Evolutionary and functional analysis of two-component system in chickpea reveals CaRR13, a TypeB RR, as positive regulator of symbiosis

The critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.), led to the formation of pseudo-nodules even in the absence of rhizobia. Hence, a genome-wide analysis of the cytokinin signalling, two-component system (TCS) genes, was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out, which revealed that histidine kinases (HKs) were highly conserved, whereas there was diversification leading to neofunctionalization at the level of response regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1 Delta mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule-deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13, was functionally characterized. Its localization in the nucleus and its Y1H assay-based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signalling molecule regulating early events of nodule organogenesis in chickpea.