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Yam is an important food staple in many Pacific Island countries, but its production is limited by several factors including virus infection. The presence of viruses also hinders the exploitation of the crop by restricting the international dissemination of yam germplasm due to quarantine concerns. In this study, a total of 224 yam accessions present in the Pacific germplasm collection at SPC-CePaCT, Fiji, were tested for the presence of badnaviruses using a combination of rolling circle amplification (RCA) and sequencing. Of these, 35 tested positive for either Dioscorea bacilliform AL virus (DBALV) or Dioscorea bacilliform AL virus 2 (DBALV2). DBALV was only detected in samples from Vanuatu and Tonga, while DBALV2 was restricted to Papua New Guinean samples. Ten complete genome sequences of DBALV and DBALV2 were obtained and pairwise comparison of the partial reverse transcriptase/ribonuclease H (RT/RNase H)-coding sequence revealed identities ranging from 89% to 100% and 92% to 100%, respectively. The results suggest that DBALV and DBALV2 are the prevalent badnavirus species infecting yams in the Pacific region.

Genetic improvement of commercially accepted banana cultivars is strongly reliant on the ability to introduce genes that encode important agro-traits such as disease resistance. In most cases this can only be achieved using a transgenic approach. Public and regulatory acceptance of these events would greatly increase with “clean” single copy integration events free of the selectable marker gene and extraneous vector backbone. This would also allow for the successive addition of new genes and traits as they become available. In this study, we used the pMarker Free 1 (pMF1) vector containing the green fluorescent protein (gfp) reporter gene to assess the effectiveness of steroid-inducible recombination and positive/negative dual selection to regenerate transgenic Cavendish banana plants that were potentially free of the selectable marker gene. By examining the interaction of two different Agrobacterium strains with two different cultivars of Cavendish banana, namely Williams and Grand Naine, we describe a transformation and regeneration strategy that successfully produced marker-free, single transgene copy, gfp-expressing events. The system will provide a useful means of serially improving banana into the future.

The complete genome of an Australian isolate of zantedeschia mild mosaic virus (ZaMMV) causing mosaic symptoms on Alocasia sp. (designated ZaMMV-AU) was cloned and sequenced. The genome comprises 9942 nucleotides (excluding the poly-A tail) and encodes a polyprotein of 3167 amino acids. The sequence is most closely related to a previously reported ZaMMV isolate from Taiwan (ZaMMV-TW), with 82 and 86 % identity at the nucleotide and amino acid level, respectively. Unlike the amino acid sequence of ZaMMV-TW, however, ZaMMV-AU does not contain a polyglutamine stretch at the N-terminus of the coat-protein-coding region upstream of the DAG motif. This is the first report of ZaMMV from Australia and from Alocasia sp.

Background Agroinfiltration is a simple and effective method of delivering transgenes into plant cells for the rapid production of recombinant proteins and has become the preferred transient expression platform to manufacture biologics in plants. Despite its popularity, few studies have sought to improve the efficiency of agroinfiltration to further increase protein yields. This study aimed to increase agroinfiltration-based transient gene expression in Nicotiana benthamiana by improving all levels of transgenesis. Results Using the benchmark pEAQ-HT deconstructed virus vector system and the GUS reporter enzyme, physical, chemical, and molecular features were independently assessed for their ability to enhance Agrobacterium-mediated transformation and improve protein production capacities. Optimal Agrobacterium strain, cell culture density and co-cultivation time for maximal transient GUS (β-glucuronidase) expression were established. The effects of chemical additives in the liquid infiltration media were investigated and acetosyringone (500 μM), the antioxidant lipoic acid (5 μM), and a surfactant Pluronic F-68 (0.002%) were all shown to significantly increase transgene expression. Gene products known to suppress post-transcriptional gene silencing, activate cell cycle progression and confer stress tolerance were also assessed by co-expression. A simple 37 °C heat shock to plants, 1–2 days post infiltration, was shown to dramatically increase GUS reporter levels. By combining the most effective features, a dual vector delivery system was developed that provided approximately 3.5-fold higher levels of absolute GUS protein compared to the pEAQ-HT platform. Conclusions In this paper, different strategies were assessed and optimised with the aim of increasing plant-made protein capacities in Nicotiana benthamiana using agroinfiltration. Chemical additives, heat shock and the co-expression of genes known to suppress stress and gene silencing or stimulate cell cycle progression were all proven to increase agroinfiltration-based transient gene expression. By combining the most effective of these elements a novel expression platform was developed capable of producing plant-made protein at a significantly higher level than a benchmark hyper-expression system.

