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

拟南芥（Arabidopsis thaliana）是最具特征的模式植物，用于基础科学的各个方面的研究。一个值得注意的例外是，涉及质体基因组工程的研究是在烟草中进行的，烟草是唯一的质体基因组工程是常规的维管植物物种。近年来，利用对大观霉素（叶绿体转化中使用的选择剂）高度敏感的植物，在拟南芥中实现了高频率的质体转化。目前拟南芥质体转化的瓶颈是难以从转质体的组织培养细胞中获得可育植株。利用农杆菌直接转化雌性配子体，并通过在选择性培养基上萌发产生的幼苗来鉴定核转基因事件，克服了拟南芥核基因转化中的组织培养限制。我们的目标是重新设计农杆菌，将T-DNA递送到叶绿体中，以直接转化雌性配子体中的质体。避开组织培养过程将消除对在拟南芥中实施质体转化的专业知识的需要。因此，本文提出的研究将导致拟南芥质体基因组工程的广泛应用，结合现有的广泛的基因组资源，将在基础科学和生物技术中的应用产生重大影响。由于VirD 2毒力蛋白的核定位信号（NLS）的存在，农杆菌T-DNA递送一直是到细胞核。VirD 2是在25个核苷酸序列处切除T-DNA的内切核酸酶。在T-DNA转移过程中，它与VirD 2蛋白物理连接，并且复合物通过IV型分泌系统（T4 SS）转移到植物细胞质中。截短的VirD 2，含有204个氨基酸的N-末端是足够的T-DNA递送到细胞核，只要一个替代的T4 SS信号提供在C-末端和替代的NLS提供在N-末端。为期两年的EAGER提案的目标是证明将VirD 2重新靶向叶绿体的可行性。我们将通过去除所有NLS并在C-末端提供T4 SS信号和在N-末端提供叶绿体靶向转运肽（TP）序列来将截短的VirD 2重新靶向叶绿体。重靶向的成功将通过由VirD 2重组酶融合蛋白切除靶序列来显示，所述VirD 2重组酶融合蛋白在叶绿体中产生永久足迹。VirD 2递送也将在裂解GFP测定中显示，其中与VirD 2融合的短（13个氨基酸）肽将补充在递送VirD 2融合蛋白时发荧光的截短的GFP蛋白。后续实验将通过构建缺乏可干扰叶绿体靶向的野生型Vir蛋白的农杆菌菌株来完成农杆菌介导的叶绿体转化，和开发新的载体，以确保T-该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响进行评估来支持审查标准。

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.