Abstract
A geminivirus was first reported to be the causative agent of tobacco leaf curl disease in Japan in 1981 (Osaki & Inouye, 1981 ). In Southern Africa, at least three symptom phenotypes in tobacco have been identified that could be classified as tobacco leaf curl (Paximadis & Rey, 1997 ). One of these symptom phenotypes has been attributed to a phytoreovirus (Paximadis et al., 1997 ; Rey et al., 1999 ) and the other to a begomovirus, Tobacco leaf curl Zimbabwe virus (TbLCZWV) (Paximadis & Rey, 1997 ; Paximadis et al., 1999 ). Four plants, showing leaf curl symptom severity variation, associated with begomovirus infections in Zimbabwe (Paximadis & Rey, 1997 ), were phylogenetically compared using coat protein gene (CP) and the common region (CR) nucleotide sequences. Isolates formed a distinct cluster and were 98% similar in their CP and CR sequences (Paximadis et al., 1999 ). The present research characterizes TbLCZWV at a molecular level and reports, for the first time, defective DNA molecules associated with TbLCZWV.
Three pairs of overlapping degenerate primers [AV494 and AC1048 (Wyatt & Brown, 1996 ); PAL1c1960 and PAR1v722 (Paximadis & Rey, 1997 ); prAV1134 (Idris & Brown, 1988) and PAR1c715 (5' GAT TTC TGC AGT TDA TRT TYT CRT CCA TCC A 3'); D. P. Maxwell, Univ. of Wisconsin, Madison, USA], designed to amplify regions of the DNA A components of most begomoviruses, were employed in PCR to amplify the DNA A from total DNA extracted (Doyle & Doyle, 1987 ) from TbLCZWV-infected tobacco. PCR fragments were blunt-end cloned into the pBluescript (KS) vector (Stratagene) and three clones of each were sequenced automatically (ABI Prism 310) to obtain the complete TbLCZWV DNA A. TbLCZWV DNA A was phylogenetically compared to the DNA A sequences of other geminiviruses (GenBank) using the DNAMAN version 4.0 (Lynnon Biosoft, Quebec, Canada) full optimal alignment and neighbour-joining method options with 1000 bootstrap replications. Open reading frames (ORFs) of TbLCZWV DNA A were identified using DNAMAN, and percentage DNA A nucleotide and ORF amino acid sequence identities between virus isolates were calculated using the distances between all pairs of sequences in the multiple alignments.
Sequencing of cloned PCR fragments spanning the complete TbLCZWV DNA A revealed it to be 2767 nucleotides in size, exhibiting the typical genome organization of begomoviruses originating from the Old World, with six conserved ORFs (Fig. 1a, b). Phylogenetic analysis of TbLCZWV DNA A placed TbLCZWV in the Old World African and Mediterranean cluster (Fig. 1c), with the highest DNA A sequence identity (78·4%) being shared with Chayote mosaic virus (ChaMV), a cucurbit-infecting begomovirus from Nigeria, reported in GenBank as having a single genomic component. TbLCZWV also clustered with ChaMV in the DNA A relationship dendrogram (Fig. 1c), sharing high amino acid sequence identity with this virus in the CP (95·3%) and C3 (77·4%) ORFs. The high CP amino acid sequence identity shared between ChaMV and TbLCZWV might suggest that these are strains of the same virus; however, the unique intergenic region (IR) putative iterons of TbLCZWV (Paximadis et al., 1999 ) and <90% DNA A sequence identity shared with ChaMV demonstrate that TbLCZWV is a distinct species within the genus Begomovirus. Begomoviruses sharing <90% DNA A sequence identity are considered to be distinct species (Rybicki et al., 2000 ). In both the DNA A and ORF amino acid comparisons, TbLCZWV generally showed high sequence identity with the monopartite Mediterranean tomato yellow leaf curl begomoviruses and with South African cassava mosaic virus (Berrie et al., 2001 ).
