(A) Stromule length was manually measured in ImageJ. Consistent with the independent measurement presented in Figure 5C, silencing of NbGCP4 (VIGS-NbGCP4) resulted in a higher percentage of stromules that were 3 μm or longer (pink) compared to VIGS empty vector (EV) control silenced plants. Total of 302, 162, 137, and 109 stromules were measured for VIGS-EV, VIGS-NbGCP4, VIGS-EV with TMV-p50, and VIGS-NbGCP4 with TMV p50, respectively. Fisher’s exact test was performed for comparison. *p<0.05 and ***p<0.0001. (B) Three types of stromule movement were manually tracked. More stable extension of stromule tips (green dot) was observed a smooth, constant motion with a linear trajectory (green line) (top row). Rapidly extending and retracting stromule tips (yellow dot) were sudden and produced an erratic trajectory (yellow line) (middle row). Stromule tips (magenta dot) moving sideways had no extension and moved tangential (magenta line) to the body of the chloroplast (bottom row). (C) Stromule movement types depicted in B were counted and the frequency of constant, smoothly extending stromules (green) was higher in NbGCP4-silenced samples. Total of 337, 186, 134, and 127 movements of stromules were recorded for VIGS-EV, VIGS-NbGCP4, VIGS-EV with TMV-p50, and VIGS-NbGCP4 with TMV p50, respectively. Chi-square test was performed to compare. *p<0.05, **p<0.001.