MdBT2 promotes ubiquitination and degradation of ApNMV 1a
Given that MdBT2 is one of the components of BTB/POZ type E3 ligase and functional in interacting with target proteins. We thus hypothesized that MdBT2 might promote the ubiqitination and degradation of its interacting partner ApNMV 1a. To verify our hypothesis, we first performed a cell-free degradation assay using the apple calli of wt,MdBT2-OE , and MdBT2-anti . The gene expression ofMdBT2 was increased in the overexpression calli but inhibited in the antisense compared to that of WT (Supplementary Fig. S4A), suggesting these calli were suitable for MdBT2 functional analysis. Then total protein extracts from these three types of apple calli were incubated with 1a-HIS fusion protein for different time points, respectively. After immunoblotted with anti-HIS antibody, we found that the degradation speed of 1a-HIS protein was faster in proteins extracted from MdBT2-OE transgenic calli (Fig. 4A, the middle section), but slower in protein extracts of MdBT2-anti (Fig. 4A, the right section) compared to that of WT (Fig. 4A, the left section). These data indicated that MdBT2 promoted 1a protein degradation in vitro. To testify whether proteasome is involved in 1a-HIS degradation, proteasome inhibitor MG132 was applied to these experiments, and the 1a-HIS protein degradation was inhibited in proteins from all the three types of apple calli (Fig. 4B), suggesting 26S proteasome pathway might be involved in MdBT2-mediated 1a-HIS protein degradation.
To further verify the role of MdBT2 in promoting ApNMV 1a protein degradation, we transformed Agrobacterium harboring pCXSN-1a-HA construct into ‘GL3’ apple plantlet leaves of WT, MdBT2-OE , andMdBT2-anti transiently via vaccum method. The transgenic apple plantlets were obtained previously from our lab, and we confirmed thatMdBT2-OE transgenic plantlet had higher MdBT2 gene expression level, while that was lower in the MdBT2-antiplantlets compared to that of WT (Supplementary Fig. S4B), suggesting these transgenic lines were suitable for MdBT2 functional analysis. Total proteins were extracted five days post-transformation and anti-HA antibody was used for immunoblotting. The results showed that 1a-HA protein level in MdBT2-OE leaves was lower than that of WT, but was higher in MdBT2-anti transgenic leaves compared with that of WT (Fig. 4C). Because the transcript level of 1a-HA was similar among the three types of leaves (Fig. 4D), we thus assumed that MdBT2 regulated the protein stability of 1a-HA in vivo.
We next tested whether ubiquitination was involved in MdBT2-mediated ApNMV 1a protein destablization. The active MdBT2-GFP proteins were first obtained via immunoprecipitating from total protein extracts ofMdBT2-OE calli using anti-GFP antibody. Then 1a-HIS protein was incubated with human E1, E2, ubi, and active MdBT2-GFP in incubation buffer, and detected by anti-HIS and anti-Ubi antibodies. GFP proteins immunoprecipitated from 35S:GFP transgenic apple calli served as control. The results showed that high-molecular mass forms of 1a-HIS, which are polyubiquitinated 1a-HIS (Ubi(n)-1a-HIS), were detected in the presence of active MdBT2 proteins when immunoblotted by anti-HIS antibody (Fig. 4E, upper image). And a higher amount of ubiquitinated proteins were also detected in the presence of active MdBT2-GFP when immunoblotted using anti-Ubi antibody (Fig. 4E, lower image). These data suggested that MdBT2 was involved in promoting the ubiquitination of 1a-HIS in vitro.
To determine whether MdBT2 promotes the 1a-HIS protein ubiquitination in vivo, we transformed the pCXSN-1a-HA into apple plantlet leaves of ‘GL3’ WT, MdBT2 -OE, and MdBT2-anti as aforementioned. After treatment with MG132, total protein extracts of the three types of apple leaves were immunoprecipitated with anti-HA antibody, then the precipitates were detected using anti-HA (Fig. 4F, upper image) and anti-Ubi (Fig. 4F, lower iamge) antibodies. We found that the amount of Ubi(n)-1a-HA were higher in MdBT2-OE leaves, but lower inMdBT2 -anti leaves compared to that of WT (Fig. 4F), indicating that MdBT2 promoted the ubiquitination of 1a protein in vivo. Therefore, these data indicated that MdBT2 was involved in promoting 1a protein ubiquitination and degradation via UPS pathways.