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Journal of General Virology (1998), 79, 1501–1508. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Defective forms of cotton leaf curl virus DNA-A that have different combinations of sequence deletion, duplication, inversion and rearrangement Yule Liu,1, 2 David J. Robinson1 and Bryan D. Harrison1 1 2 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China Tobacco and tomato plants inoculated at least 9 months previously with a Pakistani isolate of cotton leaf curl virus (CLCuV-PK), a whitefly-transmitted geminivirus, contained substantial amounts of circular dsDNA molecules that were mostly about half the size of CLCuV-PK dsDNA-A. They appeared to be derived from CLCuV-PK DNA-A by various combinations of sequence deletion, duplication, inversion and rearrangement and, in a few instances, insertion of sequences of unknown origin. Each of ten tobacco plants contained a different predominant form of such a defective molecule ; however, all Introduction Whitefly-transmitted geminiviruses in the genus Begomovirus have genomes consisting typically of two molecules of circular single-stranded DNA (DNA-A and DNA-B), each of about 2±8 kb (reviewed by Lazarowitz, 1992). However, the genomes of a few begomoviruses, such as an Israeli isolate of tomato yellow leaf curl virus (Navot et al., 1991), consist of only one molecule, which is equivalent to DNA-A. Cotton leaf curl virus (CLCuV-PK) is a begomovirus that has caused recent severe disease epidemics in cotton crops in Pakistan (Mansoor et al., 1993 ; Hameed et al., 1994). DNA-A-like molecules of this virus have been sequenced (Nadeem, 1995 ; Zhou et al., 1998) but no evidence for a DNA-B molecule has been reported. In the first step of a procedure to test plant extracts for the possible presence of a DNA-B, we conducted PCR with primers based on sequences in the large intergenic region of CLCuV-PK DNA-A (the region that in other begomoviruses is Author for correspondence : Bryan D. Harrison. Fax 44 1382 562426. e-mail djrobi!scri.sari.ac.uk The EMBL accession numbers of the sequences reported in this paper are AJ222703 to AJ222707 inclusive. the forms contained the intergenic region and part of the AC1 (Rep) gene. Some of the forms contained novel open reading frames and might have a role in the evolution of variant geminiviruses. The defective components were not detected at 3 months after the original culture of CLCuV-PK was transmitted by whiteflies (Bemisia tabaci) from cotton to tomato but were present after a further 6 months. They were transmitted, along with full-length DNA-A, between tobacco and tomato plants by grafting and by B. tabaci. shared by DNA-A and DNA-B molecules) to amplify nearly full-length DNA-A and, if present, DNA-B. However, in many of the trials, the main product was about half the expected size. The work described in this paper shows that the sequences amplified are from a series of molecules apparently derived from DNA-A of CLCuV-PK by deletion in combination with various other changes. Methods + Virus sources and propagation. Virus isolates were derived from the CLCuV-PK stock culture (Harrison et al., 1997), which had been transmitted by whiteflies (B. tabaci) from cotton to tobacco (Nicotiana tabacum cv. Samsun NN) plants in late 1993. These tobacco plants were retained for up to 3 years and were cut back at intervals to encourage the growth of new shoots. In addition, isolates were subcultured in tobacco and tomato (Lycopersicon esculentum) plants inoculated by grafting, or by B. tabaci using the procedure of McGrath & Harrison (1995). Infected plants were kept at 20–30 °C in a containment glasshouse at SCRI under licence from the Scottish Office Agriculture, Environment and Fisheries Department. + PCR. Nucleic acids were extracted from plant leaves and amplified by PCR, as described by Harrison et al. (1997), with primers based on the nucleotide sequence of DNA-A (2725 nt) of an isolate of CLCuV-PK (Nadeem, 1995). Four of the primers were used by Harrison et al. (1997) : 0001-5405 # 1998 SGM BFAB Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 