1. Volume 9, Issue 6, Pages 911-925 (June 2016)
Tobacco RING E3 Ligase NtRFP1 Mediates Ubiquitination and Proteasomal Degradation of a Geminivirus-Encoded βC1 
Qingtang Shen, Tao Hu, Min Bao, Linge Cao, Huawei Zhang, Fengmin Song, Qi Xie, Xueping Zhou 
Molecular Plant 
Volume 9, Issue 6, Pages (June 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
Identification of NtRFP1 and Its Interaction with TYLCCNB-βC1.
(A) Yeast two-hybrid assay showing NtRFP1 interactions with βC1. Y2HGold yeast strains co-transformed with the indicated plasmids were subjected to 10-fold serial dilutions and plated on synthetic dextrose (SD)/-Ade/-His/-Leu/-Trp medium to identify protein interactions. Proteins were fused to either the Gal4 DNA binding (BD-NtRFP1) or activation (AD-βC1) domains. Empty vectors containing the BD or AD were used as controls. Laminin and p53 fused to the BD were used as negative and positive controls, respectively.
(B) Phylogenetic tree based on the NtRFP1 amino acid sequences shown in Supplemental Figure 1 using Clustal analysis with a PAM250 residue weight (DNASTAR).
(C) Schematic representation of NtRFP1. The putative von Willebrand Factor type A (VWA) and RING-finger functional domains indicated were deduced from SMART online software (
(D) Subcellular localization of NtRFP1 in transgenic N. benthamiana epidermal cells expressing RFP-H2B. Micrographs showing cells co-expressing GFP with RFP-H2B (top row) or GFP:NtRFP1 with RFP-H2B (bottom row) were examined under GFP fluorescence (left), RFP fluorescence (middle), or an overlay of GFP and RFP fluorescence (right) using confocal microscopy. Arrows indicate nuclei, pentagrams show a zone of GFP-NtRFP1 accumulation at the membrane. Bars represent 20 μm.
(E) BiFC visualization of interactions between TYLCCNB-βC1 and NtRFP1 in RFP-H2B transgenic N. benthamiana leaves. a, YFP fluorescence; b, RFP fluorescence; c, YFP/RFP overlay. Arrows indicate nuclei, arrowheads show the green granular bodies in the cytoplasm, pentagrams show the YFP fluorescence signal at the membrane. Bars represent 20 μm.
(F) Co-IP analysis of GFP-βC1 and Flag3-NtRFP1 in vivo. N. benthamiana leaves were infiltrated with A. tumefaciens cells harboring Flag3-NtRFP1 and GFP-βC1 (lane 1), Flag3-NtRFP1 and GFP (lane 2), Flag3-P and GFP-βC1 (lane 3), and Flag3-P and GFP (lane 4). Leaf extracts were incubated with anti-Flag M2 magnetic beads (Sigma). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using monoclonal antibody to GFP or Flag.
(G) Schematic representation of the truncated mutants of NtRFP1 and yeast two-hybrid assays identifying regions of NtRFP1 required for interactions with βC1. Proteins were fused to either the Gal4 DNA binding (BD-NtRFP1 M1 to M4) or activation (AD-βC1) domains. Empty vector containing the AD was used as a control. Laminin (Lam) and (53) p53 fused to the BD were used as negative and positive controls, respectively.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2 NtRFP1 mRNA Levels in Tobacco Analyzed by qRT-PCR.
NtRFP1 mRNA levels in various tobacco tissues (A), TYLCCNV-, TYLCCNV/TYLCCNB-, and EHA105-infiltrated leaves at 7 DPI (B), or transgenic tobacco leaves expressing 35S:HA-βC1 (C) were analyzed by qRT-PCR. Relative mRNA levels in leaf tissue were normalized using GADPH mRNA as a reference. Values are means of three independent experiments. Different lowercase letters above the bars denote significant differences (Fisher's lease significant difference method; P < 0.05).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
NtRFP1 Functions as an E3 Ubiquitin Ligase and Mediates βC1 Protein Ubiquitination.
(A) Illustration of the NtRFP1 C3-H-C4 RING finger composition and site of our mutation in the RING finger.
(B) E3 ubiquitin ligase activity of NtRFP1. MBP-NtRFP1 and its mutant MBP-NtRFP1 (H459Y). Fusion proteins were assayed for E3 activity in the presence of wheat E1, human E2 (UbcH5b), and GST-ubiquitin (Ub). The numbers on the left denote the marker protein MWs in kilodaltons (kDa). MBP was used as a negative control. Samples were resolved by 12% (top panel) and 8% (bottom panel) SDS–PAGE. An anti-ubiquitin (Ub) antibody was used to detect GST tag ubiquitination (top panel), and anti-MBP antibody was used to detect maltose fusion proteins (bottom panel). ▴, mono-ubiquitinated E2.
(C) NtRFP1 mediates the ubiquitination of βC1 protein. The N terminus of the full-length βC1 protein was fused to a GST tag (GST-βC1) and used as a substrate for the assay. Anti-GST antibody was used in the immunoblot analysis to detect GST fusion proteins (top panel, 12% SDS–PAGE), and anti-βC1 antibody was used to detect the GST-βC1 substrate protein (bottom panel, 12% SDS–PAGE).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 Polyubiquitination and Degradation of βC1 by the UPS In Vivo.
