Here, we present a simple and rapid protocol to measure the extent of cell-to-cell movement of RNA viruses in planta. To do that, the green fluorescent protein (GFP) gene was incorporated into the genome of Melon necrotic spot virus (MNSV) as a coat protein (CP) fusion protein using the Thosea asigna virus 2A catalytic peptide (TaV 2a) (Serra-Soriano et al., 2014). TaV 2a allows the co-translational cleavage of the fusion protein resulting in the independent expression of both proteins (Kim et al., 2011). Viral infection was initiated by agro-infiltration of Cucumis melo leaves. At 6-7 days post-infiltration, fluorescent infection foci images were taken with a fluorescent stereo microscope and infection areas were measured using FIJI software.

In this protocol, we used fluorescence recovery after photobleaching (FRAP) to measure the influence that some mutations and drug treatment have on mobility of a green fluorescent protein (GFP)-fused viral transmembrane protein into endoplasmic reticulum membranes (Serra-Soriano et al., 2014). The proteins of interest were transiently expressed in Nicotiana benthamiana (N. benthamiana) epidermic cells by agro-infiltration. To minimize transient overexpression artifacts, fluorescence intensity values were gathered at 36 hpi using an inverted Zeiss LSM 780 confocal microscope. Only epidermic cells showing moderated expression levels and homogenous distribution through the ER of the GFP-tagged proteins were used for further experiments. To examine the role of actin polymerization in the mobilization of GFP-tagged proteins, we pretreated tissue samples either with latrunculin B, an inhibitor of actin polymerization, or with DMSO as control. The generated fluorescence recovery curves were used to obtain the percentage of maximum fluorescence recovery (MFR), which corresponds to the mobile fraction, and the half-time of maximum recovery (t_1/2) values.