EAGER: Re-engineering Agrobacterium for T-DNA delivery to chloroplasts

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Arabidopsis (Arabidopsis thaliana) is the best characterized model plant and is used to study all aspects of basic science. A notable exception is that studies involving plastid genome engineering are carried out in tobacco, the only vascular plant species in which plastome engineering is routine. Recently, 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. Tissue culture limitations in Arabidopsis nuclear gene transformation were overcome by using Agrobacterium to directly transform the female gametocyte, and identification of nuclear transgenic events by germinating the resulting seedlings on a selective medium. Our goal is to re-engineer Agrobacterium for T-DNA delivery to chloroplasts to directly transform the plastids in the female gametocyte. Side-stepping the tissue culture process will eliminate 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.Agrobacterium T-DNA delivery has always been to the nucleus due to the presence of nuclear localization signals (NLSs) on the VirD2 virulence protein. VirD2 is an endonuclease that excises the T-DNA at a 25-nucleotide sequence. During the T-DNA transfer, it is physically linked to the VirD2 protein and the complex is translocated to the plant cytoplasm via the Type IV secretion system (T4SS). A truncated VirD2, containing 204 amino acids of the N-terminus is sufficient for T-DNA delivery to the nucleus, as long as an alternative T4SS signal is provided at the C-terminus and alternative NLSs are provided at the N-terminus. The goal of the two-year EAGER proposal is to prove the feasibility of re-targeting VirD2 to chloroplasts. We will re-target a truncated VirD2 to chloroplasts by removing all NLSs and providing T4SS signals at the C-terminus and chloroplast targeting Transit-Peptide (TP) sequences at the N-terminus. The success of retargeting will be shown by excision of target sequences by a VirD2- recombinase fusion protein that creates a permanent footprint in chloroplasts. VirD2 delivery will also be shown in a split GFP assay, in which a short (13 amino acid) peptide fused with VirD2 will complement a truncated GFP protein that fluoresces upon delivery of the VirD2 fusion protein. Follow-up experiments will accomplish Agrobacterium-mediated chloroplast-transformation by construction of Agrobacterium strains lacking wild-type Vir proteins that could interfere with chloroplast targeting, and development of new vectors that will ensure T-DNA delivery to chloroplasts in the female gametophyte.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.