Agrobacterium carrying Flag-Myc4-βC1, Flag-Myc4-GFP, GFP-NtRFP1, or GFP-NtRFP1H459Y was separately infiltrated or co-infiltrated into N. benthamiana leaves, and tissue was harvested at 3 DPI. The numbers in the middle of (A) or on the left in (B–E), show the marker protein MWs in kDa. In (C–E), protein or DNA bands were quantified using ImageJ software, fold changes in expression for βC1 or GFP against their respective controls (set as 1) are shown under their corresponding panels. Half of each sample was taken before incubation and separated in another gel, and Ponceau staining for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used as the normalization control.
(A) Detection of βC1 ubiquitination in vivo. Flag-Myc4-βC1 or Flag-Myc4-GFP cell lysates were incubated at 25°C with agitation in an Eppendorf Thermomixer for 2 h before immunoprecipitation with a polyclonal antibody against Myc. Immunoprecipitated samples were immunoblotted with a monoclonal antibody against Myc (right) or ubiquitin (Ub) (left). ▴, represents dimeric βC1; ▵ represents ubiquitinated βC1.
(B) Effect of NtRFP1 on βC1 ubiquitination in vivo. Total protein extracts were immunoprecipitated with anti-Myc antibody and protein G beads (Sigma) and immunoblotted with a mouse anti-Myc monoclonal (top panel) or anti-ubiquitin (middle panel) antibody, respectively. Some of each sample was taken before immunoprecipitation and detected with anti-GFP antibody (bottom panel). ▴ represents dimeric βC1; ▵ represents ubiquitinated βC1.
(C) Detection of the stability of βC1 protein by semi-in vivo assay. βC1 protein levels were analyzed with anti-Myc antibody at different times following 100 μM CHX treatment in the presence or absence of 20 mM ATP. ▴ represents dimeric βC1; ▵ represents post-translation modified βC1 derivatives.
(D) Effect of MG132 on the stability of βC1 protein by semi-in vivo assay. βC1 protein levels were analyzed with anti-Myc antibody at different times after 100 μM CHX and 20 mM ATP treatment in the presence of 100 μM MG132 or an equal volume of DMSO (control).
(E) Effect of MG132 on the stability of βC1 protein by in vivo assay. 100 μM CHX, in the presence of 100 μM MG132 or an equal volume of DMSO (control) were infiltrated into samples agro-infiltrated with Flag-Myc4-βC1 or Flag-Myc4-GFP at 16 h before harvesting. βC1 proteins were detected using anti-βC1 antibody and GFP (control) proteins detected using an anti-GFP antibody. Expressed target genes and GADPH (reference) mRNA expression levels were analyzed by semi-quantitative RT–PCR.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
NtRFP1 Affects βC1 Symptoms and Promotes βC1 Degradation by the 26S Proteasome.
(A) Symptoms associated with TYLCCNV/TYLCCNB infection at 27 DPI in wild-type and transgenic tobacco plants overexpressing NtRFP1 [NtRFP1(+)] or knocked down for NtRFP1 [NtRFP1(−)] (top) or the corresponding leaf undersides (bottom). Arrowheads indicate vein thickening on the undersides of the leaves.
(B) TYLCCNV and TYLCCNB DNA levels in plants at 27 DPI with the symptoms described in (A). Total DNA from plants infected by TYLCCNV/TYLCCNB was used for DNA gel blotting. Blots were probed with sequences specific for the CP gene of TYLCCNV (top) or the full-length sequence of TYLCCNB (middle). An ethidium bromide-stained gel shown below the blots provides a DNA-loading control. The position of viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated.
(C) Effects of NtRFP1 on symptoms induced by βC1. The concentrations of agrobacteria harboring pGR106-βC1/pGR106-NtRFP1, pGR106-βC1/pGR106- NtRFP1H459Y, and pGR106-βC1/pGR106-GFP were 0.5/1.0, 0.5/1.0, and 0.5/0.5 A600, respectively. Symptoms of plants (top row) and leaves (bottom row) are shown at 30 DPI.
(D) Detection of NtRFP1-promoted βC1 protein degradation by in vivo assay. Systemic leaves displaying distinct symptoms of plants described in (C) were harvested and βC1, NtRFP1, and GFP proteins were detected with anti-βC1, anti-NtRFP1, and anti-GFP antibodies, respectively. Expressed target genes βC1, NtRFP1, GFP, and GADPH (reference) mRNA expression levels were analyzed by semi-quantitative RT–PCR. Numbers at the top of each panel indicate the concentrations of agrobacteria harboring the corresponding expression vectors used in the co-infiltrations.
(E) MG132 inhibition of NtRFP1-promoted βC1 degradation. βC1 degradation was performed by mixing proteins extracted from tobacco leaves separately infiltrated with agrobacterium capable of expressing Flag-Myc4-βC1, GFP-NtRFP1, and GFP. The Flag-Myc4-βC1 protein extract was mixed with the GFP-NtRFP1 or GFP extracts in a 1:2 volume of 100 μM CHX and 20 mM ATP, in the presence of 100 μM MG132 or an equal volume of DMSO control. The corresponding protein mixtures were then agitated for 0.5 h at 25°C in an Eppendorf Thermomixer. Flag-Myc4-βC1 was detected by anti-βC1 antibody, and GFP-NtRFP1 and GFP were detected by anti-GFP antibody.
In (D–E), protein or DNA bands were quantified using ImageJ software, fold change in expression for βC1, NtRFP1, or GFP against their respective controls (set as 1) are shown under their corresponding panels. Half of each sample was taken before incubation and separated in another gel, and Ponceau staining of Rubisco used as the loading control for normalization of βC1, NtRFP1, or GFP protein levels.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions