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Background and aims Camelina (Camelina sativa, Brassicaceae) has attracted interest in recent years as a novel oilseed crop, and an increasing number of studies have sought to enhance camelina’s yield potential or to modify the composition of its oil. The ability of camelina to cross-hybridize with its wild relative, C. microcarpa, is of interest as a potential source of genetic variability for the crop. Methods Manual crosses were performed between the crop C. sativa and its wild relative C. microcarpa; F1 and F2 progenies were obtained. Cytology was used to study meiosis in the parents and F1s and to evaluate pollen viability. Flow cytometry was used to estimate nuclear DNA amounts and fatty acid methyl ester analysis was used to evaluate the lipid composition of F3 seeds. Key Results The F1 plants obtained by interspecific crossing presented severe abnormalities at meiosis and low pollen viability, and produced very few F2 seeds. The F2s presented diverse phenotypes and in some cases severe developmental abnormalities. Many F2s were aneuploid. The F2s produced highly variable numbers of F3 seeds, and certain F3 seeds presented atypical lipid profiles. Conclusions Considering the meiotic abnormalities observed and the probability of aneuploidy in the F2 plants, the C. microcarpa accessions used in this study would be difficult to use as sources of genetic variability for the crop.

International audience; There is growing evidence that plant viruses manipulate host plants to increase transmission-conducive behaviors by vectors. Reports of this phenomenon frequently include only highly susceptible, domesticated annual plants as hosts, which constrains our ability to determine whether virus effects are a component of an adaptive strategy on the part of the pathogen or simply by-products of pathology. Here, we tested the hypothesis that transmission-conducive effects of a virus (Turnip yellows virus [TuYV]) on host palatability and vector behavior (Myzus persicae) are linked with host plant tolerance and physiological phenotype. Our study system consisted of a cultivated crop, false flax (Camelina sativa) (Brassicales: Brassicaceae), a wild congener (C. microcarpa), and a viable F1 hybrid of these two species. We found that the most tolerant host (C. microcarpa) exhibited the most transmission-conducive changes in phenotype relative to mock-inoculated healthy plants: Aphids preferred to settle and feed on TuYV-infected C. microcarpa and did not experience fitness changes due to infectionboth of which will increase viruliferous aphid numbers. In contrast, TuYV induced transmission-limiting phenotypes in the least tolerant host (C. sativa) and to a greater degree in the F1 hybrid, which exhibited intermediate tolerance to infection. Our results provide no evidence that virus effects track with infection tolerance or physiological phenotype. Instead, vector preferences and performance are driven by host-specific changes in carbohydrates under TuYV infection. These results provide evidence that induction of transmission-enhancing phenotypes by plant viruses is not simply a by-product of general pathology, as has been proposed as an explanation for putative instances of parasite manipulation by viruses and many other taxa.

International audience; Using high-throughput sequencing of small interfering RNAs (siRNAs), virion-associated nucleic acid (VANA), and double stranded RNAs (dsRNAs), we have determined the complete genome sequences of Comorian isolates of two ipomoviruses, cassava brown streak virus (CBSV) and a divergent isolate of Ugandan cassava brown streak virus (UCBSV-KM) representing a new strain of this virus. While the large ORF of CBSV shares the highest nucleotide sequence identity (95.9%) with a Tanzanian isolate of CBSV, the large UCBSV-KM ORF shares the highest nucleotide sequence identity (77.5%) with a Malawian isolate of UCBSV. This low value is near the species demarcation threshold for the family Potyviridae (

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.

International audience; 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.

International audience; Several potyviruses affect lettuce (Lactuca sativa) and chicory (Cichorium spp.) crops worldwide and are important constraints for production because of the direct losses that they induce and/or because of their seed transmission. Here, the molecular and biological properties are described of two potyviruses that were recently isolated from lettuce plants showing mosaic or strong necrotic symptoms in an experimental field in southeastern France. The first potyvirus belongs to the species Endive necrotic mosaic virus and is present in a large number of wild plant species, especially Tragopogon pratensis. It is unable to infect lettuce cultivars with a resistance to Turnip mosaic virus that is present in many European cultivars and probably conferred by the Tu gene. The second potyvirus belongs to the tentative species lettuce Italian necrotic virus and was not observed in wild plants. It infected all tested lettuce cultivars. Wild accessions of Lactuca serriola, Lactuca saligna, Lactuca virosa and Lactuca perennis were identified as resistant to one or the other potyvirus and could be used for resistance breeding in lettuce. No resistance against these two potyviruses was observed in the tested Cichorium endivia cultivars. In contrast, all tested Cichorium intybus cultivars or accessions were resistant.

paper originating from a presentation made at the 13th International Plant Virus Epidemiology Symposium held at Avignon, France June 6-10, 2016; 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.

