Nitrate treatment destablizes viral 1a protein through the UPS
pathways
Because replication proteins play key roles in viral genomic RNA
duplication, we next tested the effects of nitrate on the proteins
levels of 1a and 2apol, the two assumed replication
proteins of ApNMV, both in vivo and in vitro. By using the anti-1a
specific antibody, we found the protein levels of 1a decreased with the
increased concentration of KNO3 (Fig. 2A), which is
consistent with the RNA accumulation (Fig. 1B). Then a cell-free
degradation system was utilized to test the effect of nitrate on the
protein stability of both 1a and 2apol. Increased
transcript levels of MdNRT1.1 (Ho et al., 2009) andMdBT2 (Wang et al., 2018; An et al., 2020), two representative
genes that were reported in response to nitrate, under
KNO3 confirmed that these apple plantlets were indeed
responded to nitrate treatment (Supplementary Fig. S1). Then same amount
of 1a-HIS or 2apol-HIS fusion proteins obtained from
the prokaryotic expression system were mixed with total proteins
extracted from ‘GL3’ leaves treated with KNO3 or KCl,
and incubated for different time. Western blot analysis showed that
1a-HIS protein degradation speed was faster in proteins extracted from
KNO3-treated ‘GL3’ leaves than that from KCl treatment
(Fig. 2B), while the protein degradation of 2apol-HIS
were similar between proteins extracted from KCl- and KNO3-treated
leaves (Supplementary Fig. S2A), suggesting nitrate treatment
compromised the protein stability of 1a but not 2apol.
The UPS-mediated protein degradation is one of the most common and
powerful pathways in the protein degradation system of plants. To
determine whether the nitrate-induced 1a-HIS protein degradation is
depending on the proteasome pathway, a proteasome inhibitor MG132 was
applied in the assay, while DMSO served as control. Total proteins
extracted from KNO3-treated ‘GL3’ leaves were pretreated
with MG132 or DMSO for 30 min, and 1a-HIS fusion protein was added to
those samples and detected as aforementioned. The results showed that
addition of MG132 inhibited the 1a-HIS protein degradation largely,
while DMSO had no effect on that (Fig. 2C). These data indicated that
KNO3-induced 1a-HIS protein degradation was most likely
mediated by the proteasome pathway.
In addition, we also performed the cell-free degradation assay by using
protein extracts from KNO3- or KCl-treated apple calli
to further confirm the nitrate-induced 1a-HIS protein degradation
(Supplementary Fig. S2B and S2C). And similar results were obtained
compared to that from the ‘GL3’ leaves. Collectively, these data
indicated that nitrate treatment destablized viral protein 1a through
UPS pathways.