A Nanovirus-Like DNA Component Associated with Yellow Vein Disease of Ageratum Conyzoides: Evidence for Interfamilial Recombination Between Plant DNA Viruses

Virology 264, 142–152 (1999) Article ID viro.1999.9948, available online at http://www.idealibrary.com on

A Nanovirus-like DNA Component Associated with Yellow Vein Disease of Ageratum conyzoides: Evidence for Interfamilial Recombination between Plant DNA Viruses

Keith Saunders and John Stanley1

Department of Virus Research, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom Received May 25, 1999; returned to author for revision June 22, 1999; accepted August 3, 1999

Yellow vein disease of Ageratum conyzoides, a weed species that is widely distributed throughout Asia, has been attributed to infection by the geminivirus Ageratum yellow vein virus (AYVV). In addition to a single AYVV genomic component (DNA A), we have previously demonstrated that infected plants contain chimeric defective viral components, comprising DNA A and nongeminiviral sequences, that act as defective interfering DNAs. A database search has revealed that the nongeminiviral sequences of one such defective component (def19) show significant homology with sequences of nanovirus components that encode replication-associated proteins (Reps). Primers designed to hybridise to the nongeminiviral DNA were used to PCR-amplify a full-length nanovirus-like component, referred to as DNA 1, from an extract of infected A. conyzoides. DNA 1 is unrelated to AYVV DNA A but resembles nanovirus components that encode Reps and is most closely related (73% identity) to a nanovirus-like DNA recently isolated from geminivirus-infected cotton. DNA 1 is dependent on AYVV DNA A for systemic infection of A. conyzoides and Nicotiana benthamiana and can systemically infect N. benthamiana in the presence of the bipartite geminivirus African cassava mosaic virus. A. conyzoides plants coinfected with AYVV DNA A and DNA 1 remain asymptomatic, indicating that additional factors are required to elicit yellow vein disease. Our results provide direct evidence for recombination between distinct families of plant single-stranded DNA viruses and suggest that coinfection by geminivirus and nanovirus-like pathogens may be a widespread phenomenon. The ability of plant DNA viruses to recombine in this way may greatly increase their scope for diversification. © 1999 Academic Press

INTRODUCTION Wong, 1993; Wong et al., 1993). A. conyzoides is a weed species that occurs throughout many parts of Asia and Members of the Geminiviridae genus Begomovirus frequently exhibits this phenotype, suggesting that AYVV (Briddon and Markham, 1995) are transmitted by the or related begomoviruses are widely distributed. How- whitefly Bemisia tabaci (Gennadius) to a wide range of ever, the causal agent of A. conyzoides yellow vein dis- vegetable and fibre crops worldwide in which they cause ease remains to be established. The AYVV DNA A com- serious diseases (reviewed by Brown, 1994). The major- ponent can systemically infect Phaseolus vulgaris ity of begomoviruses have two genomic components (DNA A and DNA B), both of which are essential for (French bean) and Lycopersicon esculentum (tomato) systemic infection (reviewed by Bisaro, 1996; Hanley- when reintroduced by agroinoculation and produces leaf Bowdoin et al., 1999). However, some begomoviruses curl symptoms in these hosts (Tan et al., 1995). This isolated from tomato, for example tomato yellow leaf curl suggests that A. conyzoides, like other weed species virus (TYLCV; Kheyr-Pour et al., 1991; Navot et al., 1991) (Ho¨fer et al., 1997; Roye et al., 1997), may act as a and tomato leaf curl virus (TLCV; Dry et al., 1993), have reservoir host for geminivirus diseases. However, the only a single genomic component resembling DNA A. AYVV component is unable to induce yellow vein symp- Such viruses must compensate in some way for the lack toms when reintroduced into A. conyzoides, indicating of a DNA B component, which encodes genes respon- that additional factors contribute as pathogenic determi- sible for nuclear trafficking and cell-to-cell movement of nants of the disease (Tan et al., 1995). the viral DNA (Noueiry et al., 1994; Sanderfoot and In addition to the AYVV DNA A component, infected A. Lazarowitz, 1995; Sanderfoot et al., 1996). conyzoides plants contain a population of subgenomic- Ageratum yellow vein virus (AYVV) is another example sized defective genomic components (Stanley et al., of a monopartite begomovirus. It was first isolated from 1997). The defective DNAs retain the DNA A intergenic Ageratum conyzoides L. plants growing in Singapore that region, are replicated in trans by DNA A, and are pre- were showing typical vein yellowing symptoms (Tan and sumed to be encapsidated within AYVV coat protein to facilitate whitefly transmission. They also contain nongeminiviral DNA that was assumed to be of host origin, 1 To whom reprint requests should be addressed. Fax: (01603) although database searches failed to reveal any similar- 456844. E-mail: [email protected]. ities with established sequences and blot hybridisation

