1 Institute of Biotechnology, Zhejiang University, Hangzhou 310029, P.R. China
2 Yunnan Biotechnology Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650223, P.R. China
3 International Laboratory for Tropical Agricultural Biotechnology, Danforth Plant Science Center, St Louis, MO 63132, USA
Correspondence
Xueping Zhou
xzhou{at}mail.hz.zj.cn
Claude Fauquet
iltab{at}danforthcenter.org
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this paper are AJ42031315, AJ42148285, AJ42161923, AJ45781822 and AJ506791.
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Introduction |
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Full-length clones of monopartite begomoviruses, Ageratum yellow vein virus (AYVV) from Singapore and Cotton leaf curl Multan virus (CLCuMV) from Pakistan, although infectious, were unable to induce typical symptoms of yellow vein in Ageratum conyzoides and leaf curl in cotton, respectively, and novel molecules, named DNA, were shown to be associated with both viruses, and to be essential for induction of characteristic symptoms in Ageratum and cotton (Saunders et al., 2000
; Briddon et al., 2001
). Analysis of DNA
molecules revealed that they are approximately half the size of the genomic DNA-A and except for a conserved hairpin structure and a TAATATTAC loop sequence, have little sequence similarity to either DNA-A or DNA-B molecules of begomoviruses. DNA
requires begomovirus DNA-A for replication, encapsidation, insect transmission and movements in plants (Saunders et al., 2000
; Briddon et al., 2001
).
In China, several begomoviruses have been reported infecting squash, tobacco and tomato (Zhou et al., 2001a; Yin et al., 2001
; Xie et al., 2002
). DNA-A molecules of 15 begomovirus isolates from tobacco, tomato, squash and weed species have been sequenced in our laboratory (X. Zhou and others, unpublished results). However, attempts to find DNA-B components by PCR using DNA-B primers and Southern blotting have been unsuccessful in all samples tested. In order to determine if these begomoviruses were associated with DNA
-like molecules, DNA
-specific primers were designed and used for PCR amplification. As a result, DNA
molecules were found to be associated with many begomovirus isolates in China. In this paper, we describe the genomic structure and the molecular variation of 18 DNA
molecules, and provide evidence of co-evolution of these DNA
molecules with the DNA-A molecules of their helper viruses. In addition, we also demonstrate that the C1 ORF has evolved similarly as DNA
molecules. The role of the C1 ORF of DNA
in symptom induction is examined and the existence of species of DNA
molecules is proposed. Furthermore, recombination between DNA
molecules is documented.
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Methods |
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(a) From tobacco with stunting, leaf curl, vein swelling, vein darkening or curly shoot symptoms: Y2 Baoshan, 580 km west of Kunming (1999); Y3 Baoshan, 580 km west of Kunming (1999); Y35 Baoshan, 580 km west of Kunming (2001); Y92 Baoshan, 580 km west of Kunming (2002); Y98 Baoshan, 580 km west of Kunming (2002); Y115 Baoshan, 580 km west of Kunming (2002).
(b) From tobacco with leaf curl and enation symptoms: Y5 Dali, 400 km north-west of Kunming (1999); Y8 Honghe, 160 km south of Kunming (1999); Y10 Honghe, 160 km south of Kunming (1999); Y11 Baoshan, 580 km south of Kunming (2000); Y36 Honghe, 160 km south of Kunming (2001); Y38 Honghe, 160 km south of Kunming (2001); Y43 Dali, 400 km north-west of Kunming (2001); Y45 Honghe, 160 km south of Kunming (2001); Y87 Baoshan, 580 km west of Kunming (2002); Y88 Baoshan, 580 km west of Kunming (2002).
(c) From tomato with leaf curl and enation symptoms: Y25 Chuxiong, 180 km north of Kunming (2000).
(d) From squash with leaf curl symptom: Y23 Jinghong, 700 km south of Kunming (2000).
(e) From Malvastrum coromandelianum with yellow vein symptom: Y47 Honghe, 160 km south of Kunming (2001).
(f) From Siegesbeckia orientalis with leaf curl symptom: Y64 Honghe, 160 km south of Kunming (2001).
PCR and sequence determination.
