Search

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; 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.

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

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.

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.

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.

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.

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.

In order to investigate the effect of the rol A gene product on the plasma membrane response to auxin, a clone of rol A-transformed tobacco was prepared. Auxin sensitivity was measured by the auxin concentration which induced the highest stimulation of H+-ATPase-mediated proton translocation on isolated plasma membrane vesicles. Both transformed and control plants exhibited identical auxin-sensitivity changes during vegetative and induction to flowering periods. However the sensitivity of flowering-transformed plants Was 100-times higher than that of control plants. Consistent observations were also made when using rol A+B+C-transformed plants. These results suggest that the rol A gene product could either participate in or affect the reception/transduction pathway of auxin signal at the plasma membrane.

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.

Recombination in single-stranded RNA viruses is one of the principal mechanisms responsible for their evolution. Here we show, using a variety of different methods, that the 3' untranslated region (3'UTR) of subgroup II strains of cucumber mosaic virus [CMV(II)] is related more closely to that of tomato aspermy virus (TAV) than to those of CMV(I) strains. These results suggest that the CMV(II) 3'UTR arose by interspecific CMV/TAV recombination. The putative crossover is close to the 5' end of the 3'UTR, at a recombination hot spot previously observed in short time-frame experiments. The CMV(II) strains show divergence from TAV at specific points along the 3'UTR that most probably indicate adaptive changes due to natural selection. Thus, for the large majority of CMV(II) strains examined, the 3'UTR has two discrete regions, W (nt 1902-1971) and Y (nt 2126-2184), that are more similar to the corresponding regions of TAV than to those of CMV(I) strains.

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.

One of the communication tools of the EC-funded Biosafenet project is a series of six Biosafenet Seminars that will be held 2007–2009. Their purpose is to convene small groups of scientists to consider emerging issues related to the potential impact of genetically modified organisms (GMOs). The first Seminar was organized by the ICGEB at the MasterCampus of the Fondazione Cassamarca in Ca’ Tron di Roncade (Italy) on 6th–8th June 2007, and focused on the potential impact of both naturally occurring and transgenic viral sequences in plant genomes.

In order to better understand the role of recombination in creating the diversity of viral genomes that is acted on by selection, we have studied in detail the population of recombinant RNA3 molecules occurring in tobacco plants coinfected with wild-type strains of cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) under conditions of minimal selection pressure. Recombinant RNA3s were observed in 9.6% of the samples. Precise homologous recombination predominated since it was observed at 28 different sites, primarily in six hot spots. Imprecise homologous recombination was observed at two sites, particularly within a GU repeat in the 5′ noncoding region. Seven of the eight aberrant homologous recombination sites observed were clustered in the 3′ noncoding region. These results have implications on the role of recombination in host adaptation and virus evolution. They also provide essential baseline information for understanding the potential epidemiological impact of recombination in transgenic plants expressing viral sequences.

International audience; 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.

Recombination is considered to play a key role in RNA virus evolution; however, little is known about its occurrence under natural conditions. We inoculated tobacco plants with wild-type strains of two closely related cucumovirus species: cucumber mosaic virus (CMV) and tomato aspermy virus (TAV). RNA from the inoculated leaves of doubly-infected plants was tested for the presence of recombination events in an 0.8-kb central portion of the viral RNA3. Using a sensitive and specific RT-PCR procedure, we amplified recombinant segments of RNA3 in 3 of 82 tobacco plants infected with both viruses. In each plant in which recombinant segments were amplified, several different crossover sites were observed, all of which were located within a short stretch of high sequence similarity. Two plants had both CMV-TAV and TAV-CMV recombinants. In all cases, precise homologous recombination had occurred. To the best of our knowledge, this is the first report of interspecific recombination between wild-type plant RNA viruses under conditions of minimal selection pressure in favor of the recombinants.

In this review are presented some emerging methods to improve virus resistance in transgenic plants, based on the use of nonviral genes from various organisms. The genes discussed include ones coding for proteins involved in virus resistance: antibodies, PKR kinase and 2′-5′ oligoadenylate synthase from mammalian cells; the yeast SKI antiviral genes. Genes not clearly involved in resistance are also described, including ones encoding protease inhibitors or ribosome-inactivating proteins. The natural pathways for pathogen resistance in plants may also be enhanced, through modification of genes involved in the hypersensitive response, systemic acquired resistance, or nonhost and extreme resistance.

In order to study the expression in plants of the rolD promoter of Agrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of the gusA (uidA) marker gene under control of two rolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R1 progeny plants. In mature transformed tobacco plants, the rolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity in rolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter, domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of the rolD-gus genes was less than that of a gus gene with a 35S promoter with doubled domain B, 35S2-gus. The rolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.

In order to evaluate new possibilities for protecting plants against virus infection by interference with viral replication, two chimeric genes were constructed in which the (+) strand 3′-terminal 100 nucleotides (nt) of the noncoding region of the turnip yellow mosaic virus (TYMV) genome were placed downstream from the sense or antisense cat coding region. The two chimeric genes were then introduced into the genome of rapeseed ( Brassica napus ) using an Agrobacterium rhizogenes vector system. Plants expressing high levels of either chimeric gene showed partial protection against infection by TYMV RNA or virions. One interesting feature of the protection is that a proportion of the inoculated transgenic plants does not become infected. Protection was overcome when the inoculum concentration was increased. RNA complementary to the initial transcript was detected after infection.

