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    Interacting partners of Brassica juncea regulator of G-protein signaling protein suggest its role in cell wall metabolism and cellular signaling
    (Portland Press, 2022) Kumar, Roshan; Bisht, Naveen C.
    Heterotrimeric G-proteins interact with various upstream and downstream effectors to regulate various aspects of plant growth and development. G-protein effectors have been recently reported in Arabidopsisthaliana, however, less information is available from polyploid crop species having complex networks of G-protein components. Regulator of G-protein signaling (RGS) is a well-characterized GTPase accelerating protein, which plays an important role in the regulation of the G-protein cycle in plants. In this study, four homologs encoding RGS proteins were isolated from the allotetraploid Brassica juncea, a globally important oilseed, vegetable, and condiment crop. The B. juncea RGS proteins were grouped into distinct BjuRGS1 and BjuRGS2 orthologous clades, and the expression of BjuRGS1 homologs was predominantly higher than BjuRGS2 homologs across the tested tissue types of B. juncea. Utilizing B. juncea Y2H library screening, a total of 30 non-redundant interacting proteins with the RGS-domain of the highly expressed BjuA.RGS1 was identified. Gene ontology analysis indicated that these effectors exerted various molecular, cellular, and physiological functions. Many of them were known to regulate cell wall metabolism (BjuEXP6, Bju-α-MAN, BjuPGU4, BjuRMS3) and phosphorylation-mediated cell signaling (BjuMEK4, BjuDGK3, and BjuKinase). Further, transcript analysis indicated that the identified interacting proteins have a co-expression pattern with the BjuRGS homologs. These findings increase our knowledge about the novel targets of G-protein components from a globally cultivated Brassica crop and provide an important resource for developing a plant G-protein interactome network.
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    Pattern of expression and interaction specificity of multiple G-protein beta (Gβ) subunit isoforms with their potential target proteins reveal functional dominance of BjuGβ1 in the allotetraploid Brassica juncea
    (Elsevier B.V., 2017) Kumar, Roshan; Sharma, Aprajita; Chandel, Ishita; Bisht, Naveen C.
    Heterotrimeric G-protein, consisting Gα, Gβ and Gγ subunits, interacts with various upstream and downstream effector (target) proteins to regulate a large array of conserved and species-specific biological functions. The targets of G-protein components are recently reported in model plant Arabidopsis thaliana; however limited information is available from crop species. In this study, we utilized yeast two-hybrid (Y2H) assay to screen the diversity of interacting partners of multiple Gβ subunit isoforms from allotetraploid Brassica juncea, a globally important oilseed and vegetable crop. The three BjuGβ genes (BjuGβ1-3), resulted from whole genome triplication event in Brassica lineage, showed distinct expression profile during plant developmental stages with maximal transcript abundance during reproductive stages. Protein-protein interaction of three BjuGβ proteins (bait) against the Y2H cDNA library (prey) identified a total of 14 and 1 non-redundant targets for BjuGβ1 and BjuGβ2, whereas BjuGβ3 screening surprisingly did not yield any genuine target, thereby suggesting functional dominance of BjuGβ1. The triplicated BjuGβ isoforms showed a high degree of interaction strength and specificity with the identified target proteins, which are known to be involved in diverse biological functions in plants. qRT-PCR analysis further indicated that the expression of BjuGβ-target genes was developmentally regulated under various tissue types studied and showed a high degree of co-expression pattern with the BjuGβ genes, particularly during flower and silique development in B. juncea. Taken together, our data provides novel insights on pattern of expression and interaction specificity governing functional divergence of multiple Gβ subunit proteins in polyploid B. juncea.