Characterization of taro reovirus and its status in taro (Colocasia esculenta) germplasm from the Pacific.

Taro reovirus (TaRV) has been reported infecting taro (Colocasia esculenta) in the South Pacific, but information on the virus is limited. Here, we report the genome sequence of a reovirus infecting taro in Papua New Guinea that had 10 genomic segments ranging from 1.1 to 3.9 kilobase pairs (kbp) in length with a total genome length of 26.3 kbp. TaRV was most closely related to rice ragged stunt virus (RRSV) but did not cross-react with RRSV polyclonal antisera. TaRV was not detected in 82 germplasm accessions of taro in Hawaii, or samples collected in American Samoa, Fiji, Guam, Palau, or Vanuatu.

Characterization of an Australian isolate of taro bacilliform virus and development of an infectious clone.

The badnavirus taro bacilliform virus (TaBV) has been reported to infect taro (Colocasia esculenta L.) and other edible aroids in several South Pacific island countries, but there are no published reports from Australia. Using PCR and RCA, we identified and characterized an Australian TaBV isolate. A terminally redundant cloned copy of the TaBV genome was generated and shown to be infectious in taro following agro-inoculation. This is the first report of TaBV from Australia and also the first report of an infectious clone for this virus.

Molecular Characterization and Distribution of Two Strains of Dasheen mosaic virus on Taro in Hawaii.

Dasheen mosaic virus (DsMV) is one of the major viruses affecting taro (Colocasia esculenta) production worldwide. Whole genome sequences were determined for two DsMV strains, Hawaii Strain I (KY242358) and Hawaii Strain II (KY242359), from taro in Hawaii. They represent the first full-length coding sequences of DsMV reported from the United States. Hawaii Strains I and II were 77 and 85% identical, respectively, with other completely sequenced DsMV isolates. Hawaii Strain I was most closely related to vanilla mosaic virus (VanMV) (KX505964.1), a strain of DsMV infecting vanilla in the southern Pacific Islands. Hawaii Strain II was most closely related to a taro DsMV isolate CTCRI-II-14 (KT026108.1) from India. Phylogenetic analysis of all available DsMV isolates based on amino acid sequences of their coat protein showed some correlation between host plant and genetic diversity. Analyses of DsMV genome sequences detected three recombinants from China and India among the six isolates with known complete genome sequences. The DsMV strain NC003537.1 from China is a recombinant of KJ786965.1 from India and Hawaii Strain II. Another DsMV strain KT026108.1 is a recombinant of Hawaii Strain II and NC003537.1 from China. The third DsMV strain KJ786965.1 from India is a recombinant of Hawaii Strain II and NC003537.1 from China. To our knowledge, this is the first report of recombination events in DsMV. Both Hawaii Strains I and II of DsMV were found widespread throughout the Hawaiian islands.

Complete genome sequence of Colocasia bobone disease-associated virus, a putative cytorhabdovirus infecting taro.

We report the first genome sequence of a Colocasia bobone disease-associated virus (CBDaV) derived from bobone-affected taro [Colocasia esculenta L. Schott] from Solomon Islands. The negative-strand RNA genome is 12,193 nt long, with six major open reading frames (ORFs) with the arrangement 3'-N-P-P3-M-G-L-5'. Typical of all rhabdoviruses, the 3' leader and 5' trailer sequences show complementarity to each other. Phylogenetic analysis indicated that CBDaV is a member of the genus Cytorhabdovirus, supporting previous reports of virus particles within the cytoplasm of bobone-infected taro cells. The availability of the CBDaV genome sequence now makes it possible to assess the role of this virus in bobone, and possibly alomae disease of taro and confirm that this sequence is that of Colocasia bobone disease virus (CBDV).

Characterization by Small RNA Sequencing of Taro Bacilliform CH Virus (TaBCHV), a Novel Badnavirus.

RNA silencing is an antiviral immunity that regulates gene expression through the production of small RNAs (sRNAs). In this study, deep sequencing of small RNAs was used to identify viruses infecting two taro plants. Blast searching identified five and nine contigs assembled from small RNAs of samples T1 and T2 matched onto the genome sequences of badnaviruses in the family Caulimoviridae. Complete genome sequences of two isolates of the badnavirus determined by sequence specific amplification comprised of 7,641 nucleotides and shared overall nucleotide similarities of 44.1%‒55.8% with other badnaviruses. Six open reading frames (ORFs) were identified on the plus strand, showed amino acid similarities ranging from 59.8% (ORF3) to 10.2% (ORF6) to the corresponding proteins encoded by other badnaviruses. Phylogenetic analysis also supports that the virus is a new member in the genus Badnavirus. The virus is tentatively named as Taro bacilliform CH virus (TaBCHV), and it is the second badnavirus infecting taro plants, following Taro bacilliform virus (TaBV). In addition, analyzes of viral derived small RNAs (vsRNAs) from TaBCHV showed that almost equivalent number of vsRNAs were generated from both strands and the most abundant vsRNAs were 21 nt, with uracil bias at 5' terminal. Furthermore, TaBCHV vsRNAs were asymmetrically distributed on its entire circular genome at both orientations with the hotspots mainly generated in the ORF5 region.

