It has been previously reported that the 5' region of the rice actin 1 gene (Act1) promoted high-level expression of a beta-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauliflower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previously been attainable with alternative expression vectors.

Infection of Wheat dwarf virus (WDV) strains on barley results in dwarf disease, imposing severe economic losses on crop production. As the natural resistance resources against this virus are limited, it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to a wide range of WDV strains. Because vector insect-mediated WDV infection occurs during cool periods in nature, it is important to identify a technology which is effective at lower temperature. In this study, we designed artificial microRNAs (amiRNAs) using a barley miRNA precursor backbone, which target different conservative sequence elements of the WDV strains. Potential amiRNA sequences were selected to minimize the off-target effects and were tested in a transient sensor system in order to select the most effective constructs at low temperature. On the basis of the data obtained, a polycistronic amiRNA precursor construct (VirusBuster171) was built expressing three amiRNAs simultaneously. The construct was transformed into barley under the control of a constitutive promoter. The transgenic lines were kept at 12-15 °C to mimic autumn and spring conditions in which major WDV infection and accumulation take place. We were able to establish a stable barley transgenic line displaying resistance to insect-mediated WDV infection. Our study demonstrates that amiRNA technology can be an efficient tool for the introduction of highly efficient resistance in barley against a DNA virus belonging to the Geminiviridae family, and this resistance is effective at low temperature where the natural insect vector mediates the infection process.

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The recently achieved significant improvement of cereal transformation protocols provides facilities to alter the protein composition of the endosperm, for example, to increase or decrease the quantity of one of its protein components or to express foreign molecules. To achieve this goal, strong endosperm-specific promoters have to be available. The aim of our work was to develop a more efficient tissue-specific promoter which is currently used. A chimaeric promoter was assembled using the 5′ UTR (1,900 bp) of the gene coding for the 1Bx17 HMW glutenin subunit protein, responsible for tissue-specific expression and the first intron of the rice actin gene (act1). The sequence around of the translation initial codon was optimized. The effect of the intron and promoter regulatory sequences, using different lengths of 1Bx17 HMW-GS promoter, were studied on the expression of uidA gene. The function of promoter elements, promoter length, and the first intron of the rice actin gene were tested by a transient expression assay in immature wheat endosperm and in stable transgenic rice plants. Results showed that insertion of the rice act1 first intron increased GUS expression by four times in transient assay. The shortest 1Bx17 HMW-GS promoter fragment (173 bp) linked to the intron and GUS reporter gene provided almost the same expression level than the intronless long 1Bx17 HMW-GS promoter. Analysis of the stable transformant plants revealed that 173 nucleotides were sufficient for endosperm-specific expression of the uidA gene, despite 13 nucleotides missing from the HMW enhancer sequence, a relevant regulatory element in the promoter region.

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The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G(M1)-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

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