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

拟南芥是最具特色的模式植物，广泛应用于基础科学研究的方方面面。一个值得注意的例外是，涉及叶绿体基因组工程的研究是在烟草中进行的，烟草是唯一常规进行质体组工程的维管植物物种。近年来，利用叶绿体转化中使用的选择剂壮观霉素高度敏感的植物，实现了拟南芥的高频叶绿体转化。目前，拟南芥中叶绿体转化的瓶颈是从转体组织培养细胞中获得可育植株的困难。通过利用农杆菌直接转化雌配子体，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运送到叶绿体，直接转化雌配子体中的质体。绕过组织培养过程将不再需要专门的专业知识来实践拟南芥的叶绿体转化。因此，本文提出的研究将导致拟南芥基因组工程的广泛应用，结合现有丰富的基因组资源，将对基础科学和生物技术应用产生重大影响。由于VirD2毒力蛋白上存在核定位信号(NLSS)，农杆菌T-DNA一直被运送到细胞核。VirD2是一种在25个核苷酸序列中切除T-DNA的内切酶。在T-DNA转移过程中，它与VirD2蛋白物理连接，并通过IV型分泌系统(T4SS)将复合体转移到植物细胞质中。只要在C末端提供替代的T4SS信号，在N末端提供替代的NLSS，包含204个氨基酸的截短的VirD2就足以将T-DNA递送到细胞核。这项为期两年的急切提案的目标是证明将VirD2重新定位于叶绿体的可行性。我们将通过去除所有NLSS并在C端提供T4SS信号并在N端提供针对转运肽(TP)序列的叶绿体来将截短的VirD2重定向到叶绿体。重定向的成功将通过病毒D2-重组酶融合蛋白切除靶序列来展示，该融合蛋白在叶绿体中创造了永久的足迹。病毒D2的传递也将在裂解的GFP试验中显示，其中一个短的(13个氨基酸)肽与VirD2融合将补充截短的GFP蛋白，该蛋白在传递VirD2融合蛋白时发出荧光。后续实验将通过构建缺乏可能干扰叶绿体靶向的野生型Vir蛋白的农杆菌菌株，以及开发确保T-DNA传递到雌配子体中叶绿体的新载体来完成农杆菌介导的叶绿体转化。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.