Johan Botterman

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide‐resistant plants.

Protein secretion in plant cells can occur via a default pathway.

To study protein secretion in plant cells, we established and evaluated a model system based on transient synthesis of heterologous proteins in tobacco protoplasts. We show that the nonsecretory enzymes phosphinothricin acetyl transferase, neomycin phosphotransferase II, and beta-glucuronidase are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation. These data are consistent with the view that secretion can occur independent of active sorting mechanisms by nonspecific migration through the exocytic pathway. However, the rate of secretion differs significantly among these enzymes. Furthermore, the presence of signal sequences was found to be correlated with a reduction of the levels of the encoded gene products. This is the result of post-transcriptional events that limit either synthesis or stability of the proteins in vivo.

Differential expression of five Arabidopsis genes encoding glycine-rich proteins.

Five cDNA clones coding for glycine-rich proteins in Arabidopsis thaliana were isolated. The corresponding genes are present in the genome as single copies. The derived protein sequences contain highly repetitive glycine-rich motifs. There is, however, little homology among them, nor with previously described glycine-rich proteins from other species. All five genes are expressed in leaves and stems of 6-week-old plants but show different patterns of expression in other organ systems. Analysis of the effect of different external stimuli on the expression pattern showed that, in most cases, the transcript levels were moderately but selectively affected. With flooding stress, the accumulated level of the transcript from one of the genes was remarkably increased.

The bar gene as selectable and screenable marker in plant engineering

Publisher Summary This chapter explains the use of the bar gene as a selectable marker in plant transformation, as a screenable marker in tissue culture and plant breeding, and as a reporter gene in plant molecular biology. The bar gene has served as a useful assayable marker gene in plant molecular biology. To guarantee correct translation initiation in plants, an ATG initiation codon was introduced instead of the GTG codon used in the streptomyces strain and the second codon was changed to introduce an NcoI site at the 5’ end of the coding region. Chimeric gene constructs, containing the bar coding region under control of different promoters, have been transferred to several crops. The bar gene has also been successfully used as a selectable marker in some plant species, using PPT or bialaphos as a selective agent. Transgenic plants were resistant to herbicide applications in the greenhouse and in field conditions.

Transgenic expression of two marker genes under the control of an Arabidopsis rbcS promoter: sequences encoding the Rubisco transit peptide increase expression levels

SummaryChimeric gene constructs were made in which two reporter genes, the neo and bar genes, encoding neomycin phosphotransferase II and phosphinothricin acetyl transferase, respectively, were placed under the control of the promoter of ats1A, one of four genes encoding the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (SSU) in Arabidopsis thaliana. In one set of constructs the fusions were made at the initiation codons, while in the second set the sequences encoding the ats1 A transit peptide were included. Significantly higher steady-state levels of RNA and protein were observed in leaves of transgenic plants varrying the latter constructions. Individual transgenic plants varied in their degree of tissue specific expression of the chimeric genes as well as in absolute levels of expression. Preliminary results suggest that the ats1 A promoter may be only weakly responsive to phytochrome.

Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco

A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. Theldcgene fromHafnia alvei was therefore (a) placed under the control of the 1′ promoter of the bidirectional Tr promoter fromAgrobacterium tumefaciens Ti- plasmid, and (b) cloned behind therbcS promoter from potato fused to the coding region of therbcS transit peptide. Bothldc constructs, introduced intoNicotiana tabacum with the aid ofA. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of therbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3–1% of dry mass.

High-level production of the EcoRI endonuclease under the control of the pL promoter of bacterio-phage lambda

Abstract Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using λpL promoter expression vectors. In a first step we constructed endRI :: lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing Eco RI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the over-produced EcoRI endonuclease.

Expression of dicistronic transcriptional units in transgenic tobacco.

We investigated whether the two cistrons of a dicistronic mRNA can be translated in plants to yield both gene products. The coding sequences of various reporter genes were combined in dicistronic units, and their expression was analyzed in stably transformed tobacco plants at the RNA and protein levels. The presence of an upstream cistron resulted in all cases in a drastically reduced expression of the downstream cistron. The translational efficiency of the gene located downstream in the dicistronic units was 500- to 1,500-fold lower than that in a monocistronic control; a 500-fold lower value was obtained with a dicistronic unit in which both cistrons were separated by 30 nucleotides, whereas a 1,500-fold lower value was obtained with a dicistronic unit in which the stop codon of the upstream cistron and the start codon of the downstream cistron overlapped. As a strategy to select indirectly for transformants with enhanced levels of expression of a gene which is by itself nonselectable, the gene of interest can be cloned upstream from a selectable marker in a dicistronic configuration. This strategy can be used provided that the amount of dicistronic mRNA is high. If, on the other hand, the expression of the dicistronic unit is too low, selection of the downstream cistron will primarily give clones with rearranged dicistronic units.

Processing of a chimeric protein in chloroplasts is different in transgenic maize and tobacco plants.

Transgenic maize (Zea mays L.) and tobacco (Nicotiana tabacum Petit Havana SR1) plants have been generated, which overproduce a mitochondrial Nicotiana plumbaginifolia manganese superoxide dismutase (MnSOD) in chloroplasts. For this, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a Pisum sativum ribulose-1,5-bisphosphate carboxylase (Rubisco) gene and expression of the chimeric gene was driven by the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic MnSOD gene product was correctly targeted to the chloroplasts both in maize and tobacco. However, despite the use of the CaMV 35S promoter, the MnSOD was predominantly localized in the chloroplasts of the bundle sheath cells of maize. Furthermore, the transit peptide was cleaved off at a different position in maize and tobacco.

ENGINEERING OF GLUFOSINATE RESISTANCE AND EVALUATION UNDER FIELD CONDITIONS

Glufosinate and bialaphos are non-selective herbicides which act by inhibiting glutamine synthetase. A gene which confers resistance to bialaphos (bar) was isolated from Streptomyces hygroscopicus, the organism which produces bialaphos. The gene was shown to encode a phosphinothricin acetyl transferase. Using Agrobacterium-mediated Ti plasmid transformation methodology, the bar gene has been introduced into and expressed in several plant species. Transgenic tobacco, tomato, potato, poplar, alfalfa, oilseed rape and sugar beet plants show a complete resistance towards the commercial preparations of glufosinate and bialaphos. The strategy thus provides a successful approach to obtain herbicide-resistant plants by introducing a pathway for detoxification of the herbicide. At present, these crops are being evaluated under open field conditions. Results thus far demonstrate a complete resistance to field dose applications, no yield penalties, and confirmed that glufosinate can be applied as a selective post-emergence herbicide on engineered crops.