Taro vein chlorosis virus: characterization and variability of a new nucleorhabdovirus.

Sequencing of the monopartite RNA genome of a Fijian isolate of Taro vein chlorosis virus (TaVCV) confirmed that it is a definitive rhabdovirus with most similarity to members of the genus Nucleorhabdovirus. The TaVCV 12 020 nt negative-sense RNA genome contained six ORFs in the antigenomic sequence, equivalent to the N, P, 3, M, G and L genes that have been identified in other rhabdoviruses. The putative gene products had highest similarity to those of the nucleorhabdovirus Maize mosaic virus. A characteristic 3'-AAUUCUUUUUGGGUUGU/A-5' sequence was identified in each of the intergenic regions and the TaVCV leader and trailer sequences comprised 140 and 61 nt, respectively. Assignment of TaVCV to the genus Nucleorhabdovirus was supported by thin-section electron microscopy of TaVCV-infected taro leaves, which identified virions budding from nuclear membranes into the perinuclear space. Variability studies identified high levels of TaVCV sequence diversity. Within the L gene of 20 TaVCV isolates from Fiji, the Federated States of Micronesia, New Caledonia, Papua New Guinea, Solomon Islands and Vanuatu, maximum variability at the nucleotide level was 27.4 %. Within the N gene, maximum variability among 15 isolates at the nucleotide level was 19.3 %. The high level of TaVCV variability observed suggested that the introduction of TaVCV to the Pacific Islands was not a recent occurrence.

Sequence diversity of South Pacific isolates of Taro bacilliform virus and the development of a PCR-based diagnostic test.

We have analysed the sequence variability in the putative reverse transcriptase (RT)/ribonuclease H (RNaseH) and the C-terminal coat protein (CP)-coding regions from Taro bacilliform virus (TaBV) isolates collected throughout the Pacific Islands. When the RT/RNaseH-coding region of 22 TaBV isolates from Fiji, French Polynesia, New Caledonia, Papua New Guinea (PNG), Samoa, Solomon Islands and Vanuatu was examined, maximum variability at the nucleotide and amino acid level was 22.9% and 13.6%, respectively. Within the CP-coding region of 13 TaBV isolates from Fiji, New Caledonia, PNG, Samoa and the Solomon Islands, maximum variability at the nucleotide and amino acid level was 30.7% and 19.5%, respectively. Phylogenetic analysis showed that TaBV isolates from the Solomon Islands showed greatest variability while those from New Caledonia and PNG showed least variability. Based on the sequences of the TaBV RT/RNaseH-coding region, we have developed a PCR-based diagnostic test that specifically detects all known TaBV isolates. Preliminary indexing has revealed that TaBV is widespread throughout Pacific Island countries. A sequence showing approximately 50% nucleotide identity to TaBV in the RT/RNaseH-coding region was also detected in all taro samples tested. The possibility that this may represent either an integrated sequence or the genome of an additional badnavirus infecting taro is discussed.

A promoter derived from taro bacilliform badnavirus drives strong expression in transgenic banana and tobacco plants.

Taro bacilliform virus (TaBV) is a pararetrovirus of the genus Badnavirus which infects the monocotyledonous plant, taro ( Colocasia esculenta). A region of the TaBV genome spanning nucleotides 6,281 to 12 (T1200), including the 3' end of open reading frame 3 (ORF 3) and the intergenic region to the end of the tRNA(met)-binding site, was tested for promoter activity along with four different 5' deletion fragments (T600, T500, T250 and T100). In transient assays, only the T1200, T600, T500 fragments were shown to have promoter activity in taro leaf, banana suspension cells and tobacco callus. When these three promoters were evaluated in stably transformed, in vitro-grown transgenic banana and tobacco plants, all were found to drive near-constitutive expression of either the green fluorescent protein or beta-glucuronidase (GUS) reporter gene in the stem (or pseudostem), leaves and roots, with strongest expression observed in the vascular tissue. In transgenic banana leaves, the T600 promoter directed four-fold greater GUS activity than that of the T1200, T500 and the maize polyubiquitin-1 promoters. In transgenic tobacco leaves, the levels of GUS expression directed by the three promoters was between four- and ten-fold lower than that of the double Cauliflower mosaic virus 35S promoter. These results indicate that the TaBV-derived promoters may be useful for the high-level constitutive expression of transgenes in either monocotyledonous or dicotyledonous species.

Genomic characterisation of taro bacilliform virus.

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.