Subcellular Localization of Geminivirus Proteins by Laser Scanning Confocal Microscopy

In eukaryotic cells, the subcellular localization of proteins is inherently linked to their function. Since viruses rely on the host cellular machinery to complete their life cycle, viral proteins are expected to employ the host transport machinery to reach various compartments. Several factors, including the multifunctional nature of viral proteins, the stage of virus infection, and interactions with both viral and host proteins, influence the final destination of viral proteins. For instance, NSP (nuclear shuttle protein) from bipartite begomoviruses and CP (coat protein) from monopartite begomoviruses typically exhibit nuclear localization, yet their subcellular distribution can vary depending on coexpression partners and stage of infection. Virtually all viral proteins display dynamic subcellular distribution patterns that change under their specific functions at different stages of the virus life cycle. Thus, identifying the subcellular distribution of viral proteins is essential for comprehending their multiple roles during infection. This chapter outlines a protocol for efficiently determining the subcellular localization of viral proteins during infection or when expressed with protein partners. The protocol essentially consists of three steps: (i) cloning the viral protein and protein partners fused to fluorescent tags, (ii) transiently expressing the tagged proteins in N. benthamiana leaves, and (iii) determining the subcellular localization of the tagged proteins using confocal microscopy.