07:43:57 Y. Liu, D. J. Robinson and B. D. Harrison CL1 (nt 208–226), CL3a (complementary to nt 998–980), CL4 (nt 1169–1190) and CL8 (complementary to nt 1963–1942). Nucleotides are numbered from the origin of replication in the intergenic region. In addition, two new primers were used : CL104 : 5« AACGCTCCCGCACACTATAAGTAC 3« (24 nt) (nt 67–90) CL105b : 5« TGGCCGCTTTTTGGAGCGT 3« (19 nt) (complementary to nt 2712–2694). PCR products were analysed by electrophoresis in agarose gels or, after digestion with restriction endonuclease, in polyacrylamide gels (Deng et al., 1994). + Southern blot hybridization. Nucleic acids were extracted from leaves by method B of Harrison et al. (1997), fractionated by electrophoresis in 1 % agarose gels and transferred to nylon membranes by the capillary method (Sambrook et al., 1989). The probe consisted of cloned DNA representing the sequences between the EcoRV site at residue 297 and the BglII site at residue 2449 of CLCuV-PK DNA-A (Nadeem, 1995) and was labelled with $#P by random priming. Prehybridization and hybridization were done in a solution containing 0±9 M NaCl, 0±075 M trisodium citrate, 1 % blocking reagent (Boehringer Mannheim), 0±1 M maleic acid, 0±1 % N-lauroylsarcosine and 0±02 % SDS, pH 7±5, at 65 °C, and membranes were washed four times in 2¬ SSC0±1 % SDS at 65 °C before autoradiography. + Sequence determination and analysis. PCR products were either sequenced directly or cloned and sequenced as described by Zhou et al. (1997). Sequence data were assembled and analysed with the aid of the Wisconsin Package version 8.1 programs (Anon., 1994). Results Small DNA molecules in CLCuV-PK-infected tobacco plants Primers CL104 and CL105b were designed to amplify almost the whole sequence of CLCuV-PK DNA-A, or DNA-B if present, except for about 80 residues of the intergenic region, to give a 2±6–2±7 kbp product. The first evidence for the occurrence of unexpected DNA molecules in CLCuV-PKinfected plants was obtained when DNA from a tobacco plant (plant 0) that had been inoculated with CLCuV-PK about 2 years earlier was amplified with these primers and a product of only 1±4 kbp was consistently obtained. When similar tests were done on ten other tobacco plants (plants 1 to 10) that had been infected for comparable lengths of time, all yielded unexpectedly short products of 1±4 kbp, although in some instances (lanes 2 and 4 in Fig. 1 a) the bands were too weak to reproduce photographically. Some plants also gave products of 0±9 kbp (Fig. 1 a ; Table 1). No 2±6 kbp product was detected from any of these plants, although all yielded the expected 0±8 kbp products (Harrison et al., 1997) with primer pairs CL1 and CL3a or CL4 and CL8 (Table 1). Moreover, a 2±6 kbp product was obtained in PCR with primers CL104 and CL105b from each of five tomato plants 3 months after inoculation with CLCuV-PK by whiteflies (Table 1). Although all the 1±4 kbp products were of similar size, those from each source plant gave a different pattern of fragments when digested with restriction endonucleases ; for example, Fig. 1 (b) shows the fragments obtained by digestion with AluI. Notably, the sizes BFAC (a) 1 2 3 4 5 6 7 8 9 10 H 1·4 kbp 0·9 kbp (b) 1 2 3 4 5 6 7 8 9 10 1·0 kbp 0·5 kbp 0·3 kbp 0·15 kbp Fig. 1. (a) Agarose gel electrophoresis of PCR products amplified with primers CL104 and CL105b from extracts of tobacco plants 1–10, chronically infected with CLCuV-PK ; H, extract from virus-free tobacco. (b) PAGE of fragments obtained by digestion with AluI of the 1±4 kbp PCR products shown in (a). Approximate sizes of DNA (kbp) are indicated beside each panel. of the restriction fragments added up to no more than 1±4 kbp in each instance, suggesting that each tobacco plant contained a homogeneous population of molecules related to CLCuV-PK DNA, similar in size but differing in sequence from plant to plant. The products of replicate PCRs on extracts from the same tobacco plant yielded the same pattern of restriction fragments. Southern blots of DNA extracted from chronically infected tobacco and tomato plants exposed to a CLCuV-PK DNA-A probe