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WO1996038564A1 - Sequences d'adn du virus de la ramification de l'extremite de la banane - Google Patents

Sequences d'adn du virus de la ramification de l'extremite de la banane Download PDF

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WO1996038564A1
WO1996038564A1 PCT/AU1995/000311 AU9500311W WO9638564A1 WO 1996038564 A1 WO1996038564 A1 WO 1996038564A1 AU 9500311 W AU9500311 W AU 9500311W WO 9638564 A1 WO9638564 A1 WO 9638564A1
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component
bbtv
components
sequence
dna
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PCT/AU1995/000311
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Mirko Karan
Thomas Michael Burns
James Langham Dale
Robert Maxwell Harding
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Queensland University Of Technology
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Priority to AU25570/95A priority Critical patent/AU712544B2/en
Priority to PCT/AU1995/000311 priority patent/WO1996038564A1/fr
Publication of WO1996038564A1 publication Critical patent/WO1996038564A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • TITLE "DNA SEQUENCES OF BANANA BUNCHY TOP VIRUS" FIELD OF INVENTION
  • BBTV banana bunchy top virus
  • banana (Musa spp.) is the world's largest fruit crop by value. As with most important food crops, bananas are affected by a number of serious diseases, particularly fungal and viral diseases.
  • BBTD Banana bunchy top disease
  • the disease has always been assumed to be caused by a virus and this presumed virus was classified as a possible luteovirus based on biological properties i.e. the disease is transmitted by aphids (Pentalonia nigronervosa) in a persistent manner, the phloem of infected plants is damaged, the major symptoms are marginal yellowing, stunting and leaf bunching and the disease is not sap transmissible.
  • aphids Pieris nigronervosa
  • VLPs 18-20 nm in diameter. These VLPs contained ssDNA of about 1 kb and a single protein of Mr 20,100.
  • BBTV has characteristics different from those of any described plant virus group; it is most similar to the geminiviruses, which have ssDNA as their genomic nucleic acid but their DNA is about 2.7 to 3.0 kb, their particles are usually geminate and they are transmitted by leafhoppers or whiteflies.
  • SCSV subterranean clover stunt virus
  • CFDV coconut foliar decay virus
  • SCSV is composed of at least seven distinct ssDNA molecules each containing one large open reading frame (ORF) as reported in Chu et al., 1990, Vlllth International Congress of Virology Abstracts p125.
  • ORF open reading frame
  • Such ssDNA molecules also contain a strong stem-loop sequence as described in Waterhouse et al., 1990, Vlllth International Congress Virology, Berlin.
  • SCSV was further characterized in Surin et al., 1993, IXth International Congress of Virology Abstracts, Glasgow, Scotland whereby each component contained a conserved stem and loop structure and a non coding region in at least five of the components.
  • the sequence of one ssDNA molecule of CFDV has been determined as discussed in Rohde et al., 1990, Virology 176 648-651 and one of the ORFs encodes a putative replicase.
  • PCV porcine circovirus
  • CAV chicken anaemia virus
  • PPFDV psittacine beak and feather disease virus
  • BBTV component 1 from isolates from 10 different countries being cloned and sequenced and the sequences were subsequently aligned and compared.
  • the mean sequence difference within each group was 1.9 to 3.0% and between isolates from the two groups were approximately 10%, but some parts of the sequences differed more than others.
  • the protein encoded by the major open reading frame differed by approximately 5%.
  • ssDNA single-stranded DNA
  • component 1 is 1 ,110 nucleotides in length and shares 98% nucleotide sequence identity with the BBTV DNA component 1 of the Australian isolate as described in Harding et al. (1993) above.
  • This component contains two open reading frames (ORF) capable of encoding a protein of 33.5 kDa, which may function as a replicase, and a protein about 15.2 kDa, with unknown functions.
  • Component 3 is 1 ,057 nucleotides in length and does not contain any ORFs larger than 10 kDa.
  • Component 4 is 1 ,017 nucleotides in length and potentially encodes a protein of 18.9 kDa. All three ssDNA components have a stem-loop sequence and have a conserved non-coding region. The sequence of each of these three components is different from that of BBTV DNA components of two Taiwanese isolates. BBTV-specific clones were used in dot-blot hybridisation assays for detection of BBTV in plants using radioactive and non-radioactive probes. A polymerase chain reaction (PCR) assay was developed for detection of BBTV in banana samples and single aphids. Dot-blot hybridisation assays were as sensitive as enzyme-linked immunosorbent assay (ELISA) while PCR was 1 ,000 times more sensitive than dot-blot and ELISA assays for detection of BBTV in bananas.
