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Arabidopsis thaliana MCM2 plays role(s) in mungbean yellow mosaic India virus (MYMIV) DNA replication

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Abstract

Geminiviruses are plant pathogens with single-stranded (ss) DNA genomes of about 2.7 kb in size. They replicate primarily via rolling-circle replication (RCR) with the help of a few virally encoded factors and various host-cell machineries. The virally encoded replication initiator protein (Rep) is essential for geminivirus replication. In this study, by interaction screening of an Arabidopsis thaliana cDNA library, we have identified a host factor, MCM2, that interacts with the Rep protein of the geminivirus mungbean yellow mosaic India virus (MYMIV). Using yeast two-hybrid, β-galactosidase and co-immunoprecipitation assays, we demonstrated an interaction between MYMIV-Rep and the host factor AtMCM2. We investigated the possible role of AtMCM2 in geminiviral replication using a yeast-based geminivirus DNA replication restoration assay and observed that the AtMCM2 protein complemented the mcm2∆ mutation of S. cerevisiae. Our data suggest the involvement of AtMCM2 in the replication of MYMIV ex vivo. The role of MCM2 in replication was confirmed in planta by a transient replication assay in both wild-type and mutant Arabidopsis plants through agroinoculation. Our data provide evidence for the involvement of AtMCM2 in geminiviral DNA replication, presumably in conjunction with other host factors, and suggest its importance in MYMIV DNA replication.

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Abbreviations

At :

Arabidopsis thaliana

3-AT:

3-Amino-1,2,4-triazole

BSA:

Bovine serum albumin

CFU:

Colony-forming unit

CR:

Common region

CTAB:

Cetyltrimethylammonium bromide

DDK:

Dbf4-dependent kinase

dpi:

Days post-infiltration

ds:

Double-stranded

IPTG:

Isopropyl β-D-thio-galactopyranoside

LD-PCR:

Long-distance PCR

MCM2-7:

Minichromosome maintenance protein subunits 2-7

MYMIV:

Mungbean yellow mosaic India virus

PAGE:

Polyacrylamide gel electrophoresis

PBS:

Phosphate-buffered saline

PCNA:

Proliferating cell nuclear antigen

pre-RC:

Pre-replication complex

RBR1:

Retinoblastoma protein 1

RCR:

Rolling-circle replication

Rep:

Replication initiator protein

RF-C:

Replication factor C

RPA32:

Replication protein A 32

Sc :

Saccharomyces cerevisiae

SD:

Synthetic dextrose

SDS:

Sodium dodecyl sulfate

ss:

Single-stranded

ToLCV:

Tomato leaf curl virus

VA:

Viral amplicon

WT:

Wild type

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Acknowledgments

The financial support from the Department of Biotechnology (DBT), Government of India, is duly acknowledged. We thank Dr. Kosalai Kaliappan for her help in the experiments related to phage display.

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Authors and Affiliations

  1. Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110 067, India

    Geetika Suyal, Sunil K. Mukherjee & Nirupam R. Choudhury

  2. Department of Biotechnology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, 110 062, India

    Geetika Suyal & Prem S. Srivastava

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  1. Geetika Suyal
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  2. Sunil K. Mukherjee
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  3. Prem S. Srivastava
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  4. Nirupam R. Choudhury
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Correspondence to Nirupam R. Choudhury.

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Suyal, G., Mukherjee, S.K., Srivastava, P.S. et al. Arabidopsis thaliana MCM2 plays role(s) in mungbean yellow mosaic India virus (MYMIV) DNA replication. Arch Virol 158, 981–992 (2013). https://doi.org/10.1007/s00705-012-1563-9

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