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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 KK, Mukherjee SK, Choudhury NR - Virol. J. (2011)

Bottom Line: 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.Our studies indicate that AC3 is also a multifunctional protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Viral replicon used for in planta studies. Viral replicon was constructed with the pCAMBIA1391Z binary vector. Complete replicon contains the region spanning from CR to AC3 (pCK2 replicon) and CR region of ToLCKeV. Presence of CR on either end in the same orientation enables the completion of rolling circle replication. Rolling circle replication releases the episome that contains only one complete CR and region spanning from AC1 to AC3. Red arrows indicate the nicking site of Rep protein in hairpin loop in either CRs and the black line represents the region of the vector that forms episome. Formation of an episome can be checked by PCR amplification with the oligonucleotides indicated by blue arrows. Internal primers were designed to amplify the DNA only from the episome under standardized PCR conditions. CR-AC3 is replaced by CR-AC3M or CR-AC3M21 for generating pCK2M replicon and pCK2M21 replicons. A 300 bp PCNA fragment was cloned into the MCS region to generate pCK2M-PCNA and pCK2M21-PCNA.
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Figure 3: Viral replicon used for in planta studies. Viral replicon was constructed with the pCAMBIA1391Z binary vector. Complete replicon contains the region spanning from CR to AC3 (pCK2 replicon) and CR region of ToLCKeV. Presence of CR on either end in the same orientation enables the completion of rolling circle replication. Rolling circle replication releases the episome that contains only one complete CR and region spanning from AC1 to AC3. Red arrows indicate the nicking site of Rep protein in hairpin loop in either CRs and the black line represents the region of the vector that forms episome. Formation of an episome can be checked by PCR amplification with the oligonucleotides indicated by blue arrows. Internal primers were designed to amplify the DNA only from the episome under standardized PCR conditions. CR-AC3 is replaced by CR-AC3M or CR-AC3M21 for generating pCK2M replicon and pCK2M21 replicons. A 300 bp PCNA fragment was cloned into the MCS region to generate pCK2M-PCNA and pCK2M21-PCNA.

Mentions: CR-AC3 region is reported to be sufficient to support viral replication in plants [17,73]. Since geminiviruses replicate by rolling circle replication by nicking and religating at the viral origin of DNA replication, we constructed vectors with viral origin of replication (CR) in the vector pCAMBIA1391Z. This vector was then modified to contain CR-AC3 or CR-AC3M (AC3 mutated at start codon, Figure 2) in the same orientation as CR to generate pCK2 (Figure 3) and pCK2M plasmids respectively. These vectors were used to agroinfiltrate in the tobacco leaves and the replication was observed at 4 dpi and 10 dpi. Time course analysis of the pCK2 and pCK2M episome formation in tobacco plant leaves did not show any significant down-regulation in replication upon AC3 mutation (Data not shown). To rule out the reversion of the mutation in the start codon, we carried out sequencing of the episome and found that the mutation was preserved. Thus, the non-significant alteration in the replication efficiency might be due to various reasons: one being the minimal role of ToLCKeV AC3 in viral replication in planta unlike in protoplasts and leaf discs. It is also possible that the role of AC3 in viral replication occurs at a later stage requiring analysis of samples beyond 10 dpi. The other reason might be the permissiveness of the tobacco plant for the viral replication that masked the role of AC3. Such a conjecture gets support from an observation made in case of BCTV (California strain). When BCTV C3 was mutated, BCTV genome replicated to almost wild-type levels in tobacco plant whereas the replication was reduced in natural host plant sugar beet [74].


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 KK, Mukherjee SK, Choudhury NR - Virol. J. (2011)

Viral replicon used for in planta studies. Viral replicon was constructed with the pCAMBIA1391Z binary vector. Complete replicon contains the region spanning from CR to AC3 (pCK2 replicon) and CR region of ToLCKeV. Presence of CR on either end in the same orientation enables the completion of rolling circle replication. Rolling circle replication releases the episome that contains only one complete CR and region spanning from AC1 to AC3. Red arrows indicate the nicking site of Rep protein in hairpin loop in either CRs and the black line represents the region of the vector that forms episome. Formation of an episome can be checked by PCR amplification with the oligonucleotides indicated by blue arrows. Internal primers were designed to amplify the DNA only from the episome under standardized PCR conditions. CR-AC3 is replaced by CR-AC3M or CR-AC3M21 for generating pCK2M replicon and pCK2M21 replicons. A 300 bp PCNA fragment was cloned into the MCS region to generate pCK2M-PCNA and pCK2M21-PCNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102638&req=5

Figure 3: Viral replicon used for in planta studies. Viral replicon was constructed with the pCAMBIA1391Z binary vector. Complete replicon contains the region spanning from CR to AC3 (pCK2 replicon) and CR region of ToLCKeV. Presence of CR on either end in the same orientation enables the completion of rolling circle replication. Rolling circle replication releases the episome that contains only one complete CR and region spanning from AC1 to AC3. Red arrows indicate the nicking site of Rep protein in hairpin loop in either CRs and the black line represents the region of the vector that forms episome. Formation of an episome can be checked by PCR amplification with the oligonucleotides indicated by blue arrows. Internal primers were designed to amplify the DNA only from the episome under standardized PCR conditions. CR-AC3 is replaced by CR-AC3M or CR-AC3M21 for generating pCK2M replicon and pCK2M21 replicons. A 300 bp PCNA fragment was cloned into the MCS region to generate pCK2M-PCNA and pCK2M21-PCNA.
Mentions: CR-AC3 region is reported to be sufficient to support viral replication in plants [17,73]. Since geminiviruses replicate by rolling circle replication by nicking and religating at the viral origin of DNA replication, we constructed vectors with viral origin of replication (CR) in the vector pCAMBIA1391Z. This vector was then modified to contain CR-AC3 or CR-AC3M (AC3 mutated at start codon, Figure 2) in the same orientation as CR to generate pCK2 (Figure 3) and pCK2M plasmids respectively. These vectors were used to agroinfiltrate in the tobacco leaves and the replication was observed at 4 dpi and 10 dpi. Time course analysis of the pCK2 and pCK2M episome formation in tobacco plant leaves did not show any significant down-regulation in replication upon AC3 mutation (Data not shown). To rule out the reversion of the mutation in the start codon, we carried out sequencing of the episome and found that the mutation was preserved. Thus, the non-significant alteration in the replication efficiency might be due to various reasons: one being the minimal role of ToLCKeV AC3 in viral replication in planta unlike in protoplasts and leaf discs. It is also possible that the role of AC3 in viral replication occurs at a later stage requiring analysis of samples beyond 10 dpi. The other reason might be the permissiveness of the tobacco plant for the viral replication that masked the role of AC3. Such a conjecture gets support from an observation made in case of BCTV (California strain). When BCTV C3 was mutated, BCTV genome replicated to almost wild-type levels in tobacco plant whereas the replication was reduced in natural host plant sugar beet [74].

Bottom Line: 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.Our studies indicate that AC3 is also a multifunctional protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus