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.

Co-introduction of an antisense gene for an endogenous seed storage protein can increase expression of a transgene in Arabidopsis thaliana seeds.

We have investigated whether the expression in Arabidopsis thaliana seeds of a transgene (the Phaseolus vulgaris arcelin (arc)5-I gene) could be enhanced by the simultaneous introduction of an antisense gene for an endogenous seed storage protein (2S albumin). Seeds of plants transformed with both the arc5-I gene and a 2S albumin antisense gene contained reduced amounts of 2S albumins and increased arcelin-5 (Arc5) accumulation levels compared to lines harboring the arc5-I gene only. Arc5 production could be enhanced to more than 24% of the total seed protein content, suggesting that antisense technology could be of great utility to favor high expression of transgenes.

Analysis of the stop codon context in plant nuclear genes.

A region of 18 nucleotides surrounding the stop codon (the stop codon context) in 748 plant nuclear genes was analyzed. Non-randomness was found both upstream and downstream from the stop codon, suggesting that these sequences may help in ensuring efficient termination of translation. The UAG amber codon is the least-used stop codon and the bias in the nucleotide distribution 5' and 3' to the stop codon was more pronounced for the amber codon than for the other stop codons. This might indicate that the codon context affects termination more at UAG than at UGA or UAA stop codons.

Plant regeneration from embryo-derived callus in Phaseolus vulgaris L. (common bean) and P. acutifolius A. Gray (tepary bean).

Regeneration-competent callus of Phaseolus vulgaris and P. acutifolius was obtained from mature embryo explants on a medium containing thidiazuron and indole-3-acetic acid. For the P. vulgaris genotype Xan-159, regeneration was achieved from cotyledon explants, but not from embryonic axis explants. Both explants could be used for the P. acutifolius genotype NI 574 but embryonic axes gave the best results. In-vitro-rooted plantlets of P. acutifolius could readily be established in the greenhouse. For P. vulgaris hardening problems with in-vitro-rooted plantlets could be overcome by means of in vitro grafting. The potential of the described procedure for obtaining transgenic P. vulgaris plants is discussed.

Overexpression of arginase in Arabidopsis thaliana influences defence responses against Botrytis cinerea.

Arabidopsis possesses two arginase-encoding genes, ARGAH1 and ARGAH2, catalysing the catabolism of arginine into ornithine and urea. Arginine and ornithine are both precursors for polyamine biosynthetic pathways. We observed an accumulation of ARGAH2 mRNA in Arabidopsis upon inoculation with the necrotrophic pathogen Botrytis cinerea. Transgenic lines displaying either overexpression of ARGAH2 or simultaneous silencing of both Arabidopsis arginase-encoding genes were created and their resistance to B. cinerea infection evaluated. Overexpression of arginase resulted in changes in amino acid accumulation, while polyamine levels remained largely unaffected. Silencing lines were affected in both amino acid and putrescine accumulation. Arabidopsis plants overexpressing the arginase gene were less susceptible to B. cinerea, whereas silencing lines remained as susceptible as the wild type. We discuss how arginase might interact with plant defence mechanisms. These results provide new insights into amino acid metabolic changes under stress.

A reproducible genetic transformation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the Mexican bean weevil.

A reproducible Agrobacterium tumefaciens-mediated genetic transformation method that delivers fertile and morphologically normal transgenic plants was developed for cultivated tepary bean (Phaseolus acutifolius L. Gray). Factors contributing to higher transformation efficiencies include (1) a low initial concentration of bacteria coupled with a longer cocultivation period with callus, (2) an initial selection of callus on a medium containing low levels of the selectable agent, (3) omission of the selectable agent from the medium during callus differentiation to shoots and (4) the efficient conversion of transgenic shoots into fertile plants. All plants regenerated with this procedure (T0) were stably transformed, and the introduced foreign genes were inherited in a Mendelian fashion in most of the 33 independent transformants. Integration, stable transmission and high expression levels of the transgenes were observed in the T1 and/or T3 progenies of the transgenic lines. The binary transformation vectors contained the beta-glucuronidase reporter gene, the neomycin phosphotransferase II selectable marker gene and either an arcelin 1 or an arcelin 5 gene. Arcelins are seed proteins that are very abundant in some wild P. vulgaris L. genotypes showing resistance to the storage insect Zabrotes subfasciatus (Boheman) (Coleoptera, Bruchidae). Transgenic beans from two different cultivated P. acutifolius genotypes with high arcelin levels were infested with Z. subfasciatus, but they were only marginally less susceptible to infestation than the non-transgenic P. acutifolius. Hence, the arcelin genes tested here are not major determinants of resistance against Z. subfasciatus.

