| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
Institute of Biotechnology, Zhejiang University, Hangzhou 310029, PR China
Correspondence
Xueping Zhou
zzhou{at}zju.edu.cn
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
; Stanley et al., 2005). The majority of economically important geminiviruses belong to the genus Begomovirus, which are all transmitted by the whitefly Bemisia tabaci and infect only dicotyledonous plants (Harrison & Robinson, 1999). These viruses have genomes consisting of one or two similar-sized DNA components (Lazarowitz, 1992). Both components of the bipartite begomoviruses are required for systemic infection and symptom induction (Stanley, 1983). For some monopartite begomoviruses, such as tomato yellow leaf curl virus (Navot et al., 1991) and tomato leaf curl virus (Dry et al., 1993), the single DNA component is sufficient to produce disease symptoms. However, this is not the case for other monopartite begomoviruses such as Ageratum yellow vein virus (AYVV), Bhendi yellow vein mosaic virus, cotton leaf curl Multan virus, Eupatorium yellow vein virus and tomato yellow leaf curl China virus (TYLCCNV). A novel molecule, named DNA β, was shown to be essential for the induction of typical symptoms by these viruses (Briddon et al., 2001; Jose & Usha, 2003; Saunders et al., 2003, 2004; Cui et al., 2004; Briddon & Stanley, 2006; Mansoor et al., 2006).
In China, many begomoviruses isolated from tobacco, tomato, Ageratum conyzoides, Malvastrum coromandelianum and Siegesbeckia orientalis were found to be associated with DNA β molecules (Xie et al., 2003; Zhou et al., 2003a, b; Li et al., 2005; Wu & Zhou, 2007; Xiong et al., 2007). Phylogenetic analysis of DNA β molecules and their cognate begomoviruses suggests that DNA β molecules have co-evolved with their helper viruses (Zhou et al., 2003b). We have demonstrated previously that DNA A of TYLCCNV alone could infect Nicotiana benthamiana, Nicotiana glutinosa, Nicotiana tabacum and tomato plants systemically but produced no symptoms, and that TYLCCNV DNA β was required, in addition, for induction of leaf curl disease in these hosts (Cui et al., 2004). Here, we report that recombinant DNA molecules between DNA A and DNA β (RecDNA-Aβ) were associated with some natural TYLCCNV isolates and that such recombinants also have the ability to induce typical symptoms in tobacco, tomato and Petunia hybrida when co-inoculated with TYLCCNV DNA A.
Primers Y10 Rec/F (5'-TCTCAAACTTGGCTATGCAATCGG-3'), corresponding to part of the intergenic region of DNA A, and Y10 Beta-B/R (5'-ATGTATCATCCACAACAAATAAACATGAC-3'), complementary to part of the βC1 ORF of DNA β were designed to detect the possible recombinant. An approximately 0.85 kb fragment was obtained by PCR amplification from the TYLCCNV isolate Y10. The nucleotide sequence determined from the PCR product confirmed that a recombinant between DNA A and DNA β indeed exists in the TYLCCNV isolate Y10. Next, primers Y10 Rec/F and Y10 Rec/R (5'-GAGAGACACCAATTGACCAGTCAAT-3') were designed to amplify full-length RecDNA-Aβ. A 1.3 kb PCR product was amplified and inserted into the pGEM T-Easy vector (Promega). The RecDNA-Aβ clones were identified by using primers Y10 Rec/F and Y10 Beta-B/R, and four clones designated RecDNA-Aβ-4, RecDNA-Aβ-6, RecDNA-Aβ-9 and RecDNA-Aβ-10 were selected. These clones were sequenced using an automated DNA sequencing system (model 377; Perkin Elmer). Another two clones, designated def-1 and def-2, which contained 1.3 kb inserts but from which no band could be amplified by using Y10 Rec/F and Y10 Beta-B/R, were also sequenced. The sequences of the cloned molecules were analysed for recombination between DNA A and DNA β molecules by a BLAST search (http://www.ncbi.nlm.nih.gov/blast). This analysis revealed that clones RecDNA-Aβ-4, RecDNA-Aβ-6, RecDNA-Aβ-9 and RecDNA-Aβ-10 were indeed recombinants between TYLCCNV DNA A and DNA β and comprised 1335, 1287, 1312 and 1367 nt, respectively (GenBank accession nos AJ781297
[GenBank]
–AJ781300
[GenBank]
), whereas clones def-1 and def-2 contained only sequences from DNA A of TYLCCNV (GenBank accession nos AJ784151
[GenBank]
–AJ784152
[GenBank]
