Browsing by Author "Sinharoy, Senjuti"

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Callus induction and efficient in vitro plant regeneration protocol for Chickpea
(Springer Nature Publishing AG, 2024) Jangid, Vinod Kumar; Senthil-Kumar, Muthappa; Chandran, Divya; Sinharoy, Senjuti
The development of an efficient and consistent callus-mediated in vitro regeneration protocol is crucial for biotechnological approaches aimed at improving chickpea, an economically important crop legume. In this study, we assess the effectiveness of callus-mediated regeneration in different chickpea genotypes. Through in vitro screening of explants, we identified the Indian cultivar Pusa 240 as a favourable genotype with higher efficiency of somatic embryogenesis and in vitro plant regeneration. Building upon this finding, we have successfully established two distinct protocols for chickpea callus-mediated somatic embryogenesis, utilizing leaf and hypocotyl explants obtained from the Pusa 240 genotype. These protocols achieved plant regeneration efficiencies of 27% using leaf explants and 46.6 − 66% using hypocotyl explants. Extensive literature review and comparative analysis underscored the superiority of our current protocol. Subsequently, the regenerated plants were successfully acclimatized and transferred to the greenhouse, exhibiting normal phenotypic growth. This detailed regeneration method will provide a valuable resource for chickpea genetic transformations and the generation of large mutant populations where embryogenesis via callus formation is required. The protocol presented here establishes a powerful tool for studying the functional genomics of chickpea plants and lays the foundation for future advancements in this field.
Conserved hinge regions in SYMRK enable release of Malectin-like Domain for symbiont passage during rhizobia-legume symbiosis
(Oxford University Press, 2026) Chakrabarti, Dipanjan; Paul, Anindita; Molla, Firoz; Bhattacharyya, S; Das, Sagnik; Chakraborty, Sampurna; Ghosh, Dona; Biswas, Alokmoy; Kundu, Anindya; Sinharoy, Senjuti; DasGupta, Maitrayee
Symbiosis Receptor Kinase (SYMRK), a malectin-like-domain/leucine-rich-repeat receptor-like-kinase (MLD-LRR-RLK), is the upstream most component in the Common-Symbiosis-Signalling-Pathway. We highlight two Proline residues that were distinctly acquired by SYMRK orthologues in its hinge-regions to constitute a signalling module for allowing progress of symbionts across transcellular barriers during rhizobia-legume symbiosis. Within the Ectodomain hinge (EctoD-hinge) all MLD-LRR-RLKs have a conserved W1xnGDPCxnW2x4C motif, where SYMRK orthologues within legumes have a distinct signature with a Proline preceding W2 enabling cleavage of SYMRK ectodomain for releasing MLD. Within the kinase hinge (KD-hinge) at gatekeeper+1 position, a conserved Glutamate in MLD-LRR-RLKs is replaced by Proline in all SYMRK orthologues that enabled its dual-specific kinase activity for ensuring ectodomain cleavage. Substitution of either Proline restricted cortical progression of symbionts forming infection patches in the nodule apex without affecting epidermal invasion and nodule organogenesis. This halt was entirely overcome by ectopic expression of free MLD demonstrating the released MLD to have an active role in progress of symbionts. Overall, we show that conservation of distinct Prolines in hinge-regions of SYMRK orthologues in legumes generates a signalling module involving dimerization and optimal phosphorylation of SYMRK for releasing MLD as an active transducer of symbiosis signalling.
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.
Discovery of diverse chimeric peptides in a eukaryotic proteome sets the stage for experimental validation of the mosaic translation hypothesis
(Elsevier B.V., 2025) Çakır, Umut; Gabed, Noujoud; Koroglu, Yunus Emre; Kaya, Selen; Sinharoy, Senjuti; Benedito, Vagner A.; Brunet, Marie; Roucou, Xavier; Kryvoruchko, Igor S.
The high complexity of eukaryotic organisms enabled their evolutionary success, driven by the diversification of their proteomes. Various mechanisms contributed to this process. Alternative splicing had the largest known impact among these mechanisms. Earlier, we hypothesized that along with alternative splicing, a different but conceptually similar mechanism creates novel versions of existing proteins in all eukaryotes. However, this mechanism operates at the level of translation, where amino acid sequence novelty arises through multiple programmed ribosomal frameshifting events occurring within the same transcript. This mechanism, which is termed mosaic translation, is very difficult to demonstrate even with the most up-to-date molecular tools. Thus, it remained unnoticed so far. Using a subset of mass spectrometry proteomic data from various organs of the model plant Medicago truncatula, we took the first step toward experimental validation of this hypothesis. Our original in silico approach resulted in the discovery of two candidates for mosaic proteins (homologs of EF1α and RuBisCo) and 154 candidates for chimeric peptides. Chimeric peptides and polypeptides are produced in the course of one ribosomal frameshifting event and may correspond to parts of mosaic proteins. In addition, our analysis reveals the possibility of translation of chimeric peptides from five ribosomal RNA transcripts, ten long non-coding RNA transcripts, and one transfer RNA transcript. These findings are novel and will form the basis for future experimental validation. We also present multiple lines of indirect evidence supporting the validity of our in silico data.
