Vera Thole

A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21.

Brachypodium distachyon is a novel model system for structural and functional genomics studies of temperate grasses because of its biological and genetic attributes. Recently, the genome sequence of the community standard line Bd21 has been released and the availability of an efficient transformation system is critical for the discovery and validation of the function of Brachypodium genes. Here, we provide an improved procedure for the facile and efficient Agrobacterium-mediated transformation of line Bd21. The protocol relies on the transformation of compact embryogenic calli derived from immature embryos using visual and chemical screening of transformed tissues and plants. The combination of green fluorescent protein expression and hygromycin resistance enables early identification of transformation events and drastically reduces the quantity of tissue to be handled throughout the selection process. Approximately eight independent fully developed transgenic Bd21 plants can be produced from each immature embryo, enabling the generation of thousands of T-DNA lines. The process—from wild-type seeds to transgenic T1 seeds—takes ∼8 months to complete.

The pCLEAN Dual Binary Vector System for Agrobacterium-Mediated Plant Transformation

The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.

Distribution and characterization of more than 1000 T-DNA tags in the genome of Brachypodium distachyon community standard line Bd21

A collection of 4117 fertile T-DNA lines has been generated by Agrobacterium-mediated transformation of the diploid community standard line Bd21 of Brachypodium distachyon. The regions flanking the T-DNA left and right borders of the first 741 transformed plants were isolated by adapter-ligation PCR and sequenced. A total of 1005 genomic sequences (representing 44.1% of all flanking sequences retrieved) characterized 660 independent T-DNA loci assigned to a unique location in the Brachypodium genome sequence. Seventy-six percent of the fertile plant lines contained at least one anchored T-DNA locus (1.17 loci per tagged line on average). Analysis of the regions flanking both borders of the T-DNA increased the number of T-DNA loci tagged and the number of tagged lines by approximately 50% when compared to a single border analysis. T-DNA integration (2.4 insertions per Mb on average) was proportional to chromosome size, however, varied greatly along each chromosome with often low insertion level around centromeres. The frequency of insertion within transposable elements (5.3%) was fivefold lower than expected if random insertion would have occurred. More than half of the T-DNAs inserted in genic regions. On average, one gene could be tagged for every second fertile plant line produced and more than one plant line out of three contained a T-DNA insertion directly within or 500 bp around the coding sequence. Approximately, 60% of the genes tagged corresponded to expressed genes. The T-DNA lines generated by the BrachyTAG programme are available as a community resource and have been distributed internationally since 2008 via the BrachyTAG.org web site.

T-DNA mutagenesis in Brachypodium distachyon

During the past decade, Brachypodium distachyon has emerged as an attractive experimental system and genomics model for grass research. Numerous molecular tools and genomics resources have already been developed. Functional genomics resources, including mutant collections, expression/tiling microarray, mapping populations, and genome re-sequencing for natural accessions, are rapidly being developed and made available to the community. In this article, the focus is on the current status of systematic T-DNA mutagenesis in Brachypodium. Large collections of T-DNA-tagged lines are being generated by a community of laboratories in the context of the International Brachypodium Tagging Consortium. To date, >13 000 lines produced by the BrachyTAG programme and USDA-ARS Western Regional Research Center are available by online request. The utility of these mutant collections is illustrated with some examples from the BrachyTAG collection at the John Innes Centre-such as those in the eukaryotic initiation factor 4A (eIF4A) and brassinosteroid insensitive-1 (BRI1) genes. A series of other mutants exhibiting growth phenotypes is also presented. These examples highlight the value of Brachypodium as a model for grass functional genomics.

A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon

In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project.