Luciferase complementation imaging assay
Coding sequences of ApNMV 1a and MdBT2 were constructed into pGreenII 62-KS-cLuc and -nLuc vectors (Chen et al., 2008), respectively. The recombinants were transformed into Agrobacterium LBA4404 and co-infiltrated into N. benthamiana leaves. Two days post-infiltration, the substrate of luciferase was sprayed on the leaves and incubated in dark for 3 min. Bioluminescent signals were detected under an in vivo imaging system (IVIS, Lumina II). In the IVIS acquisition control panel, ‘Luminescent’ was selected as ‘Imaging mode’, and the exposure time was set to 30 s. After photographs were acquired, a color scale, which is shown as a color bar on the right side of the images, was adjusted to improve the contrast of the images.
For the competitive assay, coding sequences of ApNMV 1a and 2apol were inserted into the pGreenII 62-KS-nLuc and -cLuc vectors as described previously, while MdBT2 was constructed into a pCXSN-HA vector (Chen et al., 2009), and all the constructs were transformed into Agrobacterium LBA4404. The Abs600of Agrobacterium containing 1a-nLuc and cLuc-2apol was adjusted to 0.5, and combined with different ratio of pCXSN-MdBT2-HA as indicated in Fig. 6B, then co-infiltrated into N. benthamianaleaves. The Agrobacterium harboring pCXSN-HA plasmid served as control. The images were acquired as previously described.