0042-6822/99 $30.00 Copyright © 1999 by Academic Press 142 All rights of reproduction in any form reserved. AETIOLOGY OF Ageratum conyzoides YELLOW VEIN DISEASE 143 studies did not detect homologies with A. conyzoides genomic DNA (Stanley et al., 1997). Here we report that a more recent database search has revealed similarities between the nongeminiviral DNA of one defective component (def19) and nanovirus genomic components that encode replication-associated proteins (Reps). On the basis of this observation, we have isolated and characterised a nanovirus-like DNA component from symptomatic A. conyzoides. Our results demonstrate that the defective component has been produced by interfamilial recombination between a geminivirus and a nanovirus and suggest that yellow vein disease of A. conyzoides may result from a virus complex comprising components FIG. 1. Southern blot analysis of viral DNAs associated with A. conyzoides exhibiting yellow vein disease. Aliquots (1␮l) of viral scDNA that originate from both families. isolated from infected plants were either untreated (lane 1) or digested with BamHI (lane 2), PstI (lane 3), or EcoRI (lane 4). Lane 5 contains 10 RESULTS ng full-length CLCuV DNA 1 excised from pBS-CLCV1 (Mansoor et al., 1999). Blots were hybridised to probes corresponding to either AYVV A nanovirus-like DNA is associated with infected DNA A intergenic region (left) or CLCuV DNA 1 (right). The positions of A. conyzoides supercoiled (sc) and linear (lin) forms of AYVV DNA A (A) and defective DNAs (def) are shown. Note that the AYVV DNA A probe cross- AYVV defective DNA clone pAYVdef19 (Accession No. hybridises to the pBS-CLCV1 cloning vector (left, lane 5). Y14168) contains two regions of nongeminiviral DNA (Stanley et al., 1997). A database search using the nongeminiviral sequence corresponding to nucleotides 591- DNA A. The unique EcoRI restriction pattern indicates 1004 revealed homologies with virion-sense DNAs of that the CLCuV DNA 1 probe is not detecting defective milk vetch dwarf virus component 1 (Accession No. AYVV DNA A. The result indicates that a nanovirus-like AB000920; 65% identity over 214 nucleotides) and subgenomic component, of approximately half the size of the terranean clover stunt virus component 6 (Accession No. DNA A component is associated with A. conyzoides U16735; 67% identity over 171 nucleotides). However, the exhibiting yellow vein disease. nongeminiviral sequence corresponding to nucleotides Isolation and characterisation of a nanovirus-like 229–344 of pAYVdef19 showed no significant homologies component from symptomatic A. conyzoides with nanovirus DNAs. A database search using the nongeminiviral sequence (nucleotides 57–1035) of clone Partially overlapping primers V4504 and V4505, based pAYVdef17 (Accession No. Y14167), that is more repre- on nanovirus-like sequences within clone pAYVdef19, sentative of the majority of AYVV-defective DNAs (Stan- were used to PCR-amplify full-length copies of the puta- ley et al., 1997), also failed to reveal additional significant tive nanovirus-like component from purified viral scDNA. homologies with nanovirus DNAs. The primers contain two mismatches that introduce a To investigate the possible presence of a nanovirus unique SphI site to facilitate cloning. These mismatches component associated with diseased A. conyzoides occur within the Rep coding sequence (described below) plants, the purified viral scDNA used to isolate defective but do not affect its amino acid sequence. A single DNA AYVV components (Stanley et al., 1997) was analysed by fragment of ϳ1.4 kb was amplified using these primers blot hybridisation (Fig. 1). As anticipated, a probe corre- (data not shown) and used to produce clones pGEM- sponding to AYVV DNA A intergenic region sequences AYVV1/7, pGEM-AYVV1/9, and pGEM-AYVV1/12. Deriva- detected both genomic and defective scDNA (left panel, tion of their complete nucleotide sequences (Accession lane 1). The defective scDNA was unaffected by BamHI No. AJ238493 for the pGEM-AYVV1/7 insert) indicates and PstI treatment (lanes 2 and 3) but was partially several nucleotide differences between these clones linearised using EcoRI (lane 4) as previously observed (Fig. 2), but they are nonetheless very closely related (Stanley et al., 1997). The probe failed to detect a lin- (95–98% identity). Interestingly, nucleotides 920-1370/ earised cloned copy of cotton leaf curl virus (CLCuV) 1–35 of pGEM-AYVV1/9 and pGEM-AYVV1/12 are identi- DNA 1, a nanovirus-like DNA component recently iso- cal and, apart from a deletion of two nucleotides after lated from cotton (Mansoor et al., 1999) (lane 5). A CLCuV position 57, nucleotides 20–1047 of pGEM-AYVV1/7 and DNA 1 probe detected scDNA of approximately the same pGEM-AYVV1/12 are identical, suggesting that pGEM- size as the defective DNA (right panel, lane 1) that also AYVV1/12 is a recombinant of the other two DNAs. remained unaffected by BamHI and PstI treatment (lanes The cloned DNAs (collectively referred to as AYVV 2 and 3) but was completely digested with EcoRI to DNA 1) have between 43 and 51% identity with compo- produce either linearised DNA or two subfragments nents of the nanoviruses coconut foliar decay virus (lane 4). The CLCuV DNA 1 probe did not detect AYVV (CFDV; Rohde et al., 1990), faba bean necrotic yellows 144 SAUNDERS AND STANLEY

FIG. 2. Comparison of cloned AYVV DNA 1 sequences. The sequences begin immediately after the putative nick site within the conserved nonanucleotide TAGTATT2AC (bold font) located at the apex of the potential stem–loop structure (double underlined). The positions of a consensus TATA box (nucleotides 1333–1340) and polyadenylation signal (nucleotides 994–999) are underlined (numbering according to the pGEM-AYVV1/7 insert). The amino acid sequence of the Rep and amino acid heterogeneities between clones are shown above the nucleotide sequences. The AETIOLOGY OF Ageratum conyzoides YELLOW VEIN DISEASE 145 virus (FBNYV; Katul et al., 1998), subterranean clover (nucleotides 48–63 in AYVV and nucleotides 48–60 in stunt virus (SCSV; Boevink et al., 1995), milk vetch dwarf CLCuV). In addition, a consensus polyadenylation motif virus (MVDV; Sano et al., 1998), and banana bunchy top (AATAAA) occurs at the 3Ј terminus of the Rep ORF virus (BBTV; Burns et al., 1995) that encode Reps. How- (nucleotides 994–999), although none of the motifs that ever, they are most closely related to CLCuV DNA 1 (73% are located downstream of the CLCuV DNA 1 Rep ORF is identity) (Mansoor et al., 1999). All three clones amplified conserved in AYVV DNA 1. using primers V4504 and V4505 have the capacity to encode a 36.5-kDa protein that resembles nanovirus Additional nanovirus-like components have not Reps (34–48% identity, 56–65% similarity) but is most been detected closely related to CLCuV DNA 1 Rep (86% identity, 92% similarity). Of the 25 nucleotide differences that occur The isolation of AYVV DNA 1 from infected A. within the Rep coding regions of the AYVV DNA 1 clones, conyzoides raised the intriguing possibility that addi- only 6 change the encoded amino acid (Fig. 2). tional nanovirus-like components may also be present. Because of the similarity of the DNA 1 components, it To address this possibility, abutting primers V4523 and is assumed that the AYVV DNA 1 Rep is encoded on the V4524 were designed to anneal to the stem–loop se- virion-sense DNA strand as has been demonstrated for quences on the assumption that such sequences will be CLCuV DNA 1 (Mansoor et al., 1999). As observed for its conserved between components. PCR-amplification from CLCuV DNA 1 counterpart, the AYVV DNA 1 Rep has a scDNA using these primers produced a fragment of tyrosine residue at amino acid 90 that may participate in similar size to that obtained using primers V4504 and the initiation of rolling circle replication (Koonin and V4505, although with a much lower yield (data not Ilyina, 1992; Laufs et al., 1995), and a consensus NTP- shown). Of 42 clones obtained from the amplified DNA binding motif, GGEGKS (amino acids 188–193), that oc- fragment, 37 contained full-length DNA 1 as judged by curs in both nanovirus and geminivirus Reps (Gorba- restriction analysis. The clones were represented as two lenya et al., 1990; Katul et al., 1997). Other features groups in approximately equal proportions on the basis include a nonanucleotide sequence TAGTATTAC that is of their distinctive EcoRI restriction patterns, typified by found in most nanovirus DNAs (Sano et al., 1998), located pGEM-AYVV1/9 and pGEM-AYVV1/12 that have a single at the apex of a potential stem–loop structure (nucleo- EcoRI site at nucleotide 1109, and pGEM-AYVV1/7 that tides 1349–1367/1–12; due to the slight variation in the has a second site at nucleotide 509. This heterogeneity size of the clones, nucleotide numbering refers to the is consistent with the restriction pattern produced by DNA 1 insert of pGEM-AYVV1/7). A consensus TATA box, digestion of the viral scDNA with EcoRI (Fig. 1, lane 4). located within the sequence TATATATA (nucleotides The remaining clone inserts were of similar size, but 1333–1340), occurs in the same relative position as a database searches using their nucleotide sequences similar motif, TATAAATA, in CLCuV DNA 1 (nucleotides adjacent to the cloning sites indicated homologies to 1343–1350). In both cases, the TATA box occurs up- either retrotransposons (3 clones) or chloroplast DNA (2 stream of the Rep open reading frame (ORF), although clones), suggesting that they had been amplified from A. the stem–loop sequence is present in the intervening conyzoides DNA contaminants within the viral scDNA region. Interestingly, a similar arrangement occurs in preparation. CFDV (Rohde et al., 1990; Hehn and Rohde, 1998), as well Complete nucleotide sequences were established for as in some other nanovirus components that encode three randomly chosen clones (pGEM-AYVV1/5, pGEM- putative Reps (Sano et al., 1998). It is possible that such AYVV1/6, and pGEM-AYVV1/14) amplified using primers an arrangement serves to coordinate the processes of V4523 and V4524. Comparison with the sequences of Rep expression and DNA replication. A tandem repeat clones amplified using primers V4504 and V4505 consequence that occurs downstream of the stem–loop se- firmed that they contained DNA 1 inserts and that they quence in CLCuV DNA 1, that was speculated to partic- were closely related (Fig. 2). The inserts of pGEM- ipate in DNA replication or the control of Rep expression AYVV1/6 and pGEM-AYVV1/14 were identical to that of (Mansoor et al., 1999), is not present in AYVV DNA 1. pGEM-AYVV1/9 apart from lacking the SphI site intro- Indeed, the sequences of these viral components be- duced during PCR-amplification using primers V4504 tween the stem–loop and Rep ORF share little in com- and V4505. The pGEM-AYVV1/5 insert also lacked the mon except for noticeable runs of pyrimidine residues SphI site but otherwise differed from that of pGEM-

