Molecular Characterisation of Geminivirial AC3 Protein in the Context of Developing Viral

Resistance in Host Plants

Kalyan Kumar Pasumarthy, Nirupam Roy Choudhury, Sunil Kumar Mukherjee
International Centre for Genetic Engineering & Biotechnology (ICGEB), New Delhi, INDIA

1. Abstract 5. AC3 Oligomeric Studies 9. AC3 – AC1 Interaction 13. Virus Induced Gene Silencing
Geminiviruses are covalently closed single stranded DNA
containing plant viruses with a genome of ~2.7 kb. These
viruses infect economically important crop plant like tomato, pCK2-PCNA
PCNA
cotton, cassava, maize, soybean. Understanding the basic
biology, such as function(s) of viral proteins in replication of
MBP-AC1 CR AC1 AC2-----AC3 35S Promoter Poly A CR
the viral DNA and spread of viral infection in the plant host
GST pull down assay for AC3-AC1
expands the scope of development of antiviral strategies to MBP oligomerisation
GST-AC3
target the viral infection. Various studies have shown that CR
the viral proteins AC1 and AC3 are required for viral
MBP-AC3
replication. While AC1 was well characterised for its role in Input Bound
MBP
the replication, little information is available regarding the

AC1
GST-AC3 GST pull down assay
function of AC3 protein of geminiviruses. In this study, we - + + + + + + + AC1 protein
250

Relative (%) ATPase activity

have purified recombinant AC3 protein from Tomato leaf curl - - AC3 protein Aberrant mRNA of PCNA
AC2
Kerala virus-[India:Kerala II:2005], analysed its oligomeric 200
Viral episome
properties and biochemical effects of its interaction with 150 RNA silencing pathway

AC1. We have screened Arabidopsis cDNA library in yeast Input Bound 100 ATPase assay
PCNA silencing
and phage display library to identify the AC3 interacting host 50
proteins/peptides. We have also developed model systems Stunted plant growth
0
in yeast and in planta to analyse the role of AC3 in 1 2 3 4 5 6 7 8
replication and gene silencing through reverse genetics
approach.
AC3 forms a homo-oligomer in vitro AC3 interacts with AC1 and enhances the ATPase activity Virus induced gene silencing (VIGS) phenomenon
of AC1

2. Methods 6. AC3 Oligomeric Studies 10. Yeast Model System 14. VIGS of Tomato PCNA Gene
Protein expression, Oligomerisation and ATPase activity Plasmid Replication Efficiency in Yeast
assays: The methods adopted have been described earlier 158 kDa 449 kDa 669 kDa

(Choudhury et al., Nucl. Acids Res., 2006, 34, 6362-77; Singh Fraction No. 2 5 8 11 14 17 20 23 26 28 30 32 34 36 38 40 42

et al., Nucl. Acids Res., 2007, 35, 755-70). MBP-AC3

MBP Replication Efficient

URA
Yeast two hybrid analysis: The analyses were done by YCp50 XhoI
(+)
following manufacturer’s protocol using Matchmaker yeast Sucrose gradient ultra-centrifugation of MBP-AC3 protein
Bgl II
transformation kit (Clonetech).
Hind III
Phage display library screening: The microtitre plates were mAu
coated with purified MBP-AC3 of ToLCKeV and the Phage 350
display library was screened using Ph.D.-12 Phage display 300
- Replication Inefficient

URA
250 MBP-AC3 YCpO
library kit (New England Biolabs). Throglobulin Xho I (-)
200 Dextran
(2000 kDa ) (669 kDa ) Bgl II
Site directed mutagenesis: Site specific mutagenesis was 150
Aprotinin
carried out by QuickChange site directed mutagenesis kit. 100 (6.9 kDa ) CR-AC3
Hind III
50
Yeast model system for viral replication: Design of the yeast 00
0 .0 1 .0

1
2 .0

2
3 .0

3
4 .0

4ml Hind III

model system was based on principles described (Raghavan Gel-filtration of MBP-AC3 protein Replication Efficient
Ycp-CR-AC3
et al., J. Virol., 2004, 78, 2405-13). (+) pCK2-PCNA pCK2M-PCNA Vector control
(Wild type AC3) (Mutated AC3)
CEN4
In planta model system for viral replication assay and virus
induced gene silencing: Based on the RCR model of viral AC3 forms a higher order oligomer (12-14 mer) Minimal region required for viral DNA replication also PCNA silencing was enhanced in the presence of wild type
DNA replication, vectors based on episome formation were supports plasmid replication in yeast AC3 ORF
designed (Pandey et al., Virol. Journal., 2009, 6, 152).

