Browsing by Author "Mandal, Drishti"

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CRISPR-Cas9 system: A new-fangled dawn in gene editing
(Elsevier B.V., 2019) Gupta, Darshana; Bhattacharjee, Oindrila; Mandal, Drishti; Sen, Madhab Kumar; Dey, Dhritiman; Dasgupta, Adhiraj; Kazi, Tawsif Ahmed; Gupta, Rahul; Sinharoy, Senjuti; Acharya, Krishnendu; Chattopadhyay, Dhrubajyoti; Ravichandiran, V.; Roy, Syamal; Ghosh, Dipanjan
Till date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9–based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.
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.
Root-specific expression of chickpea cytokinin oxidase/dehydrogenase 6 leads to enhanced root growth, drought tolerance and yield without compromising nodulation
(John Wiley & Sons, 2020) Khandal, Hitaishi; Gupta, Santosh Kumar; Dwivedi, Vikas; Mandal, Drishti; Sharma, Nilesh Kumar; Vishwakarma, Niraj Kumar; Pal, Lalita; Choudhary, Megha; Francis, Aleena; Malakar, Paheli; Singh, Nagendra Pratap; Sharma, Kapil; Sinharoy, Senjuti; Singh, Narendra Pratap; Sharma, Rameshwar; Chattopadhyay, Debasis
Cytokinin group of phytohormones regulate root elongation and branching during post‐embryonic development. Cytokinin degrading enzymes cytokinin oxidases/dehydrogenases (CKXs) have been deployed to investigate biological activities of cytokinin and to engineer root growth. We expressed chickpea cytokinin oxidase 6 (CaCKX6) under the control of a chickpea root‐specific promoter of CaWRKY31 in Arabidopsis thaliana and chickpea having determinate and indeterminate growth patterns, respectively, to study the effect of cytokinin depletion on root growth and drought tolerance. Root‐specific expression of CaCKX6 led to a significant increase in lateral root number and root biomass in Arabidopsis and chickpea without any penalty to vegetative and reproductive growth of shoot. Transgenic chickpea lines showed increased CKX activity in root. Soil‐grown advanced chickpea transgenic lines exhibited higher root‐to‐shoot biomass ratio and enhanced long‐term drought tolerance. These chickpea lines were not compromised in root nodulation and nitrogen fixation. The seed yield in some lines was up to 25% higher with no penalty in protein content. Transgenic chickpea seeds possessed higher levels of zinc, iron, potassium and copper. Our results demonstrated the potential of cytokinin level manipulation in increasing lateral root number and root biomass for agronomic trait improvement in an edible legume crop with indeterminate growth habit.
A toolbox for nodule development studies in chickpea: a hairy-root transformation protocol and an efficient laboratory strain of Mesorhizobium sp.
(American Phytopathological Society, 2019) Mandal, Drishti; Sinharoy, Senjuti
Mesorhizobium sp. produces root nodules in chickpea. Chickpea and model legume Medicago truncatula are members of inverted repeat lacking clade (IRLC). The rhizobia after internalization into the plant cell are called 'bacteroid'. Nodule Specific Cysteine-rich (NCR) peptides in IRLC legumes guide bacteroids to a 'terminally differentiated swollen (TDS)' form. Bacteroids in chickpea are less TDS than those in Medicago. Nodule development in chickpea indicates recent evolutionary diversification and merits further study. A hairy root transformation protocol and an efficient laboratory strain are prerequisites for performing any genetic study on nodulation. We have standardized a protocol for composite plant generation in chickpea with a transformation frequency above 50 %, as shown by fluorescent markers. This protocol also works well in different ecotypes of chickpea. Localization of subcellular markers in these transformed roots is similar to the localization observed in transformed Medicago roots. When checked inside transformed nodules, peroxisomes were concentrated along the periphery of the nodules, while ER and Golgi bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated for their ability to initiate nodule development and efficiency of nitrogen fixation. Inoculation with different strains resulted in different shapes of TDS bacteroids with variable nitrogen fixation. Our study provides a toolbox to study nodule development in the crop legume chickpea.
A tripartite interaction among the basidiomycete Rhodotorula mucilaginosa, N2-fixing endobacteria, and rice improves plant nitrogen nutrition
(American Society of Plant Biologists, 2020) Paul, Karnelia; Saha, Chinmay; Nag, Mayurakshi; Mandal, Drishti; Naiya, Haraprasad; Sen, Diya; Mitra, Souvik; Kumar, Mohit; Bose, Dipayan; Mukherjee, Gairik; Naskar, Nabanita; Lahiri, Susanta; Ghosh, Upal Das; Tripathi, Sudipta; Sarkar, Mousumi Poddar; Banerjee, Manidipa; Kleinert, Aleysia; Valentine, Alexander J.; Tripathy, Sucheta; Sinharoy, Senjuti; Seal, Anindita
Nitrogen (N) limits crop yield and improvement of N nutrition remains a key goal for crop research; one approach to improve N nutrition is identifying plant-interacting N2-fixing microbes. Rhodotorula mucilaginosa JGTA-S1 is a basidiomycetous yeast endophyte of narrowleaf cattail (Typha angustifolia). JGTA-S1 could not convert nitrate or nitrite to ammonium, but harbors diazotrophic (N2-fixing) endobacteria (eg. Pseudomonas stutzeri) that allows JGTA-S1 to fix N2 and grow in a N-free environment; moreover, P. stutzeri dinitrogen reductase (nifH) was transcribed in JGTA-S1 even under adequate N. Endobacteria-deficient JGTA-S1 had reduced fitness, which was restored by reintroducing P. stutzeri. JGTA-S1 colonizes rice (Oryza sativa) significantly improving its growth, N content, and relative N-use efficiency. Endofungal P. stutzeri plays a significant role in increasing the biomass and ammonium content of rice treated with JGTA-S1; also, JGTA-S1 has better N2 fixing ability than free-living P. stutzeri and provides fixed N to the plant. Genes involved in N metabolism, N transporters, and NODULE INCEPTION (NIN)-like transcription factors were upregulated in rice roots within 24 h of JGTA-S1 treatment. In association with rice, JGTA-S1 has a filamentous phase and P. stutzeri only penetrated filamentous JGTA-S1. Together, these results demonstrate an interkingdom interaction that improves rice N nutrition.