). The derivation of the sequences that make up each of the RecDNA-Aβ molecules is shown in Table 1. Inspection of the recombinant sequences showed that all the molecules contained the βC1 gene and the A-rich region from TYLCCNV DNA β, and that the remaining sequences corresponded to the intergenic region of TYLCCNV DNA A including a few 5'-terminal nucleotides of the coding region of the Rep gene. The βC1 gene of DNA β was essential for the symptom induction of typical disease, and the A-rich region was thought to satisfy the size requirements for encapsidation or virus movement (Cui et al., 2004; Saunders et al., 2000). At the DNA A/DNA β junction site, both progenitor sequences as well as the four RecDNA-Aβ molecules have 4 nt (TGGG) in common, suggesting that sequence homology is an important factor for recombination in geminiviruses (Fig. 1). The sequence TGGG is also found at the recombination site in two RecDNA-Aβ clones (pAYVdef27 and pAYVdef41) obtained from AYVV-infected ageratum (Stanley et al., 1997), indicating that this sequence might be a hot spot for recombination. Previous studies have shown that such small sequence recurrences may determine the deletion end points in the defective DNAs of African cassava mosaic virus, tomato golden mosaic virus and beet curly top virus (Stanley & Townsend, 1985; MacDowell et al., 1986; Frischmuth et al., 1991). At the other junction site, although no similar sequence identity was found, all the RecDNA-Aβ molecules have the sequence GGT at the junction between DNA A and DNA β sequences (Fig. 1), indicating that GGT might also represent a hot spot for recombination.
|
|
). By using primers Y10 Rec/F and Y10 Beta-B/R, recombinant RecDNA-Aβ was detected in five of these TYLCCNV isolates: isolates Y5, Y6, Y40, Y43 and Y88. The partial sequences of the PCR products were determined and sequence analysis confirmed that these PCR products were recombinants between TYLCCNV DNA A and DNA β (data not shown). Sequence analysis of three RecDNA-Aβ clones from isolate Y43 revealed that they were identical in sequence and had a similar structure to those found in the Y10 isolate: the intergenic region of DNA A, the βC1 gene and the A-rich region of DNA β (RecDNA-Aβ-8; GenBank accession no. AJ781301
[GenBank]
). They also had possible hot spots for recombination: TGGG on one side and GGT on the other. The detailed information for RecDNA-Aβ-8 is shown in Table 1
.
To determine the possible biological role of RecDNA-Aβ, infectious clones of RecDNA-Aβ-9 and RecDNA-Aβ-10 were constructed. The specific primers Y10 Rec Kpn/F (5'-GGTACCTCTCAAACTTGGCTATGCAATCGG-3') and Y10 Rec/R were designed to amplify one copy of the recombinant RecDNA-Aβ molecule using clones RecDNA-Aβ-9 and RecDNA-Aβ-10 as template. The PCR products were inserted into pGEM T-Easy vector to obtain pGEM-RecDNA-Aβ-9 and pGEM-RecDNA-Aβ-10, respectively. Another copy of each recombinant RecDNA-Aβ molecule was amplified by using Y10 Rec Kpn/F and Y10 Rec Kpn/R (5'-GGTACCGAGAGACACCAATTGACCAGTCA-3'), digested with KpnI and inserted into pGEM-RecDNA-Aβ-9 and pGEM-RecDNA-Aβ-10, as appropriate. Recombinant plasmids were digested with EcoRI and inserted into the binary vector pBinPLUS, to produce plasmids pBinPLUS-RecDNA-Aβ-9 and pBinPLUS-RecDNA-Aβ-10, containing dimeric RecDNA-Aβ molecules. Plasmids pBinPLUS-RecDNA-Aβ-9 and pBinPLUS-RecDNA-Aβ-10 were mobilized into Agrobacterium tumefaciens strain EHA105 by triparental mating as described previously (Zhou et al. 2003b). Then the two clones were tested for infectivity in N. benthamiana by co-agroinoculation with the infectious clone of TYLCCNV DNA A, pBinPLUS-1.7A (Zhou et al., 2003b). Both pBinPLUS-RecDNA-Aβ-9 and pBinPLUS-RecDNA-Aβ-10 were found to be infectious in N. benthamiana and produced downward leaf curling, stunting, vein darkening, vein yellowing, curly shoots and enation symptoms following co-inoculation with TYLCCNV DNA A. Subsequently all inoculations were conducted using pBinPLUS-RecDNA-Aβ-10. N. benthamiana plants, co-inoculated with TYLCCNV DNA A and DNA β or RecDNA-Aβ, or both DNA β and RecDNA-Aβ, developed typical symptoms, including downward leaf curling, stunting, leaf crumpling, vein darkening, vein yellowing, curly shoots and enations (Fig. 2a). Downward leaf curling and leaf chlorosis symptoms were found in Petunia hybrida by DNA A and RecDNA-Aβ (Fig. 2b), while severe downward leaf curling, stunting and enation symptoms were found in N. glutinosa and Solanum lycopersicum (Fig. 2c). These symptoms were indistinguishable from those induced by DNA A and DNA β. PCR with primers Y10 Rec/F and Y10 Beta-B/R which amplified a specific band from these symptomatic plants, and the nucleotide sequence of the PCR products confirmed that the symptoms were caused by RecDNA-Aβ.
