MdCUL3A is not involved in MdBT2 mediated degradation of ApNMV 1a
In CRL3 type of E3 ligase complexes, BTB/POZ proteins usually serve as scaffold to link the target protein and CUL3 through physical protein interactions (Gingerich et al., 2005; Hua and Vierstra, 2011). Previous reports have proved the interaction between MdBT2 and MdCUL3A through multiple methods (Zhao et al., 2016; Wang et al.,2018), and we reconfirmed the interaction via a pull-down assay. Similar result was obtained that MdCUL3A-HIS protein was pulled down only in the presence of GST-MdBT2, suggesting the physical interaction of the two proteins (Supplementary Fig. S5A).
We next tested if MdCUL3A was involved in MdBT2-mediated 1a protein degradation. A cell-free degradation assay was conducted using total proteins extracted from the MdCUL3A -OE transgenic apple calli. The transcript level of MdCUL3A was higher in MdCUL3A -OE compared to that of WT apple calli (Supplementary Fig. S5B), suggesting these transgenic materials were suitable for functional analysis of MdCUL3A. After immunoblotted with anti-HIS antibody, we found the 1a-HIS protein degradation speed was similar in the proteins extracted fromMdCUL3A -OE calli with that of WT (Fig. 5A), suggesting MdCUL3A might not be closely involved in MdBT2-mediated 1a protein degradation.
To explore the possible reasons for this, a competitive pull-down assay was conducted to test the effect of MdCUL3A on the 1a-MdBT2 interactions. The results showed that addition of increased amount of MdCUL3A-HIS protein decreased the quantity of MdBT2-HIS that was pulled down (Fig. 5B), suggesting the repression effects of MdCUL3A-HIS on the interactions between GST-1a and MdBT2-HIS, which might be a possible reason that MdCUL3A had no effect in MdBT2-mediated 1a degradation.