  • ELISA
  • Component 3 consists of about 1 ,075 base pairs
  • Component 4 consists of about 1 ,043 base pairs
  • Component 6 consists of about 1 ,089 base pairs
  • the invention thus includes within its scope the sequence or part thereof of BBTV DNA component 3 of the BBTV genome and sequences that are complementary to this sequence.
  • Component 3 consists of approximately 1075 nucleotides. This sequence is shown in FIG. 1.
  • the invention also includes within its scope the sequence or part thereof of BBTV DNA component 4 of the BBTV genome and sequences that are complementary to this sequence.
  • Component 4 consists of approximately 1043 nucleotides. This sequence is shown in FIG. 2.
  • the invention also includes within its scope the sequence or part thereof of BBTV DNA component 6 of the BBTV genome and sequences that are complementary to this sequence.
  • Component 6 consists of approximately 1089 nucleotides. This sequence is shown in
  • the virion sense DNA strand of each components 3, 4 and 6 includes an open reading frame.
  • Each ORF in the components 3, 4 and 6 have a potential TATA box and one or two potential polyadenylation signals associated with it and each polyadenylation signal had an associated GC-rich region containing the trinucleotide sequence TTG.
  • a number of ORFs were identified in component 2 reported in the Xie et al. (1995) reference above but none of these had appropriately located potential TATA boxes and polyadenylation signals associated with them. None of the ORF amino acid sequences nor the full DNA sequences of any of the components 3, 4 and 6 had significant sequence homology with any known protein or nucleic acid sequences.
  • the ORF of component 3 has a nucleotide sequence of 525 nucleotides encoding an amino acid sequence of approximately 175 amino acid residues for a 20.11 KDa protein.
  • the ORF of component 3 appears to encode the BBTV coat protein.
  • the ORF of component 4 has a nucleotide sequence of 351 nucleotides encoding an amino acid sequence of 117 amino acid residues for a protein of 13.74KDa.
  • the ORF of component 4 encoded a 30 residue hydrophobic domain which may indicate that this ORF encoded a transmembrane protein.
  • the ORF of component 6 has a nucleotide sequence of 462 encoding 154 amino acid sequence of a 17.4KDa protein.
  • each of the components 3, 4 and 6 are substantially non-homologous both with each other as well as components referred to in the Xie et al. reference mentioned above. This will be clearly demonstrated by reference to FIGS. 1 , 2 and 3.
  • Each of component 3, 4 and 6 contained a conserved stem-loop structure and a nonanucleotide potential TATA box which was 5' of the large virion sense ORF.
  • the stem-loop structures were incorporated in a common region (CR-SL) of 69 nucleotides which was
  • Each of components 3, 4 and 6 also contained a major common region (CR-M) which was located 5' of the CR-SL in each component, in the non-coding region and was 76% homologous over 92 nucleotides.
  • CR-M contained a near complete 16 nucleotide direct repeat and a GC-box which was similar to the rightward promoter element found in wheat dwarf geminivirus.
  • the invention also covers DNA sequences that can hybridize to any one or part thereof of the sequences of components 3, 4, 6 or their complementary sequences wherein sequences varying within 35% can hybridise under standard stringency conditions. Suitable hybridisation procedures and stringency conditions are given in Burns et al., 1994, Arch Virol. 137 371-380.
  • the invention also includes synomonous DNA sequences that encode the same protein as encoded by components 3, 4 and 6, and amino acid sequences encoded any of the aforementioned DNA sequences.
  • a further aspect of the present invention is the use of the abovementioned sequences or part thereof, of their complementary sequences or part thereof, of variations of these sequences within 35% of any one of the sequences or part thereof, of variations of their complementary sequences within 35% of any one of the sequences or part thereof as either DNA or RNA. Use includes:-
  • nucleic acid sequences of the invention may be inserted into a plasmid such as pBin19 or pUC19 between a cauliflower mosaic virus 35S promoter and a cauliflower mosaic virus 35S terminator.