Comparison of the reported DNA sequences of AYVV and CLCuMV was performed and a conserved region (nt 12481347 of AYVV DNA
) was found. Based on the conserved nucleotide sequences, abutting primers beta01 (5'-GGTACCACTACGCTACGCAGCAGCC-3') and beta02 (5'-GGTACCTACCCTCCCAGGGGTACAC-3') were designed and used for amplification of the possible full-length DNA
; a unique KpnI restriction endonuclease site (underlined) was introduced into these primers (Briddon et al., 2001
). Additional primers designed on the basis of the subsequently determined sequences were used to amplify fragments which cover the region of abutting primers beta01/beta02. PCR was carried out as described by Zhou et al. (1998)
. PCR products were recovered, purified and cloned into pGEM-T Easy vector (Promega) as described by Zhou et al. (1998)
. Sequences were determined using the automated model 377 DNA sequencing system (Perkin Elmer).
Clone construction and plant inoculation.
DNA from begomovirus isolate Y10 (Y10
) was used for construction of an infectious DNA
clone. A complete genome unit of Y10
was amplified using beta05 (5'-GAAACCACTACGCTACGCAGCAGCC-3')/beta02 and the fragment inserted into pGEM-T Easy vector to produce clone pGEM
. Subsequently, another full-length genome copy of Y10
from clone pGEM
with beta01 and beta 02, was digested with KpnI and inserted into the unique KpnI site of pGEM
to produce pGEM2
. Clone pGEM2
was digested with EcoRI and inserted into the binary vector pBinPLUS to produce pBinPLUS-2
, yielding a tandem repeat of Y10
.
Two independent PCRs were performed with primer pair beta05/C1R and beta02/C1F using pGEM as the template for construction of a C1 in-frame ATG mutation (nt 564566). The primer sequences covering and flanking the mutation sites (underlined) were 5'-AGTTCAGTTTATTTGTTGTGG-3' (C1F, sense) and 5'-CCACAACAAATAAACTGAACT-3' (C1R, complementary). PCR products were fused and amplified using an overlap extension-PCR as described by Tao et al. (2002), and the overlapping PCR product inserted into pGEM-T Easy vector to produce clone pGEMC1m
. The same strategy was then used for construction of a tandem repeat of the C1 in-frame ATG mutation of Y10
(pBinPLUS-C1m2
).
Agrobacterium tumefaciens strain EHA105 was transformed with pBinPLUS-2 or pBinPLUS-C1m2
by triparental mating. The infectious clone pBinPLUS-1.7A, containing partial repeats of Y10 DNA-A, was constructed previously (X. Zhou and others, unpublished). Agrobacterium tumefaciens cultures were grown at 28 °C for 48 h (OD550=1), after which a fine needle was used to inject 0·2 ml of culture into stems or petioles of plants at the six-leaf stage. Nicotiana benthamiana, N. glutinosa and Lycopersicon esculentum plants were agro-inoculated, either with pBinPLUS-1.7A or with pBinPLUS-2
and pBinPLUS-1.7A, while N. benthamiana plants were also agroinoculated with pBinPLUS-C1m2
and pBinPLUS-1.7A. Inoculated plants were grown in an insect-free cabinet at a constant temperature of 25 °C with supplementary lighting corresponding to a 16 h day length.
Sequence analysis.
Sequence data were assembled and analysed with the aid of DNAStar software. Other reported DNA molecules used for comparison were DNA
of AYVV (AYVV
, AJ252072), Bhendi yellow vein mosaic virus (BYVMV) from India (BYVMV
, AJ308425), CLCuMV from Pakistan (CLCuMV
-01, AJ292769; CLCuMV
-02, AJ298903) and Cotton leaf curl Rajasthan virus (CLCuRV) from India (CLCuRV
, AY083590). The database accession numbers of the begomovirus DNA-A sequences used for comparison are listed as follows: AYVV (X74516); BYVMV (AF241479); CLCuMV (AJ132430); CLCuRV (AF363011); Malvastrum coromandelianum begomovirus isolate Y47 (AJ457824); squash begomovirus isolate Y23 (AJ420319); Squash leaf curl China virus (SLCCNV, AB027465); Siegesbeckia orientalis begomovirus isolate Y64 (AJ457823); tobacco begomovirus isolates Y5 (AJ319674), Y8 (AJ319677), Y10 (AJ319675), Y11 (AJ319676), Y36 (AJ420316) and Y38 (AJ420317); Tobacco curly shoot virus (TbCSV) isolates Y1 (AF240675), Y2 (AF240676) and Y35 (AJ420318); Tobacco leaf curl Yunnan Virus (TbLCYNV) isolate Y3 (AF240674); tomato begomovirus isolate Y25 (AJ457985) and Tomato yellow leaf curl China virus (TYLCCNV, AF311734).
