Targeted knockout of a conserved plant mitochondrial gene by genome editing.

Fusion proteins derived from transcription activator-like effectors (TALEs) have emerged as genome editing tools for mitochondria. TALE nucleases (TALENs) have been applied to delete chimaeric reading frames and duplicated (redundant) genes but produced complex genomic rearrangements due to the absence of non-homologous end-joining. Here we report the targeted deletion of a conserved mitochondrial gene, nad9, encoding a subunit of respiratory complex I. By generating a large number of TALEN-mediated mitochondrial deletion lines, we isolated, in addition to mutants with rearranged genomes, homochondriomic mutants harbouring clean nad9 deletions. Characterization of the knockout plants revealed impaired complex I biogenesis, male sterility and defects in leaf and flower development. We show that these defects can be restored by expressing a functional Nad9 protein from the nuclear genome, thus creating a synthetic cytoplasmic male sterility system. Our data (1) demonstrate the feasibility of using genome editing to study mitochondrial gene functions by reverse genetics, (2) highlight the role of complex I in plant development and (3) provide proof-of-concept for the construction of synthetic cytoplasmic male sterility systems for hybrid breeding by genome editing.

Shine-Dalgarno Sequences Play an Essential Role in the Translation of Plastid mRNAs in Tobacco.

In prokaryotic systems, the translation initiation of many, though not all, mRNAs depends on interaction between a sequence element upstream of the start codon (the Shine-Dalgarno sequence [SD]) and a complementary sequence in the 3' end of the 16S rRNA (anti-Shine-Dalgarno sequence [aSD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations in the aSD coupled with genome-wide analysis of translation by ribosome profiling, we provide a global picture of SD-dependent translation in plastids. We observed a pronounced correlation between weakened predicted SD-aSD interactions and reduced translation efficiency. However, multiple lines of evidence suggest that the strength of the SD-aSD interaction is not the only determinant of the translational output of many plastid mRNAs. Finally, the translation efficiency of mRNAs with strong secondary structures around the start codon is more dependent on the SD-aSD interaction than weakly structured mRNAs. Thus, our data reveal the importance of the aSD in plastid translation initiation, uncover chloroplast genes whose translation is influenced by SD-aSD interactions, and provide insights into determinants of translation efficiency in plastids.

A software tool for the input and management of phenotypic data using personal digital assistants and other mobile devices.

BACKGROUND: Plant breeding and genetics demand fast, exact and reproducible phenotyping. Efficient statistical evaluation of phenotyping data requires standardised data storage ensuring long-term data availability while maintaining intellectual property rights. This is state of the art at phenomics centres, which, however, are unavailable for most scientists. For them we developed a simple and cost-efficient system, the Phenotyper, which employs mobile devices or personal digital assistants (PDA) for on-site data entry and open-source software for data management. RESULTS: A graphical user interface (GUI) on a PDA replaces paper-based form sheet and data entry on a desktop. The user can define his phenotyping schemes in a web tool without in-depth knowledge of the system and thus adjust it more easily to new research aspects than in a classical laboratory information management system (LIMS). In the Phenotyper, schemes are built from controlled vocabulary gained from published ontologies. Vocabulary and schemes are stored in a database that also manages the user access. From the web page, schemes are downloaded as extended markup language (XML) files for the transfer to the PDA and the exchange between users. On the PDA, the GUI displays the schemes and stores data in comma separated value format and XML format. After manual quality control, data are uploaded via a web page to an independently hosted results database, in which data are stored in an entity-attribute-value structure to provide maximum flexibility. Datasets are linked to the original and curated data files stored on a file server. The ownership stamp, project affiliation and date stamp of a dataset are used to regulate data access, which is restricted to data belonging to the user or to his projects and data, for which the embargo period has ended. By export of standardised ASCII reports to long-term data storage facility, long-term accessibility allows searching, citing and use of raw data beyond the lifetime of the database. The Phenotyper is available to the scientific community for use and further development. CONCLUSIONS: The Phenotyper provides a well-structured, but flexible data acquisition and management structure for mobile on-site measurements for efficient evaluation and shared use of data.

Documentation system for plant transformation service and research.

BACKGROUND: In plant transformation, method compliance is critical for success. Transformation methods are complicated and tend to evolve over time. Until the complete method is published, method details are often partially orally transmitted and thus bound to a few people. Their documentation in text files are often a mixture of material and method description with many references to other sources especially to media description. These media are complex and often composed from several commercially available mixtures plus individually prepared stocks. The actual transformation experiment is generally documented in lab books, in which deviations from the methods and results are reported. Additionally, work schedules are planned in diaries. Both paper-based sources lack backup copies and miss unambiguous links to method descriptions and media recipes. DESCRIPTION: To solve the problem, we devised a standard-operation-procedure system based on a Microsoft Access database containing the interlinked modules 'Media', 'Methods' and 'Experiments'. The Media module contains all basic chemicals, stocks and complex media. In this module, complex media are composed from other elements of the Media module, thus mimicking the workflows of media preparation in the lab. The Media module is made attractive to the user by functions that generate file cards and labels. The Methods module describes each method stepwise and links the steps to the media. Copy functions allow cloning of old methods to document method evolution without alteration of the old methods. Activation and inactivation functions in the Media and the Methods module remove outdated entries from active use. The Experiments module links the method to experiment specific information. This module generates a lab-book like user interface and a work schedule, and it contains a simple result section. CONCLUSION: The system has been evolved and tested over several years in a transformation service unit, where it increased efficiency. Additionally, the system provided rapid access to data for quality control and decision making. The system can be easily modified for the use in other research environments.