  • plasmids may be introduced into cells of plants. Introduction may be achieved by using Agrobacterium tumefaciens (pBin19 construct) or microprojectile bombardment (pBin19 or pUC19 constructs). Within the plant cells, the inserted nucleic acid sequences may be suitably transcribed and/or translated.
  • sequences covered by this invention may be inserted upstream or downstream of a second nucleic acid sequence wherein the transcription and/or translation of the second nucleic acid sequence is altered or facilitated by the nucleic acid sequences of the invention.
  • FIG. 1 illustrates the nucleotide sequence of Component 3
  • FIG. 2 illustrates the nucleotide sequence of Component 4
  • FIG. 3 illustrates the nucleotide sequence of Component 6;
  • FIGS. 4a, 4b and 4c illustrate the nucleotide sequence of Components 3, 4 and 6 respectively and deduced amino acid sequences of their ORFs;
  • FIG. 5 illustrates the determination of the virion-sense orientation of BBTV DNA components 2 to 6.
  • FIG. 6 illustrates the aligned stem-loop common regions (CR-SL) of BBTV DNA component 1 to 6;
  • FIG. 7 illustrates the aligned major common regions (CR-M) of BBTV DNA components 1 to 6
  • FIG. 8 illustrates the diagrammatic representation of the proposed genome organisation of BBTV
  • FIG. 9 illustrates the N-terminal sequencing of BBTV coat protein.
  • the dsDNA was treated with mung bean nuclease (Promega) and ligated into S al digested plasmid vector pUC18 (Upcroft & Healey, 1987, Gene 51 69-75).
  • the plasmid was then used to transform Escherichia coli strain JM109 (Hanahan, 1983, Journal of Molecular Biology 166 557-580) and potential recombinant clones were identified by screening on X-gal substrate (Vieira & Messing, 1982, Gene 19259-268)
  • Plasmids were isolated using the alkaline lysis method (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory). Inserts were excised by digestion with EcoR ⁇ IHind ⁇ , electrophoresed in agarose gels and capillary blotted onto Hybond N+ (Amersham) using 0.4 M NaOH (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory). Inserts for use as DNA probes were purified from agarose gels using a Gene-Clean kit (Bresatec). DNA probes were labelled using a Ready-To-Go labelling kit (Pharmacia) as recommended by the manufacturer. Prehybridisations and hybridisations were done as described by Burns et al., 1994, Archives of Virology 137 371-380.
  • Sequenase kit (US Biochemicals) as recommended by the manufacturer. Reaction products were electrophoresed in 8% (w/v) polyacrylamide gel containing 7 M urea. Gels were fixed, dried and exposed to X-ray film. Primers used for sequencing were either universal sequencing primers or 17-30 nt primers complementary to appropriate regions of the cloned viral DNA synthesised using an Applied Biosystems (ABI) PCR Mate and processed as recommended by the manufacturer.
  • ABS Biosystems Applied Biosystems
  • PCR products for sequencing were purified from agarose gels using a Gene-Clean kit (Bresatec). DNA was sequenced using a Sequenase kit (USB) essentially as described by the manufacturer. Denaturation of template DNA (500 ng) was done by boiling following the addition of DMSO and 3 pmoles of sequencing primer.
  • USB Sequenase kit
  • Nucleotide sequences were analysed using the GCG analysis package version 8 available through the ANGIS computing facility at the University of Sydney, Australia. Nucleotide and amino acid sequences were aligned using the Clustal V software package (Higgins et al., 1991 , CABIOS 8 189-191 ).
  • primer A 5' GCATCCAACGGCCCATA 3'
  • primer B 5' CTCCATCGGACGATGGA 3'
  • primer C 5' TATTAGTAACAGCAACA 3'
  • primer D 5' CTAACTTCCATGTCTCT 3'
  • primer E 5' CGGGa/tATa/cTGATTGt/gGT 3'
  • primer F 5' TACa/tTTTGTCATAGc/tGT 3'
  • the amplified products were cloned using the TA cloning kit (Invitrogen) into the plasmid vectors pCRII or pCR2000 as recommended by the manufacturer or into T-tailed pUC19 and Bluescript (Marchuk et al., 1990, Nucleic Acids Research 19 1154).
