TRTech-PGR: Ensifer-mediated Transformation as an Alternative to Agrobacterium-mediated Plant Transformation of Model Plants and Crops

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Agrobacterium-mediated plant genetic transformation (AMT) is the most commonly used technology for generating transgenic plants and introducing genome engineering reagents in plant basic research and in agricultural biotechnology. Although this technology was developed and used for plant improvement since the 1980s, Agrobacterium-mediated transformation has numerous limitations. Key among these is that Agrobacterium-mediated transformation is limited to specific plant species and/or varieties of particular plant species. Agrobacterium is a natural plant pathogen, and it therefore frequently elicits defense reactions from infected plants, causing tissue necrosis. Such necrotic tissue cannot be genetically transformed. Ensifer adhaerens is a bacterium related to Agrobacterium. When supplied with the relevant genetic elements it can, like Agrobacterium, genetically transform plants. Ensifer is not a pathogen and therefore fails to elicit necrotic plant defense responses. However, Ensifer-mediated plant genetic transformation remains inefficient and cumbersome to perform in the average plant biology laboratory. The work described in this project will develop an Ensifer-mediated plant genetic transformation system, greatly simplifying the use of Ensifer in the laboratory and improving the efficiency of Ensifer-mediated transformation. The resulting system will greatly extend the host range for plant transformation, allowing for the genetic improvement of a wider range of crop species than that now permitted by Agrobacterium-mediated transformation. Agrobacterium-mediated plant genetic transformation has been used for transformation of many plant species. However, numerous plant species and cultivars/varieties remain highly recalcitrant to Agrobacterium-mediated transformation, in part because they react with a necrotic defense response to Agrobacterium infection. As a non-pathogen, the related bacterium Ensifer adhaerens does not elicit a strong plant defense response. Ensifer can, with the appropriate genetic alterations, transform a wide spectrum of plant species. A limitation to using Ensifer as an alternative to Agrobacterium is that the best Ensifer strain currently used for plant transformation, E. adhaerens OV14, is naturally resistant to many antibiotics commonly used to manipulate Agrobacterium. To develop Ensifer-mediated transformation, this project will first delete Ensifer genes responsible for kanamycin resistance. Once a kanamycin-sensitive strain is created, various virulence (vir) gene complements from different Agrobacterium strains will be introduced into it, including the trans-zeatin synthase (tzs) gene that directs synthesis of cytokinins, that potentiates plant transformation susceptibility, and a virG mutant (N54D) allele that permits vir gene induction in the absence of phenolic compounds such as acetosyringone. These novel Ensifer strains harboring various vir gene complements will initially be compared to equivalent Agrobacterium strains for virulence on several model plant species, including Arabidopsis and tobacco. Finally, the transformation efficiency of the best Ensifer strains will be compared with equivalent Agrobacterium strains on various cultivars of two crop species: soybean (which is highly recalcitrant to AMT) and potato (many cultivars of which react to AMT with a necrotic response).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.