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    Evolutionary and expression dynamics of LRR-RLKs and functional establishment of KLAVIER homolog in shoot mediated regulation of AON in chickpea symbiosis
    (Elsevier B.V., 2021) Tiwari, Manish; Pandey, Vimal; Singh, Baljinder; Yadav, Manisha; Bhatia, Sabhyata
    Chickpea shoot exogenously treated with cytokinin showed stunted phenotype of root, shoot and significantly reduced nodule numbers. Genome-wide identification of LRR-RLKs in chickpea and Medicago resulted in 200 and 371 genes respectively. Gene duplication analysis revealed that LRR-RLKs family expanded through segmental duplications in chickpea and tandem duplications in Medicago. Expression profiling of LRR-RLKs revealed their involvement in cytokinin signaling and plant organ development. Overexpression of KLAVIER ortholog of chickpea, Ca_LRR-RLK147, in roots revealed its localization in the membrane but showed no effect on root nodulation despite increased cle peptide levels. Two findings (i) drastic effect on nodule number by exogenous cytokinin treatment to only shoot and restoration to normal nodulation by treatment to both root and shoot tissue and (ii) no effect on nodule number by overexpression of Ca_LRR-RLK147 establishes the fact that despite presence of cle peptides in root, the function of Ca_LRR-RLK147 was shoot mediated during AON.
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    Evolutionary and functional analysis of Two-Component System in chickpea reveals CaRR13, a TypeB RR, as positive regulator of symbiosis
    (John Wiley & Sons, 2021) Tiwari, Manish; Yadav, Manisha; Singh, Baljinder; Pandey, Vimal; Nawaz, Kashif; Bhatia, Sabhyata
    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 signaling, 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 sln1Δ 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 signaling molecule regulating early events of nodule organogenesis in chickpea.
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    Expression profiling of miRNAs indicates crosstalk between phytohormonal response and rhizobial infection in chickpea
    (Springer Nature Publishing AG, 2020) Tiwari, Manish; Bhatia, Sabhyata
    Legumes develop root nodules in which bacteria fix nitrogen for plants. The phytohormones auxin and cytokinin regulate nodule organogenesis by recruiting various genes to effect symbiosis. Moreover, these genes are regulated by the action of microRNAs also. To understand the complex regulatory network involving miRNAs in response to phytohormones and rhizobial interactions in chickpea roots, a miRNA expression profiling was performed. Indole acetic acid and 6-benzylaminopurine at concentrations of 0.1, 1 and 10 lM were used for auxin and cytokinin exogenous treatment and Mesorhizobium ciceri to study rhizobial interaction with chickpea root. Expression profiling of a set of 11 miRNAs was performed. Further, the targets of the candidate miRNAs were identified, followed by functional annotation. This analysis revealed that cat-miR160, cat-miR164, cat-miR396 and cat-miR398 were responsive to auxin and cytokinin. cat-miR319 was found to be only auxin responsive and is known to regulate auxin signalling by targeting TEOSINTE BRANCHED/ CYCLOIDEA/PCF (TCP) which interacts with auxin inducible genes. Further, cytokinin elicited a response at very low concentration of 0.1 lM, and most of the miRNAs investigated were responsive to cytokinin. Interactome analysis revealed that cat-miR164 and cat-miR168 work in conjunction to regulate auxin signalling. Interestingly, cat-miR169 and cat-miR482 were low expressing during auxin treatment and M. ciceri infection but their expression spiked during cytokinin treatment, indicating a cytokinin mediated mode of action. The miRNA expression profiling in response to phytohormones and rhizobia and the reported function of their target genes suggested a crosstalk among the phytohormonal responses during chickpea nodulation.