Sequencing viral siRNAs to identify previously undescribed viruses and viroids in a panel of ornamental plant samples structured as a matrix of pools.

Ornamental plants constitute a largely unknown and potentially important source of pathogens affecting not only ornamental plants, but also major crop species. We have carried out studies using high-throughput sequencing of 21-24 nt RNAs from potentially virus-infected ornamental plants, followed by assembly of sequence scaffolds, to identify the virus and viroid genomes present in a panel of 67 plant samples representing 46 species belonging to the main sectors of the ornamental plant industry (cut flowers, pot plants, bulbs). A pilot study demonstrated that samples could be pooled (5 samples per pool), and the overall process simplified without loss of detection of important known pathogens. In a full-scale study, pools of 5 samples were organized in a 5×5 matrix to facilitate attribution of a sequence to a precise sample directly from analysis of the matrix. In the total of 67 samples analyzed in the two studies, partial sequences suggesting the presence of 25 previously unknown viruses and viroids were detected, including all types of virus and viroid genomes, and also showed four cases of known viruses infecting previously undescribed hosts. Furthermore, two types of potential mis-assembly were analyzed, and were shown to not affect the conclusions regarding the presence of the pathogens identified, but show that mis-assembly can affect the results when the objective is determining complete bona fide viral genome sequences. These results clearly confirm that ornamental plants constitute a potential source of unknown viruses and viroids that could have a major impact on agriculture, and that sequencing siRNAs of potentially virus- or viroid-infected ornamental plants is an effective means for screening for the presence of potentially important pathogens.

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.

A genetically novel, narrow-host-range isolate of cucumber mosaic virus (CMV) from rosemary.

An isolate of cucumber mosaic virus (CMV), designated CMV-Rom, was isolated from rosemary (Rosmarinus officinalis) plants in several locations near Avignon, France. Laboratory studies showed that, unlike typical CMV isolates, CMV-Rom has a particularly narrow host range. It could be transmitted by aphids Aphis gossypii and Myzus persicae, but with low efficacy compared to a typical CMV isolate. Phylogenetic analysis of the nucleotide sequences of the CMV-Rom genomic RNAs shows that this isolate does not belong to any of the previously described CMV subgroups, IA, IB, II or III.

A critical evaluation of whether recombination in virus-resistant transgenic plants will lead to the emergence of novel viral diseases.

In the evaluation of the potential impacts of first-generation genetically modified (GM) crops, one of the most complex issues has been whether the expression of viral sequences would lead to the emergence of novel viruses, which could occur through recombination between transgene mRNA and that of an infecting non-target virus. Here, we examine this issue, focusing on Cucumber mosaic virus (CMV), which is a particularly pertinent choice, as it is both a major plant pathogen and also the virus with which this question has been studied in the most detail. Using recent results on recombination in CMV, we employ a novel framework giving particular prominence to the formulation of the risk hypothesis and to hypothesis testing via examination of the potential pathway to harm. This allows us to conclude with greater certainty that the likelihood of this potential harm, the emergence of novel viruses, is low.

Narrow bottlenecks affect Pea seedborne mosaic virus populations during vertical seed transmission but not during leaf colonization.

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Populations having high Ne adapt faster, as selection acts more intensely, than populations having low Ne, where random effects of genetic drift dominate. Estimating Ne for various steps of plant virus life cycle has been the focus of several studies in the last decade, but no estimates are available for the vertical transmission of plant viruses, although virus seed transmission is economically significant in at least 18% of plant viruses in at least one plant species. Here we study the co-dynamics of two variants of Pea seedborne mosaic virus (PSbMV) colonizing leaves of pea plants (Pisum sativum L.) during the whole flowering period, and their subsequent transmission to plant progeny through seeds. Whereas classical estimators of Ne could be used for leaf infection at the systemic level, as virus variants were equally competitive, dedicated stochastic models were needed to estimate Ne during vertical transmission. Very little genetic drift was observed during the infection of apical leaves, with Ne values ranging from 59 to 216. In contrast, a very drastic genetic drift was observed during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. A simple model of vertical transmission, assuming a cumulative action of virus infectious particles and a virus density threshold required for vertical transmission to occur fitted the experimental data very satisfactorily. This study reveals that vertically-transmitted viruses endure bottlenecks as narrow as those imposed by horizontal transmission. These bottlenecks are likely to slow down virus adaptation and could decrease virus fitness and virulence.

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.

Cucumber mosaic virus.

