Mario Gils

Silencing in Arabidopsis T-DNA Transformants: The Predominant Role of a Gene-Specific RNA Sensing Mechanism versus Position Effects

Pronounced variability of transgene expression and transgene silencing are commonly observed among independent plant lines transformed with the same construct. Single-copy T-DNA lines harboring reporter genes of various kind and number under the control of a strong promoter were established in Arabidopsis thaliana for a comprehensive analysis of transgene expression. Characterization of 132 independent transgenic lines revealed no case of silencing as a result of site of T-DNA integration. Below a certain number of identical transgenes in the genome, gene copy number and expression were positively correlated. Expression was high, stable over all generations analyzed, and of a comparable level among independent lines harboring the same copy number of a particular transgene. Conversely, RNA silencing was triggered if the transcript level of a transgene surpassed a gene-specific threshold. Transcript level–mediated silencing effectively accounts for the pronounced transgene expression variability seen among transformants. It is proposed that the RNA sensing mechanism described is a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, including transgenes, and so plays an important role in genome defense.

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5′-upstream regions, coding sequences and 3′-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5′-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.

Pollination control technologies for hybrid breeding

Efforts in hybrid breeding have made this technology one of the main factors contributing to the substantial global rise in agricultural output over the last few decades. For hybrid breeding, an efficient pollination control system is necessary to avoid the unwanted self-pollination or sib-pollination of the female parental line. This review will provide a historical overview of pollination control systems and their use in hybrid crop breeding. We outline the prerequisites for commercial hybrid breeding and summarize the most important non-biological and biological technologies. Our main focus is on hybrid systems that are based on genetically engineered plants. We describe their suitability for pollination control, propagation of the male-sterile crossing partner, fertility restoration and mixed planting. Additionally, we report on the latest findings in the development of inducible sterility systems and various technologies that enable pollination control via metabolic engineering. We discuss the pros and cons of the different pollination control strategies.

Transgene excision from wheat chromosomes by phage phiC31 integrase.

The Streptomyces phage phiC31 integrase was tested for its ability to excise transgenic DNA from the wheat genome by site-specific recombination. Plants that stably express phiC31 integrase were crossed to plants carrying a target construct bearing the phiC31 recognition sites, attP and attB. In the progeny, phiC31 recombinase mediates recombination between the att sites of the target locus, which results in excision of the intervening DNA. Recombination events could be identified in 34 independent wheat lines by PCR and Southern blot analysis and by sequencing of the excision footprints. Recombinant loci were inherited to the subsequent generation. The results presented here establish the integrase-att system as a tool for catalysing the precise elimination of DNA sequences from wheat chromosomes.

Intein-mediated protein assembly in transgenic wheat: production of active barnase and acetolactate synthase from split genes

Engineering traits by the assembly of non-functional gene products is a promising tool for modern plant biotechnology. In this article, we describe the establishment of male sterility and herbicide resistance in wheat (Triticum aestivum) by complementing inactive precursor protein fragments through a split intein system. N- and C-terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from the Synechocystis sp. gene DnaB and delivered into the wheat genome via biolistic particle bombardment. Both barnase fragments were expressed under the control of a tapetum-specific promoter. High efficiency of the split barnase system was achieved by introducing GGGGS linkers between the fusion domains of the assembled protein. Depending on the vector version that was transformed, up to 51% of primary transformed plants produced sterile pollen. In the F(1) progeny, the male-sterile phenotype segregated with both barnase gene fragments. Expression of the cytotoxic barnase in the tapetum did not apparently affect the vegetative phenotype and remained stable under increased temperatures. In addition, the reconstitution of sulphonylurea resistance was achieved by DnaE intein-mediated assembly of a mutated acetolactate synthase (ALS) protein from rice. The impacts of the technical advances revealed in this study on the concepts for trait control, transgene containment and hybrid breeding are discussed.

