John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK1
Author for correspondence: John Stanley. Fax +44 1603 450045. e-mail john.stanley{at}bbsrc.ac.uk
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Abstract |
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Geminiviruses within the genus Begomovirus are transmitted by the whitefly Bemisia tabaci Genn. and have either one or two genomic components, depending on the species, each of which is approximately 27002800 nucleotides in length (reviewed by Hanley-Bowdoin et al., 1999 ). The majority of begomoviruses, typified by African cassava mosaic virus (ACMV) and Tomato golden mosaic virus (TGMV) as examples from the Old and New World, respectively, have two circular single-stranded DNA components. DNA A encodes the coat protein and replicates autonomously while DNA B is required for systemic infection and disease production. Some begomoviruses, for example Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl virus (ToLCV), lack a DNA B component yet produce a systemic symptomatic infection in plants, and the disease is transmissible between plants by the whitefly vector (Kheyr-Pour et al., 1991
; Navot et al., 1991
; Dry et al., 1993
). In contrast, the monopartite begomoviruses Ageratum yellow vein virus (AYVV; Fig. 1
) (Tan et al., 1995
) and Cotton leaf curl virus (CLCuV) (Briddon et al., 2000
) are able to systemically infect Ageratum conyzoides (ageratum) and cotton, respectively, although they are unable to induce symptoms typical of ageratum yellow vein disease (AYVD) and cotton leaf curl disease (CLCuD) in these hosts. Recently, a variety of small (
1300 nucleotide) recombinant DNAs (recDNAs) and satellite DNAs were isolated from ageratum exhibiting AYVD (Stanley et al., 1997
; Saunders & Stanley, 1999
; Saunders et al., 2001
). One such satellite, referred to as DNA
, is an essential component of the AYVD complex (Saunders et al., 2000
), and a similar satellite has since been isolated from CLCuD-affected cotton (Briddon et al., 2001
).
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In addition to a begomovirus and DNA , both AYVD and CLCuD are associated with autonomously replicating DNAs, referred to as DNA 1 (Fig. 1
) (Mansoor et al., 1999
; Saunders & Stanley, 1999
). These DNAs are related to nanovirus components that encode Rep proteins, and have probably evolved by adaptation to whitefly transmission during co-infection of plants with a begomovirus and a nanovirus. They play no direct role in the disease aetiology, but may help to modulate the accumulation of the essential begomovirus and DNA
components (Saunders et al., 2000
). It has been suggested that the single CFDV Rep-encoding component may simply be a nanovirus-like DNA that has become associated with an unidentified planthopper-transmitted virus (Mansoor et al., 1999
).
We have previously demonstrated that the systemic movement of DNA 1 in Nicotiana benthamiana can be mediated by the begomovirus ACMV (Saunders & Stanley, 1999 ). To extend this investigation, N. benthamiana plants were agroinoculated with DNA 1 and either the begomovirus TGMV, the mastrevirus BeYDV or the curtovirus BCTV, using partial or tandem repeats of these components in agrobacterium binary vectors (Stanley et al., 1986
; Briddon et al., 1989
; von Arnim & Stanley, 1992
; Tan et al., 1995
; Liu et al., 1997
; Saunders & Stanley, 1999
). Nucleic acids isolated from plants (Covey & Hull, 1981
) were analysed by blot hybridization as described by Saunders & Stanley (1999)
using oligolabelled probes (Feinberg & Vogelstein, 1983
) specific for each component. Of six plants infected with TGMV, one contained detectable levels of DNA 1 in the upper symptomatic leaves at 16 days post-infection (p.i.) (Fig. 2A
, lanes 16). In comparison, DNA 1 was found in systemically infected tissues in one out of three plants when agroinoculated with ACMV, and in two out of three plants following mechanical inoculation with this virus (Saunders & Stanley, 1999
). However, we were unable to detect DNA 1 in three samples taken from upper leaves of plants infected with BeYDV at 16 days p.i. (Fig. 2A
, lanes 911) and in two samples from plants showing severe symptoms at 35 days p.i. (Fig. 2A
, lanes 12 and 13). Dot-blot analysis (Maule et al., 1983
) of a further 15 symptomatic plants failed to detect DNA 1 in association with BeYDV (data not shown). In contrast, DNA 1 was readily detected in all three samples from plants infected with BCTV at both 16 and 35 days p.i. (Fig. 2A
, lanes 1621). Although plants containing DNA 1 accumulated less BCTV DNA (compare lanes 14 and 15 with lanes 1621), there were no obvious differences in the onset and severity of symptoms during the course of the experiment.
