Institutional Publications
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Item Rice phosphate transporter reduces the low phosphate response through jasmonate signaling(Oxford University Press, 2026) Mani, Balaji; Maurya, Kanika; Verma, Lokesh; Gupta, Priya; Kohli, Pawandeep Singh; Gupta, Gagan; Jaskolowski, Aime; Poirier, Yves; Giri, JitenderPhosphorus (P) is an essential macronutrient for plant growth, and its deficiency severely limits crop productivity. The PHOSPHATE1 (PHO1) protein family, defined by an N-terminal SPX domain, four transmembrane (4TM) domains, and a C-terminal EXS domain, mediates phosphate (Pi) loading into the xylem for root-to-shoot transport. In rice, OsPHO1;2 is critical for Pi export, and loss-of-function mutants exhibit severe growth retardation and Pi deficiency symptoms despite sufficient external Pi. To dissect the functional contributions of PHO1 domains beyond Pi transport, we generated CRISPR/Cas9 rice lines expressing either the EXS domain containing part of the SPX domain (S-EXS) or the 4TM+EXS domains (T-EXS) of OsPHO1;2. Phenotypic analyses under Pi-sufficient and Pi-deficient conditions revealed that S-EXS lines displayed improved early growth compared to ospho1;2 mutants, despite similar shoot Pi levels. These plants exhibited reduced jasmonic acid accumulation and attenuated phosphate starvation responses, resembling wild-type hormone profiles. In contrast, T-EXS lines mirrored the growth defects of ospho1;2 mutants. Transcriptome profiling confirmed that defense and phosphate starvation pathways were less activated in S-EXS lines relative to mutants. However, both S-EXS and T-EXS lines retained seed development defects and reduced seed phosphorus content, consistent with ospho1;2 phenotypes. Heterozygous plants carrying one functional OsPHO1;2 allele exhibited normal growth and seed development, confirming the recessive nature of the mutation. Collectively, these findings demonstrate that the S-EXS domain of OsPHO1;2 promotes plant growth independently of Pi transport by modulating jasmonate signaling and suppressing phosphate starvation responses. This highlights a signaling role for PHO1 domains, offering new insights into Pi homeostasis and potential strategies for breeding Pi-efficient crops.Item Editing cis-elements of OsPHO1;2 improved phosphate transport and yield in rice(John Wiley & Sons, 2025) Maurya, Kanika; Mani, Balaji; Singh, Bhagat; Sirohi, Ujjwal; Jaskolowski, Aime; Sharma, Sandeep; Tatiparthi, Harsha Vardhan; Mangrauthia, Satendra Kumar; Pandey, Renu; Poirier, Yves; Giri, JitenderIncreasing grain yield is the primary goal of crop improvement, which is globally affected by the low availability of soil phosphate (Pi). Overexpressing Pi transporters to enhance Pi uptake often results in Pi toxicity and growth retardation. Despite advances in genetic engineering, targeting the cis-regulatory motifs of Pi transporters remains underexplored for understanding plant mechanisms and improving Pi status. Here, we demonstrate that the excision of the transcription inhibitor motif from the promoter of the Pi transporter OsPHO1;2 enhances its expression and increases root-to-shoot Pi transport, leading to improved grain yield. Through in silico and DNA-protein interaction studies, we show the role of the OsWRKY6 transcription factor in negatively regulating OsPHO1;2 expression by binding to the cis-regulatory element (W-box) present in its promoter. The oswrky6 knockout lines exhibit higher OsPHO1;2 expression and improved shoot Pi levels. Furthermore, we engineered the OsPHO1;2 promoter to precisely remove the W-box and enhance OsPHO1;2 expression. Phenotypic and physiological evaluations at the vegetative stage indicate that OsPHO1;2 promoter-edited (OsPHO1;2:PE) lines have increased shoot length, plant biomass and greater root-to-shoot Pi export under both low and normal P conditions. Notably, the 33P uptake assay reveals that OsPHO1;2:PE lines display enhanced root Pi uptake, supported by higher expression of root-associated Pi transporters (OsPHTs). An extensive agronomic assessment shows that OsPHO1;2:PE lines achieve increased seed and panicle numbers, thereby raising yield without affecting seed quality. Our findings provide valuable insights into the potential of promoter editing to improve Pi use and enhance crop yield.Item Isopropylmalate synthase regulatory domain removal abolishes feedback regulation at the expense of leucine homeostasis in plants(Oxford University Press, 2025) Varghese, Mohan; Kumar, Roshan; Sharma, Aprajita; Lone, Asif; Gershenzon, Jonathan; Bisht, Naveen C.In the leucine (Leu) biosynthesis pathway, homeostasis is achieved through a feedback regulatory mechanism facilitated by the binding of the end-product Leu at the C-terminal regulatory domain of the first committed enzyme, isopropylmalate synthase (IPMS). In vitro studies have shown that removing the regulatory domain abolishes the feedback regulation on plant IPMS while retaining its catalytic activity. However, the physiological consequences and underlying molecular regulation on Leu flux upon removing the IPMS C-terminal domain remain to be explored in plants. Here, we removed the IPMS C-terminal regulatory domain using a CRISPR/Cas9-based gene editing system and studied the resulting impact on the Leu biosynthesis pathway under in planta conditions. Absence of the IPMS regulatory domain unexpectedly reduced the formation of