Transgene inactivation inPetunia hybrida is influenced by the properties of the foreign gene

Petunia mutant RL01 was transformed with maizeA1 and gerberagdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity. The sameAgrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments. Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants. However, theA1 andgdfr transformants showed clearly different phenotypes. The flowers of the primaryA1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of thegdfr transformants showed intense and highly stable coloration. The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment. As previously reported by others, the instability in pigmentation in theA1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself. In thegdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome. Only rarely was partial methylation of the 35S promoter detected, while thegdfr cDNA always remained in an unmethylated state. We conclude that the properties of the transgene itself strongly influence the inactivation process. The dicotyledonousgdfr cDNA with a lower GC content and fewer possible methylation sites is more ‘compatible’ the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.     

Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants

Silencing ofNia host genes and transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobaccoNia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whetherNii host genes and transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a transgene consisting of the tobaccoNii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of theNii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing ofNii host genes and transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of transgene loci and/or on certain allelic or ectopic combinations of transgene loci. In one transformant carrying a single transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this transgene locus is brought into the presence of a non-allelic transgene locus by crossing, suggesting that a homozygous state is not absolutely required.     

Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: role of numbers of integrated gene copies and state of methylation.

Summary ThePetunia hybrida mutant RL01 is white flowering due to a genetic block in the anthocyanin pathway. The introduction of the maize Al cDNA under the control of the CaMV 35S RNA promoter leads to the production of pelargonidin derivatives, resulting in a brick red flower phenotype. Among the transgenic petunia plants the pigmentation of the petals exhibited different expression patterns which could be categorized into the red, the variegated, and the white phenotype. This system proved to be especially suitable for the investigation of gene expression by simply looking at the pigmentation pattern of the petals. The uniformity of floral pelargonidin pigmentation is inversely correlated with the number of integrated Al copies. Furthermore, a correlation was found between the methylation status of the 35S RNA promoter and the instability of the floral pelargonidin coloration. The status of promoter methylation controlling the expression of the Al gene seems to be influenced by the copy number and the chromosomal position of the transferred gene.     

Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 × B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by thebar orpat genes, was more efficient than selection on kanamycin, mediated by thenptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants-transgene silencing, as well as poor transgene transmission to progeny, was observed in some plant lines in which the parent plants had expressed the transgene.     

Transgene Expression in the Vegetative Tissues of Apple Driven by the Vascular-specific rolC and CoYMV Promoters

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of -glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.     

Molecular characterization of the lysosomal acid phosphatase fromDrosophila melanogaster

InDrosophila, unlike humans, the lysosomal acid phosphatase (Acph-1) is a non-essential enzyme. It is also one of the most rapidly evolving gene-enzyme systems in the genus. In order to determine which parts of the enzyme are conserved and which parts are apparently under little functional constraint, we cloned the gene fromDrosophila melanogaster via a chromosomal walk. Fragments from the gene were used to recover an apparently full-length cDNA. The cDNA was subcloned into aDrosophila transformation vector where it was under the control of the 5 promoter sequence of thehsp-70 gene. Three independent transformants were obtained; in each, Acph-1 expression from the cDNA was constitutive and not dependent on heat shock, as determined by densitometric analyses of the allozymic forms of the enzyme. The pattern of expression indicates thehsp-70 and endogenousAcph-1 promoters act together in some, but not all, tissues. The sequence of the cDNA was determined using deletions made with exonuclease III, and primers deduced from the cDNA sequence were used to sequence the genomic clone. Five introns were found, and putative 5 up-stream regulatory sequences were identified. Amino acid sequence comparisons have revealed several highly conserved motifs betweenDrosophila Acph-1 and vertebrate lysosomal and prostatic acid phosphatases.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (<Emphasis Type="Italic">Populus tremula</Emphasis> L.)

Key message

Differentiation level but not transgene copy number influenced activation of a gene containment system in poplar. Heat treatments promoted CRE gene body methylation. The flower-specific transgene deletion was confirmed.

Abstract

Gene flow between genetic modified trees and their wild relatives is still motive of concern. Therefore, approaches for gene containment are required. In this study, we designed a novel strategy for achieving an inducible and flower-specific transgene removal from poplar trees but still expressing the transgene in the plant body. Hence, pollen carrying transgenes could be used for breeding purposes under controlled conditions in a first phase, and in the second phase genetic modified poplars developing transgene-free pollen grains could be released. This approach is based on the recombination systems CRE/loxP and FLP/frt. Both gene constructs contained a heat-inducible CRE/loxP-based spacer sequence for in vivo assembling of the flower-specific FLP/frt system. This allowed inducible activation of gene containment. The FLP/frt system was under the regulation of a flower-specific promoter, either CGPDHC or PTD. Our results confirmed complete CRE/loxP-based in vivo assembling of the flower-specific transgene excision system after heat treatment in all cells for up to 30 % of regenerants derived from undifferentiated tissue cultures. Degradation of HSP::CRE/loxP spacer after recombination but also persistence as extrachromosomal DNA circles were detected in sub-lines obtained after heat treatments. Furthermore, heat treatment promoted methylation of the CRE gene body. A lower methylation level was detected at CpG sites in transgenic sub-lines showing complete CRE/loxP recombination and persistence of CRE/loxP spacer, compared to sub-lines with incomplete recombination. However, our results suggest that low methylation might be necessary but not sufficient for recombination. The flower-specific FLP/frt-based transgene deletion was confirmed in 6.3 % of flowers.