Camelina [i](Camelina sativa[/i] (L.) Crantz) is a re-emergent oilseed crop that is also becoming important as a model for applied projects based on studies in[i] Arabidopsis thaliana[/i], since the two species are closely related members of the tribe [i]Camelineae[/i] of the Brassicaeae. Since camelina can be transformed genetically by floral dip, genetically modified (GM) camelina is being created in many laboratories, and small-scale field trials are already being conducted in the US and Canada. Although camelina does not cross-fertilize [i]Brassica[/i] crop species, such as oilseed rape, nothing was known about its ability to cross with other members of the tribe Camelineae, which in addition to arabidopsis includes the widespread weed, shepherd's purse ([i]Capsella bursa-pastoris[/i]). We have tested the ability of camelina to cross with arabidopsis and [i]C. bursa-pastoris[/i], as well as with the more distantly related Cardamine hirsuta, tribe cardamineae. No seeds were produced in crosses with arabidopsis, and a few seeds were obtained in crosses with [i]C. hirsuta[/i], but the embryos aborted at an early stage of development. A few seeds were also obtained in crosses with[i] C. bursa-pastoris[/i], which germinated to produce plants of a phenotype intermediate to that of the parents, but the hybrids were both male and female sterile. Therefore, the likelihood of pollen-mediated gene flow from camelina to these related species is low.

International audience; Camelina has emerged in the last decade as a multipurpose crop plant particularly suitable for engineering new lipids for diverse uses, including feed, biofuel and green chemistry. The rebirth of this ancient crop was based on several intrinsic favorable characteristics: robust agronomic qualities, attractive oil profile, genetic proximity with the model plant arabidopsis, ease of genetic transformation by floral dip. The need to increase both the production and diversity of plant oils, while improving the sustainability of agricultural systems, has been the driving forces behind the ever-increasing investment in camelina research. Worldwide interest in engineering camelina has led to the development of a remarkable pipeline that allows the rapid production and phenotyping of new lines; it includes specific tools, such as databases, collections of natural accessions, methods of genetic transformation and lipid analysis. Implementation of numerous metabolic pathways in camelina for the production of novel lipids has highlighted the potential as well as the versatility of this new "old" oilseed crop that is well on the way to becoming an ideal plant chassis for lipid synthetic biology.

International audience; Two members of the genus Ipomovirus (family Potyviridae) are known to infect cucurbits: cucumber vein yellowing virus (CVYV), which is emerging throughout the Mediterranean Basin, and squash vein yellowing virus (SqVYV), which has been described in America and the Caribbean Basin, and more recently in Israel. In this work, an ipomovirus different from CVYV and SqVYV, tentatively named coccinia mottle virus (CocMoV), was detected in a sample of the cucurbit Coccinia grandis collected in central Sudan in 2012. Sequence identity in nt was 68 % with CVYV, 59-60 % with SqVYV, cassava brown streak virus and Ugandan cassava brown streak virus, and less than 50 % with other members of the family Potyviridae. Preliminary biological and epidemiological studies indicate that CocMoV has a narrow natural host range and a low prevalence.

During a survey conducted in Sudan in 2012, a virus with spherical particles was isolated from a squash plant showing chlorotic leaf spots. The virus was transmitted mechanically and by two whitefly species, but not by aphids. RT-PCR with generic torradovirus primers yielded a band of expected size from total RNA of a symptomatic plant. Next-generation sequencing confirmed that this is tentatively a new torradovirus, for which we propose the name 'squash chlorotic leaf spot virus'. Using specific RT-PCR primers, the virus was detected in cucurbit samples collected since 1992 at different locations in Sudan.

National audience; En biologie végétale, la recherche translationnelle vise à accélérer la valorisation de découvertes scientifiques réalisées à partir de plantes modèles au bénéfice de plantes d’intérêt agronomique. Dans ce domaine, la caméline est un outil particulièrement efficace : génétiquement proche de l’espèce modèle Arabidopsis thaliana, elle permet d’évaluer simplement de nouveaux caractères associés aux plantes oléoprotéagineuses.

