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Author: search.filters.author.Sinharoy, SenjutiSubject: search.filters.subject.Root nodule symbiosis

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    Genome sequencing of Mesorhizobium Spp. NI-7, an efficient nitrogen-fixing microsymbiont of chickpea with potential to unravel the molecular mechanisms of symbiotic nitrogen fixation in legumes
    (Springer Nature Publishing AG, 2024) Srivastava, Deevita; Ghosh, Asim K; Ranjan, Aashish; Sinharoy, Senjuti
    Root nodule symbiosis is a major pillar of sustainable agriculture. The newly formed symbiotic organ in the legume root harbours rhizobacteria, which can fix atmospheric nitrogen into a bioavailable and reduced form, ammonia. Previously, we reported the isolation of an efficient Mesorhizobium spp. NI-7, from the interior of chickpea nodules. Here, we report the draft genome sequence of the Mesorhizobium spp. NI-7 and the comparative genomics among different Mesorhizobium strains that have adopted symbiosis during chickpea domestication. The draft genome of Mesorhizobium spp. NI-7 consists of a single 4.28 Mbp chromosome and a 359 Kbp plasmid. The 16 S rDNA sequence based phylogenetic analysis highlighted that Mesorhizobium spp. NI-7 belongs to a diverse Mesorhizobium clade that evolved during the domestication of chickpea. Comparative genomics among several Mesorhizobium strains identified 2193 common orthologous groups and several unique orthologous groups among the different Mesorhizobium pairs. The draft genome contains the essential nitrogen fixation genes along with the genes required for the nutrient exchange from the plant to the symbiont. Additionally, part of the symbiotic NOD-factor operon and Type III secretion system were also detected in the Mesorhizobium spp. NI-7 draft genome. The comparative genomics among the Mesorhizobium strains identified a subset of rhizobial genes that would have evolved during chickpea-Mesorhizobium adaptation to the Indian sub-continent. These genes are unique targets that can be validated in the future to understand the chickpea and Mesorhizobium adaptation. In summary, the draft genome sequencing of Mesorhizobium spp. NI-7 will equip the plant-microbe community with a chickpea-compatible Mesorhizobium strain isolated from India, suitable for both fundamental and advanced research on nodulation in chickpea, as well as for promoting sustainable agriculture in a comprehensive manner.
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    Nodule INception (NIN)-independent epidermal events lead to bacterial entry during nodule development in peanut (Arachis hypogaea)
    (John Wiley & Sons, 2022) Bhattacharjee, Oindrila; Raul, Bikash; Ghosh, Amit; Bhardwaj, Akanksha; Bandyopadhyay, Kaustav; Sinharoy, Senjuti
    Summary Legumes can host nitrogen-fixing rhizobia inside root nodules. In model legumes, rhizobia enter via infection threads (ITs) and develop nodules where infection-zone contains a mixture of infected and uninfected cells. Peanut (Arachis hypogaea) diversified from model legumes ~50-55 million years ago. Rhizobia enter through ‘cracks’ to form nodules in peanut roots where the cells of infection-zone are uniformly infected. Phylogenomic studies indicated symbiosis as a labile trait in peanut. These atypical features prompted us to investigate the molecular mechanism of peanut nodule development. Combining cell biology, genetics, and genomic tools, we visualized the status of hormonal signaling in peanut nodule primordia. Moreover, we dissected the signaling modules of Nodule INception (NIN), a master regulator of both epidermal infection and cortical organogenesis. Cytokinin signaling operates in a broad zone, from the epidermis to the pericycle inside nodule primordia, while auxin signaling is narrower and focused. NIN is involved in the nodule organogenesis, but not in the crack entry. Nodulation Pectate Lyase (NPL), which remodels cell walls during IT-formation, is not required. Whereas Nodule enhanced Glycosyl Hydrolases (AhNGHs) is recruited for cell wall modification during crack entry. While the hormonal regulation is conserved, the function of the NIN signaling modules is diversified in peanut.
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    An improvised hairy root transformation method for efficient gene silencing in roots and nodules of Arachis hypogaea
    (Springer Nature Publishing AG, 2022) Raul, Bikash; Sinharoy, Senjuti
    Peanut (Arachis hypogaea) is a major oilseed crop and is widely cultivated in tropical and subtropical climate zone worldwide. Peanut belongs to the Papilionoid family with an atypical nodule developmental program. In particular, rhizobia enter through developmental cracks and lead to the formation of aeschynomenoid subtype determinate nodules. Peanut nodules are efficient nitrogen-fixers and form swollen bacteroid containing symbiosomes. The allotetraploid genome and recalcitrance to stable transformation used to be the major bottleneck for peanut biologists. Recent genome sequencing of peanut cultivar Tifrunner has opened up a huge opportunity for molecular research. A composite plant contains transformed roots with a non-transformed shoot. The composite plant-based approach has already proven to be a tool of choice for high throughput studies in root biology. The available protocols failed to generate efficient hairy root transformation in the genome sequenced cultivar Tifrunner. Here we describe an efficient hairy root transformation and composite plant generation protocol for the peanut cultivar Tifrunner. Our protocol generated ~92% plant regeneration efficiency with between 21.8% and 58.6% co-transformed root regeneration. We also show that this protocol can be efficiently used for protein localization, promoter GUS analysis, monitoring hormone response, and RNAi mediated knockdown of the genes using genome sequenced cultivar Tifrunner.
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    Optimization of hairy root transformation for the functional genomics in chickpea: A platform for nodule developmental studies
    (Springer Nature Publishing AG, 2020) Mandal, Drishti; Srivastava, Deevita; Sinharoy, Senjuti
    Chickpea is a major protein source in low socio-economic classes and cultivated in marginal soil without fertilizer or irrigation. As a result of its root nodule formation capacity chickpea can directly use atmospheric nitrogen. Chickpea is recalcitrant to stable transformation, particularly root regeneration efficiency of chickpea is low. The composite plant-based system with a non-transformed shoot and transformed root is particularly important for root biologist and this approach has already been used successfully for root nodule symbiosis, arbuscular mycorrhizal symbiosis, and other root-related studies. Use of fluorescent marker-based approach can accurately identify the transformed root from its non-transgenic counterpart. RNAi-based gene knockout, overexpression of genes, promoter GUS analysis to understand tissue specific expression and localization of protein can be achieved using the hairy root-based system. We have already published a hairy root-based transformation and composite plant regeneration protocol of chickpea. Here we are describing the recent modification that we have made to increase the transformation frequency and nodule morphology. Further, we have developed a pouch based artificial system, large number of plants can be scored for its nodule developmental phenotype, by using this system.