Browsing by Author "Tiwari, Ruchi"

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A complex interplay of Gβ and Gγ proteins regulates plant growth and defence traits in the allotetraploid Brassica juncea
(Springer Nature Publishing AG, 2021) Arya, Gulab Chand; Tiwari, Ruchi; Bisht, Naveen C.
Plant heterotrimeric G-proteins regulate a wide range of responses despite their limited repertoire of core components. The roles and functional interactions between different G-protein subunits are quite perplexing, which get further complicated with polyploidy. Here, we show that the allotetraploid Brassica juncea comprises multiple homologs of G-protein genes, encoding six BjuGβ and ten highly divergent BjuGγ subunit proteins, later being classified into type-A1, type-A2 and type-C Gγ proteins. The encoded BjuGβ and BjuGγ proteins shared close evolutionary relationship and have retained distinct spatio-temporal expression patterns during plant developmental stages and in response to the necrotrophic pathogen, Sclerotinia sclerotiorum. RNAi based suppression of BjuGβ and BjuGγ genes suggested functional overlap and selectivity of BjuGβs with three distinct BjuGγ type subunits, to regulate plant height (BjuGβγA2 and BjuGβγC), seed weight (BjuGβGγA1 and BjuGβGγC), silique size (BjuGβGγC) and pathogen response (BjuGβGγA1 and BjuGβGγC). Further, the triplicated BjuGβ genes, formed due to Brassica specific whole-genome-triplication event, showed differential involvement during pathogen response, wherein overexpression of BjuGβ2 displayed higher resistance to Sclerotinia infection. Taken together, our study demonstrates that multiple BjuGβ and BjuGγ proteins have retained distinct spatio-temporal expression and functional selectivity to regulate specific plant growth and defence traits in the oilseed B. juncea.
Extra-large G-proteins influence plant response to Sclerotinia sclerotiorum by regulating glucosinolate metabolism in Brassica juncea
(John Wiley & Sons, 2021) Tiwari, Ruchi; Kaur, Jagreet; Bisht, Naveen C.
Heterotrimeric G-proteins are one of the highly conserved signal transducers across phyla. Despite the obvious importance of G-proteins in controlling various plant growth and environmental responses, there is no information describing the regulatory complexity of G-protein networks during pathogen response in a polyploid crop. Here, we investigated the role of extra-large G-proteins (XLGs) in the oilseed crop Brassica juncea, which has inherent susceptibility to the necrotrophic fungal pathogen Sclerotinia sclerotiorum. The allotetraploid B. juncea genome contains multiple homologs of three XLG genes (two BjuXLG1, five BjuXLG2, and three BjuXLG3), sharing a high level of sequence identity, gene structure organization, and phylogenetic relationship with the progenitors’ orthologs. Quantitative reverse transcription PCR analysis revealed that BjuXLGs have retained distinct expression patterns across plant developmental stages and on S. sclerotiorum infection. To determine the role of BjuXLG genes in the B. juncea defence response against S. sclerotiorum, RNAi-based suppression was performed. Disease progression analysis showed more rapid lesion expansion and fungal accumulation in BjuXLG-RNAi lines compared to the vector control plants, wherein suppression of BjuXLG3 homologs displayed more compromised defence response at the later time point. Knocking down BjuXLGs caused impairment of the host resistance mechanism to S. sclerotiorum, as indicated by reduced expression of defence marker genes PDF1.2 and WRKY33 on pathogen infection. Furthermore, BjuXLG-RNAi lines showed reduced accumulation of leaf glucosinolates on S. sclerotiorum infection, wherein aliphatic glucosinolates were significantly compromised. Overall, our data suggest that B. juncea XLG genes are important signalling nodes modulating the host defence pathways in response to this necrotrophic pathogen.
A guide to culturing, maintenance, and leaf inoculation methods for rapid screening and quantification of sclerotinia sclerotiorum infection in mustard
(Springer Nature Publishing AG, 2026) Malhotra, Bhanu; Tiwari, Ruchi; Varghese, Mohan; Bisht, Naveen C.
