Browsing by Author "Tiwari, Manish"

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Dynamics of miRNA mediated regulation of legume symbiosis
(John Wiley & Sons, 2021) Tiwari, Manish; Pandey, Vimal; Singh, Baljinder; Bhatia, Sabhyata
Symbiotic nitrogen fixation in legume nodules is important in soils with low nitrogen availability. The initiation and sustainability of symbiosis requires cellular reprogramming that involves the miRNA‐mediated inhibition or activation of specific nodulation genes. The high‐throughput sequencing of small RNA libraries has identified miRNAs and their targets, which are the major players in the post‐transcriptional gene regulation (PTGS) of the different stages of legume‐rhizobia symbiosis ranging from bacterial colonization and organogenesis to symbiotic nitrogen fixation. Here we present an overview of information obtained from the miRNA libraries from nodulating tissues that have been sequenced to date. The functional analysis of miRNAs has revealed roles in phytohormone homeostasis and spatio‐temporal regulation, as well as the mobility of miRNAs and their functions in shoot to root signalling that affects diverse functions, including bacterial entry, meristem division and differentiation, nitrogen fixation and senescence. Furthermore, small RNA fragments of rhizobial origin repress complementary plant mRNAs. We also consider the roles of miRNAs in determinate or indeterminate nodules. Taken together, this overview confirms that miRNAs are master regulators of the legume‐rhizobia symbiosis.
Emerging tools and paradigm shift of gene editing in cereals, fruits, and horticultural crops for enhancing nutritional value and food security
(John Wiley & Sons, 2021) Tiwari, Manish; Trivedi, Prabodh Kumar; Pandey, Ashutosh
Gene editing using sequence‐specific nucleases, particularly CRISPR/Cas ribonucleoprotein, has drawn enormous attention in plant research in recent years. Nearly a decade ago, Cas9 protein was initially discovered for a role in adaptive immunity in bacteria. Owing to vast potential, a large number of reports came out in a short span of time, comprising the identification of Cas protein from different bacterial sources, new Cas9 variants with reduced off‐targets, multiplexing, base editing, prime editing, and RNA manipulation in plants. Studies revealed that CRISPR/Cas‐based gene editing can play a major role in ensuring food security via developing resilient commercial crops with improved yield and nutritional value. Use of the CRISPR/Cas9 system for creating mutation in genes and regulatory regions of promoter generated a number of alleles with variable phenotypes, which can serve as an excellent genetic resource in the breeding program. In this review, we provide a recent overview of state‐of‐art discoveries in the CRISPR/Cas system comprised of new Cas proteins, modifications of existing Cas9, refinements in CRISPR/Cas‐induced gene editing, applications, and outcome emphasizing on major cereals and horticultural crops. We also highlight the current global policy framework for the regulation of gene‐edited crops.
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.
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.
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.
Feeding the future: role of OsAUX5 in enhancing rice nutritional value
(Springer Nature Publishing AG, 2023) Mondal, Kongkong; Tiwari, Manish; Singh, Roshan Kumar; Prasad, Manoj; Dey, Narottam
Essential amino acids (EAAs) such as valine, leucine, isole-ucine, phenylalanine, tryptophan, threonine, lysine, methio-nine, and histidine are not synthesized in the human body. They, therefore, need to be acquired from either plant or animal sources (Sá etal. 2020). Although plant proteins sup-ply the required amount of EAAs, they are often identiﬁed as nutritionally inferior compared to animal proteins.
High throughput identification of miRNAs reveal novel interacting targets regulating chickpea-rhizobia symbiosis
(Elsevier B.V., 2021) Tiwari, Manish; Singh, Baljinder; Yadav, Manisha; Pandey, Vimal; Bhatia, Sabhyata
Legumes developed symbiotic associations to meet its nitrogen requirement. The nitrogen fixation takes place in root nodules which involves bacterial colonization, organogenesis and nitrogen fixation. In order to unravel the miRNA mediated regulation of chickpea symbiosis, one microRNA and four parallel analysis of RNA ends (PARE) libraries were sequenced. Analysis of microRNA library identified a set of 91 miRNAs comprising of 84 conserved and 7 novel miRNAs. Additionally, PARE library analysis revealed 564 genes being targeted by 85 miRNAs. Phylogenetic analysis of the precursor sequences of the 91 miRNAs was carried out which revealed their ancestral relationships. Further, the mechanism of miRNAs biogenesis was predicted using the miRNAs information from other legumes. Reads from the nodule library were mapped to bacterial genomes to predict bacterial-encoded small RNAs. Real time expression analysis was used to validate the antagonistic expression pattern of important miRNA-mRNA target pairs. Four candidate miRNAs were selected for in planta study based on the antagonistic expression profiling as well as the novelty of their respective targets. miR171f, miR172c, miR394 and miR1509 targeted nodulation receptor kinase, Apetala2, histidine phosphotransferase, adenylate kinase respectively and were ectopically expressed in chickpea roots. The overexpression lines showed significant change in nodule numbers, the miR172c, miR394 and miR1509 resulted in an increase in nodule number whereas, miR171f overexpression led to a decrease in nodule number. Our analysis lays the foundation for functional characterization of novel miRNAs and their respective target pairs which control nodulation in chickpea and other leguminous crops.