1.11.50
BANANA STREAK VIRUS -- AN INTEGRATED PLANT PARARETROVIRUS?

G HARPER, J OSUJI, P HESLOP-HARRISON and R HULL

John Innes Centre, Norwich Research Park, Colney, Norwich, Norfolk, NR4 7UH, UK

Background and objectives
Banana (Musa) is the world's fourth most important commodity and its improvement and production are seriously affected by viruses including banana streak virus (BSV). First described as characteristic leaf-stripes on Musa (AAA group, Cavendish subgroup) Poyo, the causal agent was named banana streak virus [1]. Subsequently the disease has been recorded in essentially all Musa-producing regions and in many of the numerous clones of Musa and its sub-groups. BSV is a member of the badnavirus group which have non-enveloped bacilliform particles of size 30x130-150 nm, containing a double-stranded DNA genome of 7.4-8.0 kb. Features of their genomes suggest that they are pararetroviruses. The use of reverse transcriptase in their replication can potentially lead to a high degree of variation between isolates and different group members.

The episomal virus from a Nigerian BSV isolate has been cloned and its 7387 bp genome sequenced [2]. A direct PCR assay for the sensitive detection of BSV from Musa plants has been developed. Results using this PCR approach indicated the widespread if not universal presence of BSV sequences in Musa[3]. In contrast, other detection methods requiring the presence of viral particles or expression of BSV genes indicated a much lower incidence of the virus. These findings, other evidence and recent reports strongly suggested the integration of BSV sequences into the Musa genome and its possible activation under certain stresses. This situation has very significant implications for the breeding, tissue culture and movement of germplasm. The aim of this work is to characterise BSV integrants in the Musa germplasm.

Results and conclusions
Sequence-specific amplification of Musa DNA using BSV primer and arbitrary Taq1 primers gave a number of BSV-hybridising products. Sequence of one class of these products revealed an interface between Musa DNA and the site on the BSV genome corresponding to the 5' end of the viral 35S RNA. PCR with a primer based on the Musa sequence upstream of this interface and successive primers through the BSV genomic sequence show intact BSV sequence up to about nt 5500, after which the sequence becomes fragmented and inverted. Sequenced regions show very high homology (99%) to the sequenced episomal virus. Fluorescent in situ hybridization with labelled segments of BSV shows integration of virus sequence into mitotc chromosomes of Musa. The number of loci is determined to be at least 3 in the breeding line Obino L' Ewai, and it is present in varying numbers in other Musa accessions. Fluorescent in situ hybridization with labelled segments of BSV and the associated Musa sequence to fibres of Musa DNA stretched to its molecular length showed adjacent localisation of the probes. Two types of structure were observed for stretched fibres from cv. Obino L'Ewai: one spanned ca 50 kb and included 5-10 repeats of the BSV and adjacent probe, with some smaller repeated structures and inversions. The other structure spanned less than 25 kb and included 3-5 repetitions.

The data above show unequivocal evidence of the integration of BSV sequences into Musa germplasm. By analogy to retrotransposons, activation of these sequences can be induced by stresses, e.g. tissue culture. The integrant, essentially identical in sequence to the previously isolated and sequenced episomal BSV, is a possible candidate for the observed 'epidemic' of BSV encountered in the progeny of recent Musa breeding programmes. This phenomenon of integrated viral sequences giving rise to episomal infections, known in vertebrates, is so far unique in plants. The aim of future work is to characterise activatable integrant(s). This will allow the development of diagnostics which will show the potential risk of cultivars becoming infected following stress. An understanding of the integrant(s) will be important in determining approaches to eliminating or controlling the problem.

References
1. Lockhart BEL, 1986. Phytopathology 76, 995-999.
2. Harper G, Hull R, 1998. Virus Genes. 3. Harper G, Dahal G, Hull R, 1996. BCCP Proceedings 65. BCCP, Surrey, UK, pp. 47-51.