showed a complex pattern of bands, which was difficult to interpret (Fig. 2 a). None of the major bands was eliminated by treatment of the extracts with mung bean nuclease before electrophoresis, suggesting that they all represented doublestranded DNA. However, when the extracts were treated with ScaI, for which there is a single site in the intergenic region of CLCuV-PK DNA-A and which was therefore expected to linearize any circular forms of the DNA, much simpler band patterns were obtained (Fig. 2 b). DNA from a plant recently infected by viruliferous whiteflies yielded only a band of about 2±7 kbp, the expected size, whereas DNA from chronically infected plants gave a band of this size together with one or two smaller bands, often of about 1±5 kbp. None of the major Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 07:43:57 Defective forms of CLCuV DNA-A Table 1. DNA fragments amplified by PCR from extracts of CLCuV-infected tobacco and tomato plants DNA fragments obtained with indicated primers (kbp)* Plant CL1CL3a CL4CL8 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0 0±8 0±8 0±8 0 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0±8 0 0±8 0±8 0±8 0 Tobacco 1 Tobacco 2 Tobacco 3 Tobacco 4 Tobacco 5 Tobacco 6 Tobacco 7 Tobacco 8 Tobacco 9 Tobacco 10 Virus-free tobacco Tomato WFA† Tomato WFB† Tomato WFC† Virus-free tomato CL104CL105b 1±4 (0±9) (1±4) 1±40±9 (1±4)0±9 1±4 1±4 1±4 (0±9) 1±4 (0±9) 1±4 1±4 0 2±6 2±6 2±6 0 CL3aCL8 0 0 0±9 0±9 0±25 0±9 0 0±45 0 0 0 0 0 * Figures in parentheses indicate fragments produced in only small amounts ; figures in bold type indicate fragments sequenced. † Tomato plants inoculated 3 months previously with CLCuV-PK by whiteflies. , Not tested. (a) 1 2 3 4 5 6 (b) M H 1 2 3 4 5 6 1·6 kbp 0·5 kbp PCR products from chronically infected tobacco plants are not an artifact of the PCR but represent sequences amplified from less than full-sized DNA-A-related circular molecules present in the plants in substantial amounts. It is not clear why the PCR failed to amplify, in addition to the 1±4 kbp products, the expected 2±6 kbp product from the full-length DNA-A molecules detected in Southern blots. Possibly, the efficiency of amplification of the longer product from the same primer sequences was substantially less. Sequences of the small DNA molecules Fig. 2. Southern blots of (a) untreated and (b) ScaI-treated DNA extracted from : 1, tobacco plant 8 ; 2, tobacco plant 9 ; 3, tobacco plant 12 ; 4 and 5, tomato plants inoculated by grafting with scions from chronically infected tobacco ; 6, tomato recently infected by whiteflies with a CLCuVPK culture lacking defective DNA ; H, virus-free tobacco. Blots were probed with DNA representing residues 297–2449 of CLCuV-PK DNA-A. M, Linear DNA markers, whose sizes (kbp) are indicated at the left. bands in Fig. 2 (a) correspond in position to those in Fig. 2 (b), and there are generally twice as many major bands in each track of Fig. 2 (a) as in the corresponding track of Fig. 2 (b). Thus, it seems likely that the major bands in Fig. 2 (a) represent, in order of increasing mobility, open and supercoiled circular forms of the 2±7 kbp DNA and open and supercoiled circular forms of the smaller DNA(s). These results, together with the reproducibility of the PCR results, suggest that the 1±4 kbp The 1±4 kbp PCR products from four infected tobacco plants, as well as the 0±9 kbp product from one of these plants, were cloned and sequenced. In addition, the PCR products from two plants were sequenced directly. The sequences obtained for cloned or non-cloned products from the same plant were identical. Inspection of the sequences showed that the molecules from each plant contained sequences from CLCuV-PK DNA-A, but that large parts had been deleted and that the remaining sequences had been duplicated, inverted and}or rearranged in various ways (Table 2). For example, the 1±4 kbp sequence from tobacco plant 0 has three deletions, an inversion and a rearrangement when compared with CLCuVPK DNA-A. Most of the sequences from infected tobacco differed at only a few positions from the corresponding