We have characterized the complete genome of a novel circular double-stranded DNA virus, tentatively named Dioscorea nummularia-associated virus (DNUaV), infecting Dioscorea nummularia originating from Samoa. The genome of DNUaV comprised 8139 bp and contained four putative open reading frames (ORFs). ORFs 1 and 2 had no identifiable conserved domains, while ORF 3 had conserved motifs typical of viruses within the family Caulimoviridae including coat protein, movement protein, aspartic protease, reverse transcriptase and ribonuclease H. A transactivator domain, similar to that present in members of several caulimoviridae genera, was also identified in the putative ORF 4. The genome size, organization, and presence of conserved amino acid domains are similar to other viruses in the family Caulimoviridae. However, based on nucleotide sequence similarity and phylogenetic analysis, DNUaV appears to be a distinct novel member of the family and may represent a new genus.

Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs (PSY1 and PSY2), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.

The fungus Fusarium oxysporum is responsible for causing a destructive wilting disease in numerous crops including banana. To date, only two non-TIR-NBS-LRR genes that confer resistance to F. oxysporum, the I2 and Fom-2 genes from tomato and melon, respectively, have been isolated. We previously identified two partial non-TIR-NBS sequences, designated RGC2 and RGC5, from FOC race 4 resistant banana, Musa acuminata ssp. malaccensis. In the present study, we have isolated and characterized the entire cDNA sequences of RGC2 and RGC5 from the root transcriptome of M. acuminata ssp. malaccensis. The open reading frames (ORFs) of both genes were predicted to encode the typical structure of non-TIR-NBS-LRR proteins. Similarity searches using the entire ORF of RGC2 and RGC5 revealed considerable sequence similarity to the I2 gene. Further, phylogenetic analyses showed that the banana RGC2 and RGC5 clustered within the ancient non-TIR clade N1.1. Interestingly, although phylogenetically distantly related, the known Fusarium R genes I2 and Fom-2 also grouped within the clade N1.1. The fact that the I2 and Fom-2 genes cluster in the same phylogenetic clade N1.1 opens the possibility that other unknown Fusarium R genes from different plant families may share the same clade. These findings may assist future attempts to identify Fusarium R genes in banana and other plants.

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.

The death of plant cells in culture following exposure to Agrobacterium tumefaciens remains a major obstacle in developing Agrobacterium-mediated transformation into a highly efficient genotype-independent technology. Here, we present evidence that A. tumefaciens exposure induces cell death in banana cell suspensions. More than 90% of embryogenic banana cells died after exposure to A. tumefaciens and cell death was accompanied by a subset of features associated with apoptosis in mammalian cells, including DNA laddering, fragmentation, and formation of apoptoticlike bodies. Importantly, these cellular responses were inhibited in cells expressing the animal antiapoptosis genes Bcl-xL, Bcl-2 3′ untranslated region, and CED-9. Inhibition of cell death resulted in up to 90% of cell clumps transformed with Bcl-xL, a 100-fold enhancement over vector controls, approaching the transformation and regeneration of every “transformable” cell. Similar results using sugarcane, a crop plant known for recalcitrance to Agrobacterium transformation, suggest that antiapoptosis genes may inhibit these phenomena and increase the transformation frequency of many recalcitrant plant species, including the major monocot cereal crop plants. Evidence of inhibition of plant cell death by cross-kingdom antiapoptotic genes also contributes to the growing evidence that genes for control of programmed cell death are conserved across wide evolutionary distances, even though these mechanisms are not well understood in plants. Additional keywords: necrosis.