conserved tyrosine residue at position 90, which may be involved in binding to nascent DNA during replication, is indicated by a filled triangle, and the consensus NTP-binding motif at positions 188–193 is underlined. The SphI site (nucleotides 965–970) was introduced by a two nucleotide mismatch during PCR-amplification of full-length DNA 1 and is not present in the naturally-occurring viral DNAs. The sequence of the pGEM-AYVV1/14 insert is identical to that of pGEM-AYVV1/6, and they differ from the pGEM-AYVV1/9 insert only in that they lack the introduced SphI site. Asterisks indicate nucleotide deletions. 146 SAUNDERS AND STANLEY

FIG. 3. Replication of AYVV DNAs in N. benthamiana leaf disks. Leaf disks were agroinoculated with DNA 1 (lanes 1–3), DNA A (lanes 4–6), and a mixture of DNA 1 and DNA A (lanes 7–9). Samples were extracted from leaf disks harvested 3 days (lanes 1, 4, and 7), 5 days (lanes 2, 5, and 8), and 7 days (lanes 3, 6, and 9) p.i. Equivalent amounts of nucleic acids were loaded in each lane. Lane M contains one-tenth of the amount of a sample extracted from an agroinoculated N. benthamiana plant (corresponding to the sample in Fig. 4, lane 8). Blots were hybridised to probes specific for AYVV DNA A and DNA 1 as indicated. The positions of single-stranded (ss) and supercoiled (sc) DNAs are shown.

AYVV1/7 by only two additional nucleotides upstream of et al., 1997), although symptoms in coinfected plants the Rep ORF and two nucleotide substitutions in the were slightly delayed in their onset and, hence, margin- vicinity of 3Ј terminus of the ORF. ally less severe. However, systemically infected plant tissues sampled 20 days p.i. contained similar levels of AYVV DNA 1 can autonomously replicate DNA A irrespective of the presence or absence of DNA The ability of AYVV DNAs to replicate was investigated 1 (Fig. 4, lanes 4–9). DNA 1 was detected in systemically using a N. benthamiana leaf disk assay (Fig. 3). Under infected tissues after coinoculation with DNA A (lanes these conditions, DNA A autonomously replicated to 7–9) but was not detected when inoculated alone (lanes produce typical ssDNA and scDNA forms (lanes 4–6), 1–3) and plants remained asymptomatic. and its accumulation was largely unaffected by the pres- A. conyzoides plants remained asymptomatic when ence of DNA 1 (lanes 7–9). DNA 1 also replicated to inoculated with either DNA A or DNA 1 or when coin- produce both DNA forms, although the level of ssDNA oculated with both components. DNA A was detected in was relatively low in comparison with scDNA (lanes 1–3). newly developing leaves at 27 days p.i. (Fig. 5, lanes However, in the presence of DNA A, the single-stranded 7–12), although systemic spread was sporadic and the form of DNA 1 accumulated to much higher levels (lanes 7–9). This could be due to a direct effect of DNA A on DNA 1 replication, although this is unlikely in view of the highly specific nature of the interaction of Reps with their cognate replication origins. It is more likely that the accumulation of the ssDNA results from encapsidation by DNA-A-encoded coat protein that sequesters this DNA form and protects it from degradation. This is consistent with the observation that CLCuV DNA 1 is associated with CLCuV DNA-A-encoded coat protein and appears to be encapsidated in geminate particles (Man- soor et al., 1999).

