TRTech-PGR: Agrobacterium-mediated transformation of the plastid genome

High-frequency plastid transformation in Arabidopsis was achieved by using plants hyper-sensitive to spectinomycin, the selective agent used in chloroplast transformation. The current bottleneck of plastid transformation in Arabidopsis is the difficulty of obtaining fertile plants from transplastomic tissue culture cells. This tissue culture limitation will be overcome by using Agrobacterium to directly transform plastids in the oocytes and identification of transgenic events by germinating seed on selective medium. To achieve this goal, Agrobacterium will be re-engineered to enable T-DNA delivery to the plastids of maternal germline cells. This process will avoid tissue culture and tissue-culture induced genetic variation, thereby greatly simplifying the process of obtaining transplastomic plants. Side-stepping the tissue culture process eliminates the need for specialized expertise to practice plastid transformation in Arabidopsis. Therefore, research proposed here will lead to widespread applications of Arabidopsis plastid genome engineering which, combined with the available extensive genomic resources, will have a major impact on basic science and applications in biotechnology. The new protocol for plastid transformation will be applicable in any crop in which the floral dip protocol yields nuclear transgenic plants. The goal of the project is to directly transform plastids in the female gametocyte in Arabidopsis flowers by re-engineering Agrobacterium for T-DNA delivery to chloroplasts. During Agrobacterium infection, VirD2 nicks the T-DNA border sequences and covalently links to the 5’ end of the T-strand via its Tyr29. The T-strand is then guided through the Type IV secretion system (T4SS) to the plant cell, where it integrates in the plant nucleus. With support from the NSF EAGER Grant, VirD2 was reengineered by removing its nuclear localization signals (NLSs) and fusing its N-terminus with a chloroplast-targeting transit peptide (TP). The reengineered plastid-VirD2 (Pt-VirD2) was shown to localize to chloroplasts in a split GFP assay, in which VirD2 fused with 16 amino-acids of GFP (GFP11) complemented a large GFP (GFP1-10) already in chloroplasts. Agrobacterium-mediated chloroplast transformation will be obtained by constructing vectors which encode both T-DNA and Pt-VirD2 function. Pt-VirD2 will direct T-DNA containing an antibiotic resistance marker to the plastid genome. To develop the method, transplastomic events will be identified by cocultivation of Agrobacterium with tobacco leaves and selecting for antibiotic resistance encoded in the vector. Arabidopsis flowers will then be dipped in an Agrobacterium culture carrying the plastid transformation vector and transplastomic Arabidopsis plants will be identified by germinating seed on a selective medium.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.