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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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Mutations in ToLCKeV AC3. (a) Schematic diagram of the CR-AC3 region of the replicon constructs. The hyphenated arrows indicate the mutations in AC3 ORF. Numbers indicate the base positions with reference to the viral genome. (b) Sequence alignment of the AC3 mutated at start codon with the wild type AC3. The mutated base is shown against the white background at base number 2 in the AC3 ORF. Mutation was confirmed by sequencing. This mutant construct is denoted as CR-AC3M. (c) Termination mutations in AC3 were located at bases 62 and 64 of the AC3 ORF (denoted as CR-AC3M21). Details of these mutations have been explained in the text.
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Figure 2: Mutations in ToLCKeV AC3. (a) Schematic diagram of the CR-AC3 region of the replicon constructs. The hyphenated arrows indicate the mutations in AC3 ORF. Numbers indicate the base positions with reference to the viral genome. (b) Sequence alignment of the AC3 mutated at start codon with the wild type AC3. The mutated base is shown against the white background at base number 2 in the AC3 ORF. Mutation was confirmed by sequencing. This mutant construct is denoted as CR-AC3M. (c) Termination mutations in AC3 were located at bases 62 and 64 of the AC3 ORF (denoted as CR-AC3M21). Details of these mutations have been explained in the text.

Mentions: Budding yeast S. cerevisiae is known to support the replication of animal and plant RNA and DNA viruses including geminiviruses in the absence of complementing yeast autonomously replicating sequence (ARS) as an episome [70-72]. We have developed a vector system on the similar line of yeast vector developed for MYMIV [72]. The yeast vector YCp50 was modified to contain viral DNA spanning the entire viral origin of DNA replication (also called common region - CR or intergenic region - IR) region to AC3 (i.e., CR-AC3) replacing the ARS sequence (YCp-CRAC3) (Figure 1). This CR-AC3 region contains the complementary strand DNA with complete viral origin of DNA replication and viral ORFs AC1, AC2, AC3 and AC4. Another vector (YCp-CRAC3M) was constructed with a mutation (M1T) in the AC3 ORF that corresponds to the nucleotide change ATG to ACG (Figure 2). Such mutation would result only in a silent mutation in the overlapping AC2 ORF. We expected that this mutation would not produce any intact or N' terminal truncated AC3 protein since the second and only other methionine in AC3 protein is located at the C' terminus 133rd amino acid position. Both the vectors YCp-CRAC3, YCp-CRAC3M and the control YCp50 plasmids were transformed into yeast separately and the colony growth was monitored on selection medium (Ura-). Yeast transformed with YCp-CRAC3 and YCp-CRAC3M exhibited much delayed growth phenotype (0.25-0.5 mm sized colonies in 5 days) in comparison to wild type plasmid YCp50 (3-4 mm size, Additional file 2). This kind of slow growth continued even after 10 days of incubation at standard conditions. This contrasted with the observation in case of MYMIV where the yeast was growing normally [72]. In our case, the delayed growth may be due to the possible toxicity of the viral proteins expressing in yeast. With this view further analyses were done in planta.


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)

Mutations in ToLCKeV AC3. (a) Schematic diagram of the CR-AC3 region of the replicon constructs. The hyphenated arrows indicate the mutations in AC3 ORF. Numbers indicate the base positions with reference to the viral genome. (b) Sequence alignment of the AC3 mutated at start codon with the wild type AC3. The mutated base is shown against the white background at base number 2 in the AC3 ORF. Mutation was confirmed by sequencing. This mutant construct is denoted as CR-AC3M. (c) Termination mutations in AC3 were located at bases 62 and 64 of the AC3 ORF (denoted as CR-AC3M21). Details of these mutations have been explained in the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Mutations in ToLCKeV AC3. (a) Schematic diagram of the CR-AC3 region of the replicon constructs. The hyphenated arrows indicate the mutations in AC3 ORF. Numbers indicate the base positions with reference to the viral genome. (b) Sequence alignment of the AC3 mutated at start codon with the wild type AC3. The mutated base is shown against the white background at base number 2 in the AC3 ORF. Mutation was confirmed by sequencing. This mutant construct is denoted as CR-AC3M. (c) Termination mutations in AC3 were located at bases 62 and 64 of the AC3 ORF (denoted as CR-AC3M21). Details of these mutations have been explained in the text.
Mentions: Budding yeast S. cerevisiae is known to support the replication of animal and plant RNA and DNA viruses including geminiviruses in the absence of complementing yeast autonomously replicating sequence (ARS) as an episome [70-72]. We have developed a vector system on the similar line of yeast vector developed for MYMIV [72]. The yeast vector YCp50 was modified to contain viral DNA spanning the entire viral origin of DNA replication (also called common region - CR or intergenic region - IR) region to AC3 (i.e., CR-AC3) replacing the ARS sequence (YCp-CRAC3) (Figure 1). This CR-AC3 region contains the complementary strand DNA with complete viral origin of DNA replication and viral ORFs AC1, AC2, AC3 and AC4. Another vector (YCp-CRAC3M) was constructed with a mutation (M1T) in the AC3 ORF that corresponds to the nucleotide change ATG to ACG (Figure 2). Such mutation would result only in a silent mutation in the overlapping AC2 ORF. We expected that this mutation would not produce any intact or N' terminal truncated AC3 protein since the second and only other methionine in AC3 protein is located at the C' terminus 133rd amino acid position. Both the vectors YCp-CRAC3, YCp-CRAC3M and the control YCp50 plasmids were transformed into yeast separately and the colony growth was monitored on selection medium (Ura-). Yeast transformed with YCp-CRAC3 and YCp-CRAC3M exhibited much delayed growth phenotype (0.25-0.5 mm sized colonies in 5 days) in comparison to wild type plasmid YCp50 (3-4 mm size, Additional file 2). This kind of slow growth continued even after 10 days of incubation at standard conditions. This contrasted with the observation in case of MYMIV where the yeast was growing normally [72]. In our case, the delayed growth may be due to the possible toxicity of the viral proteins expressing in yeast. With this view further analyses were done in planta.

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