Universal Primers for the PCR-Mediated Amplification of DNA ?? A Molecule Associated with Some Monopartite Begomoviruses

Abstract

DNA beta is an approx 1350 nucleotide, single-stranded DNA molecule which has been shown to be associated with some monopartite geminiviruses of the genus Begomovirus. This component requires the helper begomovirus for replication in the cells of host plants and for insect transmission, possibly by trans-encapsidation. Sequence comparisons of the two available DNA beta sequences has identified a highly conserved region upstream of a predicted hairpin structure. Abutting primers designed to this conserved region allows PCR-mediated amplification of the full-length DNA beta component from total nucleic acid extracts isolated from infected plants originating from a variety of geographically distinct sources and host plants.

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MOLECULAR BIOTECHNOLOGY
Volume 20, 2002
Universal Primers for DNA
β
315
315
Molecular Biotechnology 2002 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2002/20:3/315–318/$11.00
*Author to whom all correspondence and reprint requests should be addressed: Department of Disease and Stress Biology, John Innes Centre,
Colney Lane, Norwich, NR4 7UH, UK. E-mail: rob.briddon@bbsrc.ac.wk.
1
National Institute of Biotechnology and Genetic Engineering,
P.O. Box 577, Jhang Road, Faisalabad, Pakistan.
HINTS AND TIPS
Abstract
DNA β is an approx 1350 nucleotide, single-stranded DNA molecule which has been shown to be associ-
ated with some monopartite geminiviruses of the genus Begomovirus. This component requires the helper
begomovirus for replication in the cells of host plants and for insect transmission, possibly by trans-
encapsidation. Sequence comparisons of the two available DNA β sequences has identified a highly con-
served region upstream of a predicted hairpin structure. Abutting primers designed to this conserved region
allows PCR-mediated amplification of the full-length DNA β component from total nucleic acid extracts
isolated from infected plants originating from a variety of geographically distinct sources and host plants.
Index Entries: Geminivirus; satellite; whitefly; begomovirus; DNA β
1. Introduction
Geminiviruses are the causative agents of a
number of serious diseases of crop plants in tropi-
cal and subtropical regions of the world. The
majority of the economically destructive gemini-
viruses belong to the genus Begomovirus. These
viruses typically have bipartite genomes, are
transmitted exclusively by the whitefly Bemisia
tabaci and infect only dicotyledonous hosts (1). A
small number of monopartite begomoviruses have
been identified, all of which cause problems in
tomato crops, the most prominent of which is
Tomato yellow leaf curl virus (TYLCV; 2,3),
a
serious pathogen of tomato crops across the Middle
East and the Mediterranean which has now spread
into the Carribean and North America (4–6).
Clones have been obtained for several
begomoviruses which, when reintroduced to
plants, induce symptoms atypical of the disease;
including viruses associated with Ageratum yel-
low vein disease originating from Singapore (7)
and cotton leaf curl disease originating from Paki-
stan (8). For both these diseases, in addition to a
presumed monopartite begomovirus (Ageratum
yellow vein virus and Cotton leaf curl virus,
respectively), a molecule (DNA 1) encoding a
protein related to the replication associated pro-
tein (Rep) of nanoviruses was isolated (9,10).
DNA 1 molecules were shown to be capable of
self-replication in plant cells but to require the
“helper” begomovirus for encapsidation, insect
transmission and movement in infected plants.
However, the DNA 1 molecules were found to
play no part in symptom induction.
Recently a further single-stranded DNA mol-
ecule, termed DNA β, was shown to be associated
with both diseases and to be essential for induc-
tion of the characteristic symptoms in the hosts
from which they were isolated, Ageratum and cot-
ton respectively (11,12), but to require the bego-
Universal Primers for the PCR-Mediated Amplification of DNA β
A Molecule Associated with Some Monopartite Begomoviruses
R. W. Briddon,* S. E. Bull, S. Mansoor,1 I. Amin,1 and P. G. Markham