  • Recombinant clones were selected using X-gal substrate on Luria Bertani (LB) agar containing the appropriate antibiotic and plasmids isolated using the alkaline lysis method (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory). Clones with apparent full-length inserts (approximately 1 kb) were selected for sequencing.
  • BBTV ssDNA was extracted, electrophoresed in agarose and capillary blotted onto duplicate nylon membranes (Harding et al., 1993, Journal of General Virology 74 323- 328).
  • RNA transcripts of full-length BBTV clones of each of the four components were synthesised using a riboprobe in vitro transcription kit (Promega) as recommended by the manufacturer.
  • RESULTS Cloning and sequencing ofgenomic components 3, 4 and 6 These genomic components of BBTV were cloned and sequenced from two libraries, (i) a random primed library and (ii) a PCR library, (i) Randon Primed Library
  • a random primed library was generated from BBTV ssDNA extracted from purified virions.
  • the resultant dsDNA was treated with mung bean nuclease, blunt-end ligated into Smal cut pUC18 and cloned into E. coli JM109.
  • This library was screened with 32 P-iabelled DNA from BBTV virions, healthy bananas and the insert from pBT338 which was a partial clone of BBTV DNA component 1 (Harding et al., 1991 , Journal of General Virology 72 225-230; Harding et al., 1993, Journal of General Virology 74 323-328).
  • BBTV DNA Component 6 Two clones from the random primed library, pBTRP-P1 and P2, hybridised with labelled BBTV virion DNA but not with DNA from healthy bananas or pBT338. However, the inserts of these clones, both of approximately 1 kb, were digested with EcoRV whereas neither components 1 nor 2 had EcoRV sites. The two clones were partially sequenced using universal forward and reverse primers. The sequences of both clones were identical but clearly different to those of components 1 and 2. Again, two immediately adjacent, outwardly extending primers, primers C and D, were designed from the sequence and synthesised.
  • BBTV virion ssDNA was used as a template with these two primers in a PCR reaction and the resultant product cloned into a T-tailed Bluescript vector.
  • One apparent full length clone, pBT- P2A1 was selected and sequenced in both directions from subclones generated by exonuclease III digestion and universal forward and reverse, and sequence-specific primers.
  • the final component 6 sequence of 1089 bp was then compiled and a deduced amino acid sequence of the major ORF is shown in FIG. 4c. ( ⁇ ) PCR Library
  • the first region later defined as the stem-loop common region (CR-SL) included the potential stem/loop sequence previously identified in component 1 (Harding et al., 1993, Journal of General Virology 74 323-328); the second region, which was contained within the region later defined as the major common region (CR-M), was a sequence of approximately 66 nucleotides 5' to the stem- loop sequence.
  • CR-SL stem-loop common region
  • M major common region
  • BBTV genomic components should contain a CR-M and therefore two immediately adjacent, outwardly extending degenerate primers, primers E and F, were designed from this region, synthesised and extended by PCR using BBTV virion ssDNA as a template (Burns et al., 1994, Archives of Virology 137 371-380). Seven products, each of approximately 1 kb, were resolved by polyacrylamide gel electrophoresis. The products were cloned into pCRII.
  • the resultant clones were divided into three groups, groups B, C and D, on the basis that they hybridised with BBTV virion DNA but not DNA from healthy bananas and that each group had restriction patterns different to the other two groups and to components 1 , 2 and 6 (Burns et al., 1994, Archives of Virology 137 371-380).
  • One group, group A had a restriction pattern indistinguishable from that of component 2 and it was later confirmed by sequencing that group A clones represented clones of component 2.
  • Each group of clones was assumed to represent a new and unique BBTV DNA component.
  • three clones (component 3) or four clones (components 4 and 5) were partially sequenced using universal forward and reverse primers.
  • each of these groups of clones were generated using degenerate primers covering a sequence of 34 nucleotides derived from the conserved CR-M of components 1 and 2.
  • the CR-M from components 1 and 2 was not fully conserved and thus it was expected that the hypothesised CR-M sequence would vary between other components. Therefore, converging primers unique to each component were designed and used to amplify a sequence including CR-M for each component from BBTV virion ssDNA. The resultant PCR product was sequenced directly using the two component specific converging primers.