the end product Leu but increased the levels of Leu pathway intermediates in mustard (Brassica juncea). Additionally, delayed growth was observed when IPMS devoid of the regulatory domain was introduced into IPMS-null mutants of Escherichia coli and Arabidopsis thaliana. Further, a detailed biochemical analysis showed that in the absence of the C-terminal regulatory domain, a Leu pathway intermediate (α-ketoisocaproate) could compete with the native IPMS substrate (2-oxoisovalerate) for the active site. Combining these metabolomic, biochemical, and in planta analyses, we demonstrate that the C-terminal regulatory domain of IPMS is critical for maintaining Leu-Val homeostasis in plants.Item CRISPR/Cas9 edited StbHLH47 lines exhibit altered expression profiling of iron regulating genes and increased iron content in Solanum tuberosum(Elsevier B.V., 2024) Chauhan, Hanny; Alok, Anshu; Aiana; Upadhyay, Santosh K.; Pandey, Ashutosh; Singh, KashmirIron is an essential plant nutrient, and a continuous supply of it is required as it is a key factor in various metabolic processes, including photosynthesis, chlorophyll synthesis, and respiration. Various transcription factors are known to regulate iron homeostasis in plants, and the bHLH transcription factor family is one of them. The StbHLH47 is a homologue of the Arabidopsis POPEYE (PYE), which is known to repress iron homeostasis-related genes in Arabidopsis. Potato is the most consumed vegetable in the world and is low in iron content. We have generated CRISPR/Cas9-edited StbHLH47 lines and performed a detailed analysis of these lines. The analysis revealed that the roots of StbHLH47 edited lines have decreased ferric chelate reductase (FCR) activity compared to the roots of the wild-type (WT) plant. We also observed that CRISPR/Cas9 edited lines have fewer trichomes when compared to the WT plant. The expression of genes associated with iron homeostasis was also measured. Compared to the control, the expression of StbHLH47 was downregulated in the edited lines, while the expression of StNAS4, StOPT3, and StFRO3 was upregulated. This suggests the negative regulation of StbHLH47 in modulating iron. The iron content was also quantified using inductively coupled plasma mass spectrometry (ICP-MS) and found to be increased in the generated transgenic lines when compared to WT plants. Overall, this study reveals that StbHLH47 negatively regulates the expression of iron homeostasis-related genes. StbHLH47 edited lines exhibited decreased FCR activity, changes in phenotype, and increased iron content in the potato plants.Item Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed, high-leaf glucosinolate oilseed mustard with uncompromised defence and yield(John Wiley & Sons, 2023) Mann, Avni; Kumari, Juhi; Kumar, Roshan; Kumar, Pawan; Pradhan, Akshay K.; Pental, Deepak; Bisht, Naveen C.Glucosinolate content in the two major oilseed Brassica crops-rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.Item Development of an Agrobacterium-delivered codon-optimized CRISPR/Cas9 system for chickpea genome editing(Springer Nature Publishing AG, 2023) Gupta, Santosh Kumar; Vishwakarma, Niraj Kumar; Malakar, Paheli; Vanspati, Poonam; Sharma, Nilesh Kumar; Chattopadhyay, DebasisChickpea is considered recalcitrant to in vitro tissue culture amongst all edible legumes. The clustered, regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)–based genome editing in chickpea can remove the bottleneck of limited genetic variation in this cash crop, which is rich in nutrients and protein. However, generating stable mutant lines using CRISPR/Cas9 requires efficient and highly reproducible transformation protocols. As an attempt to solve this problem, we developed a modified and optimized protocol for chickpea transformation. This study transformed the single cotyledon half-embryo explants using CaMV35S promoter to drive two marker genes (β-glucuronidase gene; GUS and green fluorescent protein; GFP) through binary vectors pBI101.2 and modified pGWB2, respectively. These vectors were delivered in the explants through three different strains of Agrobacterium tumefaciens, viz., GV3101, EHA105, and LBA4404. We found better efficiency with the strain GV3101 (17.56%) compared with two other strains, i.e., 8.54 and 5.43%, respectively. We recorded better regeneration frequencies in plant tissue culture for the constructs GUS and GFP, i.e., 20.54% and 18.09%, respectively. The GV3101 was further used for the transformation of the genome editing construct. For the development of genome-edited plants, we used this modified protocol. We also used a modified binary vector pPZP200 by introducing a CaMV35S-driven chickpea codon-optimized SpCas9 gene. The promoter of the Medicago truncatula U6.1 snRNA gene was used to drive the guide RNA cassettes. This cassette targeted and edited the chickpea phytoene desaturase (CaPDS) gene. A single gRNA was found sufficient to achieve high efficiency (42%) editing with the generation of PDS mutants with albino phenotypes. A simple, rapid, highly reproducible, stable transformation and CRISPR/Cas9-based genome editing system for chickpea was established. This study aimed to demonstrate this system’s applicability by performing a gene knockout of the chickpea PDS gene using an improved chickpea transformation protocol for the first time.Item Gene pyramiding in transgenic plant development: Approaches and challenges(Springer Nature