AYVV DNA 1 systemic movement is mediated by geminivirus DNA FIG. 4. Detection of AYVV DNAs in agroinoculated N. benthamiana. AYVV DNA A and DNA 1 were introduced into plants Samples were extracted from individual plants agroinoculated with by agroinoculation. N. benthamiana plants coinoculated DNA 1 (lanes 1–3), DNA A (lanes 4–6), and a mixture of DNA 1 and DNA A (lanes 7–9). Equivalent amounts of nucleic acids were loaded in each with DNA A and DNA 1 developed systemic leaf curl lane. Blots were hybridised to probes specific for AYVV DNA A (top) and symptoms 12 days p.i. that were qualitatively indistin- DNA 1 (bottom). The positions of single-stranded (ss) and supercoiled guishable from those induced by DNA A alone (Stanley (sc) DNAs are shown. AETIOLOGY OF Ageratum conyzoides YELLOW VEIN DISEASE 147

DNA 1 has many features in common with nanovirus components that encode Reps but is most closely related to CLCuV DNA 1, a nanovirus-like component recently isolated from geminivirus-infected cotton growing in Pakistan (Mansoor et al., 1999). Both DNA 1 components encode a Rep, and both have been shown to replicate autonomously. CLCuV DNA 1 is also whitefly transmitted, and gradient purification and immunotrap- ping data indicate that it is probably encapsidated by CLCuV DNA-A-encoded coat protein (Mansoor et al., 1999). The stimulation of accumulation of the single- stranded forms of DNA 1 in N. benthamiana leaf disks by FIG. 5. Detection of AYVV DNAs in agroinoculated A. conyzoides. DNA A suggests that this is also the case for AYVV. Samples were extracted from individual plants agroinoculated with However, the level of nucleotide sequence identity (73%) DNA 1 (lanes 1–6), DNA A (lanes 7–12), and a mixture of DNA 1 and DNA A (lanes 13–18). Equivalent amounts of nucleic acids were loaded clearly indicates that these nanovirus-like components in each lane. Lane M contains one-tenth of the amount of a sample originate from distinct virus species. AYVV DNA 1 (1367 extracted from an agroinoculated N. benthamiana plant (corresponding nucleotides in pGEM-AYVV1/7) is of similar size to to the sample in Fig. 4, lane 8). Blots were hybridised to probes specific CLCuV DNA 1 (1376 nucleotides), and both are signifi- for AYVV DNA A (top) and DNA 1 (bottom). The positions of single- cantly larger that the majority of nanovirus components stranded (ss) and supercoiled (sc) DNAs are shown. that are between 1000 and 1100 nucleotides in length (Boevink et al., 1995; Burns et al., 1995; Katul et al., 1997, viral DNA accumulated to much lower levels than in N. 1998; Sano et al., 1998). AYVV DNA 1 is approximately benthamiana. This may explain why DNA A systemic half the size of AYVV DNA A (2471 nucleotides; Tan et al., spread in this plant species was not previously observed 1995), consistent with the proposal of Mansoor et al. after agroinoculation (Tan et al., 1995). DNA 1 sporadi- (1999) for CLCuV that DNA 1 has become size-adapted cally systemically infected A. conyzoides, but only in the for encapsidation within DNA-A-encoded coat protein to presence of DNA A (lanes 13–18), and accumulated to facilitate whitefly transmission. much higher levels relative to DNA A in this host than in Despite the isolation of nanovirus-like components N. benthamiana. from infected plants, the causal agents of A. conyzoides To investigate if other geminiviruses can mediate the yellow vein disease and cotton leaf curl disease remain systemic movement of DNA 1, N. benthamiana plants to be established. Until now, only geminivirus DNA A were challenged with AYVV DNA 1 and ACMV by agro- components and their defective derivatives have been inoculation. All coinoculated plants produced systemic isolated from infected plants. Several attempts to detect symptoms indistinguishable from a normal ACMV infec- a DNA B component have been unsuccessful. AYVV DNA tion at 7 days p.i. As observed in previous experiments, A induces severe systemic leaf curl symptoms in N. DNA 1 alone was unable to move systemically (Fig. 6, benthamiana (Tan et al., 1995; Stanley et al., 1997) but A. lanes 1–3) but was detected in systemically infected conyzoides remains asymptomatic when challenged tissues in the presence of ACMV at 9 days p.i. (lanes with this component even though, as we now demon- 7–9). Unlike AYVV, ACMV can be efficiently introduced strate, it can systemically infect this plant species. We into N. benthamiana by mechanical inoculation. After have demonstrated that DNA 1 can also systemically mechanical coinoculation with ACMV, DNA 1 was de- infect both plant species, although only in the presence tected in both the inoculated leaves (lanes 10–12) and of DNA A. Nonetheless, coinfection with both compo- systemically infected leaves (lanes 13–15) at 9 days p.i. nents is insufficient to cause typical disease symptoms, but was not detectable when inoculated alone. implying that additional factors remain to be identified. Our attempts to PCR-amplify additional nanovirus-like DISCUSSION components that may contribute to the phenotype, using We have demonstrated that A. conyzoides plants grow- primers that anneal to sequences within the stem–loop ing in Singapore that exhibit yellow vein symptoms are region, produced only DNA 1 clones. However, although infected with a nanovirus-like component that we refer to stem–loop sequences are generally highly conserved as DNA 1, in addition to the previously characterised between components of individual bipartite geminivi- DNA A component of the begomovirus AYVV (Stanley et ruses, they are somewhat less conserved between those al., 1997). The viral scDNA used to isolate DNA 1 was of nanoviruses (Burns et al., 1995; Katul et al., 1998; Sano extracted from diseased A. conyzoides plants that had et al., 1998), which may explain the preference for am- been maintained using viruliferous B. tabaci, implying plification of one particular component. For this reason that the component, like DNA A, is whitefly transmitted. we are continuing to explore the possibility that addi- 148 SAUNDERS AND STANLEY