|
Three strategies were employed to extensively search for a DNA B component in TbLCZWV-infected tobacco material. The first employed PCR and non-overlapping abutting primers [pIRc2671 (5' GGG TAC CGA TAT ACC AGG AG 3') and pIRv2672 (5' AAT ATA TAG TGG GTA CCG AAT GG 3')], designed in the IR of TbLCZWV DNA A, a region that includes the TATA box and two putative iterons identified upstream and downstream of the TATA box (Paximadis et al., 1999 ). Full-length PCR fragments (Fig. 2a) were blunt-end cloned into the pMOSBlue cloning vector (Amersham Pharmacia) and PCR was used to screen clones for their full-length nature (abutting primers pIRc2671 and pIRv2672) and their DNA A or B status using DNA A-specific primers (AV494 and AC1048). Fragments of ca.1300 bp, also amplified in the abutting primer PCR (Fig. 2a), were blunt-end cloned into pBluescript (KS) and sequenced (ABI Prism 310). The second strategy employed PCR to attempt to amplify a DNA B-specific fragment using three degenerate DNA B-specific primers [prBV1855 (Idris & Brown, 1998 ); pBL1v2040 (Rojas et al., 1993 ); pBR1c800 (5' ACG ACT GCA GTT VAC MGT CCC TTT GAA ACG 3'); D. P. Maxwell], and two CR-specific primers [PCRc1 (Rojas et al., 1993 ); PCRc4 (5' GGC CAT AGA GCT TTG AGG ATC CCG ATT CAT TTC 3'; D. P. Maxwell)], in all possible combinations. In addition, abutting primers (pIRc2671 and pIRv2672) were used in combination with the DNA B-specific primers. Lastly, restriction enzyme analysis of TbLCZWV DNA A identified enzymes with single (BamHI, SalI) and with no restriction sites (EcoRV, HindIII, KspI, NcoI, NotI, SacI, SmaI and XbaI). Supercoiled replicative viral dsDNA (scDNA), presumably a mixture of DNA A and B, purified from total DNA, was digested with these ten restriction enzymes, electrophoresed on agarose gels and Southern blotted onto nylon membranes (Sambrook et al., 1989 ). Blots were probed with a digoxigenin (DIG)-labelled (Roche Molecular Biochemicals) 437 bp CR-harbouring probe. Additional bands, not corresponding to the expected restriction pattern for DNA A, would be considered to be of possible DNA B origin.
|
The three strategies employed to search for a DNA B component all failed to indicate a second genomic component associated with TbLCZWV, suggesting TbLCZWV to be a monopartite begomovirus. The search for a DNA B component using IR abutting primers unexpectedly revealed the presence of smaller molecules approximately half the size of the full-length DNA A (Fig. 2a) that proved, upon sequencing, to be subgenomic defective DNA molecules of DNA A origin (Fig. 2b, c). Subgenomic defective DNA molecules, associated with a number of begomoviruses (Stanley & Townsend, 1985 ; MacDowell et al., 1986 ; Czosneck et al., 1989 ; Stanley et al., 1997 ; Liu et al., 1998 ; Sharma et al., 1998 ), seem to be fairly uniform in structure, i.e. in addition to the IR they tend to retain a large portion (5' end) of the BC1 or C1 ORFs, with a large portion of the BV1 or CP and remaining ORFs deleted. Occasionally they have sequences of unknown origin inserted between the truncated BV1 or C1 and the remaining begomovirus-specific sequence. Similar structures were observed with the defective molecules associated with TbLCZWV in this study (Fig. 2b, c). The interfering role of the TbLCZWV defective DNA molecules has not, however, been established experimentally.
Although the failure to detect a DNA B component strongly suggested that TbLCZWV is a monopartite begomovirus, the ability of the single genomic component to move and cause disease symptoms in its original host (Nicotiana tabacum) had to be shown to unequivocally prove the monopartite nature of this virus. A full-length DNA A genomic clone (pBSZW-A) was prepared by linearizing TbLCZWV scDNA with BamHI, and cloning into the BamHI site of pBluescript (KS). A full-length head-to-tail dimer of the genomic DNA A was constructed in pBluescript (KS) and subcloned into plant transformation vectors pBin19 (Bevan, 1984 ) and pBI121 (Clontech), yielding clones pBinZW-DA and pBIZW-DA respectively. Agrobacterium tumefaciens C58C1 RifR (pMP90 GmR) (Koncz & Schell, 1986 ), transformed with pBinZW-DA and pBIZW-DA using the freezethaw method of Holsters et al. (1978) , was used to agroinoculate Nicotiana benthamiana and two cultivars of N. tabacum, namely Samsun and HG (a Zimbabwean cultivar). Lycopersicon esculentum cv. Moneymaker was also agroinoculated with A. tumefaciens C58C1 RifR (pMP90 GmR) transformed with pBIZW-DA. Exponential-phase recombinant Agrobacterium was pelleted, washed with sterile water and resuspended in 200 µl of Luria broth per ml of original culture. A 30 gauge needle was used to transfer A. tumefaciens cultures to plants by injecting the stems and pricking the leaves of seedlings at the four to six leaf stage. Plants were monitored for symptoms. All four hosts developed leaf curl symptoms similar to those noted in the field samples (Fig. 3af), and both plant transformation vectors used were equally infectious. Southern hybridizations (using a DIG-labelled full-length TbLCZWV DNA A probe) verified the presence of both double-stranded and single-stranded forms of TbLCZWV-specific DNA in the agroinoculated plants, and PCR, using TbLCZWV-specific abutting primers, amplified expected full-length fragments, as well as ca.1300 bp fragments (only in tobacco), thought to be defective DNA molecules.