Publishing AG, 2023) Rajput, Ruchika; Naik, Jogindra; Misra, Prashant; Trivedi, Prabodh Kumar; Pandey, AshutoshClimate change leads to frequent alterations in environmental factors with a reciprocal impact on crop productivity. Over the last few decades, various approaches have been used for producing more stress-tolerant and climate-flexible crops. Genetic engineering is one of the approaches used to modify multiple characters or to improve more than one agronomic trait in plants. These instances simultaneously demand simultaneous genetic manipulation of multiple genes, necessitating stacking or pyramiding of multiple genes as compared to single-gene manipulations, and the genetic engineering of plants using multiple genes is technically challenging. In the last two decades, considerable progress has been made with respect to the development and application of the methods for gene pyramiding in transgenic context. The conventional methods of gene stacking include the crossing of individual transgenic plants, co-transformation using multiple plant expression constructs, transformation with single constructs carrying multiple transgenes as well as with the constructs carrying polycistronic transgenes. These methods have been instrumental for gene stacking in several commercialized crops. The tools of targeted genome editing (ZFN, TALEN, and CRISPR) that carry out precise genetic modifications, have opened new avenues in the area of crop biotechnology for defending plants against various stresses. The present review covers the current status of biotechnological techniques used to combat biotic and abiotic stresses in crop plants and describes multiple associated challenges.Item Recent advancements and future perspectives of foxtail millet genomics(Springer Nature Publishing AG, 2023) Panchal, Anurag; Singh, Roshan Kumar; Prasad, ManojOne of the oldest domesticated crops, foxtail millet (Setaria italica (L.) P. Beauv.) is known for its enormous seed production and ability to grow in unfavourable agro-economic conditions. This understudied diploid panicoid crop plant which performs C4 type of photosynthesis, contains approximately seven folds higher proteins with macro and micro-nutrients as compared to major cereals including rice and wheat. Encompassing a small genome size of ~ 515 Mb, short life cycle, and inbred properties make this orphan crop a potential model system to study abiotic stress tolerance mechanisms adopted by plants. Rich phenotypic variation and the largest available germplasm collection of cultivated and wild species exists for foxtail millet. Release of whole genome sequence in 2012, led to acceleration of functional genomic studies and molecular characterization of Setaria genes conferring stress tolerance. Recent advancements in integrative OMICS and NGS approaches are contributing in functional analysis, understanding complex gene regulatory networks and molecular mechanisms behind its growth and development. Abiotic stress tolerance traits of foxtail millet including drought, salinity, and its survival in nutrient-poor soil can be introduced in popular crop plants by genetic engineering techniques. Stable plant transformation system and precise gene editing in foxtail millet are under optimization which can revolutionize the theme of climate resilient future crops. Overall, foxtail millet has characteristics to serve as an excellent C4 model plant to study evolution, stress physiology and biomass production for bioenergy crops to serve sustainable global food security in near future.Item RNA interference for improving disease resistance in plants and its relevance in this clustered regularly interspaced short palindromic repeats-dominated era in terms of dsRNA-based biopesticides(Frontiers Media S.A., 2022) Halder, Koushik; Chaudhuri, Abira; Abdin, Malik Z.; Majee, Manoj; Datta, AsisRNA interference (RNAi) has been exploited by scientists worldwide to make a significant contribution in the arena of sustainable agriculture and integrated pest management. These strategies are of an imperative need to guarantee food security for the teeming millions globally. The already established deleterious effects of chemical pesticides on human and livestock health have led researchers to exploit RNAi as a potential agri-biotechnology tool to solve the burning issue of agricultural wastage caused by pests and pathogens. On the other hand, CRISPR/Cas9, the latest genome-editing tool, also has a notable potential in this domain of biotic stress resistance, and a constant endeavor by various laboratories is in progress for making pathogen-resistant plants using this technique. Considerable outcry regarding the ill effects of genetically modified (GM) crops on the environment paved the way for the research of RNAi-induced double-stranded RNAs (dsRNA) and their application to biotic stresses. Here, we mainly focus on the application of RNAi technology to improve disease resistance in plants and its relevance in today’s CRISPR-dominated world in terms of exogenous application of dsRNAs. We also focused on the ongoing research, public awareness, and subsequent commercialization of dsRNA-based biocontrol products.Item Compendium of plant-specific CRISPR vectors and their technical advantages(MDPI AG, 2021) Alok, Anshu; Chauhan, Hanny; Upadhyay, Santosh Kumar; Pandey, Ashutosh; Kumar, Jitendra; Singh, KashmirCRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.