Drought stress exacerbates fungal colonization and endodermal invasion and dampens defense responses to increase dry root rot in chickpea
(American Phytopathological Society, 2022) Irulappan, Vadivelmurugan; Kandpal, Manu; Saini, Kumud; Rai, Avanish; Ranjan, Aashish; Sinharoy, Senjuti; Senthil-Kumar, Muthappa
Drought plays a central role in increasing the incidence and severity of dry root rot (DRR) disease in chickpea. This is an economically devastating disease, compromising chickpea yields particularly severely in recent years due to erratic rainfall patterns. Macrophomina phaseolina (formerly Rhizoctonia bataticola) is the causal agent of DRR disease in the chickpea plant. The infection pattern in chickpea roots under well-watered conditions and drought stress are poorly understood at present. This study provides detailed disease symptomatology and the characteristics of DRR fungus at morphological and molecular levels. Using microscopy techniques, the infection pattern of DRR fungus in susceptible chickpea roots was investigated under well-watered and drought stress conditions. Our observations suggested that drought stress intensifies the progression of already ongoing infection by weakening the endodermal barrier and overall defense. Transcriptomic analysis suggested that the plant’s innate immune defense program is downregulated in infected roots when subjected to drought stress. Further, genes involved in hormonal regulation are differentially expressed under drought stress. These findings provide hints in terms of potential chickpea genes to target in crop improvement programs to develop climate change-resilient cultivars.
Editorial: Plant-rhizobia symbiosis and nitrogen fixation in legumes
(Frontiers Media S.A., 2024) Sinharoy, Senjuti; Tian, Chang-Fu; Montiel, Jesu´s
Nitrogen (N) is essential for life, but eukaryotes lack the ability to access this element, as only prokaryotic enzymes can convert N to ammonia. The Haber-Bosch process revolutionized agriculture by enabling synthetic N-fertilizer production, but its overuse and mismanagement created significant environmental challenges (Rockstrom et al., 2009; Richardson et al., 2023). Biological Nitrogen Fixation (BNF) by diazotrophic bacteria and symbiotic nitrogen fixation (SNF) by N-fixing plants offer age-old solutions to the N-problem (Adams et al., 2018).
Engineered diazotrophs with host-inducible nitrogen supply systems: Transforming rice farming through innovative nitrogen biofertilizers
(John Wiley & Sons, 2026) Sengupta, Ahana; Sahoo, Rudra Narayan; Sinharoy, Senjuti
Nitrogen pollution represents a critical challenge in the 21st century, highlighting the urgent need for sustainable alternatives to industrial nitrogen fixation. Diazotrophic bacteria, which uniquely convert dinitrogen (N2) into bioavailable forms, offer a promising solution through biological nitrogen fixation (BNF). These bacteria typically perform nitrogen fixation under nitrogen-limited conditions. Over the past 50 years, extensive research has elucidated the molecular mechanisms and regulatory pathways governing BNF. Recent microbiome studies have revealed that wild rice accessions harbor a greater abundance of diazotrophic bacteria, whereas a substantial proportion of these beneficial microbes have been lost in modern cultivated varieties. Advancements in synthetic biology have enabled the engineering of nitrogen‑exporting diazotrophs, potentially reducing dependence on industrial nitrogen fertilizers. This review emphasizes the importance of targeted research to develop customized diazotrophic microbes in conjunction with synthetic microbial community that can serve as nitrogen exporters for rice. Furthermore, it highlights the necessity of identifying rice cultivars that are particularly responsive to these microbial interventions. Finally, it provides a comprehensive roadmap addressing key challenges and opportunities in deploying BNF to supplement plant nitrogen nutrition and advance sustainable agriculture.