Sclerotinia rot poses a significant challenge to the cultivation of oilseed Brassica crops, causing the reduction of seed number, weight, and quality leading to >95% yield losses globally. Due to its substantial economic impact on agriculture, Sclerotinia sclerotiorum has been extensively studied at the molecular level and has now been adopted as a model to investigate the host-pathogen interactions. Despite various procedures and strategies described in different reports, there exists a considerable disparity in how the pathogen is cultured, maintained, and manipulated across studies.This chapter provides a comprehensive guide to the fundamental procedures of working with S. sclerotiorum. It describes step-by-step methods for its routine culturing and maintenance over artificial media, replenishing and storing laboratory stocks, and conducting detached leaf assays-a robust method used for screening the mustard cultivars. Additionally, we outline the quantification of pathogen load in planta through qRT-PCR analysis. The methods presented in the current chapter are reproducible and can be suitably applied when working with different isolates of the pathogen.Through the present compilation, we aim to address the research gaps in methods involving the pathogen that would not only benefit the new researchers entering the field but also future research endeavors on white mold.
The multifaceted roles of heterotrimeric G-proteins: lessons from models and crops
(Springer Nature Publishing AG, 2022) Tiwari, Ruchi; Bisht, Naveen C.
Heterotrimeric G-proteins (hereafter G-proteins), consisting of G-alpha (Gα), G-beta (Gβ) and G-gamma (Gγ) subunits, are key signal transducers conserved across different forms of life. The discovery of plant lineage-specific G-protein components (extra-large G-proteins and type-C Gγ subunits), inherent polyploidy in angiosperms, and unique modes of G-protein cycle regulation in plants pointed out to a few fundamental differences of plant G-protein signaling from its animal counterpart. Over the last 2 decades, extensive studies in the model plant Arabidopsis thaliana have confirmed the involvement of G-proteins in a wide range of plant growth and development, and stress adaptation processes. The G-protein research in crop species, however, is still in its infancy, and a handful of studies suggest important roles of G-proteins in regulating plant architectural and key agronomical traits including plant’s response to abiotic and biotic factors. We propose that the advancement made in plant G-proteins research will facilitate the development of novel approaches to manage plant yield and fitness in changing environments.
XLG2 and CORI3 function additively to regulate plant defense against the necrotrophic pathogen Sclerotinia sclerotiorum
(John Wiley & Sons, 2024) Tiwari, Ruchi; Garg, Kajal; Senthil-Kumar, Muthappa; Bisht, Naveen C.
The membrane-bound heterotrimeric G-proteins in plants play a crucial role in defending against a broad range of pathogens. This study emphasizes the significance of Extra-large Gα protein 2 (XLG2), a plant-specific G-protein, in mediating the plant response to Sclerotinia sclerotiorum, which infects over 600 plant species worldwide. Our analysis of Arabidopsis G-protein mutants showed that loss of XLG2 function increased susceptibility to S. sclerotiorum, accompanied by compromised accumulation of jasmonic acid (JA) during pathogen infection. Overexpression of the XLG2 gene in xlg2 mutant plants resulted in higher resistance and increased JA accumulation during S. sclerotiorum infection. Co-immunoprecipitation (co-IP) analysis on S. sclerotiorum infected Col-0 samples, using two different approaches, identified 201 XLG2-interacting proteins. The identified JA-biosynthetic and JA-responsive proteins had compromised transcript expression in the xlg2 mutant during pathogen infection. XLG2 was found to interact physically with a JA-responsive protein, Coronatine induced 1 (CORI3) in Co-IP, and confirmed using split firefly luciferase complementation and bimolecular fluorescent complementation assays. Additionally, genetic analysis revealed an additive effect of XLG2 and CORI3 on resistance against S. sclerotiorum, JA accumulation, and expression of the defense marker genes. Overall, our study reveals two independent pathways involving XLG2 and CORI3 in contributing resistance against S. sclerotiorum.