Anni Jacobs

Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction

SummaryWe investigated whether the efficiency of transformation of plant cells by Agrobacterium tumefaciens during cocultivation is limited by the properties of the plant cells or by the infecting bacteria.Therefore, tobacco protoplasts were infected by cocultivation with two different agrobacteria strains carrying Ti plasmids with distinguishable T-DNAs. These T-DNAs cotransform plant cells at a frequency equal to the product of their independent transformation frequencies, which indicates that all plant cells are equally competent. On the other hand, when these T-DNAs are located on the same Ti plasmid vector within one bacterial strain, the cotransformation frequency is significantly higher than the product of the single transformation frequencies. We interpret these results to indicate that transformation is limited more by the establishment of effective bacteria/plant cell interaction than by (i) the process of DNA integration and (ii) by the number of plant cells capable of being transformed by Agrobacterium. We found that most plant cells are transformed by only one or a few agrobacteria. Analysis of the number of T-DNA copies in these clonally transformed lines indicates amplification of the original, infecting T-region copy.

Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences

Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production.

Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis.

The yield and assembly of an IgG1 antibody and its derived Fab fragment were compared inNicotiana andArabidopsis. The results obtained showed a lot of interclonal variability. For 45% of the primary transgenic calluses, antigen-binding entities represented less than 0.1% of the total soluble protein (TSP). Only two of the 103 analysed transformants contained more than 1% of antigen-binding protein, with 1.26% being the highest yield. Analogous amounts of complete antibody and Fab accumulated in primary callus tissue. Moreover, yields were in the same range for both species as far as primary callus tissue is concerned. However, the accumulation of the Fab fragment in leaf tissue of regenerated plants differed significantly betweenNicotiana andArabidopsis. The Fab fragment accumulated to only 0.044% of TSP inNicotiana leaves but up to 1.3% inArabidopsis leaves. Furthermore, both species showed differences in the assembly pattern of the complete antibody. WhereasArabidopsis contained primarily fully assembled antibodies of 150 kDa,Nicotiana showed an abundance of fragments in the 50 kDa range.

Plant chromosome/marker gene fusion assay for study of normal and truncated T-DNA integration events

SummaryDuring Agrobacterium tumefaciens infection, the T-DNA flanked by 24 by imperfect direct repeats is transferred and stably integrated into the plant chromosome at random positions. Here we measured the frequency with which a promoterless reporter gene is activated after insertion into the Nicotiana tabacum SR1 genome. When adjacent to the right or left T-DNA border sequences, at least 35% of the transformants express the marker gene, suggesting preferential T-DNA insertion (>70%) in transcriptionally active regions of the plant genome. When the promoterless neomycin phosphotransferase II (nptII) gene is located internally in the T-DNA, the activation frequency drops to 1% since gene activation requires T-DNA truncation. These truncation events in the nptII upstream region occur independently of the nature of the upstream sequence and of the T-DNA length. Deletion of the right border region prevents the detection of activated marker genes. Therefore, T-DNA truncation probably occurs after synthesis of a normal T-DNA intermediate during the transfer and/or integration process. In the absence of border regions, expression of the nptII selectable marker directed by the nopaline synthase promoter was detected in 1 out of 105 regenerated calli, suggesting the possibility that any DNA sequence from the Ti plasmid can be transformed into the plant genome, albeit at a low frequency.

Agrobacterium tumefaciens Transformation and Cotransformation Frequencies of Arabidopsis thaliana Root Explants and Tobacco Protoplasts

In view of the recent finding that different T-DNAs tend to ligate and integrate as repeats at single chromosomal positions, the frequency of transformation and cotransformation was determined during cocultivation of Arabidopsis thaliana root explants and Nicotiana tabacum protoplasts with two Agrobacterium strains. The transformation frequency of unselected A. thaliana shoots was lower than 1% whereas that of cocultivated tobacco protoplasts was approximately 18%. The cotransformation frequencies, defined as the frequencies with which cells transformed with a first T-DNA contained a second unselected T-DNA, were approximately 40% reproducible, irrespective of the selection, the transformation frequency, and the plant system used. Extrapolation of these results suggests that at least two independently transferred T-DNAs were present in 64% of the transformed plant cells. Molecular analysis of cocultivated N. tabacum shoots regenerated on nonselective medium showed that only a few transformants had a silenced (2/46) or truncated (1/46) T-DNA. Therefore, most integrated T-DNAs expressed their selectable or screenable markers in primary transgenic plants. Remarkably, 10 to 30% of the selected A. thaliana shoots or progenies lost the T-DNA marker they were selected on. As these regenerants contained the unselected T-DNA with a high frequency (17%), these selected plants might result from the expression of unstable, transiently expressed T-DNAs. In conclusion, a significant part of the T-DNAs is lost from the transformed cells.

