Molecular characterization of banana virus X (BVX), a novel member of the Flexiviridae family.

A novel virus was identified in banana (Musa spp). Analysis of the last 2917 nucleotides of its positive strand genomic RNA showed five open reading frames corresponding, from 5' to 3', to a truncated ORF coding for a replication-associated protein, three ORFs coding for a movement-associated triple gene block (TGB) and a capsid protein (CP) gene. This genome organization is similar to that of some members of the Flexiviridae family such as potexviruses and foveaviruses. This virus was named Banana virus X (BVX). Comparative sequence analysis showed that BVX is only distantly related to other members of the Flexiviridae family, in which it appears to define a new genus. BVX produces defective RNAs derived from its genomic RNA by non-homologous recombination. Three distinct pairs of donor/acceptor recombination sites involving short direct nucleotide repeats were characterized, accounting for deletions of 1268, 1358 and 1503 nucleotides. Contrary to the situation encountered for Potexviruses, these recombination sites are located within the TGB1 and CP genes and result in a truncated TGB1 protein.

Expression of a yeast RNase III gene in transgenic tobacco silences host nitrite reductase genes.

When a gene encoding the Schizosaccharomyces pombe dsRNA-specific RNase III, pac1, was expressed in transgenic tobacco plants, six out of thirteen transformed plants gave progeny among which were individuals displaying a distinctive chlorotic phenotype. These chlorotic plants strongly resemble those transformed with a 35S-Nii (nitrite reductase) transgene, in which both Nii host genes and the 35S-Nii transgene are silenced by co-suppression. RNA blots showed that the host Nii genes were silenced in chlorotic 35S-pac1 plants but not in individuals with a normal green phenotype. Neither the transcript levels of the other cellular genes tested nor the transcription of Nii genes was significantly affected by the expression of pac1. This is the first observation of post-transcriptional silencing of host genes by a transgene with no apparent sequence similarity to the target gene.

Synthesis of (-)-strand RNA from the 3' untranslated region of plant viral genomes expressed in transgenic plants upon infection with related viruses.

When expressed in transgenic tobacco plants, transgene mRNA that includes the 3' untranslated region (3' UTR) of Lettuce mosaic virus served as template for synthesis of complementary (-)-strand RNA following an infection by Tobacco etch virus, Tobacco vein mottle virus or Pepper mottle virus, but not when infected with Cucumber mosaic virus. Deletion of the 3' UTR from the transgene abolished the synthesis of (-)-strand transcripts. Similar results were obtained in transgenic tobacco plants expressing mRNA that includes the RNA3 3' UTR of Cucumber mosaic virus when infected with Tomato aspermy virus. These results show that the viral RNA-dependent RNA polymerase of several potyviruses and Tomato aspermy virus have the ability to recognize heterologous 3' UTRs when included in transgene mRNAs, and to use them as transcription promoters.

Sequences of the coat protein gene of five peanut stripe virus (PStV) strains from Thailand and their evolutionary relationship with other bean common mosaic virus sequences.

The coat protein gene and part of the 3' non-coding region of five strains of peanut stripe virus (PStV) from Thailand have been cloned and sequenced. Phylogenetic comparisons of these strains, known as T1, T3, T5, T6 and T7, and related sequences showed that these strains are indeed strains of PStV. Further, PStV strains appear to be related to each other according to their geographic origin. That is, the Thai strains are more closely related to each other than they are to strains from the USA or Indonesia, despite the variety of symptoms caused by these strains and the overlap of symptom types between the strains from different locations. Like other PStV strains, PStV-Thai can be considered strains of bean common mosaic virus (BCMV) but can be distinguished from bean-infecting strains of BCMV and blackeye cowpea mosaic virus (B1CMV) through sequence and host range. No evidence was found that PStV-Thai strains, unlike PStV-Ib, are recombinants of PStV and B1CMV, although the T3 strain may be a recombinant of different PStV sequences. Phylogenetic analyses of viruses of the BCMV group suggest that acquisition of the ability to infect peanut may have occurred only once.

Cloning and sequence analysis of the coat protein genes of an Australian strain of peanut mottle and an Indonesian 'blotch' strain of peanut stripe potyviruses.

We have analysed the coat protein gene sequences of two potyviruses infecting peanut. The 3' terminal 1247 nucleotides (nt) of an Australian strain of peanut mottle virus (PeMoV-AU) and the 3' terminal 1388 nt of an Indonesian 'blotch' strain of peanut stripe virus (PStV-Ib) were cloned and sequenced. Those regions included the 861 and 864 nt encoding the respective putative coat proteins as well as the 285 and 253 nt, respectively of 3' non-coding sequences. Comparison of the nucleotide sequences of PeMoV-AU and PStV-Ib revealed a sequence similarity of 64.4% for the coat protein gene and 34.6% for the 3' non-coding region. The deduced amino acid sequences of PeMoV-AU and PStV-Ib coat proteins are 66.7% identical. These results provide further evidence that PeMoV and PStV are distinct viruses. Comparisons of the 3' terminal sequences of PeMoV-AU and PStV-Ib with those of the genomic RNA of other strains of PeMoV and PStV and with other potyviruses are discussed.

Construction of a chimeric viral gene expressing plum pox virus coat protein.

The capsid-encoding gene of plum pox virus (PPV) was fused with the leader sequence of the coat protein mRNA (cp) of tobacco mosaic virus by a novel mutagenesis technique which involves reverse transcription of minus-strand RNA [synthesized by in vitro transcription of a double-stranded (ds) cDNA clone], using an ad hoc synthetic oligodeoxynucleotide as primer. The resulting cDNA was rendered ds and cloned into the plasmid, pBluescribe M13+. Transcription of this chimeric construction produced RNA molecules of 1250 nucleotides in length, which were used as messengers in the in vitro protein-synthesizing systems. The major product of this transcript consists of a 36-kDa polypeptide and was identified as the PPV coat protein (CP) by molecular weight estimation and by immunoprecipitation with a polyclonal antiserum to PPV. Transfer of this cDNA via Agrobacterium tumefaciens into plants was successfully performed. Transgenic Nicotiana plants producing the PPV CP were subsequently obtained.

Dot hybridization detection of plum pox virus using 32P-labeled RNA probes representing non-structural viral protein genes.

A cDNA library covering the complete genome of plum pox virus strain D (PPV D) has been obtained, and an endonuclease restriction map derived from it. This map was superposed on the PPV genomic organisation map, established for a nonaphid transmissible strain of PPV (Maiss et al., 1989). This allowed us to select seven probes, corresponding to different regions on the PPV genome. These probes were tested in a dot-blot hybridization assay for the detection of PPV. Probes of various lengths (0.25 to 1.5 kb) were tested and those measuring at least 0.8 kb (4 of the 7 probes selected) proved to be the most sensitive. The detection limit was of about 5 pg of purified virus per assay. Probes representing non-structural viral protein genes were equally sensitive in detecting both serotypes D and M of PPV. The previously described probe pBPPV1 (Varveri et al., 1988), covering the coat protein gene of strain D, was less sensitive, when compared to the above probes, in detecting heterologous strains of PPV. The polyvalence of probes transcribed from non-structural viral protein genes was confirmed by screening isolates of PPV, collected in infected orchards in several Mediterranean countries.