Characterisation of the replication-associated protein of South African cassava mosaic virus and elucidation of protein binding partners in cassava
Date
2018
Authors
Ayres, Frances Margaret
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Abstract
Geminiviruses are a large family of plant-infecting viruses that have severely affected a variety of economically important crops, such as cassava. Cassava is a perennial crop that plays a vital economic role in the lives of subsistence farmers of the developing world. Cassava is a salient crop in developing countries and is favoured due to its high starch content and drought tolerance. Africa has emerged as the leading producer of cassava; however, yields of cassava tuberous roots and leaves have been negatively impacted by the emergence of viral pathogens. Geminiviruses such as South African cassava mosaic virus (SACMV) introduce cassava mosaic disease which negatively impacts upon crop yields. The geminivirus-encoded replication-associated protein (Rep) is a highly conserved protein that is crucial for viral replication, and modulates a number of virus-host interactions. The aims of this study included the expression and characterisation of SACMV Rep and determining host protein binding partners of the viral protein. Expression of a soluble recombinant protein was successfully carried out. Structural analyses were undertaken by studying the secondary and tertiary structural features of the protein using circular dichroism (CD), and intrinsic fluorescence spectroscopy. Secondary structure studies were inconclusive, with intrinsic fluorescence revealing that adenosine triphosphate (ATP) binding did not induce significant protein conformational changes. Binding of 8-Anilino-1-naphthalenesulfonic acid (ANS) to SACMV Rep was observed, with the interaction not disrupted in the presence of ATP. This confirmed the lack of a persistent SACMV Rep-ATP interaction. Functional characterisation was undertaken using a DNA binding and cleavage assay. A comparison between results obtained in this study and previous geminivirus Rep research suggests cleavage of the nucleotide probe, and binding to the cleaved 5สน-end. The formation of a protein-DNA complex confirmed that the expressed recombinant protein was functionally competent. Interaction of cassava proteins with SACMV Rep were probed using a yeast two-hybrid assay (YTH). Putative cassava protein binding partners of SACMV Rep were selected based on previous research and geminivirus related literature. The putative binding partners, histone H3, cyclin D3;2 and cyclin D4;2, were used as the prey proteins with bait protein SACMV Rep utilised as the bait. Results obtained from the X-gal filter lift assay suggested that there was no direct interaction of SACMV Rep with histone H3, cyclin D3;2 or cyclin D4;2. This study presents the first investigation into the elucidation of structural and functional features of SACMV Rep, and the first effort into identifying host protein binding partners of SACMV Rep
Description
A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in the School of Molecular and Cell Biology
Johannesburg 2018
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Citation
Ayres, Frances Margaret, (2018) Characterisation of the replication-associated protein of South African cassava mosaic virus and elucidation of protein binding partners in cassava, University of the Witwatersrand, Johannesburg, https://hdl.handle.net/10539/26914.