The genome sequence of segmental allotetraploid peanut Arachis hypogaea
(Springer Nature Publishing AG, 2019) Bertioli, David J.; Jenkins, Jerry; Clevenger, Josh; Dudchenko, Olga; Gao, Dongying; Seijo, Guillermo; Leal-Bertioli, Soraya C. M.; Ren, Longhui; Farmer, Andrew D.; Pandey, Manish K.; Samoluk, Sergio S.; Abernathy, Brian; Agarwal, Gaurav; Ballén-Taborda, Carolina; Cameron, Connor; Campbell , Jacqueline; Chavarro, Carolina; Chitikineni, Annapurna; Chu, Ye; Dash, Sudhansu; Baidouri, Moaine El; Guo, Baozhu; Huang, Wei; Kim, Kyung Do; Korani, Walid; Lanciano, Sophie; Lui, Christopher G.; Mirouze, Marie; Moretzsohn, Márcio C.; Pham, Melanie; Shin, Jin Hee; Shirasawa, Kenta; Sinharoy, Senjuti; Sreedasyam, Avinash; Weeks, Nathan T.; Zhang, Xinyou; Zheng, Zheng; Sun, Ziqi; Froenicke, Lutz; Aiden, Erez L.; Michelmore, Richard; Varshney, Rajeev K.; Holbrook, C. Corley; Cannon, Ethalinda K. S.; Scheffler, Brian E.; Grimwood, Jane; Ozias-Akins, Peggy; Cannon, Steven B.; Jackson, Scott A.; Schmutz , Jeremy
Like many other crops, the cultivated peanut (Arachis hypogaea L.) is of hybrid origin and has a polyploid genome that contains essentially complete sets of chromosomes from two ancestral species. Here we report the genome sequence of peanut and show that after its polyploid origin, the genome has evolved through mobile-element activity, deletions and by the flow of genetic information between corresponding ancestral chromosomes (that is, homeologous recombination). Uniformity of patterns of homeologous recombination at the ends of chromosomes favors a single origin for cultivated peanut and its wild counterpart A. monticola. However, through much of the genome, homeologous recombination has created diversity. Using new polyploid hybrids made from the ancestral species, we show how this can generate phenotypic changes such as spontaneous changes in the color of the flowers. We suggest that diversity generated by these genetic mechanisms helped to favor the domestication of the polyploid A. hypogaea over other diploid Arachis species cultivated by humans.
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.
Genome-wide identification of auxin response factors (ARFs) in three different species of Arachis
(Springer Nature Publishing AG, 2021) Raul, Bikash; Bhattacharjee, Oindrila; Tembhare, Kunal; Khanna, Tanyya; Shaheen, Tarannum; Sinharoy, Senjuti; Bandyopadhyay, Kaustav
The phytohormone auxin is involved in the regulation of plant growth, nutrient acquisition, and response to environmental stimuli. Auxin response factors (ARFs) are transcription factors containing B3 DNA binding domain. ARFs play central role in auxin response, using Aux/IAA proteins as partners. Arachis is a genus within the Dalbergioid clade of papilionoid legumes, which out-branched from other members of papilionoids. Cultivated peanut (Arachis hypogaea L.) is an allotetraploid formed by hybridization of two parental genotypes Arachis duranensis, and Arachis ipaensis merely 10,000 years ago. We have made a genome-wide inventory of all the ARFs present in tetraploid A. hypogaea, as well as in two diploid parental genotypes. Our data show that Arachis contains more ARFs per diploid genome (around 31), compared to other legumes (around 25). We further observed few ARF-like genes which are defective in important domains. Most of the ARFs in tetraploid Arachis are redundant, representing the A and B sub-genomes. Some of the ARFs show expression bias from either A or B sub-genome, while some of the pairs are expressed from both sub-genomes. Many ARFs do not express in any of the conditions for which we have expression data. Finally, few pairs show diferential spatio-temporal expression pattern from A and B sub-genomes, indicative of diversifcation of function. This is the frst efort to list all the ARFs from an allotetraploid legume. The list of ARFs in all three species of Arachis will help the scientifc community working to understand auxin regulation in crop legumes.
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.
