Biology subjects

Parihar, D.

Publications and source records attributed to Parihar, D..

4 recordsLinked to original sources

bioRxiv2025

Promoter evolution in HIV-1C establishes latent reservoirs highly resistant to reversal

Latent viral reservoirs remain a major barrier to curing HIV-1, with the long-terminal repeat (LTR) and Tat playing crucial roles in regulating viral transcription. Subtype-specific transcription factor binding site (TFBS) variations within the LTR significantly influence latency and reservoir stability. In earlier work, we identified HIV-1C LTR variants with duplicated TFBS motifs, including NF-{kappa}B, AP1, RBEIII, and TCF-1/LEF-1. Using five cell models, including Jurkat and primary CD4 T cells, we compared canonical R-LTR and variant R2-LTR strains. Across sub-genomic reporters, single-round infections, and full-length viral vectors, we found that the balance between RBEIII and NF-{kappa}B motifs governs stability of latency. The two-viruses-one-cell system that normalized confounding environmental factors further revealed that latency is primarily controlled by intrinsic transcriptional circuits rather than external stimuli. In longitudinal studies of HIV-1 individuals from acute and chronic infection phases, we observed dominant R strains during early infection and the spontaneous emergence of R2 strains in nearly half of chronic-phase subjects, a process accelerated by ART. Upon CD4 T cell activation, R strains preferentially rebounded, while R2 strains showed strong resistance to reversal, even in subjects harbouring a co-infection. Together, these findings establish the clinical significance of LTR variation in latency regulation and identify the R2 phenotype as a critical determinant of reservoir persistence. These results underscore the importance of addressing reservoir heterogeneity in cure strategies, particularly in HIV-1C-prevalent regions.

microbiology↗

bioRxiv2024

CrGEF1, CrGAP1, and CrGDI2 function as key regulators of ROP-GTPase mediated modulation of alkaloid biosynthesis in Catharanthus roseus

ROP-GTPase Regulatory Proteins (RGRPs) have been shown to control plant morphogenesis, development and immunity, however, their role in specialized metabolism is hitherto not known. Here, we demonstrate that specific RGRPs control monoterpene indole alkaloid (MIA) biosynthesis by interacting with distinct Rho of Plants (ROP) in Catharanthus roseus. Among the five Guanine nucleotide Exchange Factors (GEFs), four GTPase-activating proteins (GAPs), and two GDP dissociation inhibitors (GDIs) identified in the C. roseus genome, only CrGEF1, CrGAP1, and CrGDI2 specifically interacted with CrROP3 and CrROP5. These RGRPs displayed distinct cytosolic and/or membrane localization patterns, with their transcripts predominantly expressed in aerial tissues. Functional studies revealed that CrGEF1 acts as a positive regulator of MIA biosynthesis, as its silencing led to a reduction in MIA production, while overexpression enhanced MIA levels. Conversely, CrGAP1 and CrGDI2 function as negative regulators, with their silencing resulting in increased MIA production and their overexpression causing reduced MIA levels. Notably, terminal truncated forms of these RGRPs showed interaction with CrROP3 or CrROP5 but failed to influence MIA biosynthesis, underscoring the importance of these domains in their regulatory functions. Overall, our findings uncover a previously unexplored mechanism by which distinct RGRPs coordinate with specific ROPs to regulate transcription factors and fine-tune MIA biosynthesis in C. roseus. SignificancePlants fine-tune their metabolic pathways to adapt to environmental and physiological cues, balancing primary and specialized metabolism. Here, we uncover a feedback regulatory mechanism by which ROP-GTPase regulatory proteins (RGRPs) control alkaloid biosynthesis in Catharanthus roseus. We found that CrGEF1, CrGAP1, and CrGDI2 specifically interact with ROP-GTPases CrROP3 and CrROP5, modulating the monoterpene indole alkaloid (MIA) pathway. This RGRP-ROP module plays a crucial role in dynamically regulating MIA levels, where CrGEF1 promotes MIA production while CrGAP1 and CrGDI2 act as negative regulators. These findings highlight the importance of RGRP-ROP circuitry in control of alkaloid biosynthesis in C. roseus, and specialized metabolism in plants.

plant biology↗

bioRxiv2024

Functional characterization of two glycosyltransferases from Withania somnifera illuminates their role in withanosides biosynthesis and defence against bacteria

The medicinal properties of Ashwagandha (Withania somnifera L. Dunal) are attributed to the presence of unique class of natural products called as withanolides and their glycosylated forms, withanosides. Withanosides are proposed to be formed from withanolides by the action of glycosyltransferases (GTs). This study reports the functional characterization of two GTs (WsGT4 and WsGT6) from W. somnifera that exhibited induced expression in response to methyl jasmonate treatment and showed highest expression in leaves compared to other tissues. Biochemical assays with recombinant WsGT proteins showed that WsGT4 and WsGT6 formed glycosylated products with four and one of the seven tested withanolides substrates, respectively. WsGT4 catalyzed product formation using withanolide A, withanolide B, withanone, and 12-deoxywithastramonolide as substrates, with UDP-glucose serving as the glucose donor, while WsGT6 catalyzed the product formation only with withaferin A as substrate employing either UDP-glucose or UDP-galactose as sugar donors. Moreover, in planta studies through virus-induced gene silencing and transient overexpression of WsGT4 and WsGT6 in W. somnifera leaves modulated the levels of withanolides and withanosides, indicating their role in withanosides biosynthesis. Furthermore, while individual silencing of both WsGT4 and WsGT6 in W. somnifera reduced the tolerance to Pseudomonas syringae DC3000 growth, their overexpression enhanced the tolerance to the bacterium in W. somnifera. Taken together, these results shed light on the roles of WsGT4 and WsGT6 in withanoside biosynthesis and defence against model bacterial pathogen in W. somnifera.

plant biology↗

bioRxiv2023

Rho of plant GTPases with geranylgeranylation motif modulate monoterpene indole alkaloid biosynthesis in Catharanthus roseus

Rho Of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpenoid indole alkaloids (MIA) in Catharanthus roseus, indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C- terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, both of their transcripts showed higher expression in most parts compared to other CrROPs. Protein- protein interaction studies revealed that both CrROP3 and CrROP5, but not CrROP2 (lacking CSIL motif), interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of {Delta}CrROP3 and {Delta}CrROP5 mutants lacking the CSIL motif failed to enhance MIA biosynthesis. Taken together, these results implicate that CrROP3 and CrROP5 have positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.

plant biology↗