The seemingly inexorable expansion of global human population size, significant increases in the use of biofuel crops and the growing pressures of multifunctional land-use have intensified the need to improve crop productivity. The widespread cultivation of high-yielding genetically modified (GM) crops could help to address these problems, although in doing so, steps must also be taken to ensure that any gene flow from these crops to wild or weedy recipients does not cause significant ecological harm. It is partly for this reason that new GM cultivars are invariably subjected to strict regulatory evaluation in order to assess the risks that each may pose to the environment. Regulatory bodies vary in their approach to decision-making, although all require access to large quantities of detailed information. Such an exhaustive case-by-case approach has been made tractable by the comparative simplicity of the portfolio of GM crops currently on the market, with four crops and two classes of traits accounting for almost all of the area under cultivation of GM crops. This simplified situation will change shortly, and will seriously complicate and potentially slow the evaluation process. Nowhere will the increased diversity of GM crops cause more difficulty to regulators than in those cases where there is a need to assess whether the transgene(s) will enhance fitness in a non-transgenic relative and thereafter cause ecological harm. Current practice to test this risk hypothesis focuses on attempting to detect increased fitness in the recipient. In this paper we explore the merits and shortcomings of this strategy, and investigate the scope for developing new approaches to streamline decision-making processes for transgenes that could cause unwanted ecological change.

The uncapped and ORF-less subgenomic RNA5 is produced in subgroup II strains of cucumber mosaic virus (CMV), but not in subgroup I strains. Its initiation nucleotide (nt 1903) is in a 21 nt conserved sequence (Box1) that is absent in CMV subgroup I. Putative non-coding RNA structural elements surrounding Box1 in the plus and minus strand were identified in silico and by in vitro RNase probing. Four main stem–loop structures (SLM, SLL, SLK and SLJ) were identified between nt 1887 and 1999 of isolate R-CMV (subgroup II), with notable differences within SLM and SLL between the two strands. Mutation of a stem–loop within SLM, even when the predicted wild-type structure was maintained, showed significant reduction in RNA5 levels in planta. Three mutants containing 3–4 nt substitutions between positions −39 and +49 showed significantly reduced levels of RNA5, while another similar mutant at positions 80–83 had RNA5 levels comparable to wild-type. Deletion of Box1 resulted in similar levels of RNA3 and 4 as wild-type, while eliminating RNA5. Insertion of Box1 into a subgroup I isolate was not sufficient to produce RNA5. However, in a mutant with an additional 21 nt of R-CMV 3′ of Box1 (positions −1 to +41), low levels of RNA5 were detected. Taken together, these results have identified regions of the viral genome responsible for RNA5 production and in addition provide strong evidence for the existence of newly identified conserved structural elements in the 5′ part of the 3′ untranslated region.

Plant genetic engineering has promised researchers improved speed and flexibility with regard to the introduction of new traits into cultivated crops. A variety of approaches have been applied to produce virus-resistant transgenic plants, some of which have proven to be remarkably successful. Studies on transgenic resistance to Cucumber mosaic virus probably have been the most intense of any plant virus. Several effective strategies based on pathogen-derived resistance have been identified; namely, resistance mediated by the viral coat protein, the viral replicase, and post-transcriptional gene silencing. Techniques using non-pathogen-derived resistance strategies, some of which could offer broader resistance, generally have proven to be much less effective. Not only do the results obtained so far provide a useful guide to help focus on future strategies, but they also suggest that there are a number of possible mechanisms involved in conferring these resistances. Further detailed studies on the interplay between viral transgene-derived molecules and their host are needed in order to elucidate the mechanisms of resistance and pathogenicity.

The intentional introduction into the environment or market of genetically modified organisms (GMOs) is nearly always governed by a framework of science-based risk assessment and risk management measures. This is usually implemented through the integration of hazard identification and characterisation of all of the elements of risk associated with a new GM crop or derived product. Typical categories of hazards arising from the introduction of transgenic crops include: possible unintended negative health effects in a susceptible subgroup of the consumer (target) population; the evolution of resistance in the targeted pest/pathogen populations when the transgene confers resistance to a pest or pathogen; non-target hazards associated directly or indirectly with the transgenic plant or transgene product outside the plant; and those associated with the integration and subsequent expression of the transgene in a different organism or species following gene flow. The consequences of likely exposure to these and other hazards are considered in this introduction to the main issues raised when evaluating the possible risks arising from the importation or cultivation of genetically modified crops.

International audience; 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

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.

