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.