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The recombinant recDNA19 (previously called def19; Stanley et al., 1997 ) contains two fragments (A and B) from AYVV DNA A separated by two distinct regions (C and D) that do not originate from the begomovirus component (Fig. 1
). PCR-amplification using viral supercoiled DNA extracted from naturally infected ageratum plants and divergent primers based on the sequence of fragment C allowed the identification of DNA 1 (Saunders & Stanley, 1999
). Because the sequences of fragments C and D show little homology, we were interested in identifying the source of the DNA from which fragment D derives. Divergent primers based on the sequence of fragment D were used to PCR-amplify a product of approximately the same size as DNA 1, suggesting that the recombinant fragment also derives from a circular DNA component. Sequence analysis of the PCR product allowed the identification of a unique EcoRI site. Divergent primers overlapping this site and corresponding to nucleotides 342371 (primer V4547) and complementary sense nucleotides 317347 (primer V4548) were used to PCR-amplify a full-length copy of the component, which was subsequently cloned into pGEM-T Easy (Promega) and sequenced (clone pGEM-AYVV2; database accession no. AJ416153). Sequences were analysed using version 8 of the program library of the Genetics Computer Group (Devereux et al., 1984
). The sequence of recDNA19 fragment D is identical to the complementary strand of the component, referred to as DNA 2, between nucleotides 11761295 (Fig. 1
). DNA 2 is similar in length to DNA 1 (1360 and 1367 nucleotides, respectively), it has a typical stemloop sequence incorporating the nonanucleotide TAGTATTAC that is the hallmark of many nanovirus components, and encodes a Rep that facilitates its autonomous replication in an N. benthamiana leaf disk assay (data not shown). Like DNA 1, it also has an extensive A-rich region located immediately downstream of the Rep coding sequence. However, DNA 2 shows only 47% nucleotide sequence identity with AYVD DNA 1 and 49% with the CLCuD DNA 1 homologue (Mansoor et al., 1999
). In contrast, the two DNA 1 homologues are much more closely related (73% nucleotide identity; Saunders & Stanley, 1999
). This diversity is also reflected at the protein level (Fig. 3A
). The Reps encoded by the DNA 1 homologues are much more closely related to each other (87% identity, 91% similarity) than to DNA 2 (AYVD DNA 1: 40% identity, 61% similarity; CLCuD DNA 1: 43% identity, 51% similarity). Nonetheless, phylogenetic analysis shows that the Reps encoded by DNA 2 and both DNA 1 homologues are more closely related to each other than to their counterparts encoded by nanovirus components (Fig. 3B
). However, they show a closer relationship with nanovirus Reps that are responsible simply for the autonomous replication of their own component than with the master Reps (Timchenko et al., 1999
, 2000
; Horser et al., 2001
) that form a cluster in this analysis.
|
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Nanovirus-like components are associated with monopartite begomovirus infections in the field, and we have demonstrated that DNA 1 can accumulate in plants when co-inoculated with the Old and New World bipartite begomoviruses ACMV and TGMV, and with the curtovirus BCTV (Mansoor et al., 1999 ; Saunders & Stanley, 1999
; this paper). This raises the intriguing possibility that the association of nanovirus-like components with geminiviruses may be a widespread phenomenon in the field. Our demonstration of adaptation to a leafhopper vector may have significant implications for the dispersal, diversity and evolution of nanovirus components, particularly in view of their propensity to undergo recombination. Certainly, this observation supports the suggestion that the CFDV component is in fact a nanovirus-like component associated with a geminivirus that has become adapted to planthopper transmission by a size increase (Mansoor et al., 1999
). Reports of nanovirus-like components associated with geminivirus diseases are so far confined to the Old World. However, it is worth noting that BCTV and, more recently, the monopartite begomovirus TYLCV have been introduced into the New World from the Middle East (Briddon et al., 1998
; Polston et al., 1994
), where geminivirus and nanovirus diseases are prevalent and provide the opportunity for mixed infections and component adaptation. It remains to be seen if additional distinct nanovirus-like components are associated with AYVD and CLCuD, and detailed surveys are required to search for nanovirus-like components associated with other geminivirus diseases to determine the full extent of their dissemination.
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Acknowledgments |
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References |
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Received 22 October 2001;
accepted 10 December 2001.
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