Electroporation-mediated DNA delivery to seedling tissues ofPhaseolus vulgaris L. (common bean).

DNA was delivered to intact embryonic axes of the legumePhaseolus vulgaris L. through electroporation. Expression of the ß-glucuronidase reporter gene was observed in hypocotyl and epicotyl tissue in a spot-like manner. Transgene expression was high when a single pulse of 260 ms at a field strength of 225 V·cm(-1) was applied but could be achieved within a wide range of electrical conditions. Linearization of plasmid DNA greatly enhanced transient expression levels. The procedure was successful for embryonic axes of all testedP. vulgaris cultivars, for similar explants of several large-seeded leguminous species, as well as for some other tissues ofP. vulgaris.

The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants.

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.

Isolation and characterisation of arcelin-5 proteins and cDNAs.

Arcelins are seed storage proteins present in some wild bean accessions (Phaseolus vulgaris). They are implicated in the resistance phenotype of these wild beans towards the Mexican bean weevil. Arcelin 5, one of six arcelin electrophoretic variants, has been characterised in detail. The purified arcelin-5 protein fraction contained two major polypeptides of 32.2 and 31.5 kDa, designated arcelin 5a and arcelin 5b, respectively, and one minor polypeptide of 30.8 kDa, designated arcelin 5c. The three polypeptides have an identical isoelectric point and are identical for their first nine N-terminal amino acids. Arcelin 5a and arcelin 5b are glycoproteins whereas arcelin 5c is not glycosylated. Native arcelin 5 has a molecular mass corresponding to a dimer form. Using amino acid sequence analysis and PCR techniques, two different arcelin-5 cDNA sequences were obtained, designated arc5-I and arc5-II. Both encode proteins of 261 amino acids with a signal peptide of 21 amino acids. The identity between the two is 99% at the DNA level and 97% at the level of the deduced amino acid sequences. The arc5-I and arc5-II cDNAs encode arcelin 5a and arcelin 5b, respectively. Sequence comparisons and protein characteristics show clearly that arcelin 5 is related to, but distinct from, other arcelin variants and lectins of P. vulgaris.

Production of antibodies and antibody fragments in plants.

Our current knowledge allows the generation of transgenic plants that efficiently produce heterologous proteins from plant, bacterial, fungal or animal origin. Among all types of recombinant proteins, antibodies are particularly attractive because of their ability to specifically recognize and bind virtually any type of antigen. Plants show several advantages as a large-scale antibody production system: they can be grown easily and inexpensively in large quantities that can be harvested, stored and processed by using existing infrastructures. Isolation and purification of plant-made antibodies, if necessary, allow fundamental, industrial, and therapeutical applications. In the past, we and others have successfully generated antibody-producing plants. The maximal accumulation levels of antibodies and antibody fragments that we observed are 1-5% of the extracted proteins. Currently, several biotechnological companies grow field crops to produce antibodies for ex planta applications on an industrial scale.

Plants as bioreactors for protein production: avoiding the problem of transgene silencing.