▪ Abstract Virus-resistant transgenic plants (VRTPs) hold the promise of enormous benefit for agriculture. However, over the past ten years, questions concerning the potential ecological impact of VRTPs have been raised. In some cases, detailed study of the mode of action of the resistance gene has made it possible to eliminate the source of potential risk, notably the possible effects of heterologous encapsidation on the transmission of viruses by their vectors. In other cases, the means of eliminating likely sources of risk have not yet been developed. When such residual risk still exists, the potential risks associated with the VRTP must be compared with those associated with nontransgenic plants so that risk assessment can fully play its role as part of an overall analysis of the advantages and disadvantages of practicable solutions to the problem solved by the VRTP.

Potyviruses, such as potato virus Y and tobacco etch virus, as well as cucumber mosaic cucumovirus, have the ability to inhibit 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 colored 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. They also suggest that virus infection could lead to loss of agronomically desirable traits obtained by PTGS in transgenic plants.

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.

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.

Recombination in RNA viruses is a general phenomenon, and is considered to play a major role as a driving force in virus variability and thus in virus evolution. An ever increasing number of RNA viruses has been shown to undergo RNA recombination, whether under natural or experimental con- ditions. More than 30 years after its discovery, the mechanisms of viral RNA recombination are only now beginning to be elucidated. Recent reports strongly suggest that RNA re- combination is linked to virus replication, and that it occurs by a copy-choice mechanism. The detection of recombination between host transcripts and infecting viral RNA genomes has given rise to new concerns about the release of virus-resistant transgenic crops, since recombination could generate viruses with properties dierent from the infecting strain. We will describe here current knowledge of recombination in RNA viruses, and then will present results showing recombination in transgenic virus-infected plants, and finally will discuss to what extent this may lead to increased variability in plant RNA viruses.

International audience; I17F and R are subgroup I and II cucumber mosaic virus (CMV) strains, respectively. Whereas I17F induces severe symptoms on all hosts so far tested, R induces generally mild symptoms, except on Nicotiana glutinosa, on which it causes leaf blistering and severe stunting. Pseudorecombinants and recombinants, based on RNAs 1 and 2 from R-CMV, were created by adding either I17F RNA 3 or one of two chimeric RNAs created by exchanging approximate halves of RNA 3 of the two strains. The viruses created were tested on different hosts of the virus. On maize, local necrotic lesions were induced by all strains with the 5′ part of RNA 3 from R-CMV, whereas only I17F-CMV induced a systemic infection. The seven solanaceous hosts tested could be classified into two main groups. In the first, RNA 3 was not directly involved in the symptoms that were systemically induced, and the extreme disease severity induced by I17F was correlated with high virus accumulation. In the same hosts, the lesser virulence of R-CMV could reflect a deficiency in long-distance movement, involving RNA 3. The second group included Nicotiana glutinosa where the symptoms induced by R-CMV were determined by the 3′ part of RNA 3.

The rolA gene is transferred naturally by Agrobacterium rhizogenes to the genome of host plants, where it induces dramatic changes in development of transformed plants, including dwarfism and leaf wrinkling. The predicted translation product of the rolA gene is a small (11.4 kDa), basic (pI = 11.2) protein, which has no clearly significant similarity to sequences in the data bases. We have introduced into the tobacco genome a gene encoding a rolA∷GUS fusion protein. Expression of this gene led to synthesis of an RNA and a protein of expected size, and the transformed plants exhibited the dwarfism and leaf wrinkling typical of rolA plants, but to a lesser degree than plants transformed with the wild-type rolA gene. The distribution of β-glucuronidase (GUS) activity was compared in subcellular fractions of leaf extracts from plants expressing either the rolA∷gus gene or a control gus construct. As expected, in the control plants, GUS activity was essentially cytosolic. In contrast, in plants expressing the rolA∷gus gene the highest specific activity was associated with the plasmalemma fraction.

Among the plant species cultivated for human consumption, tobacco is one that accumulates cadmium to a significant degree. In order to reduce the Cd levels in tobacco leaves, we have introduced into the tobacco genome a gene encoding a mammalian metallothionein, since these low-molecular-weight cysteine-rich proteins fix Cd and other heavy metals. Here we describe the Cd accumulation characteristics observed during two years of greenhouse tests and one year of field trial of tobacco plants expressing a metallothionein gene. In all three tests, leaf Cd levels were markedly decreased. For instance, in the field trial, Cd levels in the leaf lamina tissue of the transformed line were decreased by 73% compared to controls. The decrease in leaf Cd was correlated with an increase in Cd in the roots and stems. The plants had normal growth characteristics, and the distribution of other ions was not affected by the expression of the metallothionein gene. Even though the transformation/expression vector used here can cause frequent post-transcriptional gene silencing, comparison of hemizygous and homozygous individuals of the plant line expressing the metallothionein gene gave little evidence for silencing.

