In vitro and in vivo approaches to identify tomato proteins interactive with pepino mosaic virus proteins
2008
Ghita, M.
The aim of this thesis was to bring more information about a potential interaction between tomato proteins and Pepino mosaic virus capsid protein and heat shock protein 70 using in vitro and in vivo approaches. Electron microscopy observations showed a co-localization between PepMV CP and tomato HSP70, after a double-immunogold labelling with specific antisera conjugated with different sized gold particles. In vivo immunoprecipitation assays and Western blot analysis did not conclusively answer the hypothesis. It may be that these two proteins do not form any sort of complexes in infected tissue or the complexes do not reach detectable levels in the soluble fraction of infected cells or the experiment itself can be further optimized technically. In order to facilitate the visualization of the interaction between these two proteins in living cells, DNA constructs (pUC SPYNE-HSP70/ CAT and pUC SPYCE- CP/ p25) were generated to be used in bimolecular fluorescence complementation assay in protoplasts.
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