     

The effect of MAR elements on variation in spatial and temporal regulation of transgene expression

The level of transgene expression often differs among independent transformants. This is generally ascribed to different integration sites of the transgene into the plant genome in each independently obtained transformant (position effect). It has been shown that in tobacco transformants expressing, for example, a cauliflower mosaic virus (CaMV) 35S promoter-driven -glucuronidase (GUS) reporter gene, these position-induced quantitative differences among individual transformants were reduced by the introduction of matrix-associated regions (MAR elements) on the T-DNA. We have previously shown by imaging of in planta firefly luciferase (luc) reporter gene activity that quantitative differences in transgene activity can be the result of either a variation in (1) level, (2) spatial distribution and/or (3) temporal regulation of transgene expression between independent transformants. It is not known which of these three different aspects of transgene expression is affected when the transgene is flanked by MAR elements. Here we have used the firefly luciferase reporter system to analyse the influence of MAR elements on the activity of a CaMV 35S-luc transgene in a population of independently transformed tobacco plants. Imaging of in planta LUC activity in these tobacco plant populations showed that the presence of MAR elements does not result in less variation in the average level of transgene expression between individual transformants. This result is different from that obtained previously with a 35S-GUS reporter gene flanked by MAR elements and reflects the differences in the stability of the LUC and GUS reporter proteins. Also the variation in spatial patterns of in vivo LUC activity is not reduced between independent transformants when the transgene is flanked by MAR elements. However, MAR elements do seem to affect the variation in temporal regulation of transgene expression between individual transformants. The potential effects of MAR elements on the variability of transgene expression and the relation to the stability of the (trans)gene product are discussed.     

Agrobacterium rhizogenes-mediated induction of adventitious rooting fromPinus contorta hypocotyls and the effect of 5-azacytidine on transgene activity

Bipartite constructs ofAgrobacterium rhizogenes strain LBA 9402 or A4RSII induced transformed roots on the hypocotyls ofPinus contorta following inoculation, LBA 9402 being more effective. The developmental sequence of root formation and morphology following infection were studied. Furthermore, the pattern of gene expression was studied during rooting and in roots using theuidA reporter gene driven by the 35S promoter. Morphologically most of the roots were normal, whether or not they expressed the reporter gene, but extensive proliferation of lateral roots was observed in some roots with -glucuronidase (GUS) activity. All roots originated from tissues inside the endodermis, often similar to auxin-induced rooting in hypocotyl cutting as described by Grönroos and von Arnold (1987). Where the origin of GUS-positive roots could be traced, they developed from callus forming inside the endodermis. GUS activity was often observed along the root inside the endodermis, at the base of the lateral roots and at the root apex, but not in a region behind the apex. Stable integration of the transgene was verified using Southern blot analysis.To investigate wherther transgene inactivation occurs in conifer plants, root segments and calluses initiated from them were treated with 5-azacytidine. Treatment with 5-azacytidine increased the frequency of GUS-positive roots from about 20% to 50%. The effect of 5-azacytidine on calluses, however, varied among callus lines. To investigate whether methylation was the cause of transgene inactivation, DNA from 5-azacytidine-treated and untreated calluses was digested using the two isoschizomeric restriction enzymes,Hpa Il andMsp 1, which differ in their sensitivity to methylation. There was no evidence for methylation and demethylation at the cleavage sites examined.     

Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes

Previous work has shown that two homologous, unlinked transgene loci can interact in plant nuclei, leading to non-reciprocal trans-inactivation and methylation of genes at one locus. Here, we report the structure and methylation of different transgene loci that contain the same construct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence between strength of silencing activity and copy number/degree of methylation was further demonstrated by producing novel alleles of a strong silencing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target locus. The strong silencing locus, which was located close to a telomere, trans-inactivated various structural variants of the original target construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous regions, a process possibly aided by its telomeric location. Our data support the idea that epistatic trans-inactivation of unlinked, homologous transgenes in plants results from a pre-existing epigenetic difference between transgene loci, which is subsequently equalized by epigene conversion involving DNA-DNA pairing.