ApNMV 1a interacts with MdBT2
To determine the protein(s) that mediates the degradation of ApNMV 1a under nitrate treatment, we performed an Y2H assay using 1a protein as bait to screen the apple cDNA library, and MdBT2 (MDP0000643281), a known nitrate-responsive protein, was identified as a potential prey. This protein contains an N-terminal BTB domain, a BACK-like domain in the middle, and a C-terminal TAZ domain (Fig. 3A). To confirm the protein interaction between ApNMV 1a and MdBT2, an Y2H assay was conducted. The coding sequences of 1a and MdBT2 were inserted into pGBT9 and pGAD424, respectively, and they were co-transformed into yeast cells. The result showed that the colonies grew normally in SD/-Trp/-Leu/-His/-Ade defective medium, suggesting the interaction between 1a and MdBT2 (Supplementary Fig. S3). In addition, we also tested interactions between MdBT2 and other vial components (2apol, MP, and CP) using Y2H assay, and found that MdBT2 only interacted with 1a but not with other viral proteins (Supplementary Fig. S3).
To identify the domain(s) that is required for interactions of the two proteins, we first chopped the MdBT2 into several fragments depending on the distribution of domains (Fig. 3A), and constructed them into pGBD vectors, while full-length of MdBT2 served as positive control. We found that the yeast grew on SD/-Trp/-Leu/-His/-Ade defective medium only when both BACK-like and TAZ domains were present, indicating these two domains were responsible for interacting with 1a (Fig. 3A). Similarly, we next splitted 1a into N-terminal MET domain and C-terminal HEL domain and found that neither of them interacted with MdBT2 (Fig. 3B), indicating the full-length of 1a was required for 1a-MdBT2 interactions.
We next utilized a BiFC assay to verify the 1a-MdBT2 interactions. The Agrobacterium harboring the MdBT2-nYFP and 1a-cYFP constructs were co-infiltrated into N. benthamiana leaves, and strong yellow fluorescence signals were captured in the cytoplasm under a confocal microscope (Fig. 3C, upper panel). However, when combined MdBT2-nYFP with cYPF (Fig. 3C, middle panel), or 1a-cYFP with nYFP (Fig. 3C, bottom panel), no signal was observed in neither of them. These data indicted that ApNMV 1a physically interacted with MdBT2 in the cytoplasm in vivo.
To further confirm the interactions between 1a and MdBT2, a pull-down assay was conducted. The fusion protein GST-1a or a simple GST was incubated with MdBT2-HIS and GSH-attached beads. Then target proteins were eluted with GSH solution and tested with anti-GST and anti-HIS antibodies. The results showed that the MdBT2-HIS fusion protein was pulled down in the presence of GST-1a but not GST (Fig. 3D), suggesting GST-1a interacted with MdBT2-HIS in vitro.
We finally determined the 1a-MdBT2 interactions using a luciferase complementation imaging assay. The Agrobacterium harboring MdBT2-nLuci and cLuci-1a were co-infiltrated into N. benthamiana leaves, and empty vectors served as control. The results showed that luminescent signals were observed only when both MdBT2-nLuci and cLuci-1a were present, and no signal was captured in neither MdBT2-nLuci plus cLuci combination nor nLuci plus cLuci-1a combination (Fig. 3E), suggesting the interactions of 1a and MdBT2 in vivo. Collectively, all these data indicated that ApNMV 1a interacted with MdBT2 both in vivo and in vitro.