This journal club presentation summarizes a research article that investigated the interaction between the Tobacco RING E3 Ligase NtRFP1 and the betasatellite-encoded βC1 protein of the Geminivirus Tomato yellow leaf curl China virus. The presentation identifies that NtRFP1 interacts with βC1 and functions as an E3 ubiquitin ligase that mediates the ubiquitination and proteasomal degradation of βC1. This degradation of the βC1 pathogenicity determinant by NtRFP1 affects βC1-induced symptoms. In conclusion, the study finds that the βC1 protein is targeted for degradation by the host plant's ubiquitin-proteasome system through

1. JOURNAL CLUB PRESENTATION
PRESENTED BY
ROMANCE MANNA
M.SC BIOTECHNOLOGY
REG. NO- 013756
ROLL NO- 30099219010
MAULANA ABUL KALAM AZAD UNIVERSITY OF TECHNOLOGY
GUIDED BY
DR. JAYA BANDYOPADHYAY

2. Tobacco RING E3 Ligase NtRFP1 Mediates Ubiquitination and Proteasomal
Degradation of a Geminivirus-Encoded βC1
Molecular Plant 9, 911–925, June 2016 ª The Author 2016.
Qingtang Shen1,4, Tao Hu1, Min Bao1,2, Linge Cao1, Huawei Zhang3, Fengmin Song1, Qi Xie3 and Xueping Zhou1,2, * 1 State Key Laboratory of Rice Biology,
Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China 2 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of
Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China 3 State Key Laboratory of Plant Genomics, National Center for Plant Gene
Research, Chinese Academy of Sciences, Beijing 100101, China 4Present address: Department of Molecular Genetics, University of Toronto, Room 4180, 1
Kings College Circle, Toronto, ON M5S 1A8, Canada *Correspondence: Xueping Zhou (zzhou@zju.edu.cn) http://dx.doi.org/10.1016/j.molp.2016.03.008
Impact factor
Molecular Plant – 8.820
Cell Press – 32.403

3. Introduction
• Plant viruses are obligate molecular parasites that encode relatively few proteins and are
exclusively dependent on host resources for multiplication and movement.
• Monopartite begomoviruses are associated with betasatellites, known as DNA b. betasatellites consist of a
circular single stranded DNA of approximately 1350 nucleotides that are essential for induction of typical
disease symptoms
• An approximately 13.5 kDa protein (βC1) encoded by betasatellites is a pathogenicity determinant and a
suppressor of both transcriptional and post transcriptional gene silencing.
• Tomato protein, SUCROSE-NONFERMENTING1-related kinase (SlSnRK1) can also interact with and
phosphorylate βC1, and mutations in the primary phosphorylation site affect pathogenicity functions of βC1

4. Objective
• To find out the interaction between TYLCCNB-βC1 and RING finger
protein
• To find if NtRFP1 functions as an E3 ubiquitin ligase and promotes βC1
ubiquitination.

5. Materials and Methods
• Plant Material and Growth Conditions
• Vector Construction
• Agroinfection Assay in Tobacco Plants
• Yeast Two-Hybrid Interactions, qRT–PCR Analyses, and DNA Gel Blot Assays
• BiFC and Subcellular Localization Assays
• Generation of Transgenic Tobacco Plants & Protein Extraction, Immunoblot
Analyses, and Immunoprecipitation
• In Vitro Ubiquitination Assays
• Chemical Treatments, Semi-In Vivo, and In Vivo Protein
• Degradation

6. Result
Figure 1. Identification of NtRFP1 and It’s Interaction with TYLCCNB-βC1.
A) Yeast two-hybrid assay showing NtRFP1
interactions with βC1. Proteins were fused to either
the Gal4 DNA binding (BDNtRFP1) 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
C) Schematic representation of NtRFP1.
D) Subcellular localization of NtRFP1 in transgenic
N. benthamiana epidermal cells expressing
RFPH2B. 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.
E) BiFC visualization of interactions between
TYLCCNB-βC1 and NtRFP1 in RFP-H2B transgenic
N. benthamiana leaves.

7. Result
Figure 1. Identification of NtRFP1 and It’s Interaction with TYLCCNB-βC1.
(F) Co-IP analysis of GFP-βC1 and Flag3-
NtRFP1 in vivo. N. benthamiana.
(G) Schematic representation of the truncated mutants of
NtRFP1 and yeast two-hybrid assays identifying regions of
NtRFP1 required for interactions with βC1.

8. Result
Expression of NtRFP1 Is Induced by βC1
Figure 2. NtRFP1 mRNA Levels in Tobacco Analyzed by qRTPCR.
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

9. Result
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. MBPNtRFP1
and its mutant MBP-NtRFP1 (H459Y).
(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.

10. Result
NtRFP1 Is a Functional E3 Ubiquitin Ligase and Mediates βC1 Ubiquitination In Vitro
Figure 4. Polyubiquitination and Degradation of βC1 by the UPS In Vivo.

11. Result
Figure 5. NtRFP1 Affects βC1 Symptoms and Promotes βC1 Degradation by the 26S
Proteasome.

12. conclusion
• The βC1 protein encoded by the Tomato yellow
leaf curl China virus-associated betasatellite
functions as a pathogenicity determinant.
• Yeast two hybrid system- c1 interact with NtRFP1
and fluorescence complementation pathway and
also co-immune precipitation

13. References
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• Ikeda, F., and Dikic, I. (2008). Atypical ubiquitin chains: new molecular signals. ’Protein
Modifications: beyond the Usual Suspects’ review series. EMBO Rep. 9:536–542
• Jakubiec, A., Tournier, V., Drugeon, G., Pflieger, S., Camborde, L., Vinh, J., He´ ricourt, F., Redeker,
V., and Jupin, I. (2006). Phosphorylation of viral RNA-dependent RNA polymerase and its role in
replication of a plus-strand RNA virus. J. Biol. Chem. 281:21236–21249..

14. THANK YOU