Banana bunchy top virus (BBTV), a multi-component circular ssDNA virus, replicates via a dsDNA intermediate that also serves as a template for virion sense transcription. Seven virus-derived transcripts have been previously identified and analysed in BBTV-infected bananas by northern analysis and 3' rapid amplification of cDNA ends (3' RACE). In this study, we have used RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-RACE) to complete the mapping of the BBTV gene transcripts and have now fully mapped the transcribed regions of each BBTV component and effectively defined the upstream regulatory region.

Banana bunchy top virus(BBTV) has a multi-component genome of circular, single-stranded DNA. BBTV replicates via a rolling-circle mechanism, probably involving sequence-specific interaction of the replication initiation protein (Rep) with iterated sequences (iterons) within the viral genome. Three putative iterons (designated F1, F2 and R), with the sequence GGGAC, have been identified in the intergenic region of each BBTV component. To investigate their role in replication, each of the iterons was mutated, singularly and in tandem, in a BBTV DNA-N 1.1mer and the ability of these molecules to be replicated by the BBTV ‘master’ Rep was evaluated in banana cells using transient biolistic assays. All iteron mutants were replicated less efficiently than the native DNA-N. Mutation of the F1 and R iterons caused a 42 and 62 % reduction in DNA-N replication, respectively, whereas mutation of the F2 and combined F1F2 iteron virtually abolished DNA-N replication.

The complete nucleotide sequences of Fiji disease virus (FDV) genome segments S2, S4 and S7 were determined. This now completes the sequencing of all ten dsRNA genome segments of the Fijivirus type member, FDV, which comprises a total of 29339 nt. FDV S2, S4 and S7 comprised 3820, 3568 and 2194 nt, respectively. S2 and S4 each contained a single open reading frame (ORF), which encoded putative proteins of 137 and 133 kDa, respectively, while S7 contained two ORFs, which encoded putative proteins of 42 and 37 kDa. The putative amino acid sequences of FDV S2 and S4 showed most similarity to the gene products of Rice black-streaked dwarf virus (RBSDV) S2 and RBSDV S3, respectively. The putative amino acid sequences of FDV S7 ORF I and II showed most similarity to Maize rough dwarf virus (MRDV) S6 ORF I and RBSDV S7 ORF II, respectively. Phylogenetic analyses showed that FDV was most closely related to the group 2 fijiviruses.

The genome of an Australian isolate of Sugarcane bacilliform virus (SCBV-IM) was cloned, sequenced and analysed. The genome consisted of 7687 nucleotides and contained three open reading frames which were similar in size and organisation to those of other badnaviruses. SCBV-IM was found to be most similar to the SCBV-Morocco isolate with amino acid sequence similarity of 91.4 %, 83.8 % and 85.3 % in the ORF I, II and III coding regions, respectively. Phylogenetic analysis of the SCBV-IM ORF III deduced amino acid sequence showed that SCBV isolates were more closely related to each other than to other badnaviruses. Amplification of SCBV sequences from three different sugarcane varieties revealed considerable variability in the viral populations, both within single infected plants as well as between infected plants, suggesting that the SCBV isolates sequenced to date may not be representative of the range of virus variability.

Potential promoter regions of the Banana bunchy top virus (BBTV)-associated DNA components S1 and S2 were fused to the #-glucuronidase reporter gene and assessed for activity in both tobacco (Nicotiana tabacum cv. Xanthi) and banana (Musa spp. cv. Bluggoe). Transient assays indicated that all the S1- and S2-derived promoters were active and had greater expression in tobacco than banana. In stably transformed tobacco and banana, the S1- and S2-derived promoters directed expression in root meristems and trichomes. The S1 promoter was also expressed in the vascular tissue of leaves and roots, while both the S1 and S2 promoters were active in tobacco leaf trichomes and pollen. In banana, expression was significantly enhanced by the inclusion of the maize polyubiquitin intron 3' to the promoter. Interestingly, there was some evidence to indicate that S1 promoter fragments containing part of the open reading frame at the 5' end of the promoter had enhanced activity, suggesting that promoter elements may not be confined to the non-coding region.