International audience; Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) are closely related cucumoviruses, We have made pseudorecombinant viruses in which the RNAs 3 of these two viruses have been exchanged and recombinant viruses containing chimeric RNA 3 molecules, in which the coat proteins and the 3'-end regions of CMV and TAV have been exchanged, giving rise to recombinants designated RT3 and TR3, The replication properties and the cell-to-cell and long-distance movement patterns of these pseudorecombinant and recombinant viruses were examined in different hosts, All the viruses were able to replicate and accumulate RNA 4 in protoplasts, The pseudorecombinants and the R1R2RT3 recombinant infected tobacco systemically, but the R1R2TR3 recombinant was not detectable, even in the inoculated leaves, Comparison of the abilities of the viruses to replicate in protoplasts and intact cucumber plants suggests that cell-to cell movement factors are also encoded by RNAs 1 and/or 2, Major determinants of symptom severity in Nicotiana glutinosa are localized on the 3' part of RNA 3, and in Nicotiana benthamiana, more severe symptoms were observed with the T1T2R3 strain than,vith the others tested.

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.

International audience; In tackling agricultural challenges, policy-makers in sub-Saharan Africa (SSA) have increasingly considered genetically modified (GM) crops as a potential tool to increase productivity and to improve product quality. Yet, as elsewhere in the world, the adoption of GM crops in SSA has been marked by controversy, encompassing not only the potential risks to animal and human health, and to the environment, but also other concerns such as ethical issues, public participation in decision-making, socio-economic factors and intellectual property rights. With these non-scientific factors complicating an already controversial situation, disseminating credible information to the public as well as facilitating stakeholder input into decision-making is essential. In SSA, there are various and innovative risk communication approaches and strategies being developed, yet a comprehensive analysis of such data is missing. This gap is addressed by giving an overview of current strategies, identifying similarities and differences between various country and institutional approaches and promoting a way forward, building on a recent workshop with risk communicators working in SSA.

When applying risk assessment and the broader process of risk analysis to decisions regarding the dissemination of genetically modified organisms (GMOs), the process has a tendency to become remarkably complex. Further, as greater numbers of countries consider authorising the large-scale dissemination of GMOs, and as GMOs with more complex traits reach late stages of development, there has been increasing concern about the burden posed by the complexity of risk analysis. We present here an improved approach for GMO risk analysis that gives a central role to problem formulation. Further, the risk analysis strategy has been clarified and simplified in order to make rigorously scientific risk assessment and risk analysis more broadly accessible to diverse stakeholder groups.

Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus (uidA) gene ofEscherichia coli, which encodes a β-glucuronidase (E.C. 3.2.1.31; GUS), have been the most widely used during the last ten years. The apparent simplicity of the histochemical GUS assay has been a major factor in the increase in articles usinggus genes. However, over the last four years, there have been occasional reports expressing doubts concerning the specificity of the observed localizations based on discrepancies between results obtained with GUS histochemistry and immunocytochemistry and/orin situ hybridization. This brief review compares the results obtained with immunocytochemistry with those obtained with various GUS substrates for histochemical studies. Certain sources of artefact are discussed, as are the limits that should be imposed on interpretation of GUS histochemistry results at the organ, tissue and cell levels.

Chimeric genes under the control of a CaMV 35S promoter with a doubled enhancer (35S2) that encode a mammalian metallothionein (hMTII), or an Escherichia coli beta-glucuronidase (GUS), or a hMTII/GUS fusion protein were introduced into the genome of tobacco (Nicotiana tabacum cv. PBD6). Transcripts and Cd-binding proteins of expected size were observed in plants expressing either the 35S2-hMTII or the 35S2-hMTII/GUS gene, and in the latter plants a protein with GUS activity that was larger than the native GUS enzyme was observed. Thus, plants expressing the hMTII-GUS gene synthesize a bifunctional protein, with both GUS and Cd-binding activity. In an in vitro assay, seedlings expressing either one of these genes had 60-70% lower Cd concentration in their shoots than controls, and Cd translocation to the shoot system was reduced (approximately 20% of Cd absorbed was translocated), compared with that in controls expressing a 35S2-GUS gene, where approximately 50% of the Cd absorbed was translocated.

