Mark Tepfer

Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD 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, therolD-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 inrolD-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 therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S2-gus. TherolD-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.

RECOMBINATION IN RNA VIRUSES AND IN VIRUS-RESISTANT TRANSGENIC PLANTS

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.

An overview of general features of risk assessments of genetically modified crops

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.

Cadmium partitioning in transgenic tobacco plants expressing a mammalian metallothionein gene

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.

Recombinants of cucumber mosaic virus (CMV) : determinants of host range and symptomatology

SummaryI17F 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.

In situ detection of expression of thegus reporter gene in transgenic plants: ten years of blue genes

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.

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.

Virus-resistant transgenic plants : potential ecological impact

Evolution the past, a window on the future?-Systematic search for recombination events in plant viruses and viroids- Different mechanisms of homologous and nonhomologous recombination in brome mosaic virus-Studies on RNA recombination in vivo and in vitro-RNA recombination in viral protein mediated virus resistant transgenic plants- Transgenic plants expressing viral sequences create a favourable environment for recombination between viral sequences-Behaviour of cucumovirus pseudorecombinant and recombinant strains in solanaceous hosts-

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.

rol A modulates the sensitivity to auxin of the proton translocation catalyzed by the plasma membrane H+-ATPase in transformed tobacco

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.