Infection of non-host model plant species with the narrow-host-range Cacao swollen shoot virus.

Cacao swollen shoot virus (CSSV) is a major pathogen of cacao (Theobroma cacao) in Africa, and long-standing efforts to limit its spread by the culling of infected trees have had very limited success. CSSV is a particularly difficult virus to study, as it has a very narrow host range, limited to several tropical tree species. Furthermore, the virus is not mechanically transmissible, and its insect vector can only be used with difficulty. Thus, the only efficient means to infect cacao plants that have been experimentally described so far are by particle bombardment or the agroinoculation of cacao plants with an infectious clone. We have genetically transformed three non-host species with an infectious form of the CSSV genome: two experimental hosts widely used in plant virology (Nicotiana tabacum and N. benthamiana) and the model species Arabidopsis thaliana. In transformed plants of all three species, the CSSV genome was able to replicate, and, in tobacco, CSSV particles could be observed by immunosorbent electron microscopy, demonstrating that the complete virus cycle could be completed in a non-host plant. These results will greatly facilitate the preliminary testing of CSSV control strategies using plants that are easy to raise and to transform genetically.

Deep sequencing of recombinant virus populations in transgenic and nontransgenic plants infected with Cucumber mosaic virus.

Recombination is a major source of virus variability, and the question of whether novel recombinant viruses would emerge in transgenic plants expressing viral sequences has been a biosafety issue. We describe the results of pyrosequencing the recombinant viral RNAs appearing in transgenic plants expressing the coat protein (CP) gene and 3' noncoding region of Cucumber mosaic virus RNA3, as well as in nontransgenic controls. The populations of recombinants in both transgenic and nontransgenic plants were similar to those previously described from Sanger sequencing but many more recombinant types were observed, including a novel class of large deletions removing all or nearly the entire CP gene. These results show that populations of recombinant viral genomes arising de novo can be characterized in detail by pyrosequencing, and confirm that the transgenic plants did not harbor novel recombinants of biosafety concern.

Tobacco mosaic virus as an AFM tip calibrator.

The study of high-resolution topographic surfaces of isolated single molecules is one of the applications of atomic force microscopy (AFM). Since tip-induced distortions are significant in topographic images the exact AFM tip shape must be known in order to correct dilated AFM height images using mathematical morphology operators. In this work, we present a protocol to estimate the AFM tip apex radius using tobacco mosaic virus (TMV) particles. Among the many advantages of TMV, are its non-abrasivity, thermal stability, bio-compatibility with other isolated single molecules and stability when deposited on divalent ion pretreated mica. Compared to previous calibration systems, the advantage of using TMV resides in our detailed knowledge of the atomic structure of the entire rod-shaped particle. This property makes it possible to interpret AFM height images in term of the three-dimensional structure of TMV. Results obtained in this study show that when a low imaging force is used, the tip is sensing viral protein loops whereas at higher imaging force the tip is sensing the TMV particle core. The known size of the TMV particle allowed us to develop a tip-size estimation protocol which permits the successful erosion of tip-convoluted AFM height images. Our data shows that the TMV particle is a well-adapted calibrator for AFM tips for imaging single isolated biomolecules. The procedure developed in this study is easily applicable to any other spherical viral particles.

Biological properties and relative fitness of inter-subgroup cucumber mosaic virus RNA 3 recombinants produced in vitro.

In vitro reverse transcription of a mixture of total RNA from plants infected with the I17F or R strains of cucumber mosaic virus (CMV), representative of subgroups IA and II, respectively, results in viral cDNA populations including rare recombinant RNA 3 molecules, some of which also have point mutations. The biological properties of 17 recombinants in the capsid gene or the 3' non-coding region of RNA 3 were evaluated when associated with I17F RNAs 1 and 2. Six viruses displayed deficiencies (non-viability, deficiencies for movement and/or replication, delayed infection, loss of aphid transmissibility). Nine induced symptoms close to those of I17F-CMV on tobacco and pepper plants. All recombinants bearing the movement protein (MP) of R-CMV and part or most of the capsid protein (CP) of I17F-CMV, as well as the recombinant created in vitro by exchanging the corresponding open reading frames, also induced filiformism on tobacco, but induced only faint symptoms on melon. Two recombinants induced atypically severe symptoms on both tobacco and pepper. Most of the recombinants generally accumulated to lower levels than the wild-type I17F strain in tobacco. Three recombinants, however, including one responsible for severe symptoms, accumulated to generally higher levels than I17F-CMV. When two of these were tested in co-infection experiments with I17F RNA 3, they proved to be poorly competitive, suggesting that they would be unlikely to emerge in the field.

Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum.

The recessive resistance genes pot-1 and pvr2 in Lycopersicon hirsutum and Capsicum annuum, respectively, control Potato virus Y (PVY) accumulation in the inoculated leaves. Infectious cDNA molecules from two PVY isolates differing in their virulence toward these resistances were obtained using two different strategies. Chimeras constructed with these cDNA clones showed that a single nucleotide change corresponding to an amino acid substitution (Arg119His) in the central part of the viral protein genome-linked (VPg) was involved in virulence toward the pot-1 resistance. On the other hand, 15 nucleotide changes corresponding to five putative amino acid differences in the same region of the VPg affected virulence toward the pvr2(1) and pvr2(2) resistances. Substitution models identified six and five codons within the central and C terminal parts of the VPg for PVY and for the related potyvirus Potato virus A, respectively, which undergo positive selection. This suggests that the role of the VPg-encoding region is determined by the protein and not by the viral RNA apart from its protein-encoding capacity.