sequences in CLCuV-PK DNA-A but, in the sequences from Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 07:43:57 BFAD Y. Liu, D. J. Robinson and B. D. Harrison Table 2. Affinities of nucleotide sequences in defective DNA molecules of CLCuV Source plant Primers Product size Sequencing (kbp) procedure* Tobacco 0 104105b 1±4 C Tobacco 1 104105b 1±4 C(2) Tobacco 4 104105b 1±4 C, D Tobacco 10 104105b 1±4 C, D Tobacco 4 104105b 0±9 C, D Tobacco 4 3a8 0±9 D Tobacco 8 3a8 0±45 D Tobacco 5 3a8 0±25 D Origin of sequence elements† 1–441 (67–507) 1–406 (66–471) 1–202 (80–281) 1–163 (66–228) 1–194 (88–281) 1–169 (894–735) 8–350 (983–641) 1–35 (983–949) 442–751 (1844–2153) 407–648 (416–657) 203–400 (1208–1011) 164–452 (1203–915) 195–350 (1033–1188) 170–196 ? 351–464 (1839–1952) 36–180 ? 752–1068 (1239–923) 649–892 ? 401–1232 (1881–2712) 453–562 ? 351–472 (1957–2078) 197–545 (2461–1–84) 1069–1324 (2457–2712) 893–1246 (2359–2712) Calculated size (nt)‡ 1403 1324 1324 563–810 811–1233 (1937–2186) (2279–2701) 473–857 (2327–2711) 545–821 (1615–1892) 1322 958 181–239 (1897–1955) * PCR product cloned and sequenced (C) or sequenced directly (D). Two separate clones of the 1±4 kbp product from tobacco 1 were sequenced. † Figures are nucleotide positions in the sequence obtained ; figures in parentheses are the equivalent sequences in CLCuV DNA-A. ? indicates the sequence was not recognized. ‡ Calculated assuming the sequence of the intergenic region was complete in the defective molecules. Fig. 3. Nucleotide sequences around novel conjunctions of CLCuV-PK DNA-A sequences in defective DNA. Junction J1 is between the first and second sequence elements listed in Table 2 for each PCR product, J2 between the second and third, etc. For each junction, the middle line shows the sequence of the defective DNA and the upper and lower lines the sequences of the regions of CLCuV-PK DNA-A that have apparently been joined, with residues they share with the defective DNA capitalized. Residues shared by all three sequences at the junction point are underlined. tobacco plants 1 and 10, segments of unrecognized sequence not from CLCuV-PK DNA-A were present. All the sequences began and ended in the intergenic region, close to the expected priming sites, and all contained parts of the two adjacent genes, BFAE AV2 and AC1. Although the sequences that complete the circle in the small DNA molecules by bridging between the 5« ends of the primer sites were not determined, they were probably the same as the equivalent sequences in DNA-A, i.e. residues Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 07:43:57 Defective forms of CLCuV DNA-A Table 3. ORFs in defective DNA molecules of CLCuV ORFs in defective DNA extracted from : Related CLCuV ORF† Reading sense‡ Tobacco 0 1±4 kbp Tobacco 1 1±4 kbp Tobacco 10 1±4 kbp Tobacco 4 1±4 kbp Tobacco 4 0±9 kbp AV2 (103) V 121* (101 from AV2) 188 (101 from AV2) 47 (39 from AV2) 39 (39 from AV2) AV1 (CP) (256) AC1 (Rep) (361) V 92 (77 from AV1) 62 (42 from AC1) 67 (65 from AV1) 130 (74 from AC1) – 107 (55 from AV2 52 from AC3) – 162 (146 from AC1) 245 (234 from AC1) AC4 (100) None None C – – Complete C C 168 – 47 (23 from AC4) 126 61 92 – – – 171 (126 from AC1 45 from AC3) 80 (35 from AC4) – – C – * Figures are the number of amino acid residues in the predicted protein product of the ORF. The number of residues derived from the N terminus of the related CLCuV-PK ORF product is given in parentheses together with, in the two instances where they occur, the number of residues derived from the C terminus of the AC3 protein. † The number of amino acid residues in the predicted product of the CLCuV-PK ORF is given in parentheses. ‡ V indicates that the ORF is read from viral sense DNA, C that it is read from the complementary sense. 