  • Component 3 (Group C clone pBTP-64) was sequenced in both directions from the original clone and from subclones generated by exonuclease III digestion or restriction fragments using universal forward and reverse primers and three sequence-specific primers. Two additional converging primers were designed from this sequence to amplify a 380 bp product including the CR-M. The sequence of this product was identical to that of pBTP-64 except for five single nucleotide changes, four of which were in the sequence covered by the original degenerate primers and one outside this sequence at nucleotide 947. The final component 3 sequence of 1075 bp was then compiled as shown in FIG. 4a.
  • Component 4 (Group D clone pBTP-62) was sequenced in both directions from the original clone and from subclones generated by exonuclease III digestion using universal forward and reverse primers and three sequence-specific primers. Two additional converging primers were designed from this sequence to amplify a 350 bp product including the CR-M. The sequence of this product was identical to that of pBTP-62 except for two single nucleotide changes in the sequence covered by the original degenerate primers. The final component 4 sequence of 1043 bp was then compiled as shown in FIG. 4b.
  • BBTV genome is encapsidated as single-stranded DNA (Harding et al., 1991 , Journal of General Virology 72 225-230; Harding et al., 1993, Journal of General Virology 74 323-328).
  • strand-specific DNA or RNA probes specific for each component were synthesised and hybridised with BBTV virion DNA.
  • Component 2 specific probes were two 3' end-labelled 30mer oligonucleotides whereas probes specific for components 3, 4, 5 and 6 were SP6, T3 or T7 promoted 32 P-labelled RNA transcripts.
  • the probes whose sequences were complementary to the component sequences presented in FIGS.
  • FIGS. 4a, 4b and 4c hybridised strongly to BBTV virion DNA whereas the probes whose sequences were the same as the FIGS. 4a, 4b and 4c sequences did not hybridise (FIG. 5). This indicated that each component was encapsidated as ssDNA and only in one orientation, that presented in FIGS. 4a, 4b and 4c.
  • strand- and component-specific probes that hybridised with BBTV virion ssDNA were used as probes to demonstrate that each component was associated with banana bunchy top disease.
  • Plant DNA extracts from three (for component 2) or four (for components 3, 4, 5 and 6) different BBTV isolates and DNA from four healthy bananas was Southern blotted and hybridised with each probe.
  • Each component- specific probe hybridised with a low molecular weight DNA of expected size in all the extracts from BBTV-infected bananas but did not hybridise with the extracts from healthy bananas (results not shown). This indicated that each component was clearly associated with the disease and the virus.
  • FIG. 5 the data for component 5 is shown for the sake of comparison. Analysis of the BBTV genomic components
  • sequences of the BBTV genomic components presented here and the sequence of component 1 were aligned and compared. Each of the six sequences were different except for two significant regions which had varying degrees of homology between all six components.
  • Stem-loop common repion We have previously identified a potential stem-loop structure in BBTV component 1 (Harding et al., 1993, Journal of General Virology 74 323-328) which had a loop sequence almost identical to the invariant loop sequence of geminiviruses (Lazarowitz, 1992, Critical Reviews in Plant Sciences.11 327-349). An equivalent stem/loop structure was also found in components 2 to 6 (FIG. 6). Each component had an 11 nucleotide loop sequence of which 9 consecutive nucleotides were conserved between all components. Each component also had a 10 bp stem sequence of which 14 nucleotides were fully conserved.
  • the region of homology extended up to 25 nucleotides 5' of the stem-loop structure and up to 13 nucleotides 3' of the stem/loop structure.
  • the 5' 25 nucleotides were fully conserved between components 1 , 3, 4 and 5. There were apparently two deletions in both components 2 and 6.
  • eight nucleotides were fully conserved with components 1 , 3, 4 and 5 whereas in component 6, 16 nucleotides were conserved with these other components.
  • the 13 nucleotides 3' of the stem-loop were fully conserved between all six components except for an apparent single nucleotide deletion in component 2.
  • the sequence of up to 69 nucleotides including the stem-loop sequence was termed the stem-loop conserved region or CR-SL.
  • the second common region was located at various distances 5' of the CR-SL and was called the Major Common Region or CR-M. This region varied in size from 65 nucleotides in component 1 to 92 nucleotides in component 5 (FIG. 7).
  • Component 1 apparently had the first 26 nucleotides of the CR-M deleted as well as a further single nucleotide deletion.