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Results |
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Sequence analysis of DNA
The complete nucleotide sequences of DNA molecules associated with 18 virus isolates were determined to be 1333 to 1355 nt in length. These sequences have been submitted to GenBank, and are available under accession nos AJ42031315, AJ42148285, AJ42161923, AJ45781822 and AJ506791. Sequences of the DNA
s obtained from the above samples are named corresponding to their associated begomovirus: thus Y2
refers to DNA
from isolate Y2, etc. Nucleotide numbering for DNA
, as for geminivirus genomic components, proceeds from the 3' A residue in the nonanucleotide sequence TAATATT/AC.
Comparison of these sequences shows that they are of three main types (Table 1). Type 1 includes 13 virus isolates from tobacco, tomato and Siegesbeckia orientalis. The sequences in this type show 7299 % overall nucleotide sequence identity, while the isolates Y8
, Y36
, Y38
, Y45
and Y64
show 9799 % sequence identity with each other. Type 2 includes four virus isolates from tobacco, for which the overall nucleotide sequence identity is 8398 %. Type 3 includes the virus isolated from Malvastrum coromandelianum. Only 5257 % overall nucleotide sequence identity was found between Type 1 and Type 2 DNA
molecules. Type 3 is relatively different from the other DNA
molecules with less than 42 % overall nucleotide sequence identity with DNA
molecules within Type 1 and Type 2. Comparison between Type 1, Type 2 and Type 3 DNA
and reported DNA
molecules (AYVV
, CLCuMV
, CLCuRV
and BYVMV
) shows low sequence identity (3543 %), except that Y47
has relatively high overall sequence identity (6267 %) with CLCuMV
and CLCuRV
(Table 1
).
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Infectivity and symptoms induced by DNA
To determine if the DNA molecules associated with begomovirus isolates in China are also involved in symptom induction, a tandem repeat of Y10
was inserted into the binary vector pBinPLUS, and an infectious clone, pBinPLUS-2
, obtained. This plasmid was shown to be infectious in N. benthamiana, N. glutinosa and L. esculentum when co-inoculated by agro-inoculation with the previously constructed infectious clone pBinPLUS-1.7A, containing partial repeats of Y10 DNA-A. Symptoms in these plants were compared with those induced by inoculation of pBinPLUS-1.7A alone. All plants co-inoculated with DNA-A and DNA
produced systemic symptoms of severe downward leaf curl, vein darkening, stunting and enations, which are identical to those observed in the field. No symptoms were obtained in N. glutinosa or L. esculentum while only very mild symptoms were observed in N. benthamiana when agro-inoculation took place with DNA-A alone (Fig. 2
A). These results clearly show that the leaf curl disease induced by isolate Y10 in tobacco is a result of co-infection by DNA-A and DNA
. It is possible that other DNA
molecules associated with Chinese begomovirus isolates may have the same function, but this remains to be proven.
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Co-evolution of DNA molecules with their helper virus genomes
Complete and partial DNA-A sequences of several Chinese begomovirus isolates are available. Comparison of these sequences shows that all the DNA-A sequences associated with Type 1 DNAs are related to the recently reported TYLCCNV, with 8895 % nucleotide sequence identity (Yin et al., 2001
). Consequently, these virus isolates are named as TYLCCNV isolates (X. Zhou and others, unpublished results). All Type 2 DNA
s are associated with TbCSV DNA-A molecules (Xie et al., 2002
), whereas Type 3 DNA
is associated with a new begomovirus species, for which the name Malvastrum yellow vein virus (MYVV) is proposed (X. Zhou and others, unpublished results).
A phylogenetic tree of DNA sequences associated with 18 Chinese isolates of begomoviruses AYVV, CLCuMV, CLCuRV and BYVMV was constructed and compared with that of the complete nucleotide sequences of their cognate DNA-A molecules (Fig. 3
A). From Fig. 3(A)
, we can recognize the three main DNA
Types associated with the three Chinese begomoviruses described above. Type 1 molecules can form a further four separate branches, one branch containing six isolates (Y8, Y10, Y36, Y38, Y45, Y64) originally from Honghe district, the second branch consisting of two isolates (Y5, Y43) from Dali district, the third branch containing one isolate (Y25) from Chuxiong district and the fourth branch containing four isolates (Y11, Y87, Y88, Y92) from Baoshan district. Type 2 molecules include four isolates from Baoshan district, while Type 3 contains one isolate from Honghe district (Y47), which also clusters with CLCuMV
and CLCuRV
. In addition, comparison shows that the variability of DNA
sequences is highly related to the variability of DNA-A sequences, and clustering of DNA-A sequences corresponds to the clustering of DNA
sequences (Fig. 3A
). The linear correlation (R2=0·8772) between pairwise nucleotide sequence identities of DNA-A and DNA
among these Chinese isolates clearly demonstrates that DNA
molecules have co-evolved with their cognate DNA-A molecules (Fig. 4
A).
