Functional Characterization Of Proteins Involved In Cell To Cell Movement Of Cotton Leaf Curl Kokhran Virus- Dabawali

Abstract

Viruses are submicroscopic obligate parasites and depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. As exception, viruses belonging to the family Geminiviridae are DNA viruses and infect both mono and dicotyledonous plants causing a large economic loss. These viruses are smaller in size encoding fewer proteins and employ the host cell machinery for successful infection and spread. Geminiviruses undergo frequent recombinations due to mixed infection resulting in vast diversity across the family and account for driving evolution in these viruses. Movement in these viruses is complex since they have to cross two important barriers, nuclear and cell wall barrier to establish systemic spread. All these factors play very important role while designing control measures against these viruses. Thus a detailed understanding of these processes at molecular level is essential. Cotton is the major cash crop in Indian subcontinent with huge export values. India has become the second largest producer of cotton in the world. However, the major constraint in cotton cultivation has been crop loss due to diseases caused by viruses, particularly the cotton leaf curl disease (CLCuD) caused by begomoviruses. Present thesis deals with the analysis of genetic variability of CLCuD in India and functional characterization of proteins involved in the movement of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab). CLCuKV-Dab belongs to family Geminiviridae and subgroup Begomovirus. A review of the literature on Geminiviridae classification, genome organization, virus entry, replication, transcription, translation, assembly and movement is presented in Chapter 1. This chapter also includes the review of host factors involved in replication, geminiviral proteins involved in gene silencing and a detailed report on CLCuD complexes and sub viral DNAs that are associated with CLCuD. The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in both bacterial and baculovirus expression systems, Protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology and cell biology methods are described in detail in Chapter 2. Previous study has reported the complete genomic sequences of CLCuKV-Dab and Tomato leaf curl Bangalore virus-cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequence of CLCuKV-Gang and the Cotton leaf curl Rajasthan virus (CLCuRV-Ban). Phylogenetic analysis of DNA-A sequences of these viruses with other CLCuD causing viruses is discussed in detail in Chapter 3. Chapter 4 deals with overexpression, purification and functional characterization of CLCuKV-Dab CP in terms of its interaction with DNA, the kinetics and its role in cell to cell movement. The proposed partners to CP in the cell to cell movement of monopartite begomoviruses are AV2 and AC4. Thus the Chapter 5 describes the functional characterization of recombinant AV2 of CLCuKV-Dab. Chapter 6 deals with expression of CP and AV2 as GFP fusion proteins in insect cells using baculovirus expression system to study the localization patterns of these proteins. Chapter 7 describes functional characterization of CLCuKV-Dab AC4. Bioinformatic analysis of AC4 showed that it belongs to the rare group of natively unfolded proteins that are functionally active In conclusion, there is a large genetic variability that exists among the begomoviruses and in particular, among the CLCuD causing begomoviruses in India. Functional characterization of the proteins involved in the cell to cell movement in CLCuKV-Dab led to a possible model for its movement; the CP translated in the cytoplasm is targeted the nucleus via its NLS and there binds to progeny ssDNA and exports the ssDNA out of nucleus through its export signals. AC4 or some other host proteins yet to be identified transports the ssDNA-CP complex from the nuclear periphery to AV2 present at the cell periphery. The complex is then transported from one cell to the neighboring cell via plasmodesmata. AC4 being an ATPase/NTPase could provide energy for the process.