International audience; Modification of the gravitropic response of seedling roots of rapeseed {Brassica napus) transformed by Agrobacterium rhizogenes A4 V. Legue, F. Vilaine, M, Tepfer and G, Perbal Legue. V,. Vilaine, E. Tepler. M. and Perbal. G. 1994. Modification ofthe gravitropic response of seedling roots of rapeseed \Bra.ssica napii.\i transformed by Agrobacieiium rhi:ngem's A4,-Physiol, Plant, 91: 559-566. We have e.xamlued the growth and gra\ itropic response of seedling roots of rapeseed {Brassica napus. CrGC5-l) transformed by AgrobacWiiiini rhizogenes .\4. in order to evaluate if this could constitute a new model system for the study of gravitropism. The transformed clone chosen for study had integrated full-length TL-and TR-D.NA from pRi (the root inducing piasmid). and thus included all of the agrobacteriai genes potentially involved in the modified phenotype of transformed plants. In the vertical position, the gnnvth rate of transformed roots was higher than controls. During 24 h of continuous stimulation, the optimal angle for gravitropic bending in normal roots was 135° twith respect to the gravity a.sis). with decreasing response at 90° and 45^, Eor transformed roots, slight curvature developed at 45"' and at 90°. and stronger curvature was observed at 135. though transformed root tips never reached the vertical position. The minimum stimulation time necessary to elicit a response (presentation time) was also determined: it was significantly shorter in normal roots t80 si than in transformed ones * 120 s). The results show that pRi transformed roots are less sensitive to gravity than normal roots.

International audience; Both positive [(+)] and negative [(-)] sense versions of two satellite RNA (satRNA) genes from cucumber mosaic virus (CMV), the necrogenic I17N and the non-necrogenic R, have been introduced into the genome of tobacco plants. On infection with satRNA-free CMV, satRNA was amplified in plants expressing each of the four genes. All four genes confer protection against CMV infection. However, co-inoculation of plants with viral RNA and CMV satRNA transcripts synthesized in vitro showed that (-) sense transcripts were less active than the corresponding (+) sense transcripts. This is the first report that(-) sense CMV satRNA transcripts can serve as a template for satRNA replication.

International audience; Severe symptoms of cotton leaf curl disease (CLCuD) are caused by the association of a single-stranded circular DNA satellite (betasatellite) with a helper begomovirus. In this study, we analyzed 40 leaf samples (primarily cotton with CLCuD symptoms and other plants growing close by) from four sites between New Delhi and the Pakistan/India border, using rolling-circle amplification (RCA) and PCR. In total, the complete sequences of 12 different helper viruses, eight alphasatellites, and one betasatellite from five different plant species were obtained. A recombinant helper virus molecule found in okra and a novel alphasatellite-related DNA from croton are also described. This is the first report of the presence of both DNA components (helper virus and betasatellite) associated with resistance-breaking CLCuD in India, and it highlights the need for further work to combat its damage and spread.

Summary When transferred to the plant genome, the rolA gene of Agro- bacterium rhizogenes induces numerous changes in plant phenotype, including prominent leaf wrinkling and dwarfism. Cytological, physiological and molecular studies were carried out on tobacco plants expressing rolA, with the goal of explaining these developmental modifications.

Viral diseases of cultivated crops are responsible for the worldwide loss of billions of US dollars in agricultural productivity every year. Historically, this loss has been reduced or minimized principally by the implementation of specific agricultural/phytosanitary measures, and by the introduction of naturally occurring virus-resistance genes into appropriate cultivars by plant breeding. Since the first report of virus-resistant transgenic plants (VRTPs) in 1986, a remarkable diversity of virus-resistance transgenes has been developed. Despite this, to a large part due to controversy surrounding the use of genetically modified organisms, the number of commercially available VRTPs remains small. However, since the potential risks associated with VRTPs were first formulated in the early 1990s, fundamental research on plant-virus interactions and also research specifically aimed at resolving biosafety issues have greatly circumscribed the potential impact of the risks envisaged. Yet, in spite of the advances, both in strategies for creating VRTPs and in the assessment of potential risks, it remains remarkably difficult to weigh the risks/costs and benefits of different means to manage plant viral diseases, and even to make scientifically well-founded choices of the most appropriate strategy for creating VRTPs. Many of the outstanding issues concern the lack of sufficient knowledge of the breadth and durability of the resistance of VRTPs under field conditions. VRTPs will only take their appropriate place in modern agriculture when their potential users will be able to base their choices on realistic assessments of their efficacy, durability, and safety.