Figure legends
Fig. 1 Nitrate treatment inhibits ApNMV viral RNA accumulation in Malus domestica. A. Schematic model of infectious clone construction of ApNMV. The three RNA segments were constructed into binary vector, respectively.In the constructions, the three segments are driven by double CaMV 35S promoter, and are followed by a ribozyme (Rz) and a Nos terminator (Tnos). B. ApNMV RNA accumulation in leaves of ‘GL3’ treated with KNO3. The infectious clone of ApNMV was used to agro-infiltrate the ‘GL3’ leaves under indicated concentration of KNO3, and the leaves were collected for RNA extraction 4 days post-infiltration. DIG-labeled probes targeting the CP-coding sequence was used to test the RNA levels of (+)RNA3 and (+)RNA4 in a Northern blot assay with two duplicates for each treatment. The digits (Mean±SD) indicate the signal intensity of (+)RNA3 and the band intensity of 0 mM were set as 100. The rRNA served as loading control. The experiment was repeated three times.
Fig. 2 Nitrate treatment destablizes viral protein 1a through the UPS pathways. A. Protein levels of 1a in leaves of ‘GL3’ treated with KNO3. The infectious clone of ApNMV was used to agro-infiltrate the ‘GL3’ leaves under indicated concentration of KNO3, and the leaves were collected for protein extraction 4 days post-infiltration. Anti-1a specific antibody was used in a Western blot assay with two duplicates for each treatment. MdACTIN served as loading control. The digits (Mean±SD) indicate the signal intensity and the band intensity of 0 mM were set as 1.00. B. 1a-HIS protein degradation in a cell-free degradation assay. 1a-HIS protein obtained from prokaryotic expression were embedded with total proteins extracted from apple callus treated with KCl or KNO3 at the indicated time points. The protein samples were then subjected to a Western blot detection with anti-HIS antibody. MdACTIN served as loading control. C. 1a-HIS protein degradation in the proteins extracted from KNO3-pretreated aplle callus in the absence or presence of MG132. The samples were collected at the indicated time points and subjected to a Western blot detection with anti-HIS antibody. MdACTIN served as loading control. The charts on the right side of indicated the degradation trends in (B) (upper chart) and (C) (bottom chart), respectively. The intensity of protein bands at 0 h was set as 1.00. All experiments were repeated three times independently.
Fig. 3 MdBT2 interacts with ApNMV 1a in vivo and in vitro. (A) and (B) illustrated the interactions between MdBT2 and 1a. MdBT2 (A) and 1a (B) was truncated into different fragments based on the domain structure, and different color on the left side indicates different domains that were included in the assay. Different combinations of constructs were transformed in yeast cells and cultured on selective medium SD-Trp/-Leu (SD-T-L), and interactions were tested on selective medium SD-Trp/-Leu/-His/-Ade (SD-T-L-H-A). The images were taken 3 d after incubation at 30 ℃. C. BiFC assay indicated the interactions of MdBT2 and 1a in N. benthamiana cells. The nuclear was stained with DAPI (4’,6-diamidino-2-phenylindole). BF, bright filed. Scale bar, 10 μm. D. Pull-down assay showed the in vitro interactions between MdBT2 and 1a. MdBT2-HIS protein was incubate with GST-1a or only GST proteins and went through the GST-attached column. Anti-GST and anti-HIS antibodies were used to detect the target proteins. GST-1a, GST, MdBT2-HIS bands are indicated by arrows on the left side. The ladder on the right side indicated the molecular weight (KDa) of target proteins. E. Luciferase complementation imaging assays indicated the interactions between MdBT2 and 1a in N. benthamiana cells. The bar on the right side indicates the intensity of the signals captured. Empty nLuci and cLuci vectors served as control. The images displayed here are representative of three independent experiments.
Fig. 4 MdBT2 promotes the ubiquitinatin and degradation of 1a in vivo and in vitro. 1a protein degradation in total proteins extracted from different transgenic apple calli in the absence (A) or presence (B) of MG132 in a cell-free protein degradation assay. 1a-HIS protein was incubated with protein extracts from transgenic (MdBT2-OE and MdBT2-anti) and WT apple calli with the indicated time. Anti-HIS was used to detect the target proteins. MdActin served as loading control. The charts on the right side showed the protein degrading trends in (A) (upper chart) and (B) (lower chart), and the intensity of protein bands of 0 h was set as 1.00. The 35S:1a-HA construct was transiently expressed in WT, MdBT-OE, and MdBT2-anti ‘GL3’ leaves, and the leaves were collected for protein (C) and RNA (D) extraction 5 d post agro-infiltration. Anti-HA antibody was used to detect the 1a-HA protein in a Western blot assay (C). MdACTIN served as loading control. qRT-PCR was used to detect the transcript level of 1a-HA (D). The 1a transcript level of the first duplicate in WT was set as 1.00. E. MdBT2 mediated the 1a protein ubiquitination in vitro. Active MdBT2-GFP protein was immunoprecipitated from 35S ::MdBT2-GFP transgenic apple calli using anti-GFP antibody, and incubated with 1a-HIS protein, E1, E2, and ubiquitin in vitro. GFP protein immunoprecipitated from35S ::GFP transgenic apple calli served as control. Anti-HIS (upper panel) and anti-Ubi (lower panel) antibodies were used to detect the target proteins. Anti-GFP antibody was used to detect the input of GFP and active MdBT2-GFP proteins. The 1a-HIS and Ubi(n)-1a-HIS were labeled on the left side. F. MdBT2 mediated the 1a-HA protein ubiquitination in vivo. 35S::1a-HA construct was transiently expressed in WT, MdBT2-OE and MdBT2-anti ‘GL3’ leaves, and the samples were collected for protein extraction 4 d post-infiltration. Anti-HA antibody was used for immunoprecipitation (IP), anti-HA (upper panel) and anti-Ubi (lower panel) antibodies were used for immunoblot (IB). Input indicated the samples collected before IP and detected with anti-ACTIN antibody. The 1a-HA and Ubi(n)-1a-HA were labeled on the left side. All the images showed here are representative of three independent experiments.