|
A perplexing feature of tobacco leaf curl since its earliest reports has been the observed variation in symptom severity (Storey, 1932 ; McClean, 1940 ). The begomovirus-infected tobacco plants used in the current study also showed variation in symptom severity ranging from mild to severe (Paximadis & Rey, 1997 ), and a variation of symptoms was also seen following agroinoculation of the cloned TbLCZWV DNA A, with N. tabacum cv. Samsun exhibiting symptoms that closely resemble the mild form B described by McClean (1940) and N. tabacum cv. HG exhibiting symptoms more typical of the mild form C (McClean, 1940 ). The severe form of the disease was not seen in N. tabacum, but N. benthamiana did exhibit severe leaf curl symptoms. Although the full-length genomic DNA A used for the agroinoculations was derived from a field tobacco plant exhibiting mild symptoms in N. tabacum, we have previously shown, using CP and CR nucleotide sequence comparisons between the four isolates (Paximadis et al., 1999 ), that different viral strains or viruses are unlikely to be found in the four plants. The discovery of defective DNA molecules in leaf curl-affected tobacco plants may possibly explain the variable symptom severity. It was noted that the defective DNAs from two different plants (Mild and HG; Paximadis & Rey, 1997 ) were different in structure (Fig. 2b, c), and since defective DNAs are known to cause symptom amelioration, it could be hypothesized that the defective DNAs may be playing a role in symptom modifications. Both Storey (1932) and McClean (1940) suggested that environmental factors contributed to symptom severity. One cannot therefore exclude the possibility that the environmental stresses experienced by an individual plant, the type of cultivar and defective DNAs present may all play a role in the severity of the symptoms displayed. One also cannot rule out the possibility however that other unidentified factors, like a satellite virus similar to DNA β associated with Ageratum yellow vein virus (Saunders et al., 2000 ) and Cotton leaf curl virus (Briddon et al., 2001 ), may be contributing to symptom severity.
In conclusion, TbLCZWV is a distinct, previously uncharacterized new member of the monopartite begomoviruses, associated with subgenomic defective DNA molecules that originate from its single genomic component. A monopartite genome organization has also been reported for Tobacco leaf curl Japan virus (Shimizu & Ikegami, 1999 ) and Tobacco leaf curl China virus (GenBank); however, these viruses are phylogenetically distantly related to TbLCZWV (Fig. 1c) and furthermore, the infectious nature of their single genomic components has not been reported.
References
Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12, 711-8721.
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.[Medline]
Czosneck, H., Ber, R., Navot, N., Antignus, Y., Cohen, S. & Zamir, D. (1989). Tomato yellow leaf curl virus DNA forms in the viral capsid, in infected plants and in the insect vector. Journal of Phytopathology 125, 47-54.
Doyle, J. J. & Doyle, J. L. (1987). A rapid isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19, 11-15.
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. Journal of General Virology 74, 147-151.
Fauquet, C. M., Maxwell, D. P., Gronenborn, B. & Stanley, J. (2000). Revised proposal for naming geminiviruses. Archives of Virology 145, 1743-1761.[Medline]
Frischmuth, T. & Stanley, J. (1991). African cassava mosaic virus DI DNA interferes with the replication of both genomic components. Virology 183, 539-544.[Medline]
Holsters, M., de Waele, D., Depicker, A., Messens, E., van Montagu, M. & Schell, J. (1978). Transfection and transformation of Agrobacterium tumefaciens. Molecular and General Genetics 163, 181-187.
