Abstract
Many Aeschynomene sp. belonging to the Dalbergioids family, like Arachis hypogea, can develop a nitrogen-fixing root symbiosis with photosynthetic bradyrhizobia. Among them, certain strains are lacking the nod genes operon which is essential for Nod Factor biosynthesis. First genetic analysis in Aeschynomene evenia revealed that many genes involved in bacterial perception in model legumes are not functionals in the Nod-independent symbiosis whereas the genes involved in signal transduction are mainly conserved.In the aim to better understand how Nod-independent nodulation has been set up, we searched to identify new genes using forward genetics on A. evenia. An EMS mutagenized population has been generated leading to the isolation of 250 stable mutant lineages affected in the nodulation process. Among these mutants, half of them are totally unable to nodulate and are qualified by « Nod- ».A Targeted-Sequence-Capture analysis has been realized on the Nod- mutants and allows the identification of mutations in many known genes of the « Nod signaling pathway » This kind of analysis is possible thanks to a reference genome which has been sequenced and annotated recently. Mutations have been found in AePOLLUX, AeCCaMK, AeCYCLOPS, AeNIN, and AeNSP2 genes whereas no mutations have been found in any LysM-RLK (Nod factor receptors). Gene expression analysis by Q-PCR on different mutants have been realized to observe eventual deregulation of other symbiotic genes.Concerning Nod- mutants for which no known symbiotic gene has been associated, we performed a Mapping-by-Sequencing approach that is combining cartography and whole-genome sequencing to localize homozygous mutations. For some mutants with the same phenotype, we identify mutations on a gene coding a Receptor-Like Cytoplasmic Kinase (RLCK). This gene underwent a specific duplication event that is only observed in Nod-independent Aeschynomene species, suggesting a role of this cytoplasmic kinase during the Nod-independent symbiosis. This kind of protein devoid of the extracellular domain and belonging to a family known for associates with Receptor-Like Kinase (RLK) to transduce the signal and activation of genetic programs of development, reproduction, growth, and immunity. However, this is one of the first times that a protein of this family is shown to be involved in nitrogen-fixing symbiosis (except for LjNiCK4 in 2019).Furthermore, the Mapping-by-sequencing approach allows the identification of 3 other new symbiotic genes (AeCRK, AeRINRK, AeMLRR-RLP) that are not described in model legumes (except LjRINRK1 in 2019). In this manuscript, the results of these analyses will be presented and discussed in order to understand the molecular mechanisms governing the nodulation in the absence of Nod factors.