|
). A similar recombinant DNA β was also found to be associated with cotton leaf curl disease; however, it was not shown if this recombinant has a biological function (Briddon et al., 2001). The recombinant RecDNA-Aβ in TYLCCNV and recombinant DNA β in AYVV all contain the A-rich region and βC1 gene of DNA β. Evidence has shown that an ATG in-frame mutation in βC1 abrogates the induction of typical symptoms by the βC1 gene (Zhou et al., 2003b; Cui et al., 2004; Saunders et al., 2004). Our further study has demonstrated that the βC1 gene alone in transgenic tobacco plants is sufficient to induce the leaf curl symptoms (Cui et al., 2004). These results indicate that the typical symptoms induced by recombinant RecDNA-Aβ are attributable to the intact βC1 gene in the RecDNA-Aβ molecule. Why the TYLCCNV RecDNA-Aβ induced severe symptoms or why the AYVV recombinant DNA β induced milder symptoms remains unknown.
To investigate the relative amounts of DNA A, DNA β and RecDNA-Aβ in infected plants, nucleic acids were extracted from individual symptomatic N. benthamiana plants 30 days after inoculation and analysed by Southern blot hybridization using the TYLCCNV coat protein (CP) gene and the full-length clone of DNA β as probes (Cui et al., 2004). The results show that the RecDNA-Aβ replicated stably in N. benthamiana plants. The intergenic region of DNA A in the RecDNA-Aβ, which contains the iterons involved in Rep binding during the initiation of replication, may be responsible for the trans-replication of the recombinant RecDNA-Aβ molecule. Relatively lower levels of accumulation of RecDNA-Aβ were observed in plants co-inoculated with DNA A and RecDNA-Aβ as compared with those co-inoculated with DNA A and DNA β. For DNA A, relatively high levels of accumulation were found in plants co-inoculated with DNA A and RecDNA-Aβ. In plants co-inoculated with all three components, DNA A accumulated to a relatively lower level (Fig. 2d). The viral DNA accumulation levels in N. benthamiana plants could not be directly correlated with the severity of symptoms.
To examine if RecDNA-Aβ is transmissible by whitefly, N. glutinosa plants, which had been agroinoculated with TYLCCNV DNA A and RecDNA-Aβ and showing clear symptoms of the virus infection, were used as the virus source for whitefly transmission as described previously (Jiu et al., 2006). Three of seven N. glutinosa plants began to show downward leaf curling symptoms 12 days after inoculation by the viruliferous adults, and the presence of RecDNA-Aβ in the symptomatic plants was verified by PCR with primers Y10 Rec/F and Y10 Beta-B/R and sequencing of the PCR products (data not shown). The result implies that RecDNA-Aβ is encapsidated in TYLCCNV virions and has the potential to be maintained in the field.