Idris, A. M. & Brown, J. K. (1998). Sinaloa tomato leaf curl geminivirus: Biological and molecular evidence for a new subgroup III virus. Phytopathology 88, 648-657.[Medline]
Kheyr-Pour, A., Bendahmane, M., Matzeit, V., Accotto, G. P., Crespi, S. & Gronenborn, B. (1991). Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus. Nucleic Acids Research 19, 6763-6769.
Koncz, C. & Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Molecular and General Genetics 204, 383-396.
Lazarowitz, S. G. (1992). Geminiviruses: genome structure and gene function. Critical Reviews in Plant Sciences 11(4), 327349.
Liu, Y., Robinson, D. J. & Harrison, B. D. (1998). Defective forms of cotton leaf curl virus DNA-A that have different combinations of sequence deletion, duplication, inversion and rearrangement. Journal of General Virology 79, 1501-1508.[Abstract]
McClean, A. D. P. (1940). Some leaf-curl diseases in South Africa. Part I. Leaf-curl disease of tobacco. Science Bulletin, Department of Agriculture and Forestry, South Africa 225, 7-60.
MacDowell, S. W., Coutts, R. H. A. & 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 Research 14, 7967-7984.
Navot, N., Pichersky, E., Zeidan, M., Zamir, D. & Czosneck, H. (1991). Tomato yellow leaf curl virus: a whitefly transmitted geminivirus with a single genomic component. Virology 185, 151-161.[Medline]
Noris, E., Hidalgo, E., Accotto, G. P. & Moriones, E. (1994). High similarity among the tomato yellow leaf curl virus isolates from the West Mediterranean Basin: the nucleotide sequence of an infectious clone from Spain. Archives of Virology 135, 165-170.[Medline]
Osaki, T. & Inouye, T. (1981). Tobacco leaf curl virus. CMI/AAB Descriptions of Plant Viruses, no. 232.
Paximadis, M. & Rey, M. E. C. (1997). Aetiology of tobacco leaf curl in southern Africa. Annals of Applied biology 131, 449-457.
Paximadis, M., Dusterwald, G., Duyver, L. & Rey, M. E. C. (1997). Preliminary investigation of the causal agent(s) of a disease causing leaf curl of tobacco in South Africa. Plant Pathology 46, 282-290.
Paximadis, M., Idris, A. M., Torres-Jerez, I., Villarreal, A., Rey, M. E. C. & Brown, J. K. (1999). Characterization of tobacco geminiviruses in the Old and New World. Archives of Virology 144, 703-717.[Medline]
Rey, M. E. C., DAndrea, E., Calvert-Evers, J., Paximadis, M. & Boccardo, G. (1999). Evidence for a phytoreovirus associated with tobacco exhibiting leaf curl symptoms in South Africa. Phytopathology 89, 303-307.[Medline]
Rojas, M. R., Gilbertson, R. L., Russel, D. R. & Maxwell, D. P. (1993). Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. Plant Disease 77, 340-347.
Rybicki, E. P., Briddon, R. W., Brown, J. K., Fauquet, C. M., Maxwell, D. P., Harrison, B. D., Markham, P. G., Bisaro, D. M., Robinson, D. & Stanley, J. (2000). Family Geminiviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 285297. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
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. Proceedings of the National Academy of Sciences, USA 97, 6890-6895.
Sharma, A., Murayama, A., Osaki, T. & Ikegami, M. (1998). Characterization of virus-specific DNA forms from tomato tissues infected by tobacco leaf curl virus: evidence for a single genomic component producing defective DNA molecules. Plant Pathology 47, 787-793.
Shimizu, S. & Ikegami, M. (1999). Complete nucleotide sequence and the genome organization of tobacco leaf curl geminivirus from Japan. Microbiology and Immunology 43, 989-992.[Medline]
Stanley, J. & Townsend, R. (1985). Characterisation of DNA forms associated with cassava latent virus infection. Nucleic Acids Research 13, 2189-2206.
Stanley, J., Frischmuth, T. & Ellwood, S. (1990). Defective viral DNA ameliorates symptoms of geminivirus infection in transgenic plants. Proceedings of the National Academy of Sciences, USA 87, 6291-6295.
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.[Medline]
Storey, H. H. (1932). Leaf curl of tobacco in southern Rhodesia. Rhodesian Agricultural Journal 29, 186-192.
Wyatt, S. D. & Brown, J. K. (1996). Detection of subgroup III geminivirus isolates in leaf extracts by degenerate primers and polymerase chain reaction. Phytopathology 86, 1288-1293.
Received 18 June 2001; accepted 24 August 2001.