The presence of tomato leaf curl Kerala virus AC3 protein enhances viral DNA replication and modulates virus induced gene-silencing mechanism in tomato plants
© Pasumarthy et al; licensee BioMed Central Ltd. 2011
Received: 29 June 2010
Accepted: 18 April 2011
Published: 18 April 2011
Geminiviruses encode few viral proteins. Most of the geminiviral proteins are multifunctional and influence various host cellular processes for the successful viral infection. Though few viral proteins like AC1 and AC2 are well characterized for their multiple functions, role of AC3 in the successful viral infection has not been investigated in detail.
We performed phage display analysis with the purified recombinant AC3 protein with Maltose Binding Protein as fusion tag (MBP-AC3). Putative AC3 interacting peptides identified through phage display were observed to be homologous to peptides of proteins from various metabolisms. We grouped these putative AC3 interacting peptides according to the known metabolic function of the homologous peptide containing proteins. In order to check if AC3 influences any of these particular metabolic pathways, we designed vectors for assaying DNA replication and virus induced gene-silencing of host gene PCNA. Investigation with these vectors indicated that AC3 enhances viral replication in the host plant tomato. In the PCNA gene-silencing experiment, we observed that the presence of functional AC3 ORF strongly manifested the stunted phenotype associated with the virus induced gene-silencing of PCNA in tomato plants.
Through the phage display analysis proteins from various metabolic pathways were identified as putative AC3 interacting proteins. By utilizing the vectors developed, we could analyze the role of AC3 in viral DNA replication and host gene-silencing. Our studies indicate that AC3 is also a multifunctional protein.
Geminiviruses are circular ssDNA containing plant viruses with a genome size of ~ 2.7 kb . Geminiviruses have an atypical genomic content. They are either monopartite with a single genomic component , monopartite with a satellite DNA that is around half the size of the genome  or bipartite with two genomic components of ~2.7 kb encoding different genes on both components . Monopartite viruses encode all the genes required for successful infection, replication and movement on the single genome. In case of monopartite viruses with satellite DNA and bipartite viruses, the DNA A contains the genes necessary for replication while the cognate genome component encodes genes for infectivity and movement within the plants [3, 5].
Whiteflies and leaf-hoppers are the vectors that transmit geminiviruses from one plant to other. These viruses replicate their DNA via rolling circle replication mechanism by utilizing the host plant cellular machinery [5–7]. Geminiviral proteins expressed after a successful viral infection in a plant cell induce the expression of host cell replication machinery from the differentiated plant cells [8–11]. The induced replication machinery is then diverted on to the viral DNA through the protein-protein interactions by the viral proteins for the productive replication [12–17].
Geminiviral proteins are often multi-functional in nature. Complementary strand of the geminiviruses encode four ORFs, viz., AC1, AC2, AC3 and AC4. Replication initiator protein (Rep/AC1/C1) is an essential viral protein for replication . It binds the viral DNA in a sequence specific manner by recognizing the iterons at the origin of replication on the viral DNA [19–22]. Rep functions as a site-specific endonuclease by recognizing the hairpin loop structure and sequence at the viral origin of replication to initiate the viral replication. It also functions as a ligase to terminate the replication of viral DNA [23–27]. Rep has the unique ability to act as a repressor of its own transcription [28, 29] and thereby regulates the expression of down-stream AC2 and AC3 genes . Rep is also an ATPase [26, 31, 32] and a helicase [32, 33]. In addition, it interacts with various host proteins [9, 13–16, 34, 35] and viral proteins [36, 37]. Similarly, the C2/AC2 protein of geminiviruses can bind to the DNA  and control the coat protein gene expression [39, 40] either by activation or derepression . AC2 is also known for its ability to suppress post-transcriptional gene-silencing mechanism [42–44] inside the host plant by inhibiting adenosine kinase [45, 46] or by reducing genome wide cytosine methylation . AC2 also inhibits SNF1 kinase to reduce the basal defense . Likewise, AC4/C4 protein from geminiviruses was also shown to have multiple functions with roles in post-transcriptional gene-silencing [49, 50], movement of virus inside the host cells [51, 52], cell division , transcription  and interacts with host protein AtSKeta - a protein from brassinosteroid signaling pathway .