Cucumber mosaic virus (CMV) is an important virus because of its agricultural impact in the Mediterranean Basin and worldwide, and also as a model for understanding plant-virus interactions. This review focuses on those areas where most progress has been made over the past decade in our understanding of CMV. Clearly, a deep understanding of the role of the recently described CMV 2b gene in suppression of host RNA silencing and viral virulence is the most important discovery. These findings have had an impact well beyond the virus itself, as the 2b gene is an important tool in the studies of eukaryotic gene regulation. Protein 2b was shown to be involved in most of the steps of the virus cycle and to interfere with several basal host defenses. Progress has also been made concerning the mechanisms of virus replication and movement. However, only a few host proteins that interact with viral proteins have been identified, making this an area of research where major efforts are still needed. Another area where major advances have been made is CMV population genetics, where contrasting results were obtained. On the one hand, CMV was shown to be prone to recombination and to show high genetic diversity based on sequence data of different isolates. On the other hand, populations did not exhibit high genetic variability either within plants, or even in a field and the nearby wild plants. The situation was partially clarified with the finding that severe bottlenecks occur during both virus movement within a plant and transmission between plants. Finally, novel studies were undertaken to elucidate mechanisms leading to selection in virus population, according to the host or its environment, opening a new research area in plant-virus coevolution.

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.

Evaluation of potential risks associated with recombination in transgenic plants expressing viral sequences.

Virus-resistant transgenic plants have been created primarily through the expression of viral sequences. It has been hypothesized that recombination between the viral transgene mRNA and the RNA of an infecting virus could generate novel viruses. As mRNA/viral RNA recombination can occur in virus-resistant transgenic plants, the key to testing this risk hypothesis is to compare the populations of recombinant viruses generated in transgenic and non-transgenic plants. This has been done with two cucumoviral systems, involving either two strains of cucumber mosaic virus (CMV), or CMV and the related tomato aspermy virus (TAV). Although the distribution of the sites of recombination in the CMV/CMV and TAV/CMV systems was quite different, equivalent populations of recombinant viruses were observed in both cases. These results constitute the first comparison of the populations of recombinants in transgenic and non-transgenic plants, and suggest that there is little risk of emergence of recombinant viruses in these plants, other than those that could emerge in non-transgenic plants.

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.

Different mutations in the genome-linked protein VPg of potato virus Y confer virulence on the pvr2(3) resistance in pepper.

Five different amino acid substitutions in the VPg of Potato virus Y were shown to be independently responsible for virulence toward pvr2(3) resistance gene of pepper. A consequence of these multiple mutations toward virulence involving single nucleotide substitutions is a particularly high frequency of resistance breaking (37% of inoculated plants from the first inoculation) and suggests a potentially low durability of pvr2(3) resistance. These five mutants were observed with significantly different frequencies, one of them being overrepresented. Genetic drift alone could not explain the observed distribution of virulent mutants. More plausible scenarios were obtained by taking into account either the relative substitution rates, the relative fitness of the mutants in pvr2(3) pepper plants, or both.

A novel strategy for creating recombinant infectious RNA virus genomes.

Reverse transcriptases with RNase H activity are particularly apt to switch templates and generate recombinant molecules in vitro. This property has been exploited for the first time to create a library of recombinant RNAs 3 between two strains of Cucumber mosaic virus (CMV) or between CMV and Tomato aspermy virus (TAV), which share 75 and 63% sequence identity, respectively. The recombination events were almost entirely of the precise homologous type, and occurred at the same sites as those previously identified in co-infected plants, making it possible to use this strategy to create numerous cDNA fragments with crossovers similar to those occurring in vivo. Sub-cloning of recombinant fragments into an infectious full-length clone was accomplished by homologous recombination in yeast, alleviating the need for in vitro ligation at common restriction sites. Most of the recombinant genomes were infectious. Association of these two methods constitutes an efficient and practical means for generating numerous infectious viral genomes equivalent to ones that might arise by precise homologous recombination between two parental viral genomes in nature.

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.

Evidence for diversifying selection in Potato virus Y and in the coat protein of other potyviruses.

The modes of evolution of the proteins of Potato virus Y were investigated with a maximum-likelihood method based on estimation of the ratio between non-synonymous and synonymous substitution rates. Evidence for diversifying selection was obtained for the 6K2 protein (one amino acid position) and coat protein (24 amino acid positions). Amino acid sites in the coat proteins of other potyviruses (Bean yellow mosaic virus, Yam mosaic virus) were also found to be under diversifying selection. Most of the sites belonged to the N-terminal domain, which is exposed to the exterior of the virion particle. Several of these amino acid positions in the coat proteins were shared between some of these three potyviruses. Identification of diversifying selection events in these different proteins will help to unravel their biological functions and is essential to an understanding of the evolutionary constraints exerted on the potyvirus genome. The hypothesis of a link between evolutionary constraints due to host plants and occurrence of diversifying selection is discussed.