MdBT2 interferes with the interactions between 1a and 2apol
We have previously identified the protein interactions between ApNMV 1a and 2apol, the homologous of both are required and sufficient to support viral genomic RNA replication in BMV caseĀ (Diaz and Wang, 2014; Zhang et al., 2020). Given that 1a also interacted with MdBT2 (Fig. 3), we next asked whether MdBT2 affected the interaction between 1a and 2apol. We first performed a luciferase complementation imaging assay to explore the relationship of the three proteins. The interaction between 1a and 2apol was reconfirmed in the assay, and strong luminescent signals were observed only in the presence of both 1a-nLuci and cLuci-2apol (Fig. 7A). Then pCXSN-MdBT2-HA was introduced and co-infiltrated with 1a-nLuci and cLuci-2apol and observed under an in vivo imaging system. The results indicated that luminescent signals were decreased progressively with the increased ratio of pCXSN-MdBT2-HA (Fig. 7B), suggesting that MdBT2-HA competed with 2apol and interfered with its interactions with 1a in vivo.
To further verify the interference of MdBT2 on the 1a-2apol interactions, we next conducted a competitive pull-down assay using fusion proteins of GST-2apol, MdBT2-HIS, and 1a-HIS obtained from prokaryotic expression system. GST-2apol and 1a-HIS were mixed and incubated with MdBT2-HIS and went through GST-attached column. With the addition of increasing amount of MdBT2-HIS protein, the amount of 1a-HIS protein pulled down decreased progressively, indicating that MdBT2-HIS competed with GST-2apol to interact with 1a-HIS in vitro (Fig. 7C). In addition, we also observed that MdBT2-HIS was not pulled down by GST-2apol (Fig. 7C), indicating they did not interact with each other, which was similar to what we found in Y2H assay (Supplementary Fig. S3). Thus, in addition to promoting 1a ubiquitination and degradation, MdBT2 might also inhibit ApNMV viral genomic RNA replication through interfering with the interactions between viral replication components 1a and 2apol.