Development and Application of a Self-Assembling Split-Fluorescent Protein Toolkit to Monitor Geminiviral Movement and Infection in Plant

Geminiviruses are a group of circular single-stranded DNA viruses that cause severe diseases in many crop plants. However, there is still no fluorescent protein tag suitable for labeling viral proteins endogenously due to the limited genomic space and structure of geminiviruses for foreign gene fragment insertion. Here, we established a split super-folder green fluorescent protein (sfGFP)-based imaging system that provides a platform to visualize the subcellular localization of geminiviral proteins in Nicotiana benthamiana . A short fragment of the GFP- coding sequence (GFP11) was inserted into a specific locus of the geminiviral genome, while the remainder of the GFP (GFP1–10) was transiently or constitutively expressed in N. benthamiana . Consequently, complementation fluorescence enables the examination of the subcellular localization of viral proteins in particular cells. Using this split sfGFP system, we examined the subcellular localization of the coat protein and BV1 protein of tomato golden mosaic virus (TGMV) and further monitored its intercellular and long-distance movement in N. benthamiana during viral infection. This approach allows us to study endogenously the subcellular localization and viral movement of geminiviruses in N. benthamiana . This new split sfGFP system may also hold the potential to provide orthogonal fluorescent proteins usable for geminiviral genome tagging in plants.