Such a battery of multiple functions in viral proteins is most of the time brought out by their ability to form hetero-oligomer or homo-oligomer. In case of the geminiviral proteins, Rep/AC1 is able to bind, nick and ligate DNA as a monomer. However, its helicase activity is strictly dependent on its ability to form a higher order homo-oligomer [32, 33]. One possible way by which Rep is able to induce the replication machinery is through formation of a hetero-oligomer by interacting with retinoblastoma protein [9, 56]. Similarly, AC2 protein is capable of interacting with ADK and suppresses local gene-silencing as a monomer whereas it can transactivate the virion sense strand genes as an oligomer only . These observations indicate that the ability to form oligomers and to interact with other host proteins confers unique properties to the viral proteins which they cannot perform as monomers.
AC3 protein was shown to interact with viral protein AC1 [36, 58]. It was also shown to interact with host proteins like pRBR , PCNA  and SlNAC1 . AC3 was shown to enhance viral DNA replication by an unknown mechanism [60–65]. Preliminary studies on AC3 oligomerization suggested that AC3 also forms a higher order oligomer like AC1 [58, 66]. Together, these hetero and homo-oligomerization studies observed in case of AC3 suggest that it might also have multiple functions in addition to its role in replication which is unexplored as yet. In this study we tried to address the roles of Tomato leaf curl Kerala virus-[India:Kerala II:2005] (DQ85263) AC3 protein in the viral life cycle. We have performed an exhaustive phage display analysis to find out the interacting peptides of AC3 protein. These interacting peptides were observed to be homologous to proteins from various metabolisms indicating the likely role of AC3 in these cellular pathways. Since replication of viral DNA and gene-silencing are the two important phenomena that determine the progress of viral infection, we have chosen to investigate the role of AC3 in these biological processes. We have designed vectors to analyze the role of AC3 in replication and virus induced gene-silencing in both yeast and plants.
Results and Discussion
Phage display analysis for AC3 interacting peptides
AC3 protein of geminiviruses is a highly hydrophobic protein containing around 62% aromatic amino acids . This property poses difficulty in isolating the AC3 protein (with small tags or without tag) in the soluble fraction in sufficient quantities from bacterial cells . Although it is possible to express the TGMV-AC3 protein in soluble fraction in insect cell lines but purification in high quantities becomes uneconomical . Bioinformatic analysis indicated that AC3 proteins lack similarity to any known enzymatic motifs [58, 68]. All these factors hindered the exploration of the mechanistic role of AC3 on enhancing viral DNA replication and the existence of any other role in viral infection. In order to find the AC3 interacting peptides which could indirectly point towards the likely role of AC3 in other cellular processes, we have employed phage display analysis.
Putative interacting proteins of ToLCKeV AC3 from RNAi pathway
Interacting Sequence in Peptide(s)
Repressor of Silencing 1 (ROS1)
Suppressor of gene silencing 3 (SGS3)
Hua Enhancer 1 (HEN1)
Dicer-like 1 (DCL1)
Dicer-like 2 (DCL2)
Argonaute1 (AGO1), AGO6
AGO7, Pinhead like protein, zippy
NP_177103.1 AAG60096.1 AC073178_7
List of putative ToLCKeV AC3 interacting DNA and histone modifying enzymes
Interacting Sequence in Peptide(s)
Histone acetyl transferase
Variant in methylation 2 (VIM2), VIM3, VIM4, VIM5
NP_176091.2 NP_176779.2 NP_176778.1
Decreased methylation to DNA (MET1)
List of putative ToLCKeV AC3 interacting proteins from DNA recombination and cell cycle pathway
Interacting Sequence in Peptide(s)
TLTWHTKTPVRP HFKHQHSYARPP AYSPISTVTQPY SHWWARVPFYPP
Replication protein A1(RPA1)
Geminivirus Rep interacting kinase 1 (GRIK1), GRIK2
Retinoblastoma like protein (pRBR)
Anti silencing function 1b (ASF 1b), ASF1a
FHKHSPRSPIFI YALKHLPESTIP LLHAPYDHSVSP
RecQ Helicase, RecQ sim, RecQ4A
Werner Helicase - interacting protein
LITNNPGRLPPQ SHEIYVGSDGFR FHKEWRTHFQQR
List of putative ToLCKeV AC3 interacting DNA and RNA polymerases
Interacting Sequence in Peptide(s)
RNA dependant RNA polymerase (RDR1), RDR2
DNA pol γ2
DNA pol ε subunit
DNA pol α subunit
DNA pol ζ catalytic subunit
DNA pol l
DNA pol δ small subunit
The proteins with at least two unique hits from different peptides and each with a minimum identity/similarity of five amino acids continuously or with one mismatch or gap were considered as putative interacting proteins. These interacting proteins were observed to belong to various metabolic and cellular processes, viz., transcription activation, cell cycle, kinases, replication, RNAi, histone and DNA modification (Tables 1,2,3, & 4 and Additional file 1b). Identification of proteins from various cellular processes suggests that AC3 is likely to play role in these cellular processes. Since these putative interactions are only indicative, assays to investigate the impact of AC3 in these cellular processes is necessary for confirmation of its role.