3. Geminivirus Genome Structure 7. Phage Display Methodology 11. In planta Model System 15. Summary
• Tomato leaf curl Kerala II virus AC3 protein was expressed
and purified as a fusion protein with GST and MBP
A library of phage, each
AV2 displaying a different peptide pCK2 Replicon (Binary vector)
sequence, was exposed to a plate
• AC3 protein forms a higher order oligomer in the range of
MCS
AC4 coated with the target 12 -14 mer
CR AC1 AC2 AC3 35S CR
• AC3 interacting peptides identified through phage display
AV1 indicates that AC3 might interact with proteins from various
metabolic pathways including DNA replication, DNA/Histone
Unbound phage was
washed away
modification enzymes, RNAi pathway
CR

Specifically-bound phage was eluted • AC3 interacts with AC1 and enhances the ATPase activity of
with an excess of a known ligand for Degenerate oligos will AC1
the target, or by lowering pH. amplify the DNA only
AC1

AC3
when episome is • Ex vivo model in yeast and in planta model system were
Tomato leaf curl Kerala II Virus Genome (NCBI Accn No. DQ852623) formed
The eluted pool of phage was amplified
developed to assay the role of AC3 protein in viral DNA
and the process is repeated for a total
of 3–4 rounds AC2 replication and virus induced gene silencing of host gene
(VIGS)
Viral Episome
• AC3 enhances viral DNA replication by 3-4 fold in planta
CR AC1 AC2 AC3
After 3–4 rounds, individual clones • AC3 enhances virus induced gene silencing of host PCNA
were isolated and sequenced.
Minimal region required for viral DNA replication gene in tomato plant

4. AC3 Protein Expression 8. AC3 Interacting Proteins 12. Role of AC3 in Viral Replication 16. Acknowledgements
S FT W 1 3 5 7
Proteins involved in RNA silencing Histone/DNA modifying enzymes Financial support from CSIR and Dept. of
CR AC1 AC2-----AC3
Repressor of Silencing 1 (ROS1) H3-K9 Methyltransferase Biotechnology, Govt. of India is acknowledged
• MBP is 43kDa Supressor of gene silencing 3 Histone Methyltransferase
Hua Enhancer 1 Histone acetyl transferase AC3 Mutation site
• AC3 is 402bp ie 134Aa (~15kDa)
Hua Enhancer 4 Decreased DNA methylation 1
• Fusion protein is ~ 58kDa Dicer-like 1 Variant in methylation 2 AC3 mutated viral DNA in pCK2M replicon
• Expressed at 20°C, 0.2mM IPTG Dicer-like 2 Increase in Bonsai methylation 1
Argonaute 1 Maintanance of methylation
Agronaute 2 Decreased methylation to DNA 1
MBP-AC3

RNA/DNA polymerases Replicaion proteins/Helicases

I FT W 1 2 3 4 5
RNA dependant RNA polymerase 1 Replication protein A1
RNA dependant RNA polymerase 2 Geminivirus Rep interacting Kinase 1
• GST is 26kDa RNA dependant RNA polymerase 6 RecQ Helicase Replicon
DNA polymerase γ2 Werner Helicase
• Fusion protein is ~ 41kDa RAD1
DNA polymerase ε subunit
• Expressed at 20°C, 0.2mM IPTG DNA polymerase α subunit RAD4
DNA polymerase η splice variant RAD5 Actin
DNA polymerase ζ catalytic subunit RAD23-3
GST-AC3 DNA polymerase λ RAD50
5dpi 10dpi 15dpi
DNA polymerase δ subunit Anti silencing factor 1

AC3 was expressed as fusion protein and purified to Putative AC3 interacting proteins are majorly from AC3 enhances viral DNA replication by 3-4 fold in planta Plant Molecular Biology Group, ICGEB
homogeneity Replication and RNAi silencing pathway