Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses

L. C. Berrie1, E. P. Rybicki2 and M. E. C. Rey1

Department of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, PO Wits 2050, Johannesburg, South Africa1
Department of Microbiology, University of Cape Town, Private Bag, Rondebosch 7701, Cape Town, South Africa2

Author for correspondence: Leigh Berrie. Present address: Department of Microbiology, University of Cape Town, Private Bag, Rondebosch 7701, Cape Town, South Africa. Fax +27 21 689 7573. e-mail Leigh{at}molbiol.UCT.ac.za


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Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses. SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus. One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component. Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana). This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.


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Three distinct groups of species of cassava-infecting whitefly-transmitted geminiviruses (WTGs, genus Begomovirus) have been distinguished based on their nucleotide sequences (Stanley & Gay, 1983 ; Hong et al., 1993 ; Zhou et al., 1997 ) and by their reactions with a panel of monoclonal antibodies (Harrison & Robinson, 1988 ; Swanson & Harrison, 1994 ). Group A, African cassava mosaic virus (ACMV), is found in West Africa, including Burundi, the western parts of Kenya, Chad, Uganda and recently, Cameroon (Fondong et al., 1998 ). Group B, East African cassava mosaic virus (EACMV), occurs in the eastern parts of Kenya, and in Madagascar, Malawi, Tanzania, Zimbabwe (Swanson & Harrison, 1994 ; Zhou et al., 1998a ), and has recently been detected in Uganda (Zhou et al., 1997 ), Zambia (Ogbe et al., 1997 ), Nigeria (Ogbe et al., 1999 ) and Cameroon (Fondong et al., 1998 ). Group C, Indian cassava mosaic virus (ICMV), occurs in India and Sri Lanka. Recently, we have identified yet another distinct cassava-infecting geminivirus in South Africa, South African cassava mosaic virus (SACMV), by coat protein (CP) amino acid and common region (CR) nucleotide sequence comparisons (Berrie et al., 1997 , 1998 ). It is now well accepted that recombination may play an important role in the evolution of begomoviruses (Fondong et al., 2000 ; Zhou et al., 1998a , b ). Indeed, a documented natural recombinant (EACMV-UG), consisting of most of the CP gene of ACMV inserted in an EACMV-like A component, has played a major role in the cassava mosaic epidemic which has spread through Uganda and into neighbouring countries (Zhou et al., 1997 ; Deng et al., 1997 ). As a step towards determining the extent and nature of the variation in cassava-infecting begomoviruses, we report the thorough characterization of a novel begomovirus (SACMV) by sequence analysis of both DNA components and comparisons with those of other cassava-infecting begomoviruses, as well as biological characterization by host-range studies.

Healthy cassava plants and plants infected with SACMV were obtained from Mpumalanga Province in South Africa and maintained in a greenhouse at 25 °C with a day length of 16 h and a dark period of 8 h. Total DNA was extracted as previously described (Berrie et al., 1998 ) from young leaves of SACMV-infected cassava plants. Full-length DNA-A and DNA-B genomic clones of SACMV were generated by digestion of 30 µg of total DNA with SalI and EcoRI, respectively. Fragments of 2·8 kbp were gel purified, cloned into the SalI or EcoRI sites of pBluescript KS(+), respectively, and transformed into E. coli JM109 cells. Positive clones were selected by dot blot hybridizations using DIG-labelled DNA-A- or DNA-B-specific probes (DIG DNA labelling and detection kit, Roche): a 310 bp SACMV DNA-A-specific probe was generated using PCR primers PAV1978 and PAC496 (Rojas et al., 1993 ) followed by digestion with PstI and Bam HI. The resulting 310 bp fragment consisted of sequences contained within the Rep gene of SACMV, being specific for SACMV DNA-A sequences only. A 687 bp DNA-B probe was generated using PCR primers PCRC2 and PBV2039 (Wyatt & Brown, 1996 ). The resulting fragment consisted of 596 bp of the BC1 ORF, as well as 91 bp of the 3'-end of the IR, allowing the probe to hybridize to both SACMV DNA-A and DNA-B sequences. Genomic clones hybridizing to both DNA probes were accepted to contain DNA-A sequences, whereas those hybridizing to the DNA-B probe only were assumed to contain DNA-B sequences.

Complete sequences of infectious clones of SACMV DNA-A and -B (GenBank accession nos AF155806 and AF155807, respectively) were determined in both directions by automatic sequence analysis with the ALF Express system (Amersham Pharmacia), according to the manufacturer’s instructions. Sequence data were assembled and analysed, and both pairwise and multiple sequence alignments were generated using DNAMAN versions 2.6 and 3.0 for Windows (Lynnon Biosoft, Quebec, Canada). Phylogenetic trees were constructed from the multiple alignments by the neighbour-joining method using DNAMAN and drawn using Treeview (Page, 1996 ) (1000 bootstrap replicates).