  • Components 2, 3 and 4 had two single nucleotide deletions and component 6 had one single nucleotide deletion. Forty-five nucleotides were conserved between all components and 23 of the first 26 nucleotides, deleted in component 1 , were conserved between components 2 to 6.
  • components 2 to 6 there was an almost complete 16 nucleotide direct repeat (ATACAAc/gACa/gCTATGA) from nucleotides 4 to 20 and 21 to 36. Further, a 15 nucleotide GC rich sequence (average of 86% GC) was located from nucleotides 78 to 92 and was 93% conserved between all components. The sequence between the last nucleotide of the CR-M and the first nucleotide of the CR-SL varied in length from 22 nucleotides in component 1 to 233 nucleotides in component 2 (FIG. 6). Interestingly, this sequence of 175 nucleotides in components 3 and 4 was 97% conserved between these two components.
  • ATACAAc/gACa/gCTATGA 16 nucleotide direct repeat
  • a potential TATA box was identified in BBTV component 1 and was located 20 nucleotides 3' of the last nucleotide of the stem-loop and 43 nucleotides 5' of the start codon of the putative replicase gene (Harding et al., 1993, Journal of General Virology 74 323-328). Similar potential TATA boxes were also identified in components 2 to 6. In each of these components, the potential TATA box was a nine nucleotide sequence, CTATa/ta/tAt/aA, and was located downstream from the stem-loop sequence (FIGS. 4a, 4b and 4c).
  • ORF coding for a putative replicase was identified in the virion sense of BBTV component 1 which had a potential TATA box 43 nucleotides 5' of the ATG start codon and a polyadenylation signal 13 nucleotide 5' of the stop codon (Harding et al., 1993, Journal of General Virology 74 323-328).
  • Components 2 to 6 were therefore analysed for ORFs in both the virion and complementary sense that could code for proteins of more than 25 amino acids. Numerous such ORFs were identified in both orientations in all five components. However, only four ORFs were identified that had associated with them a potential 5' TATA box and an appropriately located polyadenylation signal.
  • Components 3 to 6 each had one such ORF in the virion sense. These ORFs were (i) 525 nucleotides potentially coding for a 175 amino acid protein of 20.11 K in component 3, (ii) 351 nucleotides potentially coding for a 117 amino acid of 13.74K in component 4, (iii) 483 nucleotides potentially coding for a 161 amino acid protein of 18.97K in component 5 and (iv) 462 nucleotides potentially coding for a 154 amino acid protein of 17.4K in component 6 (FIGS. 4a, 4b and 4c).
  • ORFs were identified in component 2, four in the virion sense and five in the complementary sense (Table 1 ). However, none of these ORFs had appropriately located nonanucleotide potential TATA boxes and polyadenylation signals and therefore were unlikely to be transcribed.
  • Virology 176 648-651 A model for implicating the loop sequence in rolling circle replication has been described for - geminiviruses (Saunders et al., 1993, DNA forms of the geminivirus - African cassava mosaic virus - consistent with the rolling circle mechanism of replication. IXth International Congress of Virology, Glasgow, August, 1993. Abstract P60-18). It is possible that the loop sequence in BBTV has a similar function. The stem-loop sequences were also highly conserved in all BBTV components and contained the pentanucleotide sequence TACCC which has been shown to be the site for initiation of viral strand DNA synthesis in wheat dwarf geminivirus (Heyraud et al., 1993, EMBO Journal 12 4445-4452).
  • the major common region was identified in all components and was located 3' of the major ORF (except for component 2 where no major ORF was identified) and 5' of the CR-SL (FIG. 8). Hexanucleotide repeats were identified within the CR-M in all three components. However, no function could be directly attributed to these repeats but they may be associated with, or part of promoter sequences.
  • the CR-M also contained a 15 nt GC-rich sequence located at the 3' end and had the potential to form a small stem-loop structure.
  • This GC-rich sequence also contained two direct GC-repeats which resembled the Sp1 binding sites found in promoters of genes in animal cells and viruses (Fenoll et al., 1990, Plant Molecular Biology 15 865-877).
  • a similar promoter in the monocot-infecting maize streak geminivirus has been shown to be required for maximal rightward transcription and also appeared to bind maize nuclear factors in a non-cooperative manner (Fenoll et ai, 1990, Plant Molecular Biology 15 865-877).