Induced post-invasive defenses in the nonhost plant Parthenium hysterophorus L. prevent root cortical colonization by Macrophomina phaseolina and impart resistance to dry root rot
(Elsevier B.V., 2025) Mirchandani, Rishabh; Kandpal, Manu; Ranjan, Aashish; Sinharoy, Senjuti; Senthil-Kumar, Muthappa
Dry root rot (DRR) of chickpea is caused by the broad-range necrotrophic fungus Macrophomina phaseolina. Chickpea germplasm does not provide durable resistance to DRR, which is particularly devastating under drought. Even moderately resistant chickpea varieties become susceptible under combined stress. We hypothesized that nonhost resistance (NHR) is durable even under combined stress. Using the blotter paper assay and stereomicroscopic observations, we identified the asterid weed Parthenium hysterophorus as a potential nonhost of M. phaseolina among 82 potential nonhosts. Epidermal necrotic lesions were prevented in P. hysterophorus. In planta fungal load was 0.195 and 0.007 ng/ng total DNA in chickpea and P. hysterophorus, respectively. M. phaseolina could not colonize the P. hysterophorus root while up to 6 cortical cell layers were colonized in chickpea. Further, NHR was durable under combined stress. Dual RNA sequencing revealed that M. phaseolina actively attempted to infect the nonhost and activated specific genes in the xenobiotics degradation pathway. P. hysterophorus also showed an active defense response with1958 and 2294 differentially expressed genes at 2 and 4 DAI, respectively, with 363 upregulated at both time points. Differential expression of cell wall synthesis, phytohormone signaling, and other defense response pathways likely contributes to NHR. Few genes in the phenylpropanoid biosynthesis pathways in P. hysterophorus were also upregulated, possibly because these metabolites are linked to the distinct changes in the fungus during nonhost infection. We therefore conclude that P. hysterophorus exhibits post-invasive NHR to M. phaseolina and that general defense, phytohormone signaling and secondary metabolic pathways contribute to NHR.
An iron-activated citrate transporter, MtMATE67, is required for symbiotic nitrogen fixation
(American Society of Plant Biologists, 2018) Kryvoruchko, Igor S.; Routray, Pratyush; Sinharoy, Senjuti; Torres-Jerez, Ivone; Tejada-Jiménez, Manuel; Finney, Lydia A.; Nakashima, Jin; Pislariu, Catalina I.; Benedito, Vagner A.; González-Guerrero, Manuel; Roberts, Daniel M.; Udvardi, Michael K.
Iron (Fe) is an essential micronutrient for symbiotic nitrogen fixation in legume nodules, where it is required for the activity of bacterial nitrogenase, plant leghemoglobin, respiratory oxidases, and other Fe proteins in both organisms. Fe solubility and transport within and between plant tissues is facilitated by organic chelators, such as nicotianamine and citrate. We have characterized a nodule-specific citrate transporter of the multidrug and toxic compound extrusion family, MtMATE67 of Medicago truncatula. The MtMATE67 gene was induced early during nodule development and expressed primarily in the invasion zone of mature nodules. The MtMATE67 protein was localized to the plasma membrane of nodule cells and also the symbiosome membrane surrounding bacteroids in infected cells. In oocytes, MtMATE67 transported citrate out of cells in an Fe-activated manner. Loss of MtMATE67 gene function resulted in accumulation of Fe in the apoplasm of nodule cells and a substantial decrease in symbiotic nitrogen fixation and plant growth. Taken together, the results point to a primary role of MtMATE67 in citrate efflux from nodule cells in response to an Fe signal. This efflux is necessary to ensure Fe(III) solubility and mobility in the apoplasm and uptake into nodule cells. Likewise, MtMATE67-mediated citrate transport into the symbiosome space would increase the solubility and availability of Fe(III) for rhizobial bacteroids.
Microscopic and transcriptomic analyses of Dalbergoid legume peanut reveal a divergent evolution leading to Nod Factor dependent epidermal crack-entry and terminal bacteroid differentiation
(American Phytopathological Society, 2022) Raul, Bikash; Bhattacharjee, Oindrila; Ghosh, Amit; Upadhyay, Priya; Tembhare, Kunal; Singh, Ajeet; Shaheen, Tarannum; Ghosh, Asim Kumar; Torres-Jerez, Ivone; Krom, Nick; Clevenger, Josh; Udvardi, Michael; Scheffler, Brian E.; Ozias-Akins, Peggy; Sharma, Ravi Datta; Bandyopadhyay, Kaustav; Gaur, Vineet; Kumar, Shailesh; Sinharoy, Senjuti
Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS come from model legumes that exhibit a root-hair mode of bacterial infection in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step towards understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated a comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen scavenging mechanisms in the Papilionoid family. Finally, absence of cysteine-rich motif-1 containing-NCRs, but the recruitment of defensin like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobial symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.
The mysterious non-arbuscular mycorrhizal status of Brassicaceae species
(John Wiley & Sons, 2023) Sharma, Aprajita; Sinharoy, Senjuti; Bisht, Naveen C.
