Pierre-Yves Teycheney

The classification and nomenclature of endogenous viruses of the family Caulimoviridae

Endogenous members of the family Caulimoviridae have now been found in the genomes of many plant species. Although these sequences are usually fragmented and rearranged and show varying degrees of decay, the genomes of the ancestral viruses can often be reassembled in silico, allowing classification within the existing viral taxonomic framework. In this paper, we describe analyses of endogenous members of the family Caulimoviridae in the genomes of Oryza sativa, Nicotiana tabacum and Solanum spp. and on the basis of phylogeny, genome organization and genetic distance within the pol gene, propose two new virus genera called Orendovirus and Solendovirus. A system of nomenclature for endogenous virus sequences in plants is also proposed.

Virus-specific spatial differences in the interference with silencing of the #chs-A# gene in non-transgenic petunia

Potyviruses, such as potato virus Y and tobacco etch virus, as well as cucumber mosaic cucumovirus, interfere with post-transcriptional gene silencing (PTGS). When RedStar-type Petunia hybrida cultivars, whose flowers have alternating white and pigmented sectors, were infected with these viruses, each virus induced a different pattern of restoration of floral anthocyanin pigmentation. Local reversion to coloured phenotypes in the white sectors, which occurred through interference with PTGS of the chalcone synthase A (chs-A) gene, was correlated with locally increased levels of chs-A mRNA and virus concentration. Our results show that virus infection can interfere with PTGS of a native plant gene, and that this can have profound effects on symptom expression.

Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes

Tobacco plants expressing a transgene encoding the coat protein (CP) of a subgroup I strain of cucumber mosaic cucumovirus (CMV), I17F, were not resistant to strains of either subgroup I or II. In contrast, the expression of the CP of a subgroup II strain, R, conferred substantial resistance, but only towards strains of the same subgroup. When protection was observed, the levels of resistance were similar when plants were inoculated with either virions or viral RNA, but resistance was more effective when plants were inoculated with viruliferous aphids. Resistance was not dependent on inoculum strength and was expressed as a recovery phenotype not yet described for plants expressing a CMV CP gene. Recovery could be observed either early in infection (less than one week after inoculation) or later (4 to 5 weeks after inoculation). In plants showing early recovery, mild symptoms were observed on the inoculated leaves, and in some cases symptoms developed on certain lower systemically infected leaves, but the upper leaves were symptomless and virus-free. Late recovery corresponded to the absence of both symptoms and virus in the upper leaves of plants that were previously fully infected. Northern blot analyses of resistant plants suggested that a gene silencing mechanism was not involved in the resistance observed.

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.

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

Summary.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.

Molecular characterization of yam virus X, a new potexvirus infecting yams (Dioscorea spp) and evidence for the existence of at least three distinct potexviruses infecting yams

The genome of yam virus X (YVX), a new member of the genus Potexvirus from yam (Dioscorea trifida), was completely sequenced. Structural and phylogenetic analysis showed that the closest relative of YVX is nerine virus X. A prevalence study found YVX only in plants maintained in Guadeloupe and showed that it also infects members of the complex D. cayenensis rotundata. This study provides evidence for the existence of two additional potexviruses, one of which infects D. nummularia in Vanuatu and the other, D. bulbifera and D. rotundata in Haiti and D. trifida and D. rotundata in Guadeloupe. This work also shows that existing potexvirus-specific degenerate primers targeting the ORF1-encoded polymerase domain are well suited for the identification of the three potexviruses reported here.

The genome sequence of Dioscorea bacilliform TR virus, a member of the genus Badnavirus infecting Dioscorea spp., sheds light on the possible function of endogenous Dioscorea bacilliform viruses

The complete genome sequence of Dioscorea bacilliform TR virus (DBTRV) was determined. The closest relatives of DBTRV are Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform RT virus 1 (DBRTV1). Specific primers were designed and used to determine the prevalence of DBTRV in a yam germplasm collection. It was found that this virus infects Dioscorea alata and D. trifida plants in Guadeloupe and French Guyana. DTRBV was not detected in any of the tested D. cayenensis-rotundata accessions. In silico analysis provided evidence for the presence of DBTRV-like endogenous sequences in the genome of D. cayenensis-rotundata, pointing to a possible role of these sequences in antiviral defense.

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.

Tracheophyte genomes keep track of the deep evolution of the Caulimoviridae

Endogenous viral elements (EVEs) are viral sequences that are integrated in the nuclear genomes of their hosts and are signatures of viral infections that may have occurred millions of years ago. The study of EVEs, coined paleovirology, provides important insights into virus evolution. The Caulimoviridae is the most common group of EVEs in plants, although their presence has often been overlooked in plant genome studies. We have refined methods for the identification of caulimovirid EVEs and interrogated the genomes of a broad diversity of plant taxa, from algae to advanced flowering plants. Evidence is provided that almost every vascular plant (tracheophyte), including the most primitive taxa (clubmosses, ferns and gymnosperms) contains caulimovirid EVEs, many of which represent previously unrecognized evolutionary branches. In angiosperms, EVEs from at least one and as many as five different caulimovirid genera were frequently detected, and florendoviruses were the most widely distributed, followed by petuviruses. From the analysis of the distribution of different caulimovirid genera within different plant species, we propose a working evolutionary scenario in which this family of viruses emerged at latest during Devonian era (approx. 320 million years ago) followed by vertical transmission and by several cross-division host swaps.

Marker-assisted breeding of Musa balbisiana genitors devoid of infectious endogenous Banana streak virus sequences

Breeding new interspecific banana hybrid varieties relies on the use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of Musa balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus in interspecific hybrids harboring both Musa acuminata and M. balbisiana genomes. Here we provide evidence that seedy M. balbisiana diploids display diverse eBSV allelic combinations and that some eBSVs differ structurally from those previously reported. We also show that segregation of infectious and non-infectious eBSV alleles can be achieved in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. Our work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.