated. A procedure for protoplasting root cell was established and optimized.to protoplast isolation and single cell sequencing. in order to identify novel regulators of infection. This has yielded an exceptionally detailed map of the infection process in different cell types of the root and root nodules. To optimize characterization of regulator genes identified in the single cell approach an effort to optimize the procedure for stable transformation and for generating CRISPR mutants was initiated. Stable transformed Lotus lines expressing TurBoID tagged receptors has been established and the first step towards biochemical identification of important protein-protein interactions taken.
To expand identification of genes controlling intercellular infection an EMS mutagenesis and mutant screening program was initiated on Lotus burttii. More than 5000 M1 plants were propagated in greenhouses and seeds were harvested from individual plants and in bulk. The M2 is screened for root nodule phenotypes after inoculation with Sinorhizobium fredii that infects Lotus burttii intercellularly. This unbiased screen has the potential to uncover a new layer of genes and mechanisms regulating rhizobial infection and endophyte colonization will also be monitored in plant mutants identified to assess the possible conservation of intercellular infection mechanisms. Single cell sequencing using the 10X system will further support this approach.
Research on intercellular infection initiated in RINFEC is continued in other projects in order to complete the many promising lines of investigation. The approaches and results obtained in RINFEC has established a foundation for scientific progress in understanding plant microbe-interaction and will have an impact on application of biologicals that is an emerging industry. RINFEC research was dissiminated at international and national conferences and through national and international media.