Banana bunchy top nanovirus (BBTV) has a multicomponent circular single-stranded DNA (cssDNA) genome consisting of at least six components. We have cloned, sequenced and analysed two additional cssDNA components, designated BBTV-S1 and S2, associated with a Taiwanese BBTV isolate. The sequences of BBTV-S1 and S2 comprised 1109 and 1095 nucleotides (nt), respectively, and like BBTV DNA-1, potentially encoded replication initiation proteins (Reps). However, the genome organisation of BBTV-S1 and S2 differed from that of BBTV DNA-1 in that (i) the stem sequence of the CR-SL was not conserved, (ii) the internal gene was absent and (iii) the probable TATA boxes were located 5' of the stem-loop. Further, sequence and phylogenetic analysis of the Rep genes indicated that BBTV DNA-S1 and S2 were distinct from BBTV DNA-1. When different geographical isolates of BBTV were tested for the presence of BBTV-S1/S2, these components were detected in various isolates from Vietnam, Taiwan, the Philippines, Tonga and Samoa but were not detected in isolates from Australia, Egypt, Fiji, and India. Based on these results, BBTV-S1 and S2 do not appear to be integral components of the BBTV genome and represent additional Rep-encoding DNAs associated with BBTV.

The 5′ and 3′ terminal sequences of the plus strand of Fiji disease fijivirus (FDV) segments 2, 3, 9 and 10 possess the conserved terminal sequences, 5′AAGUUUUU… . .CAGCAGAUGUC 3′. The 5′ sequence is identical to that of maize rough dwarf fijivirus (MRDV) and rice black-streaked dwarf fijivirus (RBSDV), whereas the FDV 3′ sequence shares the consensus, CAGCNNNNGUC, with MRDV and RBSDV. The FDV terminal sequences, and the amino acid sequences from FDV segment 9, are more closely related to those from MRDV and RBSDV than to those from oat sterile dwarf fijivirus (OSDV) and Nilaparvata lugens reovirus (NLRV; a putative Fijivirus).

Banana bunchy top virus (BBTV) genomic ssDNA is capable of complementary strand synthesis in vitro without the addition of exogenous primers. We have demonstrated that the self-priming of BBTV can be attributed to a population of endogenous primers which are bound to the genomic DNA within the virions. The primer molecules appeared to be composed entirely of DNA and are heterogeneous in size. The primers were cloned, sequenced and shown to map to a region within the major common region and extend 5' of this conserved region. These primers were found to be associated with multiple components of the genome and were capable of full-length complementary strand synthesis in vitro. Interestingly, most of the cloned primers appeared to be derived from BBTV DNA-5; no function has yet been determined for the putative protein of the large ORF within this component.

We have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1. Northern hybridization and 3' RACE analysis identified two poly-adenylated RNAs associated with BBTV DNA-1. Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep). An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF. Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF. This encoded a putative 5 kDa protein of unknown function. Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1. This Rep ORF was inserted 3' of a cauliflower mosaic virus 35S promoter and 5' of a vegetable storage protein terminator. The transcripts mapped from these tobacco plants were identical at the 3' end to the transcripts from BBTV infected banana plants. The site of polyadenylation for the Rep ORF was at base 963 immediately 3' of the translational stop codon confirming that the polyadenylation signals for this transcript were all within the ORF. However, the internal ORF had a large untranslated region of 272 bases with its site of polyadenylation at nucleotide 803 and a polyadenylation signal 3' of the translational stop codon. A possible upstream termination signal (A/TTGTAA) was identified and was conserved within BBTV DNA-1 sequences from different international isolates.