DNA B and DNA A of bipartite begomoviruses share a common region of approximately 200 nt. This region contains cis-acting elements for replication and gene expression, including the nonanucleotide motif (TAATATTAC), which contains the nick site for initiation of rolling circle replication (Laufs et al., 1995) and the Rep-binding site (Fontes et al., 1994; Arguello-Astorga & Ruiz-Medrano, 2001). We have demonstrated that the RecDNA-Aβ recombinants essentially contain the DNA A intergenic region and the 5' terminus of the Rep ORF. DNA β satellites and begomovirus DNA B have some common properties. They are both trans-replicated and encapsidated by begomovirus-encoded proteins, and an important symptom determinant is located in the complementary-sense strand of both molecules (Noueiry et al., 1994; Duan et al., 1997; Briddon et al., 2001; Saunders et al., 2000, 2004; Cui et al., 2004; Briddon & Stanley, 2006). The BV1 gene of DNA B has similar sequence identity with the CP gene of DNA A, indicating that the BV1 gene probably resulted from gene duplication of CP and subsequent divergence (Kikuno et al., 1984). It is possible that DNA A may have donated its intergenic region to DNA β which subsequently acquired a component for nuclear trafficking, resulting in the common region, a nuclear trafficking gene and a symptom induction gene in the DNA B component.
In conclusion, although RecDNA-Aβ is clearly a derivative of DNA β, it can induce the disease phenotype and it has the potential to exist in nature in the absence of DNA β. Our findings indicate that recombinants between the begomovirus and DNA β occur frequently in the field.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Briddon, R. W. & Stanley, J. (2006). Subviral agents associated with plant single-stranded DNA viruses. Virology 344, 198–210.[CrossRef][Medline]
Briddon, R. W., Mansoor, S., Bedford, I. D., Pinner, M. S., Saunders, K., Stanley, J., Zafar, Y., Malik, K. A. & Markham, P. G. (2001). Identification of DNA components required for induction of cotton leaf curl disease. Virology 285, 234–243.[CrossRef][Medline]
Cui, X., Tao, X., Xie, Y., Fauquet, C. M. & Zhou, X. (2004). A DNA β associated with tomato yellow leaf curl China virus is required for symptom induction. J Virol 78, 13966–13974.
Dry, I. B., Rigden, J. E., Krake, L. R., Mullineaux, P. M. & Rezaian, M. A. (1993). Nucleotide sequence and genome organization of tomato leaf curl geminivirus. J Gen Virol 74, 147–151.
Duan, Y. P., Powell, C. A., Purcifull, D. E., Broglio, P. & Hiebert, E. (1997). Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins. Mol Plant Microbe Interact 10, 1065–1074.[Medline]
Fontes, E. P. B., Gladfelter, H. J., Schaffer, R. L., Petty, I. T. D. & Hanley-Bowdoin, L. (1994). Geminivirus replication origins have a modular organization. Plant Cell 6, 405–416.[Abstract]
Frischmuth, S., Frischmuth, T. & Jeske, H. (1991). Transcript mapping of abutilon mosaic virus, a geminivirus. Virology 185, 596–604.[CrossRef][Medline]
Harrison, B. D. & Robinson, D. J. (1999). Natural genomic and antigenic variation in whitefly-transmitted geminiviruses (Begomoviruses). Annu Rev Phytopathol 37, 369–398.[CrossRef][Medline]
Jiu, M., Zhou, X. P. & Liu, S. S. (2006). Acquisition and transmission of two begomoviruses by the B and a non-B biotype of Bemisia tabaci from Zhejiang, China. J Phytopathol 154, 587–591.[CrossRef]
Jose, J. & Usha, R. (2003). Bhendi yellow vein mosaic disease in India is caused by association of a DNA β satellite with a begomovirus. Virology 305, 310–317.[CrossRef][Medline]
Kikuno, R., Toh, H., Hayashida, H. & Miyata, T. (1984). Sequence similarity between putative gene products of geminiviral DNAs. Nature 308, 562[CrossRef][Medline]
Laufs, J., Traut, W., Heyraud, F., Matzeit, V., Rogers, S. G., Schell, J. & Gronenborn, B. (1995). In-vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus. Proc Natl Acad Sci U S A 92, 3879–3883.
Lazarowitz, S. G. (1992). Geminiviruses: genome structure and gene function. Crit Rev Plant Sci 11, 327–349.[CrossRef]
Li, Z., Xie, Y. & Zhou, X. (2005). Tobacco curly shoot virus DNAβ is not necessary for infection but intensifies symptoms in a host-dependent manner. Phytopathology 95, 902–908.[Medline]
MacDowell, S. W., Coutts, R. H. & Buck, K. W. (1986). Molecular characterization of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus. Nucleic Acids Res 14, 7967–7984.