Construction of yeast vectors for analyzing the viral DNA replication
Construction of plant vectors for analyzing the viral DNA replication
ToLCKeV AC3 enhances viral replication in young tomato plants
To exclude the possibility of permissiveness of viral replication in tobacco, we performed an agroinoculation experiment with pCK2 and pCK2M21 (with additional mutations in AC3 ORF) in the natural host tomato. Additional mutations in AC3 ORF corresponds to the amino acid positions 20 and 21 which are mutated to consecutive termination codons (Figure 2c). Since AC2 and AC3 ORFs overlap each other, we checked if these mutations have any effect on the AC2 protein sequence. While the mutation corresponding the 20th amino acid in AC3 ORF is a silent mutation in AC2 ORF, the mutation in the 21st amino acid of AC3 confers a change in the overlapping AC2 (G70V) ORF. Since 70th amino acid of AC2 does not lie in any of the known functional domains (C'-terminal nuclear localisation signal, Zn finger motif and N'-terminus acidic transcription activation domain) required for silencing activity or transcriptional activation activity, we argued that such a mutation would not affect the functions of AC2.
Our observation suggested that AC3 enhances replication but is not essential for replication. This is in line with earlier observation . Role of AC3 was evident at 10-15 dpi. However, our results differed from published reports on the level of AC3 influence on viral replication. This might be due to the differences in the experimental design or the assay system. Earlier reports on AC3's role in replication were based on the analysis by mutating AC3 after the AC2 stop codon. This resulted in truncated AC3 with 80 amino acids in case of TGMV AC3 and more than 100 amino acids in other viruses [60, 61, 63–65, 74]. In these studies it is possible that the truncation in the AC3 protein rendered it non-functional. It is also likely that the truncated AC3 interfered with the cellular pathways involved in replication. With its N'-terminus and middle region being intact, AC3 could titrate various proteins that interact with AC1 (like PCNA, pRBR, etc.). In such a case, the signal perceived by the N'-terminus of AC3 gets abruptly terminated being unable to relay the signal through a functional C'-terminus, thereby affecting replication. Our mutation strategy assured that AC3 is not expressed since we had mutated the start codon and included two stop codons at 20th and 21st amino acid positions. It is possible that in complete absence of AC3, another protein or an alternate pathway might rescue the viral replication . This hypothesis gets considerable support from an experiment performed with transgenic plants. In their work Hayes et al.  raised various transgenic plants expressing DNA A ORFs and tandem repeats of DNA B genome. Plants expressing DNA A ORFs were crossed with transgenic plants containing DNA B as tandem repeats (2DNA B). When DNA from two such plants: AC1 × 2DNA B and AC1AC3 × 2DNA B were analyzed, the difference in the replication of DNA B in the presence and absence of AC3 was observed to be less than 1.5 fold indicating that the replication in planta was sustainable without AC3. Delay and amelioration of symptoms and reduced systemic movement of the virus in case of AC3 mutations observed in planta by agroinoculation experiments [18, 60, 63–65, 74] suggest that AC3 has a more important role in systemic spread. Thus, the observed reduction in DNA levels at systemic locations is an indirect effect rather than its direct involvement in replication. Having a multitude of interacting partners that were discovered [12, 14, 59, 76] and are being discovered, large multimer forming ability  that enables interaction with multiple partners indicate that AC3 is an important protein with multi-functional capability. Thus, further examination of its involvement in various cellular processes is needed.
The phage display data indicated that various ToLCKeV AC3 interacting peptides are homologous to the proteins of RNAi pathway. Interestingly, we found that few of these proteins (MOM1, MET1, DCL1, DCL2, AGO1, AGO2, AGO7, and HEN4) have multiple hits from different peptide sequences identified from phage display (Tables 1 and 2). We believed these proteins to be likely interacting partners of ToLCKeV-AC3. Hence, we investigated if AC3 could influence the RNAi pathway(s). One way to examine the role of AC3 in RNAi pathway is to study the silencing of an endogenous gene through the virus induced gene-silencing mechanism (VIGS) in the presence and absence of functional AC3 ORF.
