Research Groups

Plant Biology: Plant Molecular Biology

Research Interests and Description

Staff Research Scientist: Sunil Kumar Mukherjee

Research Interests

Replication machinery and RNAi suppression activities of geminiviruses.

Description of Research

Geminiviruses are plant pathogens which infect a large number of crop plants worldwide resulting in enormous financial loss. These are characterized by the presence of single stranded (ss) DNA genomes which replicate in the plant nucleus. Understanding the molecular basis of the replication of geminiviruses is likely to shed light on the complex process of replication of plant DNA. Such studies are also essential to design any strategy to contain infection of various crops by geminiviruses, and its subsequent spreading.
We have chosen Mungbean yellow mosaic India virus (MYMIV) and Tomato Leaf Curl Virus( ToLCV) as the model systems for elucidating the mechanism of geminiviral DNA replication. MYMIV contains two ssDNA genomes: DNA-A and DNA-B. DNA-A is solely responsible for its replication and it encodes only a few proteins. Of these, the Replication Initiator protein (Rep, AC1) has been found to be essential for its replication through Rolling Circle mode (RCR). We had previously established that Rep acts at the initiation of the replication stage by creating a nick at the replication origin. We have also demonstrated that the intrinsic ATPase and helicase activities of Rep play a pivotal role at the elongation stage as well. At the termination stage, Rep’s involvement through its ligation activity has been implicated.
However, other than Rep and a few other viral ORFs, geminivirus is largely dependent on various host factors. We have reported the involvement of a few host proteins such as Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA). Current research focuses on the identification and characterization of such host proteins. We are employing Yeast two Hybrid (Y2H) and Phage Display Library techniques to identify the Rep interacting host proteins. More than fifty important proteins which interact strongly with the Rep protein have already been identified. We have recently characterized the Rad54 protein, and showed its involvement during geminivirus DNA replication. We are investigating the role of the Rad51 and NAC proteins in replication. Another focus is to constitute an in vitro replication assay system to study geminivirus DNA replication. We have devised protocols to isolate nuclear extract from Saccharomyces cerevisiae. Our preliminary studies show its efficiency in supporting replication of vectors constructed based on the geminiviral genome. We are extrapolating the work with nuclear extracts prepared from various yeast mutants and directly checking the geminiviral DNA replication efficiency in such extracts. This convenient technique offers us an opportunity to identify the host proteins involved in the replication process. Involvement of the proteins identified in the geminiviral DNA replication are to be validated through in planta assays. Experiments are underway using Arabidopsis thaliana as the model plant, and replication efficiencies of geminiviral DNA in the wild type plants as well as in the mutant lines are being investigated.
Six assays to screen for RNAi suppressors encoded by plant viruses have been devised. Of 24 suppressors screened, MYMIV-AC2 and FHV-B2 have been selected to study the mechanism of suppression. These interact with host RNAi factors and inactivate RNAi activities. MYMIV-AC2 has been used for molecular farming, preparation of the robust VIGS vector and assisting in planta ribozyme activities. We have also raised transgenics overexpressing micro RNAs that target viral replication proteins for severe downregulation.

Recent Publications

Karjee, S., Islam, M.N., Mukherjee, S.K. 2008. Screening and identification of virus encoded RNA silencing suppressors. Met. Mol. Biol. In Press

Sanan-Mishra, N., Kumar, V., Sopory, S.K., Mukherjee, S.K. 2009. Cloning and Validation of novel miRNA from basmati rice indicates cross-talk between abiotic and biotic stresses. Mol. Gen. Genomics In press

Singh, G., Popli, S., Yukti, H., Palhotra, P. Mukherjee, S.K., Bhatnagar, R.K. 2009. Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of the Dicer. FASEB J In press

Teotia, P.S., Mukherjee, S.K., Mishra, N.S. 2008. Fine tuning of Auxin signalling by miRs. Physiol.Mol.Biol.Plants 14, 81-90

Karjee, S., Islam, M.N., Mukherjee, S.K. 2008. Screening and identification of virus encoded RNA silencing suppressors. Met. Mol. Biol. 442, 187-203