  • the nucleotide length and sequence between the CR-M and CR-SL was dissimilar in four of the six components. However, in components 3 and 4, this 175 nucleotides region was 97% homologous and the 334 nucleotides from the 5' end of the CR-M to the 3' end of the CR-SL were 98% homologous.
  • a similar large common region of 300 nucleotides has been found in geminiviruses and is identical between the A and B components of individual bipartite geminiviruses (Lazarowitz, 1992, Geminiviruses: genome structure and gene function. 2).
  • Components 3, 4 and 6 each had one large ORF in the virion sense, 3' of the CR-SL. Each of these ORFs had potential conserved TATA boxes and polyadenylation signals associated with them (FIG. 8).
  • the potential TATA boxes highly conserved with the nonanucleotide sequence CTATa/ta/tAa/tA which was essentially similar to that described by Bucher et al., 1990, Journal of Molecular Biology 212 563-578.
  • the distance between the potential TATA box and the translation initiation codon varied in each component from 13 nucleotides in component 3 to 102 nucleotides in component 1.
  • An ATGG translation initiation codon was identified in the five components encoding large ORFs.
  • ORFs of components 3, 4 or 6 None of major ORFs of components 3, 4 or 6 had significant sequence homology either at the DNA or protein level with any other available sequences and no functions could be assigned to the putative proteins.
  • the ORFs of components 3 and 5 encoded proteins of a size similar to that of the BBTV coat protein, 20.1 KDa (Harding et al., 1991 , Journal of General Virology 72 225-230). Further, a sequence of 30 hydrophobic amino acids were identified near the N-terminal end of the putative protein of the component 4 ORF which is characteristic of in- or trans-membrane domains.
  • BBTV coat protein in semi-purified virus preparation 1.
  • Semi-purified BBTV was prepared from 1 kg of infected mid ⁇ rib tissue using the extraction method of Harding et al., 1991 , Journal of General Virology 72 225-230, omitting the final casesium sulphate density gradient step.
  • the virus preparation was mixed with SDS-gel sample buffer (Tris, %glycerol) and electrophoresed in duplicate 4%/12% polyacrylamide gels (BioRad Mini-Protean II). 3.
  • the BBTV-specific band was identified by mapping the immunoreactive band with the banding pattern on coomassie-stained polyacrylamide gel.
  • the semi-purified BBTV was electrophoresed on 4%/12% polyacrylamide gel.
  • the PVDF was stained lightly with coomassie blue R-250 (BioRad) and destained with 12% v/v methanol.
  • BBTV-specific band was then excised and sequenced by Edman degradation method.
  • the amino acid sequence obtained was compared to the major open reading frames (ORF) of the six known BBTV component. This comparison revealed very high homology between the amino acid sequence obtained and one component of BBTV.
  • ORF major open reading frames
  • the 18 residues obtained from direct sequencing has 60% identity to residues 2-19 of the protein encoded by BBTV-3 major ORF. Up to 85% identity level was obtained when the first residue of the unknown sequence, which is always the least accurate residue, was discounted (see FIG. 9).
  • the protein encoded by BBTV-3 major ORF was used in a search of protein database using Blast (Altschul et al., 1990, Journal Molecular Biology 215 403- 410).
  • SCSV5 subterranean clover stunt virus DNA component-5
  • BBTV-3 encodes the coat protein of BBTV.
  • BBTV-3 encoded protein also show considerable homology to the reported coat protein of subterranean clover stunt virus, a virus probably belonging to the same group of new ssDNA plant viruses as BBTV.
  • Table 1 The open reading frames contained within BBTV component 2 potentially coding for proteins of more than 2K.
  • FIG. 1 complementary strand
  • FIG. 1 The nucleotide sequence of component 3 (Queensland)
  • the potential TATA boxes are in bold and double underlined; the potential polyadenylation signals are in bold and underlined; the stem- loop structure is in italics and underlined, with the stem sequence arrowed; the CR-SL is underlined; the CR-M is in bold and italics; and the
  • FIG. 4a - corresponds to Component 3
  • FIG. 4b - corresponds to Component 4
  • FIG. 4c - corresponds to Component 6.
  • blots were separately probed with either 32 P-labelled oligonucleotides (component 2) or full length RNA transcripts (components 3 to 6) specific for the virion- or complementary-sense strands of each respective component.