AC3 strongly manifests the phenotype associated with PCNA gene-silencing
Plant height and inter-nodal distance of the tomato plants agroinfiltrated with VIGS vectors at 45 dpi
Number of Plants
Average Height (in cm)
Number of Internodes
Average Internodal Distance (in cm)
PCNA gene is required for the replication of DNA. It is expressed in meristematic tissues that rapidly divide and grow. PCNA is absent in the mature leaves . So, silencing of endogenous PCNA would hamper the DNA replication in the rapidly growing tissues resulting in stunted growth - an easily recognisable phenotype [78, 79]. In our case plant growth was severely retarded which was evident from the reduced plant height, flowering and absence of fruits. Another advantage of our VIGS construct is the absence of virion sense strand ORFs AV1 and AV2. Absence of these proteins prevents virion packaging and movement of virion particle. So, by design, our VIGS vector is movement defective and cannot produce disease symptoms [73, 80]. Thus, the observed deformities in the plant growth are due to the silencing of PCNA.
Growth retardation observed in our experiments in the presence of AC3 indicates that AC3 could have strong influence on virus induced gene-silencing of endogenous gene PCNA in this experiment. However, it is difficult to ascertain the exact role of AC3 in RNAi and with which proteins it actually interacts from our experiment in isolation.
In this study we have identified various ToLCKeV AC3 interacting peptides through phage display analysis. Few of these interacting peptides were found to be homologous to proteins from replication process, RNAi pathway, histone and DNA modifying enzymes indicating the role of AC3 in these pathways. In order to verify if ToLCKeV AC3 has any role in any of these metabolisms, we have developed vectors to investigate its role in replication and gene-silencing. We observed that ToLCKeV AC3 effectively functions in the viral replication at an intermediate stage and enhances replication in host plant tomato. In the gene-silencing mechanism, the phenotype associated with the host gene PCNA silencing was strongly manifested in the presence of functional AC3 ORF. These observations indicate that the role of AC3 extends to RNAi pathway in addition to its role in DNA replication.
Phage Display analysis
We have used the 'Ph.D-12 phage display library' kit (New England Biolabs) for analyzing the various peptides that interact with AC3 protein. The protocol was followed as per the technical bulletin of the kit. In brief, the panning was carried out by incubating a library of phage-displayed peptides with a plate coated with the purified MBP-AC3 or MBP  in the TBST (100 mM Tris-HCl, 150 mM NaCl, pH 7.5, 0.1% Tween20) binding buffer (1.5 × 1011 phage diluted in 1ml buffer). Unbound phages were removed by washing with TBST. Bound phages were eluted with elution buffer (0.2 M Glycine-HCl, pH 2.2; 1 mg/ml BSA) and neutralized with 1 M Tris-HCl (pH 9.1). The eluted phages were then amplified with E. coli ER2738 bacterial strain. Amplified phages were then subjected to two more rounds of panning and taken through additional binding/amplification cycles to enrich the pool in favor of binding sequences. After three rounds, individual clones were characterized by DNA sequencing. Exclusive phage sequences were obtained after removing the M13 phage sequences. These DNA sequences were translated as per the reduced genetic code for M13 phage in E. coli ER2738. The sequence of the peptides was analyzed by 'BioEdit' software and the peptides common in MBP-AC3 and MBP interacting peptides were removed. Each peptide sequence thus obtained was then searched for homologous peptide sequences in proteins against the Arabidopsis non-redundant protein database at NCBI through 'blastp' programme adjusted for small sequence analysis. Initially we have searched for the known AC3 interacting proteins in the blast hits and have taken the E-value of pRBR (blast hit observed for the peptide sequence "FPKAFHHHKIYK" as the threshold for filtering the blast results. Further, we have shortlisted only those proteins with at least two hits from the same or different peptides or those with E-value less than the blast hit of GRIK1, another protein known to interact with Rep.