2713–2725}1–66. This would provide each of the small DNAs with a complete intergenic region, which contains all the cis-acting sequences essential for replication of geminivirus DNAs (Lazarowitz et al., 1992 ; Orozco et al., 1997). None of the sequences contained nt 658–914 or 1240–1843 of DNAA, so all lacked part of the AV1, AC1, AC2 and AC3 genes. Eleven sites in the five small DNA molecules sequenced represent novel junctions of identifiable DNA-A sequences, presumably produced by recombination. Fig. 3 shows the nucleotide sequences around these junctions aligned with the sequences in DNA-A from which they have apparently been derived. Nine of the junctions correspond to points at which the aligned progenitor sequences have from one to five nucleotides in common, but at the remaining two sites there are no such common residues. In most, but not all instances, the aligned progenitor sequences also have residues in common in the region flanking the recombination site. At one site, junction 3 in the 0±9 kbp product from tobacco plant 4, the sequence of the small DNA contains a residue different from that expected from the progenitor sequences. No obvious pattern or characteristic feature emerges from this analysis of the recombination junctions. Analysis of the coding potential of the small DNA molecules (Table 3) showed that one of them contains a complete AC4 gene. Various other open reading frames (ORFs) are also present, some of which are entirely novel and others of which include portions of in-frame coding sequences of CLCuV-PK genes, notably, in every case, the 5« part of the AC1 coding sequence. Not all of these ORFs are associated with plausible promoter and polyadenylation signals and it is unclear which, if any, of them are expressed. The presence in some of the small DNA molecules of sequences that are inverted with respect to their orientation in full-length DNA-A suggested that it should be possible to amplify fragments from them by PCR with suitably chosen pairs of primers that do not amplify any sequences from DNAA itself. Table 1 shows the results of an experiment with primers CL3a and CL8, which are both complementary to sequences in DNA-A (Harrison et al., 1997). Amplification products were obtained from DNA extracted from five of the ten chronically infected tobacco plants, with sizes ranging from about 0±25 to 0±9 kbp. Thus these plants contain DNA molecules in which the sequences corresponding to primers CL3a and CL8 are in opposite orientation, whereas in DNA-A they are in the same orientation. Products of 0±25, 0±45 and 0±9 kbp obtained with primers CL3a and CL8 were sequenced directly ; each contained non-contiguous segments of DNA-A, one of which was inverted, as well as, in some cases, unrecognized sequences (Table 2). These molecules are further examples of the variety of rearranged viral DNA molecules that occurred in the chronically infected tobacco plants. Occurrence of small DNA molecules in relation to time after infection, and their transmission The small DNA-A-related molecules were first detected and analysed in extracts from tobacco plants that had been inoculated with CLCuV-PK about 2 years previously. Grafting Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 07:43:57 BFAF Y. Liu, D. J. Robinson and B. D. Harrison (a) 1 2 3 4 5 8 H 1·4 kbp (b) A B C D E F H 2·6 kbp 1·4 kbp Fig. 4. (a) Agarose gel electrophoresis of products amplified by PCR with primers CL104 and CL105b from extracts of tomato plants, grafted with scions from tobacco plants 1, 2, 3, 4, 5 or 8, or from uninfected tobacco (H). (b) As (a), but extracts from tomato plants inoculated by whitefly transmission from tobacco plant 5 (lanes A and C) or tobacco plant 8 (lanes B and D), or by grafting from tobacco plant 5 (lane E) or tobacco plant 8 (lane F). Approximate sizes of DNA (kbp) are indicated beside each panel. of scions from these plants onto tomato plants resulted in transmission of the small DNA molecules to the tomato plants (Fig. 4 a). Similarly, the small molecules were transmitted by grafting, together with CLCuV-PK, from tomato to tobacco. When tomato seedlings were inoculated with the original culture of CLCuV-PK using whiteflies, small DNA molecules were not detected in extracts made 3 months after inoculation, but by 9 months after inoculation they were detectable. When tomato plants containing the small DNA molecules were used as virus sources in whitefly transmission experiments, the virus isolate