We have sequenced the NIb coding region of sugarcane mosaic potyvirus strain SC (SCMV-SC) and eight field isolates of SCMV from Australia. This region comprised 1563 nucleotides and encoded a putative protein of 521 amino acids containing the consensus motif GDD. The protease cleavage sites between the NIa/NIb and the NIb/coat protein were found to be Q/C and Q/A, respectively. The SCMV sequences were most similar to sorghum mosaic potyvirus with identities of 70% and 78% at the nucleotide and amino acid levels, respectively. When the sequences were compared to each other, there was a maximum of 3.3% variation between isolates at the nucleotide level and a maximum of 0.8% at the amino acid level. Phylogenetic analysis of the sequences indicated the field isolates were grouped according to their geographical location. The SCMV sequence with most homology to all other isolates has been selected to generate constructs for replicase-mediated resistance.

We have cloned, sequenced and analysed an additional five circular ssDNA components of banana bunchy top virus (BBTV) which we have called components 2, 3, 4, 5 and 6. These components were present in all BBTV infections tested. Four of these components (components 3, 4, 5 and 6) had one large open reading frame (ORF) in the virion sense located 3′ of a stem-loop structure. Each ORF had 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 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 had significant sequence identity with any known protein or nucleic acid sequences. However, the ORF of component 4 encoded a 30 residue hydrophobic domain which may indicate that this ORF encoded a trans-membrane protein. Further, the ORFs of components 3 and 5 potentially encoded proteins of about 20 kDa, the size of the BBTV coat protein. There were two regions of sequence identity between the five components described here and the previously described component 1. Each component contained a conserved stem-loop structure and a nonanucleotide potential TATA box which was 5′ of the large virion-sense ORF in five of the components. The stem-loop structures were incorporated in a common region (CR-SL) of 69 nucleotides which was 62% identical between components. All six BBTV components 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% identical over 92 nucleotides. Each 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. From these results, BBTV appears to belong to an undescribed plant virus group which could also include subterranean clover stunt virus, coconut foliar decay virus, faba bean necrotic yellows virus and milk vetch dwarf virus.

Four cDNA clones were generated from the genomic dsRNA of an Australian isolate of pangola stunt Fijivirus (PaSV). Each clone hybridized with nucleic acid extracts from PaSV infected plants but not healthy plants. Further, each clone hybridized with more than one segment of the PaSV dsRNA genome. One clone was used to demonstrate that homology existed between the Australian isolate of PaSV and a South American isolate of PaSV although the isolates differed in the sizes of the genomic dsRNAs and in the vector species. The clone also hybridized with some segments of the maize rough dwarf Fijivirus genome.

Retrotransposons are a class of transposable elements that represent a major fraction of the repetitive DNA of most eukaryotes. Their abundance stems from their expansive replication strategies. We screened and isolated sequence fragments of long terminal repeat (LTR), gypsy-like reverse transcriptase (rt) and gypsy-like envelope (env) domains, and two partial sequences of non-LTR retrotransposons, long interspersed element (LINE), in the clonally propagated allohexaploid sweet potato (Ipomoea batatas (L.) Lam.) genome. Using dot-blot hybridization, these elements were found to be present in the ~1597 Mb haploid sweet potato genome with copy numbers ranging from ~50 to ~4100 as observed in the partial LTR (IbLtr-1) and LINE (IbLi-1) sequences, respectively. The continuous clonal propagation of sweet potato may have contributed to such a multitude of copies of some of these genomic elements. Interestingly, the isolated gypsy-like env and gypsy-like rt sequence fragments, IbGy-1 (~2100 copies) and IbGy-2 (~540 copies), respectively, were found to be homologous to the Bagy-2 cDNA sequences of barley (Hordeum vulgare L.). Although the isolated partial sequences were found to be homologous to other transcriptionally active elements, future studies are required to determine whether they represent elements that are transcriptionally active under normal and (or) stressful conditions.

One DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component-predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.

The complete sequences of Fiji disease virus (FDV) genome segments 5 (S5), S6, S8 and S10 were obtained and comprised 3150 nt, 2831 nt, 1959 nt and 1819 nt, respectively. Each segment contained a single ORF which encoded putative proteins of 115 kDa, 97 kDa, 69 kDa and 63.0 kDa, respectively. The putative amino acid sequences encoded by S5 and S6 contained putative leucine zipper motifs while FDV S5 and S8 each contained an ATP-GTP-binding motif. At the amino acid level, FDV S5, S6, S8 and S10 showed most similarity to the corresponding segments of Rice black-streaked dwarf virus. Based on sequence similarities, it is predicted that FDV S8 encodes a minor core protein, while FDV S10 encodes an outer capsid protein. The evolutionary relationships of FDV to other reoviruses are discussed.

Sugarcane bacilliform virus (SCBV) DNA molecules larger than the complete genome length of 7.6 kbp were detected in infected plants and in virions. We have confirmed that these high molecular weight nucleic acids were open circular DNA and viral in origin. Due to their open circular conformation, accurate size determination of the DNA molecules was not possible using conventional electrophoresis. Using field inversion gel electrophoresis (FIGE), however, the DNA appeared to increase in genome size increments, with sizes ranging from 1 to 4 genomes (31 kbp) detected. The DNA was packaged into virions, which may explain the observation of purified virions with lengths corresponding to one, two or three times the modal length of 130 nm. The DNA products were possibly concatamers formed during replication as a result of a terminal overlap on the sense strand, and were shown to be overlapped individual genome-length molecules and not covalently-bonded continuous DNA strands. Southern analysis indicated that SCBV sequences are not integrated into the sugarcane genome and that the high molecular weight DNA observed in the sugarcane accessions analysed represents SCBV concatamers.

Taro bacilliform virus (TaBV) has been classified as a putative badnavirus based on its non-enveloped, bacilliform virion morphology and transmission by mealybugs. The complete nucleotide sequence of a Papua New Guinea isolate of TaBV has now been determined and comprises 7458 bp. The genome contains four open reading frames (ORFs) on the plus-strand that potentially encode proteins of 17, 16, 214 and 13 kDa. The size and organisation of TaBV ORFs 1-3 is similar to that of most other badnaviruses, while the location of ORF 4 is similar to that of ORF 4 and ORF X of the atypical badnaviruses Citrus yellow mosaic virus and Cacao swollen shoot virus, respectively. The putative amino acid sequence of TaBV ORF 3 contained motifs that are conserved amongst badnavirus proteins including aspartic protease, reverse transcriptase (RT) and ribonuclease H (RNase H). The highly conserved putative plant tRNA(met)-binding site was also present in the 935 bp intergenic region of TaBV. Phylogenetic analysis using the amino acid sequence of ORF 3 showed that TaBV branched most closely to Dioscorea bacilliform virus. These results confirm that TaBV is a pararetrovirus of the genus Badnavirus, family Caulimoviridae.

Banana bunchy top nanovirus has a multicomponent, circular single-stranded DNA genome comprising at least six integral components, BBTV DNA-1 to -6, which have been consistently associated with bunchy top disease worldwide. At least three other components, BBTV S1, S2 and Y, which have been isolated from Taiwanese BBTV isolates, do not appear to be integral components. We show here that both BBTV DNA-1 and S1, which encode replication initiation (Rep) proteins, were capable of self-replication when bombarded into banana embryogenic cell suspensions. However, only BBTV DNA-1 was capable of directing the replication of two other BBTV genomic components, namely BBTV DNA-3 which encodes the coat protein, and DNA-5 which encodes a retinoblastoma binding-like protein. These results indicate that (i) BBTV DNA-1 is the minimal replicative unit of BBTV and encodes the ‘master’ viral Rep and (ii) BBTV S1 is possibly a satellite DNA which is unable to replicate integral BBTV components.