Mansoor, S., Zafar, Y. & Briddon, R. W. (2006). Geminivirus disease complexes: the threat is spreading. Trends Plant Sci 11, 209–212.[CrossRef][Medline]
Navot, N., Pichersky, E., Zeidan, M., Zamir, D. & Czosnek, H. (1991). Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. Virology 185, 151–161.[CrossRef][Medline]
Noueiry, A. O., Lucas, W. J. & Gilbertson, R. L. (1994). Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport. Cell 76, 925–932.[CrossRef][Medline]
Padidam, M., Beachy, R. N. & Fauquet, C. M. (1995). Classification and identification of geminiviruses using sequence comparisons. J Gen Virol 76, 249–263.
Saunders, K., Bedford, I. D., Briddon, R. W., Markham, P. G., Wong, S. M. & Stanley, J. (2000). A unique virus complex causes ageratum yellow vein disease. Proc Natl Acad Sci U S A 97, 6890–6895.
Saunders, K., Bedford, I. D. & Stanley, J. (2001). Pathogenicity of a natural recombinant associated with ageratum yellow vein disease: implications for geminivirus evolution and disease aetiology. Virology 282, 38–47.[CrossRef][Medline]
Saunders, K., Bedford, I. D., Yahara, T. & Stanley, J. (2003). The earliest recorded plant virus disease. Nature 422, 831[CrossRef][Medline]
Saunders, K., Norman, A., Gucciardo, S. & Stanley, J. (2004). The DNA β satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (βC1). Virology 324, 37–47.[CrossRef][Medline]
Stanley, J. (1983). Infectivity of the cloned geminivirus genome requires sequences from both DNAs. Nature 305, 643–645.[CrossRef]
Stanley, J. & Townsend, R. (1985). Characterization of DNA forms associated with cassava latent virus infection. Nucleic Acids Res 13, 2189–2206.
Stanley, J., Saunders, K., Pinner, M. S. & Wong, S. M. (1997). Novel defective interfering DNAs associated with ageratum yellow vein geminivirus infection of Ageratum conyzoides. Virology 239, 87–96.[CrossRef][Medline]
Stanley, J., Bisaro, D. M., Briddon, R. W., Brown, J. K., Fauquet, C. M., Harrison, B. D., Rybicki, E. P. & Stenger, D. C. (2005). Geminiviridae. In Virus Taxonomy: Eighth Report of the ICTV. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/Academic Press.
Wu, J. B. & Zhou, X. P. (2007). Siegesbeckia yellow vein virus is a distinct begomovirus associated with a satellite DNA molecule. Arch Virol 152, 791–796.[CrossRef][Medline]
Xie, Y., Zhou, X. & Li, G. (2003). Molecular characterization of tomato yellow leaf curl China virus and its satellite DNA isolated from tobacco. Chin Sci Bull 48, 766–770.
Xiong, Q., Fan, S., Wu, J. & Zhou, X. (2007). Ageratum yellow vein China virus is a distinct begomovirus species associated with a DNAβ molecule. Phytopathology 97, 405–411.[Medline]
Zhou, X., Xie, Y., Peng, Y. & Zhang, Z. (2003a). Malvastrum yellow vein virus, a new begomovirus species associated with satellite DNA molecule. Chin Sci Bull 48, 2205–2209.[CrossRef]
Zhou, X., Xie, Y., Tao, X., Zhang, Z., Li, Z. & Fauquet, C. M. (2003b). Characterization of DNAβ associated with begomoviruses in China and evidence for co-evolution with their cognate viral DNA-A. J Gen Virol 84, 237–247.
Received 16 August 2007;
accepted 11 September 2007.
This article has been cited by other articles:
|
|
 |
|
  M. S. Nawaz-ul-Rehman, S. Mansoor, R. W. Briddon, and C. M. Fauquet Maintenance of an Old World Betasatellite by a New World Helper Begomovirus and Possible Rapid Adaptation of the Betasatellite J. Virol., September 15, 2009; 83(18): 9347 - 9355. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Ding, L. Qing, Z. Li, Y. Liu, Y. Qian, and X. Zhou Genetic Determinants of Symptoms on Viral DNA Satellites Appl. Envir. Microbiol., August 15, 2009; 75(16): 5380 - 5389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Saunders, R. W. Briddon, and J. Stanley Replication promiscuity of DNA-{beta} satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-{beta} satellite localizes sequences involved in replication J. Gen. Virol., December 1, 2008; 89(12): 3165 - 3172. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