FIG. 6. Detection of ACMV and AYVV DNA 1 in agroinoculated and mechanically inoculated N. benthamiana. Samples were extracted from individual plants either agroinoculated with AYVV DNA 1 (lanes 1–3), ACMV (lanes 4–6), and a mixture of DNA 1 and ACMV (lanes 7–9) or mechanically inoculated with a mixture of DNA 1 and ACMV (lanes 10–15). Samples from mechanically inoculated plants were from either inoculated leaves (lanes 10–12) or upper leaves showing systemic symptoms of ACMV infection (lanes 13–15). Equivalent amounts of nucleic acids were loaded in each lane. Blots were hybridised to probes specific for ACMV DNA A and DNA B, and AYVV DNA 1 as indicated. The positions of single-stranded (ss) and supercoiled (sc) DNAs are shown. tional nanovirus-like components are associated with cloned directly from viral scDNA, it must represent a infected A. conyzoides. bona fide recombinant that is replicated in trans by the Our data demonstrate that DNA 1 is dependent on geminivirus component, as has been demonstrated for AYVV DNA A for systemic infection of A. conyzoides and def17 (Stanley et al., 1997) as well as for defective com- N. benthamiana. DNA 1 can autonomously replicate, ponents of other geminiviruses such as ACMV and beet implying that DNA A facilitates its cell-to-cell and sys- curly top virus (BCTV) (Stanley and Townsend, 1985; temic movement. The coat protein and/or the product of Frischmuth and Stanley, 1992). The def19 recombinant is the upstream virion-sense gene V1 may participate in 1343 nucleotides in length, similar in size to the DNA 1 this process as both have been implicated in virus move- component, and so may be encapsidated by the DNA-A- ment in monopartite begomoviruses (Rigden et al., 1993; encoded coat protein for whitefly transmission. Def19 Wartig et al., 1997). We have also demonstrated that the contains a discontinuous region of 813 nucleotides of bipartite begomovirus ACMV can mediate DNA 1 move- DNA A that flanks 116 nucleotides of unknown origin ment in N. benthamiana, suggesting that the ACMV (Fig. 7). The DNA A sequences in def19 are identical to movement proteins encoded by DNA B genes BV1 and nucleotides 2403–2741/1–228 and 497–747 of the full- BC1 (Noueiry et al., 1994; Sanderfoot and Lazarowitz, length genomic component except for nucleotide substi- 1995; Sanderfoot et al., 1996) can functionally interact tutions within the intergenic region at positions 87 and with the nanovirus-like component. DNA 1 remained un- 92. The DNA A sequences also flank 414 nucleotides that detectable when mechanically inoculated onto N. are identical to nucleotides 784-1197 of DNA 1 (clones benthamiana, although it presumably produced a sub- pGEM-AYVV1/6 and pGEM-AYVV1/14). Def19 must there- liminal infection in the inoculated leaves. However, it was fore have been produced by a double-recombination readily detected in inoculated leaves when mechanically event between AYVV DNA A and DNA 1 and between coinoculated with ACMV. It has previously been shown DNA A and an unknown progenitor DNA. The recombi- that ACMV DNA A remains undetectable in inoculated nant was not produced by homologous recombination as leaves unless coinoculated with DNA B (von Arnim and there are no extensive sequence similarities between Stanley, 1992). Hence, our results are consistent with DNA A and DNA 1, although a small repeat sequence ACMV DNA-B-mediated cell-to-cell movement of DNA 1, (GGATT), that occurs in both DNAs at one recombination allowing the nanovirus-like component access to addi- junction (Fig. 7), may have contributed to defining the tional tissues in which to proliferate to a detectable level. cross-over point at this position. This is reminiscent of AYVV DNA 1 was isolated after the identification of the small repeat sequences that have been observed in nanovirus-like sequences within the defective DNA A defective DNAs of AYVV (Stanley et al., 1997) as well as component, def19 (Stanley et al., 1997). As def19 was those associated with ACMV, tomato golden mosaic vi- AETIOLOGY OF Ageratum conyzoides YELLOW VEIN DISEASE 149