The Brassicaceae family is unique in not fostering functional symbiosis with Arbuscular Mycorrhiza (AM). The family is also special in possessing glucosinolates -, a class of secondary metabolites predominantly functioning for plant defence. We have reviewed what effect the glucosinolates of this non-symbiotic host have on AM or vice-versa . Isothiocyanates, the toxic degradation product of the glucosinolates, particularly the indolic and benzenic glucosinolates, are known to be involved in the inhibition of AM. Interestingly, AM colonization enhances glucosinolate production in two AM-host in the Brassicales family- Moringa oleifera and Tropaeolum spp. PHOSPHATE STARVATION RESPONSE 1 (PHR1), a central transcription factor that controls phosphate starvation response also activates the glucosinolate biosynthesis in AM non-host Arabidopsis thaliana. Recently, the advances in whole-genome sequencing, enabling extensive ecological microbiome studies have helped unravel the Brassicaceae microbiome, identifying new mutualists that compensate for the loss of AM symbiosis, and reporting cues for some influence of glucosinolates on the microbiome structure. We advocate that glucosinolate is an important candidate in determining the mycorrhizal status of Brassicaceae and has played a major role in its symbiosis-defence trade-off. We also identify key open questions in this area that remain to be addressed in the future.
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.
The nodule-specific PLAT-domain protein NPD1 is required for nitrogen-fixing symbiosis
(American Society of Plant Biologists, 2019) Pislariu, Catalina I.; Sinharoy, Senjuti; Torres-Jerez, Ivone; Nakashima, Jin; Blancaflor, Elison B.; Udvardi, Michael K
Symbiotic nitrogen fixation by rhizobia in legume root nodules is a key source of nitrogen for sustainable agriculture. Genetic approaches have revealed important roles for only a few of the thousands of plant genes expressed during nodule development and symbiotic nitrogen fixation. Previously, we isolated over one hundred nodulation and nitrogen fixation mutants from a population of Tnt1-insertion mutants of Medigaco truncatula (Pislariu et al., 2011). Using Tnt1 as a tag to identify genetic lesions in these mutants, we discovered that insertions in a nodule-specific PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain-encoding gene, MtNPD1, resulted in development of ineffective nodules. Early stages of nodule development and colonization by the nitrogen fixing bacterium Sinorhizobium meliloti appeared to be normal in the npd1 mutant. However, npd1 nodules ceased to grow after a few days, resulting in abnormally small, ineffective nodules. Rhizobia that colonized developing npd1 nodules did not differentiate completely into nitrogen-fixing bacteroids and quickly degraded. MtNPD1 expression was low in roots but increased significantly in developing nodules four days post-inoculation (DPI), and expression accompanied invading rhizobia in the nodule infection zone and into the distal nitrogen fixation zone. A functional MtNPD1:GFP fusion protein localized in the space surrounding symbiosomes in infected cells. When ectopically expressed in tobacco (Nicotiana tabacum) leaves, MtNPD1 co-localized with vacuoles and the endoplasmic reticulum. MtNPD1 belongs to a cluster of 5 nodule-specific single PLAT domain-encoding genes, with apparent non-redundant functions.
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.
Organized peripheral vascular strand development in nodules is controlled by a bHLH/HLH heterodimer
(John Wiley & Sons, 2026) Srivastava, Deevita; Bhadu, Vikash; Sahoo, Rudra Narayan; Ghosh, Asim Kumar; Upadhyay, Priya; Bhardwaj, Akanksha; Udvardi, Michael K; Ranjan, Aashish; Sinharoy, Senjuti
The Leguminosae family can develop root nodules with symmetrical peripheral vascular-strands (PVSs). Medicago truncatula forms indeterminate nodules with PVSs. The PVSs elongate directly from the root toward the nodule apex, maintaining a symmetrical organization and facilitating the formation of the cylindrical nodule structure. By combining genetic, biochemical, and genomic tools, we have shown that two basic Helix-Loop-Helix groups of transcription factors, MtbHLH1 (renamed Nodule Vascular bundle Development 1 (NVD1)) and NVD2, control the development of symmetrical PVSs in M. truncatula. In nvd1 nodules, PVSs drift toward the infection zone, generating aberrantly shaped nodules. NVD1 activates its expression along with NVD2, a transcriptional regulator. NVD1 functions downstream of auxin signaling. Transcriptome sequencing of nvd1 and nvd2 nodules, combined with visualization of auxin and cytokinin (CK) signal outputs, revealed disrupted auxin and CK signaling in nvd nodules. Furthermore, ectopic expression of the auxin biosynthetic enzyme (MtYUCCA8) under pMtNVD1 and pMtNVD2 resulted in defective PVSs. Mutant nvd2 nodules display asymmetric PVSs. NVD2 regulates the transcriptional activity of NVD1 by forming heterodimers with it. The formation of symmetrical PVSs depends on the balanced presence of NVD1 and NVD2. Our findings highlight the pivotal role of the NVD1-NVD2 interaction in shaping the development of symmetrical PVSs.
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.