  • component 2 full length clone of each respective component
  • Lane 3 DNA extracted from purified BBTV virions.
  • CR-SL The aligned stem-loop common regions (CR-SL) of BBTV DNA component 1 to 6.
  • the stem-loop structure in each component is underlined and the loop sequence is in italics.
  • Asterisks indicate nucleotides that are conserved between all components. Dots have been included in some sequences to maximise sequence alignment.
  • the 15 nucleotide GC-rich sequence is underlined.
  • Asterisks indicate nucleotides that are conserved between all components and triangles indicate nucleotides that are conserved between components 2 to 6 in the first 26 nucleotides covering the deletion in component 1. Dots have been included in some sequences to maximise sequence alignment and the imperfect repeat sequences are shown in italics.
  • FIG. 9 N-terminal sequencing of BBTV coat protein. Translation of BBTV-3 major ORF (170 amino acids). Amino acid sequence obtained from Edman degradation is displayed in italics. Identical residues are marked with (:).

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Abstract

L'invention concerne le virus de la ramification de l'extrémité de la banane (BPTV) et les séquences d'ADN des constituants 3, 4 et 6. Ces séquences d'ADN peuvent servir à détecter l'infection par le BPTV et permettent l'expression de gènes dans les plantes.
PCT/AU1995/000311 1995-05-30 1995-05-30 Sequences d'adn du virus de la ramification de l'extremite de la banane WO1996038564A1 (fr)

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AU25570/95A AU712544B2 (en) 1995-05-30 1995-05-30 DNA sequences of banana bunchy top virus
PCT/AU1995/000311 WO1996038564A1 (fr) 1995-05-30 1995-05-30 Sequences d'adn du virus de la ramification de l'extremite de la banane

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0785999A4 (fr) * 1994-08-30 1998-05-06 Commw Scient Ind Res Org Regulateurs de transcription vegetale issus de circovirus
EP0832212A4 (fr) * 1995-05-31 2002-06-12 Univ Queensland Regions intergeniques du virus de ramification de l'extremite de la banane (bbtv)
CN103290138A (zh) * 2012-02-23 2013-09-11 陈艳 香蕉束顶病毒的实时pcr快速检测方法

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
ARCHIVES OF VIROLOGY, Volume 137(3-4), issued 1994, BURNS et al., "Evidence that Banana Bunchy Top Virus has a Multiple Component Genome", pages 371-380. *
JOURNAL OF GENERAL VIROLOGY, Volume 72, No. 2, issued 1991, HARDING R. et al., "Virus Like Particles Associated with Banana Bunchy Top Disease Contain Small Single Stranded DNA", pages 225-230. *
JOURNAL OF GENERAL VIROLOGY, Volume 72, No. 2, issued 1991, THOMAS J. et al., "Purification, Characterisation and Serological Detection of Virus-Like Particles Associated with Banana Bunchy Top Disease in Australia", pages 217-224. *
JOURNAL OF GENERAL VIROLOGY, Volume 74, No. 3, issued 1993, HARDING et al., "Nucleotide Sequence of One Component of the Banana Bunchy Top Virus Genome Contains a Putative Replicase Gene", pages 323-328. *
JOURNAL OF GENERAL VIROLOGY, Volume 75(12), issued 1994, KARAN et al., "Evidence for Two Groups of Banana Bunchy Top Virus Isolates", pages 3541-3546. *
PHYTOPATHOLOGY, Volume 84, No. 9, issued 1994, WU et al., "Nucleotide Sequences of Two Circular Single Stranded DNA's Associated with Banana Bunchy Top Virus", pages 952-958. *
VIROLOGY, Volume 198(2), issued 1994, YEH et al., "Genome Characterization and Identification of Viral Associated dsDNA Component of Banana Bunchy Top Virus", pages 645-652. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0785999A4 (fr) * 1994-08-30 1998-05-06 Commw Scient Ind Res Org Regulateurs de transcription vegetale issus de circovirus
EP0832212A4 (fr) * 1995-05-31 2002-06-12 Univ Queensland Regions intergeniques du virus de ramification de l'extremite de la banane (bbtv)
CN103290138A (zh) * 2012-02-23 2013-09-11 陈艳 香蕉束顶病毒的实时pcr快速检测方法

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AU712544B2 (en) 1999-11-11

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