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A phylogenetic tree based on alignment of C1-encoded 118 amino acid sequences was also constructed and compared with that of the sequences of their DNA molecules. Comparisons show that the variability of C1-encoded 118 amino acid sequences is extremely high related to the variability of DNA
sequences, and clustering of C1-encoded 118 amino acid sequences corresponds to the clustering of DNA
sequences (Fig. 3B
). The polynomial correlation (R2=0·9668) between pairwise amino acid identities of C1-encoded 118 amino acid and nucleotide sequence identities of DNA
among these Chinese isolates clearly demonstrates that the C1 ORF has evolved similarly as DNA
molecules (Fig. 4B
).
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Discussion |
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The complete nucleotide sequences of 18 DNA molecules were determined. Comparison showed that three Types of DNA
molecule were present among the isolates and that they are associated with the three begomovirus species TYLCCNV, TbCSV and MYVV. Within one Type, sequence variation was geographically related, and DNA
molecules associated with virus isolates from the same region are clustered closely, while virus isolates from different regions were more distantly related (Fig. 4A
). Geographically related antigenic and molecular variation in begomoviruses has been well-documented (Harrison & Robinson, 1999
; Padidam et al., 1995
), but this is the first such report for DNA
molecules. Nucleotide sequence variation among DNA-B components of begomoviruses is greater than that of their associated DNA-A components (Harrison & Robinson, 1999
). Sequence comparisons carried out in this study also show that sequence variation of DNA
is greater than that of DNA-A molecules (Table 1
). The data also indicated that the DNA
molecules reported here are unrelated to other known geminivirus or nanovirus components, and that their evolutionary origin remains to be determined. DNA
of Type 1 and Type 2 was found in different years and Type 1 molecules were found in different crops. We conclude, therefore, that several distinct forms of DNA
occur in Yunnan, China, and have reached some genetic stability. It will be interesting to see if TYLCCNV originating from Guangxi province (Yin et al., 2001
) also contains a DNA
.
All the DNA molecules isolated in this study were found to be approximately half the length of their associated DNA-A molecules. Comparison among these DNA
molecules, together with those reported and deposited in GenBank, allowed their structural features and their relationships to be explored in more depth. Overall nucleotide sequence of different kinds of DNA
molecules share less identity than their cognate DNA-A molecules. However, all the DNA
molecules possess a very conserved 115 nt region (Fig. 1
). This region should have important function for DNA
trans-replication and gene expression or for interaction with their cognate DNA-As. It is most important, therefore, to determine what functions this region may have, and how it interacts with virus- or host-encoded proteins. The two genomic molecules (DNA-A and DNA-B) of bipartite begomoviruses share a common region of approximately 200 nt including the nonanucleotide motif (TAATATT/AC), which contains cis-acting elements for replication and gene expression. Unlike DNA
, this region is particularly prone to variation among Begomovirus species, and no conserved region similar to that in DNA
is found among them. Different begomovirus DNA-A sequences possess different iteron sequences in their intergenic region, which facilitates sequence-specific Rep binding to initiate rolling circle replication (Fontes et al., 1994
; Chatterji et al., 1999
, 2000
). Available evidence indicates that DNA
s do not contain iteron sequences, and yet they still depend on DNA-A for their replication (Saunders et al., 2000
; Briddon et al., 2001
). The DNA-A components of the bipartite begomoviruses African cassava mosaic virus and Indian cassava mosaic virus are able to trans-replicate AYVV
in N. benthamiana, but were unable to functionally interact with AYVV
to produce a symptomatic systemic infection (Saunders et al., 2002
), indicating that there is less replication specificity for DNA-A-mediated replication and that many species of Begomovirus could potentially replicate many kinds of DNA
. It is important to know how DNA
s are recognized by the DNA-A-encoded Rep proteins and how they control DNA-A-mediated replication.
A-rich regions were found in all DNA molecules, but their positions and arrangements varied. It is proposed that such variation may have originated from sequence duplications in order to satisfy size requirements for encapsidation (Saunders et al., 2000
). The fact that DNA
and defective DNAs associated with geminiviruses have a size maintained at approximately half that of the genomic components suggests a stringent size selection for encapsidation, and that smaller DNA molecules have adapted their size to allow encapsidation within geminate particles (Zhou et al., 2001b
).