derived from tobacco plant 5 was transmitted to 2 out of 11 tomato seedlings and the isolate derived from tobacco plant 8 to 2 out of 7. Of the infected seedlings, one with each isolate contained the 1±4 kbp molecules (Fig. 4 b, lanes B and C). However, primers CL104 and CL105b detected only near full-length DNA in a third seedling (Fig. 4 b, lane D) and two DNA species of about 1±5–1±6 kbp, which have not been studied further, in the fourth (Fig. 4 b, lane A). Thus, generation of the small DNA molecules seems to be a feature of long-term, chronic infection of tobacco or tomato plants, but once formed they can be transmitted by whiteflies. Discussion Our results show that tobacco and tomato plants, chronically infected with CLCuV-PK, contain a range of small circular DNA species derived predominantly from CLCuV-PK DNA-A. The few sequence elements that were not identifiable may have originated from CLCuV-PK DNA-B, if it exists, or from the tobacco genome. However, in an initial attempt to BFAG detect it, no evidence for DNA-B was found ; when amplification of DNA extracts with primers CL104 and CL105b gave a prominent 2±6 kbp product, digestion of that product with several different restriction endonucleases gave fragments that added up to no more than 2±6 kbp (data not shown). The small DNA molecules, which appear in plants at relatively long times after they are inoculated, seem to be generated by illegitimate recombination, which results in deletions, duplications, inversions and rearrangements of the sequences. Each chronically infected plant contains one predominant small DNA species, but the predominant molecule in each plant is different. Indeed, it is possible that different small DNA species may predominate in the same plant at different times. All the molecules seem to include the intergenic region, but recombinants that had lost this region would probably have lost the ability to be replicated. Including the bridging region between the 5« ends of the PCR primers, the size of the small molecules is most commonly about 1±4 kbp, although molecules of about 1±0 kbp also occur. Circular single-stranded DNA molecules of about half the size of the genomic DNA have also been detected in plants infected with other begomoviruses, including tomato golden mosaic virus (TGMV ; MacDowell et al., 1986), African cassava mosaic virus (ACMV ; Stanley & Townsend, 1985), tomato yellow leaf curl virus (TYLCV ; Czosnek et al., 1989) and ageratum yellow vein virus (AYVV ; Stanley et al., 1997). Those in ACMVinfected and TGMV-infected plants were shown to be derived exclusively from DNA-B. In contrast, the defective DNA molecules of AYVV are derived from DNA-A (the only genomic component detected), they are about the same size as those of CLCuV-PK, and they too contain the intergenic region and the 5« part of the Rep gene (Stanley et al., 1997). The AYVV molecules differ somewhat from those of CLCuV-PK in all containing large stretches of non-viral sequence, and only one of the sequenced AYVV molecules contains part of a noncontiguous viral gene (the CP gene). TYLCV, like AYVV, has only a single DNA component, equivalent to DNA-A, from which its small DNA was presumably derived. Moreover, the TYLCV small DNA was detected in extracts from purified particle preparations, implying that it was encapsidated (Czosnek et al., 1989). In addition to the characteristic geminate particles, plants infected with geminiviruses frequently contain a small proportion of isometric particles, and Sequeira (1982) showed that fractions of purified ACMV preparations that were enriched for the isometric particles were also enriched for small circular ssDNA molecules. Taken together, these observations suggest that geminivirus coat protein can encapsidate circular ssDNA molecules of 1±4–1±5 kb, about half the size of genomic DNA, to form isometric particles. However, the possibility that two small circular DNA molecules can be packaged in one geminate particle is not excluded. Encapsidation would be consistent with our observation (and that of Stanley et al., 1997 for AYVV) that the small DNA molecules can