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.