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

EAGER：重新设计农杆菌，将 T-DNA 传递到叶绿体

• 批准号: 2037155
• 负责人: Pal Maliga
• 金额: $ 29.95万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2037155&HistoricalAwards=false
• 关键词: EAGER; Re; engineering; Agrobacterium; DNA

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.

拟南芥（Arabidopsis thaliana）是最具特征的模式植物，被用于研究基础科学的各个方面。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，烟草是唯一一个质体基因组工程是常规的维管植物物种。最近，利用对大观霉素（一种用于叶绿体转化的选择性剂）高度敏感的植物，在拟南芥中实现了高频质体转化。目前拟南芥质体转化的瓶颈是难以从质体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，并在选择培养基上萌发后鉴定核转基因事件，克服了拟南芥核基因转化的组织培养局限性。我们的目标是重新设计农杆菌的T-DNA传递到叶绿体，直接转化雌性配子体中的质体。回避组织培养过程将消除对拟南芥质体转化实践的专业知识的需要。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，结合现有的广泛基因组资源，将对生物技术的基础科学和应用产生重大影响。由于VirD2毒力蛋白上存在核定位信号（NLSs），农杆菌的T-DNA一直是向细胞核传递的。VirD2是一种切除T-DNA 25个核苷酸序列的内切酶。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）将复合物转移到植物细胞质中。只要在c端提供替代的T4SS信号，在n端提供替代的nls信号，一个包含204个n端氨基酸的截断的VirD2就足以将T-DNA传递到细胞核。这项为期两年的EAGER提案的目标是证明将VirD2重新靶向到叶绿体上的可行性。我们将截断的VirD2重新定位到叶绿体上，通过去除所有NLSs，在c端提供T4SS信号，在n端提供叶绿体靶向转运肽（TP）序列。重靶向的成功将通过VirD2-重组酶融合蛋白在叶绿体中产生永久足迹的靶序列切除来证明。VirD2的传递也将在分裂GFP试验中显示，其中与VirD2融合的短肽（13个氨基酸）将补充截断的GFP蛋白，该蛋白在VirD2融合蛋白传递时发出荧光。后续实验将通过构建农杆菌介导的叶绿体转化，构建缺乏干扰叶绿体靶向的野生型Vir蛋白的农杆菌菌株，并开发新的载体，确保T-DNA传递到雌性配子体的叶绿体中。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Prospects for Reengineering Agrobacterium tumefaciens for T-DNA Delivery to Chloroplasts

重组根癌农杆菌将 T-DNA 递送至叶绿体的前景

• DOI: 10.1093/plphys/kiab081
• 发表时间: 2021
• 期刊: Plant Physiology
• 影响因子: 7.4
• 作者: Matsuoka, Aki;Maliga, Pal
• 通讯作者: Maliga, Pal