be transmitted by whiteflies, and the need for Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 07:43:57 Defective forms of CLCuV DNA-A encapsidation could explain why a particular size of small DNA molecule seems to be favoured. Thus, we envisage the occasional generation from DNA-A, by illegitimate recombination, of circular 1±4 kb molecules, which can be packaged in viral coat protein and which retain the origin of replication as well as sequences that interact with the viral Rep protein, and perhaps with the N-terminal part of the Rep protein that the recombinant molecules themselves encode. These molecules would then be replicated in substantial amounts by the normal mechanism for viral DNA-A. Defective molecules are also described for the leafhoppertransmitted beet curly top virus (BCTV ; Frischmuth & Stanley, 1992 ; Stenger et al., 1992), the genome of which contains elements similar to those in begomovirus DNA-A. Moreover, the BCTV defective molecules resemble those of CLCuV-PK in retaining the intergenic region and part of the Rep gene. However, they differ in that the BCTV molecules contain only a single deletion, and lack sequence inversions, rearrangements and unidentified stretches. With both these viruses, and with the defective molecules derived from DNA-B of ACMV and TGMV (Stanley & Townsend, 1985 ; McDowell et al., 1986), most of the recombination events needed to produce the defective molecules have occurred between points where there are a few (one to seven) shared nucleotides in the original sequence. What role the small DNA molecules might play in the biology of CLCuV-PK in nature is unclear and we have not ascertained whether or not they occur in naturally infected plants. It seems unlikely that, in a short-lived annual crop plant like cotton, they could have much effect on the development or persistence of leaf curl symptoms, at least until a late stage of infection. However, the means by which CLCuV-PK is perpetuated between cotton seasons is uncertain and the small DNA molecules might be involved in chronic infection of an alternative host plant. Interestingly, some biological effects of defective DNA molecules are reported for other begomoviruses. Defective forms of DNA-B of ACMV decrease replication of viral genomic DNA and attenuate symptoms (Stanley et al., 1990) as also do defective forms of DNA-A of AYVV (Stanley et al., 1997). Because they contain novel conjunctions of virus genetic material, the small CLCuV-PK DNA molecules may serve as raw material for evolution of new viruses or virus variants. It is noteworthy that several variant forms of CLCuV-PK, which have different combinations of nucleotide sequences, occur in Pakistan (Zhou et al., 1998) and the small DNA molecules may have had a role in the generation of these or other novel geminivirus variants. Another intriguing possibility is that small circular DNA molecules, similar to those described here, might have been precursors of the geminivirus-related DNA sequences found integrated into the genomes of some Nicotiana species (Bejarano et al., 1996). Like the small circular DNAs, the integrated sequences always include parts of the intergenic region and of the AC1 gene. Whatever their biological significance, the small circular CLCuV-PK-related DNA molecules provide yet another example of the facility with which illegitimate recombination can occur in geminivirus DNA. The work reported in this paper was supported financially by the European Commission (contract CI1*-CT94-0052). Y. L. was a holder of the Sino-Scottish Fellowship in Tropical Plant Virology. SCRI is grantaided by the Scottish Office Agriculture, Environment and Fisheries Department. References Anon. (1994). Program Manual for the Wisconsin Package, Version 8. Madison : Genetics Computer Group. Bejarano, E. R., Khashoggi, A., Witty, M. & Lichtenstein, C. (1996). Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution. Proceedings of the National Academy of Sciences, USA 93, 759–764. Czosnek, H., Ber, R., Navot, N., Antignus, Y., Cohen, S. & Zamir, D. (1989). 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