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.