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.