Site directed mutagenesis
AC3 ORF was mutated at three sites - one at base position 2 and others at bases 62 and 64 of AC3 ORF in two steps. Initially, the first mutation was carried out at the second base of AC3 ORF with overlapping oligos for both strands (ACM Fwd and ACM Rev). These oligos were used to amplify the whole pGEMT-Easy vector containing the wild type CR-AC3 region of the virus. The resulting amplified vector was incubated with T4 polynucleotide kinase (MBI Fermentas) along with T4- DNA ligase (MBI Fermentas) in the ligation reaction mix. The ligated products were transformed into E. coli DH5α. Plasmids were isolated from each bacterial colony and sequenced to confirm the site-directed mutation. This plasmid containing mutated AC3 ORF at start codon (CR-AC3M) was utilized to generate two more site-directed mutations at bases 62 and 64 with the oligos AC3M21 Fwd, AC3M21 Rev. The resulting construct was named CR-AC3M21. Sequence of the oligos used was:
AC3M Fwd: 5'- GTTCTGCAACGTGCACGGATTCACGCACAGG-3'
AC3M Rev: 5'- CCTGTGCGTGAATCCGTGCACGTTGCAGAAC-3'
AC3M21 Fwd: 5'- GGCGTGTTTATCTAGTAAATTCAAAATCCC-3'
AC3M21 Rev: 5'- GGGATTTTGAATTTACTAGATAAACACGCC-3'
Construction of yeast replicons
ARS containing yeast plasmid YCp50 was subjected to restriction digestion with Xho I and Bgl II to delete part of the ARS sequence rendering it replication deficient. The resulting plasmid is ligated by end filling and is called YCpO-. pGEMT-Easy clones containing CR-AC3 or CR-AC3M region were digested with Hind III restriction enzyme. The resulting CR-AC3 and CR-AC3M were cloned into Hind III site of YCpO- to generate YCp-CRAC3 or YCp-CRAC3M.
Construction of plant replicons and VIGS vectors
Hind III and EcoR I digested CaMV 35S cassette from pBI121 plasmid was cloned into Hind III and EcoR I digested plant binary vector pCAMBIA1391Z. EcoR I digested CR region of the ToLCKeV genome was cloned adjacent to the 35S cassette to generate pC. CR-AC3 or CR-AC3M21 was cloned into the Hind III site of the pC vector to generate pCK2 or pCK2M21 respectively. These plasmids were transformed into Agrobacterium tumefaciens (LBA4404). Cultures from single colonies of agrobacterium were grown and used for agroinfiltration studies. VIGS vectors were constructed by cloning 300 bp tomato PCNA into the BamH I site of the pCK2 or pCK2M21. Oligos used to amplify the PCNA fragment were:
PCNA Fwd: 5'- ACGGATCCGTTCTAGAATCGATTAAGGATCTGG- 3'
PCNA Rev: 5'- GGGGATCCCCATTAGCTTCATCTCAAAATCAG- 3'
126.96.36.199 Transient replication assay in plant leaves
The binary plasmid containing pCK2 replicon or pCK2M21 replicon containing agrobacterium was grown in YEM at 30°C till OD600 ≈ 1.0-2.0. Cells were harvested and washed with sterile YEM to remove antibiotic. Agrobacterium cells were resuspended in YEM to an OD600 ≈ 1.0-2.0 and then agroinfiltrated by injecting into tobacco or tomato leaves. Infiltrated leaves were collected at various intervals (5, 10, 15 days post inoculation) and genomic DNA was extracted. This genomic DNA was subjected to Dpn I treatment to remove the episomal DNA originated from agrobacterium. To quantitate the episomal DNA, PCR was done with following divergent primers (ACM Fwd, AC1 Rev119) and the amplification was visualized through agarose gel electrophoresis. Actin amplification (using Actin Fwd, Actin Rev oligos) was used as control.
AC3M Fwd: 5'- GTTCTGCAACGTGCACGGATTCACGCACAGG-3'
AC1 Rev119: 5'- AGCTCGAGCTAATCGACTTGGAAAAC-3'
Actin Fwd: 5'- ATGCCATTCTCCGTCTTGACTTG-3'
Actin Rev: 5'- GAGTTGTATGTAGTCTCGTGGATT-3'
Financial assistance from CSIR to KKP is highly acknowledged. Part of the research was supported from the DBT grant to SKM. We thank Dr. Vikash Kumar and Dr. Kosalai Kaliappan, for their suggestions during the phage display experiments. We also thank Dr. Prerna Pandey for her guidance during the replication and gene-silencing experiments in planta.
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