FIG. 7. Structure of defective viral DNA def19 produced by recombination between AYVV DNA 1 and DNA A. (A) Organisation of AYVV DNA 1 and DNA A. The positions of the replication-associated protein (Rep) genes, encoded on either the virion-sense strand (DNA 1) or complementary-sense strand (DNA A), the geminivirus coat protein (CP) gene, and the stem–loop structures in which a nick is introduced during the initiation of rolling circle replication are indicated. The position of primers V4504 and V4505, designed on the basis of def19 nongeminiviral DNA sequences and used to amplify a full-length copy of DNA 1, are shown as black triangles. (B) Organisation of def19 and sequences at the recombination junctions. Sequences within the hatched region are of unknown origin. DNA 1 and DNA A nucleotides that are identical to the recombinant DNA at the recombination junctions are indicated by asterisks, and a short sequence present at one junction in both parental DNAs is underlined. rus (TGMV), and BCTV infections (Stanley and occurred at some stage during their evolution. Complete Townsend, 1985; MacDowell et al., 1986; Frischmuth and nucleotide sequences for a large number of begomovi- Stanley, 1992). Def19 has an extensive ORF produced by ruses have been established, demonstrating the consid- fusion of the central region of the DNA A coat protein erable genetic diversity within the genus (Padidam et al., ORF to the 3Ј terminus of the DNA 1 Rep ORF (Fig. 7), 1995). Although the gradual accumulation of point muta- although the first in-frame initiation codon occurs 70 tions within in a population can account for much of this amino acids downstream of the ORF start. Although variation, the recent dramatic increase in geminivirus there is no reason to believe that a fusion protein ex- diseases provides the opportunity for rapid evolution by pressed from this ORF is biologically functional in this interspecific (Hou and Gilbertson, 1996; Zhou et al., 1997) instance, it clearly illustrates the potential of interfamilial and intergeneric recombination (Stanley et al., 1986; Brid- recombination to generate chimeric genes. don et al., 1996; Klute et al., 1996). Interfamilial recombi- Recombination plays a major evolutionary role by cre- nation is much less likely to occur due to the need to ating genetic diversity within virus populations, providing coinfect single cells with evolutionarily divergent viruses the potential for individuals to rapidly adapt to new hosts which must nonetheless retain the potential to exchange and environmental conditions (reviewed by Domingo and their genetic information, and would be difficult to detect Holland 1997). Interspecific recombination is well docu- unless the recombinants are able to proliferate. The mented for many virus families (reviewed by Roossinck isolation of def19, a naturally occurring recombinant DNA 1997), and the presence of gene homologues in viruses that is maintained by replication in trans by a helper representative of diverse families provides indirect evi- virus, provides direct evidence that such recombina- dence that interfamilial recombination events could have tional events can occur, on this occasion between two 150 SAUNDERS AND STANLEY distinct plant single-stranded DNA viruses, a geminivirus to full-length DNA 1 was purified by agarose gel electro- and a nanovirus. phoresis and cloned into pGEM-T Easy. The existence of the recombinant implies that both the The sequences of both strands of the DNA 1 clones geminivirus and nanovirus-like components are capable were derived using an ABI PRISM Big Dye Terminator, of coinfecting the same cell, a phenomenon that has Cycle Sequencing, Ready Reaction Kit (Perkin–Elmer) important implications for plant DNA virus evolution and together with either standard T7 and SP6 sequencing epidemiology. It supports the contention that these com- primers or DNA 1-specific primers based on established ponents, together with one or more additional factors sequence. Sequence reactions were resolved using an that remain to be defined, are responsible for A. ABI 373 automated sequencer. Sequences were anal- conyzoides yellow vein disease. It also raises the possi- ysed using version 7 of the program library of the Ge- bility that autonomous whitefly-transmitted