A negative selection scheme for tobacco protoplast-derived cells expressing the T-DNA gene 2

The amido hydrolase encoded by the T-DNA gene 2 catalyzes the conversion of indole-acetamide, α-naphthalene acetamide, and other substrate analogues into the corresponding auxins. As a result, only gene 2-expressing protoplast-derived tobacco cells can grow in medium containing low concentrations (0.2–1 μM) of α-naphthalene acetamide as auxin precursor. However, in a mixture of SR1 and SR1, gene 2+ protoplast-derived cells, cross-feeding occurs and consequently no positive selection for gene 2 is obtained. A 100-times higher concentration of α-naphthalene acetamide (between 30 and 300 μM) provides a negative selection scheme. Only the tobacco cells expressing gene 2 are sensitive to the high naphthalene acetamide concentration and cannot grow to colonies, while cells lacking the gene 2 product regenerate calli even in mixed gene 2+ and gene 2− cell populations. Thus, gene 2 might provide a unique biochemically defined marker to investigate mutations and gene inactivation.

The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

Transgenic loci obtained after Agrobacterium tumefaciens-mediated transformation can be simple, but fairly often they contain multiple T-DNA copies integrated into the plant genome. To understand the origin of complex T-DNA loci, floral-dip and root transformation experiments were carried out in Arabidopsis thaliana with mixtures of A. tumefaciens strains, each harboring one or two different T-DNA vectors. Upon floral-dip transformation, 6-30% of the transformants were co-transformed by multiple T-DNAs originating from different bacteria and 20-36% by different T-DNAs from one strain. However, these co-transformation frequencies were too low to explain the presence of on average 4-6 T-DNA copies in these transformants, suggesting that, upon floral-dip transformation, T-DNA replication frequently occurs before or during integration after the transfer of single T-DNA copies. Upon root transformation, the co-transformation frequencies of T-DNAs originating from different bacteria were similar or slightly higher (between 10 and 60%) than those obtained after floral-dip transformation, whereas the co-transformation frequencies of different T-DNAs from one strain were comparable (24-31%). Root transformants generally harbor only one to three T-DNA copies, and thus co-transformation of different T-DNAs can explain the T-DNA copy number in many transformants, but T-DNA replication is postulated to occur in most multicopy root transformants. In conclusion, the comparable co-transformation frequencies and differences in complexity of the T-DNA loci after floral-dip and root transformations indicate that the T-DNA copy number is highly determined by the transformation-competent target cells.

Gene silencing results in instability of antibody production in transgenic plants

Abstract The stability of antibody and Fab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed.

Transcriptional interference in transgenic plants

When a promoterless marker gene is transformed into the plant genome using the Agrobacterium vector system, on average 30% of the T-DNA inserts produce gene fusions. This suggests that the T-DNA is preferentially integrated into transcribed regions. Here, we proposed that this transcriptional activity is responsible for some of the variation in expression frequently observed among independent transformants. Using hybrid gene constructions, we show that transcriptional readthrough into a downstream gene with opposite orientation substantially reduces expression of this gene both in transient expression and in transgenic plants. Furthermore, a poly(A) signal/terminator can block readthrough and restore the expression of the gene. Finally, enzymatic analysis of calli suggests that less variation in neomycin phosphotransferase II synthesis is observed when the gene is separated from plant DNA by promoter and terminator elements.

Effect of T-DNA configuration on transgene expression

SummaryT-DNA vectors were constructed which carry a β-glucuronidase (gusA) gene fused to the promoter of the nopaline synthase (nos) gene and the 3′ end of the octopine synthase (ocs) gene. This reporter gene was cloned at different locations and orientations towards the right T-DNA border. For each construct, between 30 and 60 stably transformed calli were analysed for β-glucuronidase activity. Depending on the T-DNA configuration, distinct populations of gusA-expressing calli were obtained. Placing the reporter gene in the middle of the T-DNA results in relatively low expression levels and a limited inter-transformant variability. Placing the gene with its promoter next to the right border led to an increase in both the mean activity and the variability level. With this construct, some of the calli expressed the gusA gene at levels four to five times higher than the mean. In all these series, at least 30% of the calli contained reporter gene activities that were less than half of the mean expression level. Separating the gusA gene from the right T-DNA border by an additional 3′-untranslated region, derived from the nos gene, resulted in an increase in the mean expression to a level almost four times higher than that of constructions carrying the reporter gene in the middle of the T-DNA. Moreover, the number of transformants with extremely low activities decreased by at least 50% and this resulted in significantly lower inter-transformant variability independently of the orientation of the reporter gene on the T-DNA.