Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses

https://doi.org/10.1016/j.jviromet.2006.01.017Get rights and content

Abstract

Better, easier and cheaper than polymerase chain reaction (PCR) or antibody detection, rolling circle amplification (RCA) using the bacteriophage ϕ 29 DNA polymerase allows for a reliable diagnosis of geminiviruses and presumably all viruses with small single-stranded circular DNA genomes. The results show the efficiency of this technique in characterizing viral DNA components of several geminiviruses from experimental and natural host plant sources. The advantages are: (a) that no expensive devices are necessary, (b) simple handling, (c) detection of all infecting circular DNA components without any knowledge of sequence information in a single step, and (d) low costs per reaction. In addition, RCA-amplified viral DNA can be characterized by restriction fragment length polymorphism analysis and directly sequenced up to 900 bases in a single run circumventing cloning and plasmid purification. This shortcut will considerably accelerate genomics of at least gemini-, nano- and circoviruses in the future.

Introduction

Geminiviruses have devastated large areas of crop plants on several continents mainly in tropical and subtropical countries but also in the USA and around the Mediterranean. The constant spread of those epidemics (Moffat, 1999) demands efficient diagnostic tools for combat by means of quarantine measures. Specific antibodies (Harrison et al., 2002) and polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) (Briddon and Markham, 1995) have been proven to be highly suitable for this purpose. However, since mainly developing countries have been attacked by geminiviruses, low-priced procedures feasible for less well-equipped laboratories would widely improve diagnosis and thereby help to implement quarantine measures.

A possible solution for the affected regions is now provided by the use of the DNA polymerase of the Bacillus subtilis bacteriophage ϕ 29 (Blanco et al., 1989, Dean et al., 2001). The enzyme possesses both, polymerase and strand displacement-activity, thus allowing circular DNA to be replicated to nearly unlimited extent using a rolling circle amplification (RCA) mechanism. Actively replicating geminiviruses are ideal substrates for this enzyme, because they produce various DNA intermediates during complementary strand synthesis (CSR) of their circular single-stranded (ss) DNA, as well during rolling circle replication (RCR), and recombination-dependent replication (RDR) (Alberter et al., 2005, Jeske et al., 2001, Preiss and Jeske, 2003). Moreover, costly sets of primer pairs amplifying only subsets of geminiviral genomic or satellite-like molecules are dispensable since cheap random hexamer primers can be applied directly with the viral ssDNA. Here we report on the usefulness of this approach to detect and characterize geminiviruses and related subgenomic components with easily usable and with recalcitrant host plants. The technique may be used similarly for other plant viruses, for instance for members of the Nanoviridae family (Gronenborn, 2004, Stanley, 2004), and for animal viruses from the family Circoviridae (Mankertz et al., 2004), especially the widespread human annelloviruses (Hino, 2002).

Section snippets

Virus source and maintenance

Agroinfectious clones of Abutilon mosaic virus (AbMV; NC_001928, NC_001929) (Frischmuth et al., 1990), African cassava mosaic virus (ACMV; NC_001467, NC_001468), Beet curly top virus (BCTV; NC_001412), Tomato golden mosaic virus (TGMV; NC_001507, NC_001508, M73794) common strain and yellow vein strain (all originally kindly provided by Dr. John Stanley, Norwich, and used as described before (Preiss and Jeske, 2003), and Tomato yellow leaf curl Sardinia virus (TYLCSV; L27708) (Morilla et al.,

Results

To evaluate the diagnostic potential of RCA, several geminiviruses from distant countries and belonging to monopartite or bipartite species were compared in easy-to-handle laboratory host plants (N. benthamiana) as well as recalcitrant field-collected cassava plants (M. esculenta). The influence of different purification procedures on the method's reliability was assessed by comparing the respective results to those obtained with standard total nucleic acid preparation using phenol. Fig. 1

Discussion

RCA techniques have been used to clone geminiviral DNAs (Inoue-Nagata et al., 2004) and commercial kits employing ϕ 29 polymerase are now available. Here we have shown that RCA in combination with RFLP is a highly reproducible tool for geminivirus diagnosis, largely independent of viral genome organization, source plant type and origin, and sample preparation. The combination of RCA/RFLP is vastly superior to PCR-based procedures. Neither specific primers are needed to start the reaction, nor

Acknowledgments

This work was supported by Vater-und-Sohn-Eiselen-Stiftung, Ulm. The authors wish to thank Dr. Christina Wege and Prof. Dr. Robin Ghosh for critical reading the manuscript and helpful discussions, and Mr. D. Gotthardt for taking care of the plants.

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