nanoviruses netics Computer Group (Devereaux et al., 1984). Nano- may exist (none has yet been reported) or could evolve virus components used for DNA and Rep comparisons as a consequence of the current rapid dissemination of (database accession numbers in parentheses) were both geminivirus and nanovirus diseases. Certainly, the CFDV DNA (M29963), FBNYV component 1, (AJ005968), isolation of closely related nanovirus-like components SCSV component 6 (U16735), MVDV component 1 from distinct plant species (A. conyzoides and cotton) (AB000920), BBTV component 1 (S56276), and CLCuV growing in widely separated locations (Singapore and DNA 1 (AJ132345). Pakistan) suggests that coinfection of geminivirus and nanovirus-like components may be a common occur- rence. It remains to be seen if such coinfections are Plant inoculation confined to monopartite begomoviruses resembling The construction of pHNBin419, pBin1.3A, and pBin2B, AYVV and CLCuV or extend to bipartite begomoviruses containing partial repeats of AYVV DNA A and ACMV and even to leafhopper-transmitted viruses of the Curto- DNA A, and a tandem repeat of ACMV DNA B, respec- virus and Mastrevirus genera. tively, in the binary vector pBin19, has been described (Klinkenberg et al., 1989; Tan et al., 1995). A partial repeat MATERIALS AND METHODS of AYVV DNA 1 was constructed by cloning the SphI(210)- Cloning and characterisation of viral DNA SalI(965) fragment of DNA 1 from pGEM-AYVV1/7 into components pUC19 (Yanisch-Perron et al., 1985). The full-length insert of pGEM-AYVV1/7 was then cloned into the unique SphI The purification of viral scDNA from A. conyzoides L., site of this construct to produce pUC-AYVV1/7. The pUC- originating from Singapore and showing typical yellow vein symptoms, has been described (Stanley et al., 1997). AYVV1/7 insert was cloned into pBinPlus (van Engelen et The construction and characterisation of clones pHN419 al., 1995) as a HindIII/BamHI fragment to produce pBin- and pAYVdef19, containing full-length copies of AYVV AYVV1/7. Agrobacterium tumefaciens containing the Ti DNA A and a defective viral DNA (def19), respectively, plasmid pGV3850 (Zambryski et al., 1983) was trans- also has been described (Tan et al., 1995; Stanley et al., formed with pHNBin419, pBin1.3A, pBin2B, and pBin- 1997). AYVV1/7 by triparental mating (Ditta et al., 1980). N. A nanovirus-like DNA (DNA 1) was PCR-amplified from benthamiana and A. conyzoides plants were agroinocu- the viral scDNA using partially overlapping primers GGT- lated as described by Tan et al. (1995). N. benthamiana GCaTGcTTGAAGCATCATATTGG (primer V4504; corre- plants were mechanically inoculated with ACMV clones sponding to def19 nucleotides 962–987) and CAAgCAtGC- pCLV1.3A and pCLV2B, containing a partial repeat of ACCACAAGGAATACACGGG (primer V4505; comple- DNA A and a tandem repeat of DNA B, respectively mentary to nucleotides 946–973). Lower case letters (Klinkenberg et al., 1989; Etessami et al., 1991), and clone indicate nucleotide changes introduced to create a pUC-AYVV1/7, containing a partial repeat of AYVV DNA 1. unique SphI site (underlined). The amplified ϳ1.4-kb All inoculated plants were screened daily for symptom DNA fragment was purified using a Wizard PCR Preps development. DNA purification system (Promega), and was cloned into pGEM-T Easy (Promega). Recombinants were screened Leaf disk assay for viral DNA replication by restriction analysis using SphI and EcoRI. Three full- length DNA 1 clones (pGEM-AYVV1/7, pGEM-AYVV1/9 The ability of cloned viral DNA components to repli- and pGEM-AYVV1/12) were isolated and characterised. cate was tested using N. benthamiana leaf disks as In later experiments, DNA 1 was amplified using abutting described by Klinkenberg et al. (1989). Leaf disks were primers TATTACCCCGCCTCGA (primer V4523; corre- agroinoculated with AYVV DNA A and DNA 1 using A. sponding to nucleotides 1364–1367/1–12) and CTATGC- tumefaciens containing the Ti plasmid pGV3850 and CCGCCTCGA (primer V4524; complementary to nucleo- transformed with pHNBin419 and pBin-AYVV1/7, respec- tides 1349–1363). The amplified fragment corresponding tively. Leaf disks were harvested 3, 5, and 7 days p.i. AETIOLOGY OF Ageratum conyzoides YELLOW VEIN DISEASE 151

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