Like AYVV and CLCuMV
, Y10
has been shown to be indispensable for the induction of typical disease. It would be informative to determine if the three kinds of DNA
, corresponding to different Types of DNA
, could be trans-replicated by different species of DNA-A, and/or if their interaction could extend host range and/or change symptoms. It has already been demonstrated that the interaction between DNA-A of Sri Lankan cassava mosaic virus (SLCMV), a bipartite begomovirus, and AYVV
can induce severe stunting, leaf curl and chlorotic symptoms in N. glutinosa and extend the host range of SLCMV DNA-A to include A. conyzoides with the symptoms resembling those associated with AYVV (Saunders et al., 2002
).
The position and size of the C1 ORF are conserved in all 23 DNA molecules. C1 of CLCuMV
and AYVV
were predicted by TESTCODE to be functional for DNA
molecules (Briddon et al., 2001
). A natural recombinant associated with AYVV, encompassing nt 1977 of AYVV
and including C1 ORF, was sufficient to induce yellow vein symptoms in A. conyzoides (Saunders et al., 2001
). The correlation between pairwise amino acid identities of C1-encoded 118 amino acid sequences and nucleotide sequence identities of DNA
implies that C1 is important for the function of DNA
molecules. Our preliminary results show that mutation of the start codon of C1 of Y10
results in very mild symptoms on N. benthamiana. The very mild symptoms associated with this mutant could be attributed to leaky expression of the C1 gene translated from the upstream or/and downstream start codons. Further expression of C1 in tobacco plants and protoplasts is being carried out in order to elucidate the function of this ORF in pathogenicity.
We demonstrated here that there is a high correlation between the variability of the DNA molecules and their cognate DNA-A components. This has two implications. Firstly, there has been a co-evolution of these two types of molecule to become functionally dependent on each other in the same host, and secondly, we could apply to DNA
molecules the species concept that has been applied to their cognate geminiviruses. Comparison of DNA-A sequences shows that begomoviruses are grouped into three clusters, the stain/isolate cluster with 89100 % sequence identity, the species cluster with 6089 % sequence identity when viruses belong to the same geographical region and a species cluster with 4259 % sequence identity when viruses originate from different parts of the world (Fauquet, 2002
). Thirteen DNA
molecules are now known to be associated with TYLCCNV, four with TbCSV and two with CLCuMV, respectively. DNA
molecules of the same virus species share 7299 % sequence identity, while usually 3657 % sequence identity was found between DNA
molecules belonging to different virus species, with the exceptions of MYVV-Y47
(6267 %) and the recombinant CLCuRV
. We here propose that DNA
sequence identity could be used as a taxonomic criterion for species demarcation of DNA
molecules and that the nomenclature to be used will be the name of the virus species followed by the Greek letter
: for example, TYLCCNV
, TCSV
, MYVV
, etc. For different isolates of the same virus species, it will be the name of the DNA
species followed by a dash and the isolate name: for example, TbCSV
-Y2, TbCSV
-Y35, etc.
Begomoviruses are highly recombinogenic with interspecific recombination events and recombination within members of other genera and families having been reported (Briddon et al., 1996; Zhou et al., 1997
; Padidam et al., 1999
; Saunders & Stanley, 1999
). A natural recombination event between AYVV DNA-A and AYVV
has been reported to produce a viable DNA
recombinant (Saunders et al., 2001
). In addition, either CLCuRV
or CLCuMV
has apparently arisen by recombination between another unidentified DNA
molecule and CLCuMV
or CLCuRV
. This is the first example of such an event having occurred between DNA
molecules, indicating that recombination is a powerful factor in evolution of DNA
s.
DNA molecules depend on the helper geminivirus for replication and for transmission via trans-encapsidation in geminivirus particles. Begomoviruses are known to cause disease in plants either as a monopartite or a bipartite virus. However, there is also a new category of monopartite begomoviruses associated with DNA
. We hypothesize that those kinds of monopartite begomoviruses are capable of a low level of replication but do not induce symptoms unless a satellite DNA
molecule is also present. The DNA
permits the high levels of DNA-A accumulation required to induce disease and subsequently, whitefly transmission. The vectors then transmit both molecules maintaining a mechanism for co-evolution. The fact that the C1 ORF has the same degree of variability as the entire DNA
molecule and that DNA
molecules and DNA-A components are known to have a similar evolution implies that they were subjected over time to the same types of pressure, including mutation and recombination.
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ACKNOWLEDGEMENTS |
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Received 24 May 2002;
accepted 4 September 2002.