Nitrate treatment decreases ApNMV viral RNA accumulation
In addition to be a critical nutrient for plant growth and development, nitrate also functions as an important signal molecule to regulate expression of multiple genes in response to various environmental factors (Scheible et al., 2004; Ho et al., 2009) and in regulating plant resistance against pathogens (Dordas, 2008; Gupta et al., 2013; Wendehenne et al., 2002). To determine if nitrate has any effect in ApNMV infection, we first constructed the infectious clone of ApNMV by inserting the full length of ApNMV RNA1, RNA2, and RNA3 into a binary vector that driven by a double CaMV 35S promoter, respectively (Fig. 1A). The recombinant constructs were then introduced into Agrobacterium separately and co-transformed into apple plantlets leaves under different nitrate concentrations via vacuum method. Then the viral RNA accumulation were tested by Northern blot using DIG-labeled probes targeting the CP-coding sequence, and found that the RNA levels decreased with the increased concentration of KNO3 (Fig. 1B). When the concentration of KNO3 increased to 10 mM, the (+)RNA3 accumulation decreased to around 20% compared to that of 0 mM (Fig. 1B), suggesting high level of nitrate inhibited ApNMV viral genomic RNA replication.