MOLECULAR BIOTECHNOLOGY
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316 Briddon et al.
movirus for replication, encapsidation, insect trans-
mission and movement in plants. Although the evo-
lutionary origin of DNA β remains uncertain,
sequence comparisons of the two DNA β molecules
available at this time show them to have only very
limited overall nucleotide sequence identity (53%).
However, just upstream of a predicted hairpin
structure (with the loop sequence TAATATTAC, a
motif that DNA β shares with geminiviruses and,
for geminiviruses, shown to be the origin of virion
strand DNA replication [13]) the DNA β molecules
have a conserved region of approx 80 nucleotides
(Fig. 1). This “conserved sequence” has been sug-
gested to be important in trans-replication of DNA
β by the begomovirus Rep, possibly containing
cryptic Rep binding sites (12).
A pair of abutting oligonucleotide primers
(Beta01/Beta02) were designed to the “conserved
sequence” of the DNA β molecules (Fig. 1). Prim-
ers Beta01 and Beta02 are 25 nucleotides in length
and nondegenerate, annealing to a region just up-
stream of the conserved hairpin loop structure.
This region is highly conserved between CLCuD
DNA β and AYVD DNA β(Fig. 1) and for all
DNA β molecules which have thus far been
cloned and sequenced (R.W. Briddon manuscript
in preparation). The primers introduce a KpnI
restriction endonuclease recognition site which
allows recovery of the complete amplified mol-
ecule, intact without additions, following cloning
into T-vectors. Thus far the introduced KpnI site
has been found to be unique for all DNA β mol-
ecules cloned.
Total nucleic acid extracts were produced by
standard, well established methods; typically the
CTAB method (14) for cotton and Ageratum and
the method of Covey and Hull (15) for tobacco
and other plant species. Amplification conditions
utilized were typically 35 cycles of melting at
94°C for 1 min, annealing at 50°C for 1 min and
extension for 1.5 min at 72°C. For some samples
a lower annealing temperature (45°C) proved
advantageous, although this usually lead to a dra-
matic increase in non-specific, background ampli-
fication yielding a smear on ethidium bromide
stained agarose gels.
The results of PCR amplifications with Beta01
and Beta02 from extracts produced from field
infected plants is shown in Fig. 2. PCR with these
primers typically produces a major band at 600–
700 bp and a minor band at approx 1350 bp.
Amplification with nucleic acid extracts produced
from healthy plants (lane 4) produce no products
although some nonspecific bands can be seen at
lower (below 50°C) annealing temperatures
(results not shown). When used with nucleic acid
templates extracted from plants experimentally
infected with clones of the helper geminivirus and
DNA β, only a single product is detected (lane 5).
The smaller products amplified from field col-
lected material are believed to emanate from
defective DNA β molecules (deletion mutants) and
molecules which result from recombination be-
tween DNA β, DNA 1 and the helper geminivirus.
A number of such molecules have been character-
ized for both AYVD and CLCuD (8,11, 12). These
less than full length molecules, although not de-
tected in young recently experimentally inoculated
plants, rapidly build up as the infection progresses
and the plant ages. For several diseases including
CLCuD and AYVD originating from Pakistan,
clones of the approx 1350 bp PCR product, pro-
duced with primers Beta01 and Beta 02, have
proven infectious to host plants yielding typical
disease symptoms when inoculated with clones of
the respective helper begomovirus (R.W. Briddon
manuscript in preparation).
Utilizing primers Beta01 and Beta02 we have
begun to analyze the diversity and geographic dis-
tribution of diseases associated with DNA β. Crop,
weed, and ornamental species including tomato,
tobacco, okra, Sida spp., hollyhock, hibiscus, and
honeysuckle from areas including Japan, Asia,
Africa, and Europe have thus far been shown to be
affected by DNA β associated disease complexes.
Acknowledgments
This work was funded by the Common Fund
for Commodities under contract number ICAC/07
and the Ministry of Agriculture Fisheries and
Food under project PH 01 47T. The authors also
gratefully acknowledge the support of the Biotech-

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Universal Primers for DNA
β
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Fig. 1. Sequences of primers Beta01 and Beta 02 shown in comparison to the sequence of CLCuD DNA β (clone pCLCβ02;12). Nucle-
otides which differ between CLCuD DNA β and AYVD DNA β are shown in lower case. The position of the presented sequence on the map
of CLCuD DNA β is shown. The orientation and position of predicted open reading frames are shown by black arrows within the circle. The
position of the predicted hairpin structure, with the loop sequence TAATATTAC, is shown by a small circle.

MOLECULAR BIOTECHNOLOGY
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318 Briddon et al.
nology and Biological Sciences Research Council.
Viruses were held and manipulated with the
authority of the Ministry of Agriculture, Fisheries
and Food under the Plant Health (Great Britain)
Order 1993 (SI no.1993/1320); license numbers
PHF 1419/819/113 and PHF 49/123(103).
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Fig. 2. Ethidium bromide stained agarose gel of
PCR products resulting from amplifications with prim-
ers Beta01 and Beta02. Samples were the results of
amplifications from nucleic acids extracted from cot-
ton (Gossypium hirsutum) field infected with CLCuD
originating from Pakistan (lane 1), hollyhock (Althea
rosea) field infected with hollyhock leaf crumple dis-
ease originating from Egypt (lane 2) and Ageratum
conyzoides field infected with Ageratum yellow vein
disease originating from Pakistan (lane 3). The nucleic
acid templates used in lanes 4 and 5 were extracted
from a healthy Nicotiana tabacum plant and an N.
tabacum plant experimentally infected with the cloned
components of CLCuD (CLCuV and CLCuD DNA β)
respectively. The approximate sizes (in base pairs) of
co-electrophoresed marker bands (left) and the
expected position of linear, full-length DNA β mol-
ecules (arrow) are shown.