Detection of pineapple bacilliform virus using the polymerase chain reaction

A polymerase chain reaction (PCR) assay has been developed to detect pineapple bacilliform virus (PBV) in extracts from infected plants. Initially, degenerate primers were designed from conserved badnavirus amino acid sequences and used in PCR with partially purified PBV preparations. A 448 bp DNA fragment was amplified from a region in the reverse transcriptase and ribonuclease H genes. The nucleotide sequence of the cloned PCR product indicated that PBV was related to, but distinct from, other badnaviruses. Specific primers designed from the PBV sequence yielded a 403 bp fragment when used in PCR with extracts from infected pineapple plants, but not from plants infected with sugarcane bacilliform or banana streak viruses (genus Badnavirus). The specificity of the PCR product was confirmed by Southern hybridisation using a digoxigenin labelled DNA probe. PBV appears to be present in all pineapple growing areas along the east coast of Australia. PBV was detected in plants grown from seeds, plants propagated through meristem tip culture and in mealybugs which were collected from infected plants. PBV was detected in crown, leaf and root tissue from infected pineapple plants. PCR results of all field-infected samples were confirmed by immunoelectron microscopy

Identification of functional sequences in the pregenomic RNA promoter of the Banana streak virus Cavendish strain (BSV-Cav)

The promoter regions of plant pararetroviruses direct transcription of the full-length viral genome into a pregenomic RNA that is an intermediate in the replication of the virus. It serves as template for reverse transcription and as polycistronic mRNA for translation to viral proteins. We have identified functional promoter elements in the intergenic region of the Cavendish isolate of Banana streak virus (BSV-Cav), a member of the genus Badnavirus. Potential binding sites for plant transcription factors were found both upstream and downstream of the transcription start site by homology search in the PLACE database of plant cis-acting elements. The functionality of these putative cis-acting elements was tested by constructing loss-of-function and regain-of-function mutant promoters whose activity was quantified in embryogenic sugarcane suspension cells. Four regions that are important for activity of the BSV-Cav promoter were identified: the region containing an as-l-like element, the region around-141 and down to -77, containing several putative transcription factor binding sites, the region including the CAAT-box, and the leader region. The results could help explain the high BSV-Cav promoter activity that was observed previously in transgenic sugarcane plants and give more insight into the plant cell-mediated replication of the viral genome in banana streak disease. (C) 2004 Elsevier B.V. All rights reserved

A variable region of the sugarcane bacilliform virus (SCBV) genome can be used to generate promoters for transgene expression in sugarcane

Four promoters derived from sugarcane bacilliform virus (SCBV) were compared and characterised. Three were obtained by PCR amplification of purified virion DNA extracted from three sugarcane cultivars. The fourth promoter was obtained by subcloning from an almost genome-length clone of SCBV. All promoters were able to drive stable expression of b-glucuronidase in sugarcane. The PCR-derived promoter sequences shared more DNA homology with banana streak virus than to the subcloned SCBV. The subcloned promoter was the strongest expressing and was able to drive reporter gene expression in vitro and in the leaves, meristems and roots of glasshouse-grown sugarcane. Expression levels were at least equal to or higher than those measured for the maize polyubiquitin promoter

Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids

Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi-persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80-150 × 25-30 nm, similar to those of badnaviruses. A 1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR-amplified viral DNA fragment, RYNV-specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus-like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV-infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field-grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV-infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes