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）是最有特征的模型植物，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，烟草是唯一的血管植物物种常规的研究。最近，通过使用对叶绿体转化中使用的选择性剂对Spectinomycin的植物过敏，可以实现拟南芥中的高频质体转化。拟南芥中质体转化的当前瓶颈很难从移植组织培养细胞中获得肥沃的植物。通过使用农业杆菌直接转化雌性配子细胞，并通过在选择性培养基上发芽的幼苗来克服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生农分杆，以将T-DNA递送至叶绿体，以直接转化雌性配子细胞中的质体。侧向组织培养过程将消除对拟南芥中质体转化的专业知识的需求。因此，这里提出的研究将导致拟南芥质体基因组工程的广泛应用，加上可用的广泛基因组资源，将对基础科学和生物技术中的基础科学产生重大影响。grobacteriumt-DNA tna的递送始终是由于存在核位置信号（NLSS）的核心，即Virence virence virence protence。 VIRD2是一种核酸内切酶，可在25-核苷酸序列下切除T-DNA。在T-DNA转移期间，它与VIRD2蛋白进行物理链接，并通过IV型分泌系统（T4SS）将复合物转移到植物细胞质中。截短的VIRD2，含有N末端的204个氨基酸足以使T-DNA向细胞核递送，只要在C末端提供替代T4SS信号，并且在N末端提供了替代NLSS。为期两年的渴望提议的目的是证明将VIRD2重新定位为叶绿体的可行性。我们将通过去除所有NLSS并在N末端的C端和叶绿体靶向转运肽（TP）序列上提供T4SS信号，将截短的VIRD2重新定位为叶绿体。通过VIRD2-重组酶融合蛋白切除目标序列的重新定位的成功将显示出，该蛋白会在叶绿体中产生永久的足迹。 VIRD2递送也将显示在分裂的GFP测定中，其中与VIRD2融合的短（13个氨基酸）肽将补充截短的GFP蛋白，该蛋白在递送Vird2融合蛋白后荧光。随访实验将通过构建缺乏野生型VIR蛋白的农杆菌菌株来完成农杆菌介导的叶绿体转化，这些蛋白可能会干扰叶绿体的靶向，并开发新的矢量，并确保通过对女性游戏的质量培养的质量来确保t-dna递送的质量。利用基金会的知识分子和更广泛的影响审查标准。

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