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.

Adapting plants to sustainable cropping systems is a major challenge for facing climate change and promoting agroecological transition. Camelina sativa is an emerging oilseed crop species with climate-resilient properties that could be used in double-cropping systems, in particular as a summer catch crop. The availability of early-flowering camelina is essential in such cropping systems to allow full completion of the growth cycle during summer. Targeted induced gene variation (TIGV) was used in camelina on several flowering repressor genes identified in Arabidopsis to obtain early-flowering lines. Multiplex editing of 15 target genes representing FLOWERING LOCUS C, SHORT VEGETATIVE PHASE, LIKE HETEROCHROMATIN PROTEIN 1, TERMINAL FLOWER 1 and EARLY FLOWERING LOCUS 3 induced combinatorial mutations that were screened for early-flowering phenotypes. Certain mutants showing a stable early-flowering trait after five generations also presented additional phenotypes: determinate flowering, shorter stature and/or basal branching. Different combinations of mutations had a positive or negative impact on yield. This work demonstrates that efficient multiplex CRISPR is achievable in hexaploid plants like camelina, providing valuable genetic diversity for better selecting lines adapted to new cropping systems.

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.

Over the last ten years, numerous laboratories have introduced viral coat protein (cp) genes into plant genomes as a means of creating novel sources of virus resistance. More recently, there has been concern about the possibility that large scale cultivation of plants expressing viral genes could lead to ecological risks. One of the most problematic potential risks is that recombination between the viral sequence expressed by the plant and the RNA of the genome of an infecting virus could result in the creation of a novel virus genome. It has been shown experimentally that recombination in transgenic plants can occm, and thus one of the key questions concerning recombinant virus genomes is whether recombinants can have a competitive advantage or aggravate disease symptoms, in comparison to the parent strains. Using cucumber mosaic virus (CMV) and the closely related tomato aspermy virus (TA V), we have created recombinant viral genomes, including those in which the CMV cp gene has been replaced by the TAV cp gene, and vice versa. Such recombinants theoretically could arise by recombination upon infection of plants expressing a CMV or TAV cp gene. We present here an evaluation of the biological characteristics of the recombinant viruses as compared to the parent strains, including their ability to replicate, to spread within host plants, and to induce symptoms.

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 infection—both 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.

Understanding epidemiology is central to the development of effective and sustainable strategies for the control of infectious diseases, and epidemiology requires and is related to knowledge of pathogen ecology. This chapter reviews studies on the epidemiology and ecology of cucumber mosaic virus (CMV), an important pathogen with a broad host range, including the following topics: the natural host range of CMV; the occurrence and incidence of CMV infection; CMV population structures in the field; and the occurrence of highly virulent isolates of CMV, their effects, and their population dynamics. The present knowledge regarding CMV ecology and epidemiology fails to explain outstanding features of its biology, such as its extremely broad host range, its ubiquity, and the intrinsic variability of its abundance in plant populations. The use of new molecular and bioinformatics tools in field studies is expected to lead to major advances regarding the major outstanding issues of CMV epidemiology.

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.

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 x 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.

Az idei evben jelentős virusfertőzottseget tapasztaltunk szamos papri katermesztő korzetben. A hajtatasbol es a szabadfoldről szarmazo mintak uborka mozaik vir us fertőzest mutattak. A korabbi tapasztalattol (a CMV II csoportba tartozo izolatum ok dominaltak) elterően c. egyforma aranyban fordultak elő a CMV I. es II. csoportjaba tartozo izolatumok. Vizsgalatunk soran megallapitottuk, hogy egy ujabban termesztesbe kerulő CMV rez isztens fajta es az ismert, de rezisztenciaval nem rendelkező etkezesi fajta kozott nem talalt unk lenyegi kulonbseget a CMV tolerancia tekinteteben sem a tunetek megjeleneseben, sem a vir uskoncentracioban. Azt tapasztaltuk, hogy a CMV rezisztens fajtat idősebb korban fertőz ve a novenyben a virus lassabban szisztemizalodik. Erdekes tapasztalat volt, hogy a to bamovirus rezisztencia gent tartalmazo fűszerpaprika fajtak erzekenyebbek voltak az ubor ka mozaik virus fertőzesevel szemben, mint a rezisztencia gent nem tartalmazo fajtak.

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.

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 5x5 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 virusor viroid-infected ornamental plants is an effective means for screening for the presence of potentially important pathogens.

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.

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.

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.