International audience; Cacao swollen shoot virus is classified as a badnavirus based on its nonenveloped, bacilliform particle morphology and double-stranded DNA genome. A complete copy of the genome was cloned into a plasmid vector and the sequence was determined from 75 overlapping subclones covering both strands. The genome contains 7161 base pairs and possesses an intergenic region and five putative open reading frames (ORF) capable of coding for proteins > 10 kDa. All of the ORFs are present on the plus-strand. ORF 1 (17 kDa) and ORF 2 (14 kDa) encode proteins of unknown function. The large ORF 3 (211 kDa) encodes a polyprotein that can be divided into three regions. Based on distant homologies with viral movement proteins, region 1 may encode a protein involved in cell-to-cell spread, while region 2 encodes the viral capsid protein. Region 3 contains consensus sequences for viral aspartyl proteinase, reverse transcriptase, and ribonuclease H characteristic of pararetroviruses. The last two ORPs (13 and 14 kDa) overlap ORF 3 and are not present in the other badnaviruses described.

We have used the wild-type Agrobacterium rhizogenes strain A4 to induce roots on cucumber stem explants. Cultures of transformed roots obtained that were capable of hormone-autonomous growth could be grouped in three phenotypic classes. Of particular interest were extremely thick roots of a type not previously described. Characterization of the transferred DNA and of the expression of the corresponding genes allowed us to determine that the genes rolABC of the TL region of the Ri plasmid are sufficient to induce thin roots similar to those observed in other species, while the aux genes of the TR region are sufficient to induce thick roots. Among clones bearing the aux genes, there was a correlation between level of expression of aux2 and root phenotype.

International audience

This article describes several new expression vectors that capitalize on the ability of Agrobacterium rhizogenes to transfer DNA from its Ri plasmid to the plant nuclear genome. The intermediate vectors described include an expression cassette based on one of the three following promoters: the nopaline synthase promoter, or the cauliflower mosaic virus (CaMV) promoters responsible for transcription of either the 19S or 35S CaMV RNA. The termination and polyadenylation signals are either from the nopaline synthase gene or from CaMV. The expression micro-Ri plasmid described bears a selectable marker gene and an expression cassette cloned between the borders of the TL-region of the Ri plasmid of A. rhizogenes A4. Different strategies for using these vectors to introduce chimeric genes into plants are described, and the advantages and disadvantages of the two types of vectors are discussed.

Extensibility characteristics of Avena stem segments treated with gibberellic acid (GA) were investigated in living internodes using a microgrowth method and in partially extracted cell walls subjected to Instron extensometer analysis. Both techniques showed that treatment with GA greatly increases internodal plasticity, but has virtually no effect on internodal elasticity. The increase in plasticity occurred 1 to 2 hours after the initiation of hormone treatment, which is similar to the time of onset of GA-enhanced growth and cell wall synthesis. Cycloheximide was shown to inhibit the effect of GA on plasticity.

The acid-induced loosening of cell walls of Valonia ventricosa has been compared to that of frozen-thawed oat coleoptiles. The two acid extension responses are similar in regard to the shape of the pH response curve and the increase in plastic compliance induced by acid treatment. In both systems the acid response can be inhibited by Ca2+ and in both the removal of the protons leads to a rapid termination of wall loosening. The two responses differ in several significant ways, however. The acid-induced extension of Valonia walls is more rapid than that of coleoptile walls, but of smaller total magnitude. Acid-induced loosening can occur in Valonia without the wall being under tension, but not in coleoptiles. The acid-induced extension of Valonia walls is not inhibited by 8 molar urea, whereas the response in oat coleoptiles is completely inhibited by this treatment. Ethylenediaminetetraacetate (EDTA) can cause wall loosening in Valonia comparable to that produced by low pH, whereas in coleoptiles EDTA causes a much smaller response. These results with Valonia are consistent with a mechanism of acid-induced wall loosening in which a central role is played by the displacement of Ca2+ from the wall, while the larger part of acid-induced wall loosening in oat coleoptiles appears to be via a different mechanism.

Three gene constructions based on a mouse metallothionein I gene (mMT-I) were introduced into tobacco using an Ri plasmid vector system to test the effectiveness of animal gene regulatory signals in plant cells. No transcription from the native mouse gene was observed. In plant cells bearing chimeric mMT-I genes in which transcription was driven by the nopaline synthase promoter, neither polyadenylation nor splicing of mMT-I pre-mRNA was observed. Detailed comparisons of mMT-I sequences with those of known plant genes were carried out; slight differences in regions of known consensus sequences may be at least partly responsible for the non-recognition of mMT-I gene regulatory signals in plant cells, though other as yet unidentified, potentially necessary sequences may also be involved.

When frozen-thawed Avena sativa L. coleoptile and Cucumis sativa L. hypocotyl sections, under tension, are acid-treated, they undergo rapid elongation (acid-extension). The acid-extension response consists of two concurrent phases: a burst of extension which decays exponentially over 1-2 h (ExE), and a constant rate of extension (CE) which can persist for at least 6h. The extension (delta L) is closely represented by the equation: delta L = a-a e(-kt) + C t where a is the total extension of the exponential phase, k is the rate constant for ExE, and c is the rate of linear extension (CE). Low pH and high tension increased a and c, whereas temperature influenced k. The magnitude of the CE (over 50% extension/10 h), the similarity in its time course to auxin-induced growth, and the apparent yield threshold for CE indicate that CE is more likely than ExE to be the type of extension which cell walls undergo during normal auxin-induced growth.

It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L. CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legue et al. 1994, Physiol Plant 91: 559-566). After stimulation at 90 degrees C or 135 degrees, the transformed root tips curve. but never reach a vertical orientation. In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips. First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation. The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length. In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls. Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied. Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone. In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application. Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction. The level of endogenous IAA was lower in transformed roots. Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.