Plasmid construction and Agrobacterium-mediated transformation
All PCR amplified fragments were first inserted into pEASY-Blunt-Simple
(TransGen Biotech) cloning vector, and then constructed into expression
vectors. The coding sequences of viral proteins and MdBT2 were inserted
into pGAD424 and pGBT9 for yeast-two-hybrid (Y2H) assay. 1a, MdBT2, and
2apol were constructed into pGEX-4T-1 and pET-32a to
obtain the GST- and -HIS tagged fusion proteins for pull-down assay. For
the bimolecular fluorescence complementation (BiFC) assay, 1a and MdBT2
were constructed into 35S::SPYCE-cYFP and 35S::SPYNE-nYFP to obtain the
1a-cYFP and MdBT2-nYFP, respectively (Walter et al., 2004).
Similarly, the coding sequences of 1a, MdBT2, and
2apol were inserted into pGreenII 62-SK-nLuc/-cLuc for
luciferase complementation imaging assay (Chen et al., 2008). The
coding sequence of 1a and MdBT2 were inserted into pCXSN-HA to obtain
the pCXSN-1a-HA and pCXSN-MdBT2-HA (Chen et al., 2009). The plant
binary vectors used this work were driven by cauliflower mosaic virus
(CaMV) 35S promoter. All the primers used for plasmid construction were
listed in Table S1.
The three segments of ApNMV were amplified from the ApNMV-Lw we isolated
previously (Zhang et al, 2020), and were constructed into modified
pCAMBIA1300 binary vector, in which the viral genes were driven by
double 35S promoter.
For Agrobacterium-mediated transformation, the appropriate binary
constructs were transformed into Agrobacterium tumefaciens strain
LBA4404 and cultured in Lysogeny Broth medium supplemented with
corresponding antibiotics.