Banana bunchy top virus (BBTV) DNA-3 to 6 have each previously been shown to contain one large open reading frame in the virion sense, whereas no large ORF had been identified in BBTV DNA-2. RNAs transcribed from the BBTV genome were mapped using northern hybridisation and 3′ RACE. One mRNA was transcribed from each of BBTV DNA-2 to 6 and four of these mRNAs mapped to the ORFs previously identified in BBTV DNA-3 to 6. The mRNA of BBTV DNA-2 was transcribed from a virion sense ORF probably using a TATA box sequence different to that in BBTV DNA-1, and DNA-3 to 6. This ORF encoded a 10 kDa protein of unknown function. The 3′ untranslated region of the five mRNAs varied from 25 nucleotides (BBTV DNA-6) to 167 nucleotides (BBTV DNA-4) and each contained putative polyadenylation signals with associated GT rich sequences together with a possible termination signal (C/T/A)TGTAA conserved in all five mRNAs.

We have sequenced the coat protein (CP) coding region of 11 field isolates of SCMV from Australia, USA and South Africa. The differences between the nucleotide sequences of the isolates was 0.2 to 4.1% and the encoded amino acid sequences differed by 0.0 to 3.5%. Phylogenetic analysis of the CP coding sequences of the SCMV isolates and the related potyviruses SCMV-MDB, JGMV, SrMV, MDMV-A and PVY showed that the SCMV isolates formed a tightly clustered group, with SCMV-MDB forming a separate branch. This indicated that (i) the SCMV isolates are of one strain (SCMV-A) and not geographically distinct species and (ii) SCMV-MDB is clearly distinct, and may represent another potyvirus species.

A 93 nucleotide sequence was found to be strongly conserved between two ssDNA genomic components of banana bunchy top virus (BBTV). Two outwardly extending degenerate primers were designed from this sequence and used in a polymerase chain reaction (PCR) with DNA extracted from purified BBTV virions. PCR amplified products consisting of at least seven distinct bands all approximately 1 kb and possibly representing full-length BBTV dsDNA were resolved. The PCR amplified products were cloned and the clones screened by restriction enzyme analysis. Four distinct restriction analysis groups were identified. These results confirm that the genome of BBTV contains at least five components and that it belongs to a previously undescribed group of plant viruses which may also contain subterranean clover stunt virus.

The movement protein coding region of six isolates of Tobacco yellow dwarf virus (TYDV), collected over a 30 year period from various regions throughout Australia, was analysed using PCR and DNA sequencing. Four isolates of Bean summer death virus, considered a synonym of TYDV, were also analysed. Phylogenetic analysis showed that all 10 isolates were greater than 95% homologous to the published TYDV sequence (GenBank accession number M81103). The weed Raphanus raphanistrum was identified as an alternative host of TYDV for the first time.

A transgenic line of the sugarcane cultivar Q124 with significantly enhanced resistance to Fiji disease was produced by microprojectile-mediated transformation with a transgene encoding a translatable version of Fiji disease virus (FDV) segment 9 ORF 1 under the control of the maize polyubiquitin promoter. Sixty-four transgenic lines were tested in glasshouse trials by caging the plants with viruliferous Perkinsiella saccharicida planthoppers. After 2 weeks, the planthoppers were removed and the plants monitored for symptoms. One transgenic line showed significantly enhanced resistance to Fiji disease compared with the Q124 parent and other lines showed varying levels of resistance. The molecular phenotypes of the transgenic plants at both the DNA and RNA levels were not entirely consistent with a resistance mechanism based on post-transcriptional gene silencing but were consistent with reports from other sugarcane-virus resistance systems. This is the first report of transgenic sugarcane containing an FDV-derived synthetic resistance gene showing resistance to FDV, although the mechanism of resistance has not yet been elucidated.