Evidence of recombination in the evolutionary history of SACMV was detected using the pairwise scanning-based recombination analysis program, RDP (version 1.07; Martin & Rybicki, 2000 ), with the following settings: window size=10, highest acceptable probability=0·0001, internal reference sequences. The recombinant origin of regions of SACMV indicated by RDP was verified by constructing neighbour-joining trees using the aligned sequences in the putatively recombinant region. Over 60% bootstrap support for location of the putatively recombinant region of SACMV in a divergent branch of the neighbour-joining tree was accepted as confirmation that the region had been acquired through recombination. Geminivirus DNA sequences used for comparison and their database accession numbers are presented in Table 1.


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Table 1. Geminiviruses used for comparisons, their genomic sequence accession numbers and assigned abbreviations

 
Full-length genomic clones of DNA-A and DNA-B of SACMV were found to be 2800 and 2760 nucleotides in length, respectively. As with other begomoviruses, the sequences of the two components are different except for a CR of 176 bp in length, with only nine nucleotide differences between DNA-A and DNA-B (similarity 94·9%). Genome organization was similar to that of other begomoviruses, with a total of eight conserved ORFs being identified: DNA-A had six ORFs, AV1 (258 aa) and AV2 (116 aa) in the virion-sense direction and AC1 (354 aa), AC2 (135 aa), AC3 (134 aa) and AC4 (98 aa) in the complementary-sense direction; DNA-B consisted of two ORFs, one in each of the virion and complementary sense (BV1, 258 aa and BC1, 307 aa, respectively).

Phylogenetic comparisons of SACMV DNA-A with other bipartite geminiviruses revealed a close relationship with geminiviruses from the Old World (Fig. 1a). SACMV is most closely related to EACMV type 2 isolates (-MH and -MK, 85%) (Zhou et al., 1998 a ), and less closely to EACMV type 1(-KE and -TZ, 79%) and EACMV-UG isolates (79%) (data not shown). Complete DNA-B sequence comparisons showed SACMV clustering with EACMV-UG-Mld and EACMV-UG-Svr (Fig. 1b), with sequence similarities of 90%, meaning that SACMV could be considered a strain of EACMV. As SACMV DNA-A shares sequence similarities of <90% with other begomoviruses, we feel that SACMV should be considered a distinct virus, given that the A-component is the ‘master’ sequence upon which replication depends (Padidam et al., 1995 ).



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Fig. 1. Phylogenetic trees obtained from the alignment of (a) full-length DNA-A nucleotide sequences of SACMV and 26 other begomoviruses, and (b) full-length DNA-B nucleotide sequences of SACMV and 14 other begomoviruses. Vertical distances are arbitrary; horizontal distances reflect number of nucleotide differences between branch nodes. Numbers at nodes indicate percentage bootstrap scores.

 
One significant recombination event, from nt 2135 to 2515, spanning the entire AC4 ORF of SACMV, was identified. Here, the SACMV sequence formed part of a group consisting of ICMV, ACMV, ToLCV-AU and TYLCV isolates from Sardinia and Sicily, rather than clustering with EACMV isolates (Fig. 2). Recently, a detailed analysis revealed that recombination among begomoviruses is frequent and contributes significantly to virus evolution (Padidam et al., 1999 ). In EACMV isolates -MH and -MK, virion-sense genes (CP, AV2) differ substantially from those of other EACMV isolates, and more closely resemble TYLCV-IS (Zhou et al., 1998a ). Substantial differences in the 5' and 3' ends of IRs have been found to occur in geminiviruses isolated from cotton and okra in Pakistan (Zhou et al., 1998b ). In addition, possible recombination in the N-terminal half of the Rep protein gene of certain cotton leaf curl virus isolates has been shown. In SACMV, a difference in evolutionary origins of virion-sense genes and certain complementary-sense genes is evident; however, it is difficult to pinpoint the evolutionary origin of the AC4 ORF due to the substantial amount of recombination amongst begomoviruses. This region of the geminivirus genome appears to be a hot-spot for recombination, revealed in our analyses by the presence of possible recombination events in several other viruses in this region. Recombination has been found to occur in the recently identified EACMV isolate from Cameroon, in both its DNA-A (AC2 and AC3 ORFs) and DNA-B (BC1 ORF) components (Fondong et al., 2000 ), indicating the possibility for recombination in both genomic components of begomoviruses. No obvious recombination was observed, however, in the DNA-B component of SACMV. Sequencing of the DNA-B components of other cassava-infecting geminiviruses, particularly EACMV isolates, may provide more information as to the relationships between these viruses. The data suggest that SACMV DNA-A and DNA-B may have originated from at least two different virus species. The DNA-A component of SACMV appears to be a novel recombinant where a unique viral sequence has derived certain sequences from other as yet unknown begomoviruses by recombination.



