Evaluation of the progeny produced by interspecific hybridization between Camelina sativa and C. microcarpa.

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.

High-throughput sequencing of complete genomes of ipomoviruses associated with an epidemic of cassava brown streak disease in the Comoros Archipelago.

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 (<76%). Phylogenetic analysis confirms that UCBSV-KM represents a new lineage that is genetically distinct from the currently described UCBSV strains.

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.

Characterization of a new cucurbit-infecting ipomovirus from Sudan.

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.

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.

Evaluation of the potential for interspecific hybridization between Camelina sativa and related wild Brassicaceae in anticipation of field trials of GM camelina.

Camelina (Camelina sativa (L.) Crantz) is a re-emergent oilseed crop that is also becoming important as a model for applied projects based on studies in Arabidopsis thaliana, since the two species are closely related members of the tribe Camelineae 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 Brassica 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 (Capsella bursa-pastoris). We have tested the ability of camelina to cross with arabidopsis and C. bursa-pastoris, 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 C. hirsuta, but the embryos aborted at an early stage of development. A few seeds were also obtained in crosses with C. bursa-pastoris, 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.

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.

A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India.

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.

Genome-wide specificity of plant genome editing by both CRISPR-Cas9 and TALEN.

CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.

Analysis of recombination between viral RNAs and transgene mRNA under conditions of high selection pressure in favour of recombinants.

One possible environmental risk related to the utilization of virus-resistant transgenic plants expressing viral sequences is the emergence of new viruses generated by recombination between the viral transgene mRNA and the RNA of an infecting virus. This hypothesis has been tested recently for cucumber mosaic virus (CMV) by comparing the recombinant populations in transgenic and non-transgenic plants under conditions of minimal selection pressure in favour of the recombinants. Equivalent populations were observed in transgenic and non-transgenic plants but, in both, there was a strongly dominant hotspot recombinant which was shown recently to be nonviable alone in planta, suggesting that its predominance could be reduced by applying an increased selection pressure in favour of viable recombinants. Partially disabled I17F-CMV mutants were created by engineering 6 nt deletions in five sites in the RNA3 3'-non-coding region (3'-NCR). One mutant was used to inoculate transgenic tobacco plants expressing the coat protein and 3'-NCR of R-CMV. A total of 22 different recombinant types were identified, of which 12 were, as expected, between the transgene mRNA and the mutated I17F-CMV RNA3, while 10 resulted from recombination between the mutated RNA3 and I17F-CMV RNA1. Twenty recombinants were of the aberrant type, while two, including the dominant one detected previously under conditions of minimal selection pressure, were homologous recombinants. All recombinants detected were very similar to ones observed in nature, suggesting that the deployment of transgenic lines similar to the one studied here would not lead to the emergence of new viruses.

Twenty years of transgenic plants resistant to Cucumber mosaic virus.

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.

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.

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.

Risk assessment of virus-resistant transgenic plants.

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.

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.

Putting problem formulation at the forefront of GMO risk analysis.

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.

Experiences in sub-Saharan Africa with GM crop risk communication: outcome of a workshop.

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.

Assessment of the benefits and risks for engineered virus resistance.

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.

Fitness and beyond: preparing for the arrival of GM crops with ecologically important novel characters.

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.