Expression of active Streptomyces phage phiC31 integrase in transgenic wheat plants

Site-specific recombination systems are becoming an important tool for the genetic modification of crop plants. Here we report the functional expression of the Streptomyces phage-derived phiC31 recombinase (integrase) in wheat. T-DNA constructs containing a phiC31 integrase transgene were stably transformed into wheat plants via particle gun bombardment. A plant-virus-based assay system was used to monitor the site-specific recombination activity of the recombinant integrase protein in vivo. We established several independent doubled haploid (DH) inbred lines that constitutively express an active integrase enzyme without any apparent detrimental effects on plant growth and development. The potential of phiC31 integrase expression in crop plants related to transgene control technologies or hybrid breeding systems is discussed.

Native-sized spider silk proteins synthesized in planta via intein-based multimerization

The synthesis of native-sized proteins is a pre-requisite for exploiting the potential of spider silk as a bio-based material. The unique properties of spider silk, such as extraordinary tensile strength and elasticity, result from the highly repetitive nature of spider silk protein motifs. The present report describes the combination of spider silk flagelliform protein (FLAG) production in the endoplasmic reticulum of tobacco plant leaf cells with an intein-based posttranslational protein fusion technology. The repeated ligation of FLAG monomers resulted in the formation of large multimers. This method avoids the need for highly repetitive transgenes, which may result in a higher genetic and transcriptional stability. Here we show, for the first time, the production of synthetic, high molecular weight spider silk proteins larger than 250xa0kDa based on the assembly of protein monomers via intein-mediated trans-splicing in planta. The resulting multimeric structures form microfibers, thereby demonstrating their great potential as a biomaterial.

Split-gene system for hybrid wheat seed production

Significance Global food security demands the development of new technologies to increase and secure cereal production on finite arable land without increasing water and fertilizer use. Although the use of heterosis through hybrid breeding has produced tremendous economic benefits in worldwide crop production, less than 1% of the global wheat area is planted with hybrids. One of the greatest bottlenecks in breeding hybrid wheat is the lack of an efficient sterility system to block self-pollination. This report describes a recessive system for pollination control in wheat. We demonstrate the implementation and feasibility of the system for generation and maintenance of the male-sterile parent, hybrid seed production and full restoration of fertility in the hybrid wheat seed. Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore “linked in repulsion.” Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner.

Quantitative Assessment of Wheat Pollen Shed by Digital Image Analysis of Trapped Airborne Pollen Grains

The objective of the present study was to develop a technique for quantifying the dynamics of wheat pollen shed under field conditions. Pollen traps with an adhesive film were used to assess the relative pollen shed of 12 winter wheat lines. Quantitative measurements were performed in 2012 and 2013 over a period of up to 20 days. The amounts of trapped pollen were automatically determined using a customized image analysis program. We demonstrated that this method is suitable for the assessment of wheat pollen shed. The possible impact of the technical advances revealed in this study for the selection of pollinators in routine wheat breeding programs is discussed.

The auxins centrophenoxine and 2,4-D differ in their effects on non-directly induced chromosome doubling in anther culture of wheat (T. aestivum L.)

Doubled haploid technologies have become key tools for plant breeding. Using these techniques, the speed and efficiency of plant improvement processes can be significantly enhanced. Anther culture-based technologies have the potential to regenerate large numbers of doubled haploid plants without colchicine treatment. In an attempt to elucidate the influence of phytohormones on non-directly induced chromosome doubling, two synthetic auxins, 2,4-D and centrophenoxine, were tested in a wheat anther culture approach. Whereas the induction of androgenic embryo-like structures (ELSs) was efficient for both auxins, we observed a significantly higher frequency of chromosome doubling when using 2,4-D than when using centrophenoxine. When 2,4-D was added to the induction medium, a positive correlation between the size of ELSs and their ploidy level was detected by flow cytometry. The morphological selection of ELSs, a process that was included in routine operations of the method without significantly extending the input of time and effort, facilitates the production of fertile DH plants with a frequency of 60xa0%. Our findings may contribute to a more efficient production of doubled haploid wheat plants using a colchicine-free anther culture approach.