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Fig. 2. Phylogenetic tree obtained using alignments of nucleotides 2135–2515 in the recombinant region of SACMV and the corresponding region of 26 other begomoviruses. Vertical distances are arbitrary; horizontal distances reflect number of nucleotide differences between branch nodes. Numbers at nodes indicate percentage bootstrap scores.

 
In order to assess the infectivity and host range of SACMV, full-length head-to-tail dimers of the DNA-A and DNA-B components of SACMV were constructed by digestion of the genomic clones with ScaI and partial digestion with SalI or EcoRI, respectively. Resultant fragments consisting of a full-length genomic component of SACMV and either side of pBluescript KS(+) were gel-purified and re-ligated to form DNA-A and DNA-B dimers in pBluescript KS(+). Dimers were then subcloned into pBIN19 (pBINSA-A and pBINSA-B, respectively) and the clones mobilized into Agrobacterium tumefaciens strain C58C1(pMP90) (Koncz & Schell, 1986 ) by the freeze–thaw method of Holsters et al. (1978) . Cloned genomic components were introduced into Nicotiana benthamiana Domin. plantlets by agroinoculation using an adapted method of Hayes et al. (1988) . Exponential-phase recombinant Agrobacterium was pelleted at 8000 r.p.m., washed in sterile dH20 and resuspended in 200 µl Luria broth per ml of original culture. Ten N. benthamiana plants at the two-to-four leaf stage were agroinoculated three or four times with a Hamilton syringe along the stems of each plant with a total of 20 µl of the recombinant Agrobacterium (109 cells). Control plants were inoculated with Agrobacterium containing pBINSA-A only or pBINSA-B only. Following agroinoculation, plants were incubated in a growth chamber at 25 °C. After 19 days incubation, N. benthamiana plants developed symptoms of viral infection including stunting, leaf curling and crinkling, and chlorosis. Total DNA was extracted from the uppermost leaves of each plant, and the presence of viral DNA in all symptomatic plants was detected by Southern blot hybridizations, using DIG-labelled SACMV DNA-A or DNA-B components as probes. Control plants inoculated with pBINSA-A or pBINSA-B alone appeared symptomless and were negative in the Southern blot analyses.

Agroinoculations of Nicotiana tabacum L. ‘Samsun’, Lycopersicon esculentum Mill. ‘Poleboy Burpee’, Capsicum annuum L. ‘Anaheim’, Phaseolus vulgaris L. ‘Tenderpod green bush snap’, Malva parviflora L., Solanum melongena L. ‘Burpee’s black beauty’, Datura stramonium L. and cassava (Manihot esculenta Crantz) were then carried out. Ten plants of each test species were agroinoculated with SACMV dimers as mentioned previously, and the experiment conducted in triplicate. Plants from the species Phaseolus vulgaris, Malva parviflora and cassava (Manihot esculenta Crantz) were successfully inoculated with SACMV, and displayed symptoms of leaf curling, chlorosis and stunting. These results are in contrast to the experimental host-range of ACMV-KE which is largely restricted to the Solanaceae, within which the virus is more or less readily transmitted to several species in the genera Nicotiana and Datura (Bock et al., 1978 ). The host-range of EACMV has not yet been defined. DNA extracted from plants showing symptoms tested positive in Southern blot hybridizations with an SACMV-specific probe. No symptoms were evident in Nicotiana tabacum, Lycopersicon esculentum, Capsicum annuum, Solanum melongena and Datura stramonium, and no viral DNA was detected in these plants by Southern blot hybridizations.

In cassava, whitefly-transmission experiments have proved to be restricted by the feeding preferences of the vector and due to biotype incompatibility with test plants. Mechanical inoculation experiments using SACMV-infected cassava plant sap have also been unsuccessful (Berrie et al., 1997 ). Recently, cassava has been successfully inoculated with full-length genomic clones of ACMV-NG using a hand-held biolistics device (Briddon et al., 1998 ). This work demonstrated the first successful inoculation of a cloned geminivirus to cassava. Similar attempts have been made to infect cassava with full-length genomic clones of ACMV-CM and EACMV-CM, as well as inoculation by tissue wounding (V. Fondong, personal communication). Clones of both viruses were shown to be biolistically infectious; however, no success was obtained by tissue wounding. Here, we propose an alternative strategy for the inoculation of cassava with geminiviral clones by agroinoculation, and describe the first successful report of infection of cassava with a geminivirus using Agrobacterium tumefaciens. Successful infection of cassava with full-length genomic clones of SACMV, with appearance of characteristic symptoms, indicated that SACMV is almost certainly the causative agent of cassava mosaic disease in South Africa.


   Acknowledgments
 
The authors thank D. P. Martin for the use of his Recombination Detection Program and for his help in the analysis of sequence recombination data.


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Received 10 July 2000; accepted 10 October 2000.