Double transformation of Medicago truncatula and analysis of biotin ligase production in root cells: towards the creation of a mycorrhiza-induced INTACT system Open Access

Hatch, Kathryn (Spring 2018)

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Symbiosis between plants’ root systems and mycorrhizal fungi is known to be very beneficial for both organisms. Mycorrhizae tend to benefit from a reliable source of carbon, while plants benefit with increased growth rates and resistance to many types of stressors. The most common type of mycorrhizae is arbuscular mycorrhizae (AM), which form structures within as well as around cells, and so make changes to the plants at a cellular level. One way to study these changes would be to use the Isolation of Nuclei TAgged in specific Cell Types (INTACT) method to isolate nuclei from the cells interacting with AM fungi. In this method, there are two main transgenes: NTF, which binds GFP and a biotin ligase recognition peptide (BLRP) to the nuclear envelope, and BirA which codes for biotin ligase production. When both are expressed, biotin ligase biotinylates the NTF protein, allowing for the isolation of nuclei with streptavidin-coated magnetic beads. Usually in INTACT, NTF is controlled by a cell type specific promotor while BirA is constitutive, allowing for the isolation of the nuclei of the cells in which the promotor is active. However, to study mycorrhizal colonization of roots and isolate colonized specific cell types, BirA would need to be controlled by a promotor induced by mycorrhizal infection. To test whether the biotinylation of nuclei is quick enough for this system to be effective, the production of inducible BirA needs to be studied in plant roots. This study chose to make BirA estrogeninducible, as estrogen is not produced normally in plants, so the exposure could be controlled. This paper focuses on the creation of the estrogen-inducible BirA construct and the creation of doubly transgenic plants with constitutive NTF and estrogen-inducible BirA that can be used to test the viability of INTACT with two selective promotors. This work serves as a foundation for developing a method to allow detailed studies of gene expression and chromatin changes that take place as plant cells interact with AM fungi.

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