Fig. 5 MdBT2 promoted 1a degradation in an MdCUL3A-independent pathway. A. In vitro degradation of 1a-HIS in proteins extracted from MdCUL3A overexpression (MdCUL3A-OE) and WT apple calli. Prokaryotic expression system-obtained 1a-HIS protein was incubated with protein extracts from WT or MdCUL3A-OE for the indicated time in the presence of translation inhibitor cycloheximide (CHX). MdACTIN served as loading control. The chart on the right side indicates the protein degradation trend, and protein band intensity of 0 h was set as 1.00. B. Competitive pull-down assay showed the effect of MdCUL3A-HIS on the interactions between GST-1a and MdBT2-HIS. GST-1a and MdBT2-HIS proteins were incubated with different amount of MdCUL3A-HIS protein, and went through the GST-attached column. A simple HIS-tag protein served as control when MdCUL3A-HIS was absent. For the amount of MdCUL3A-HIS, single ‘+’ means 2 μg protein, and ‘+ + +’ indicates 6 μg protein. Anti-HIS antibody was used to detect both the MdCUL3A-HIS and MdBT2-HIS proteins. All the experiments were repeated three times and the images displayed were the representatives.
Fig. 6 MdBT2 inhibits ApNMV genomic RNA replication by promoting the degradation and ubiquitination of viral protein 1a. Infectious clone of ApNMV was agro-infiltrated into leaves of WT, MdBT2-OE, and MdBT2-anti, and total proteins (A and B) and RNAs (C) were extracted 5 d post-infiltration for further investigation. A. 1a protein levels in wt,MdBT2-OE , and MdBT2-anti transgenic plantlets leaves. Anti-1a specific antibody was used to detect 1a protein. MdACTIN served as loading control. The digits indicated the intensity of bands and the WT were set at 1.00. B. MdBT2 mediated the 1a protein ubiquitination in vivo. Total proteins were extracted and anti-1a antibody was used for immunoprecipitation (IP); anti-1a (top panel) and anti-Ubi (bottom panel) antibodies were used for immunoblot (IB) to test the target proteins. Input indicated the samples collected before IP and detected with anti-ACTIN antibody. The 1a and poly-ubiquitinated 1a (Ubi(n)-1a) were labeled on the left side. C. Detection of ApNMV genomic RNA accumulation in wt, MdBT2-OE , and MdBT2-anti transgenic plantlets leaves via Northern blot. Total RNAs were extracted using hot phenol method. DIG-labeled probes targeting the CP-coding sequence was used to test the (+)RNA3 and (+)RNA4 of ApNMV. The digits indicated the signal intensity of (+)RNA3, and the band intensity of WT were set as 100. The rRNA served as loading control. All the images displayed are representative of three independent experiments.
Fig. 7 MdBT2 interferes with the interactions between 1a and 2apol. A. 1a interacts with 2apolin N. benthamiana cells in a luciferase complementation imaging assay. Agrobacterium harboring different combinations were co-infiltrated into different parts of the same N. benthamianaleaf, and the iamges were taken 3 d post-infiltration under an in vivo imaging system after the leaves were incubated with the substrate of luciferase in dark for 3 min. B. A luciferase complementation imaging assay showed the affect of MdBT2-HA protein on the interactions between 1a and 2apol. Agrobacterium harboring 1a-nluci and cluci-2apol were mixed and co-infiltrated with agrobacteriun containing pCXSN-MdBT2-HA or pCXSN-HA. Increased ratio of pCXSN-MdBT2-HA was added into the mix from 1:1:1 (indicating the OD600 of the three are the same) to 1:1:5 (indicating the OD600 of pCXSN-MdBT2-HA was five time of 1a-nluci and cluci-2apol). Different combinations were illustrated on the right side of the image. The bars on the right side of (A) and (B) indicate the intensity of the signals captured. The empty nLuci and cLuci vectors served as control. C. Competitive pull-down assay showed the effect of MdBT2-HIS on the interactions between GST-2apol and 1a-HIS. Same amount of GST-2apol and 1a-HIS were mixed and incubated with different amount of MdBT2-HIS protein and went through GST-attached column. For the amount of MdBT2-HIS, ‘+’ indicated 2 μg protein, and ‘+ + +’ indicates 6 μg protein. A simple HIS-tag protein served as control when MdBT2-HIS was absent. Anti-HIS antibody was used to detect the 1a-HIS and MdBT2-HIS proteins. All the experiments were repeated three times and the images displayed were the representatives.