Plants are particularly attractive as large-scale production systems for proteins intended for therapeutical or industrial applications: they can be grown easily and inexpensively in large quantities that can be harvested and processed with the available agronomic infrastructures. The effective use of plants as bioreactors depends on the possibility of obtaining high protein accumulation levels that are stable during the life cycle of the transgenic plant and in subsequent generations. Silencing of the introduced transgenes has frequently been observed in plants, constituting a major commercial risk and hampering the general economic exploitation of plants as protein factories. Until now, the most efficient strategy to avoid transgene silencing involves careful design of the transgene construct and thorough analysis of transformants at the molecular level. Here, we focus on different aspects of the generation of transgenic plants intended for protein production and on their influence on the stability of heterologous gene expression.

Crystal structure of arcelin-5, a lectin-like defense protein from Phaseolus vulgaris.

In the seeds of the legume plants, a class of sugar-binding proteins with high structural and sequential identity is found, generally called the legume lectins. The seeds of the common bean (Phaseolus vulgaris) contain, besides two such lectins, a lectin-like defense protein called arcelin, in which one sugar binding loop is absent. Here we report the crystal structure of arcelin-5 (Arc5), one of the electrophoretic variants of arcelin, solved at a resolution of 2.7 A. The R factor of the refined structure is 20.6%, and the free R factor is 27.1%. The main difference between Arc5 and the legume lectins is the absence of the metal binding loop. The bound metals are necessary for the sugar binding capabilities of the legume lectins and stabilize an Ala-Asp cis-peptide bond. Surprisingly, despite the absence of the metal binding site in Arc5, this cis-peptide bond found in all legume lectin structures is still present, although the Asp residue has been replaced by a Tyr residue. Despite the high identity between the different legume lectin sequences, they show a broad range of quaternary structures. The structures of three different dimers and three different tetramers have been solved. Arc5 crystallized as a monomer, bringing the number of known quaternary structures to seven.

Analysis of bruchid resistance in the wild common bean accession G02771: no evidence for insecticidal activity of arcelin 5.

Arcelins are abundant seed storage proteins thought to be implicated in the resistance of wild Phaseolus vulgaris (L.) genotypes against Zabrotes subfasciatus (Boheman), an important storage insect pest of common bean. Here, the insecticidal activity of the arcelin-5 variant that is present in the highly resistant P. vulgaris accession G02771 was investigated. No correlation could be established between the presence of arcelin 5 and the insecticidal effects observed in G02771 seeds. Insect feeding assays with artificial seeds into which purified arcelin-5 protein was incorporated and with transgenic P. acutifolius (A. Gray) seeds in which the arcelin-5 genes were expressed, showed that the presence of arcelin-5 proteins, even at elevated levels, was not sufficient to achieve adequate resistance against Z. subfasciatus. The same might apply to other arcelin variants. Nevertheless, as resistance is clearly closely linked to the presence of the arcelin-1 or arcelin-5 locus, arcelins remain useful markers in breeding programmes aimed at introgressing high levels of resistance to Z. subfasciatus in P. vulgaris cultivars.

Gastrodianin-like mannose-binding proteins: a novel class of plant proteins with antifungal properties.

The orchid Gastrodia elata depends on the fungus Armillaria mellea to complete its life cycle. In the interaction, fungal hyphae penetrate older, nutritive corms but not newly formed corms. From these corms, a protein fraction with in vitro activity against plant-pathogenic fungi has previously been purified. Here, the sequence of gastrodianin, the main constituent of the antifungal fraction, is reported. Four isoforms that encoded two different mature proteins were identified at the cDNA level. Another isoform was detected in sequenced peptides. Because the antifungal activity of gastrodianins produced in and purified from Escherichia coli and Nicotiana tabacum was comparable to that of gastrodianin purified from the orchid, gastrodianins are the active component of the antifungal fractions. Gastrodianin accumulation is probably an important part of the mechanism by which the orchid controls Armillaria penetration. Gastrodianin was found to be homologous to monomeric mannose-binding proteins of other orchids, of which at least one (Epipactis helleborine mannose-binding protein) also displayed in vitro antifungal activity. This establishes the gastrodianin-like proteins (GLIPs) as a novel class of antifungal proteins.

High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida.

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.