TRTech-PGR: Ensifer-mediated Transformation as an Alternative to Agrobacterium-mediated Plant Transformation of Model Plants and Crops

农杆菌介导的植物遗传转化（Agrobacteriummediatedplantgenetictransformation，AMT）是植物基础研究和农业生物技术中最常用的转基因植物和基因组工程试剂导入技术。 虽然该技术自20世纪80年代以来被开发并用于植物改良，但农杆菌介导的转化具有许多局限性。 其中关键的是农杆菌介导的转化限于特定植物物种和/或特定植物物种的变种。 农杆菌是一种天然的植物病原体，因此它经常激发受感染植物的防御反应，导致组织坏死。 这种坏死组织不能通过基因转化。 粘粒菌是一种与土壤杆菌相关的细菌。 当提供相关的遗传元件时，它可以像农杆菌一样对植物进行遗传转化。 Ensifer不是病原体，因此不能引起坏死植物防御反应。 然而，Ensifer介导的植物遗传转化在普通植物生物学实验室中仍然是低效和繁琐的。 本项目所描述的工作将开发Ensifer介导的植物遗传转化系统，大大简化Ensifer在实验室中的使用，提高Ensifer介导的转化效率。 由此产生的系统将大大扩展植物转化的宿主范围，允许比现在允许的农杆菌介导的转化更广泛的作物物种的遗传改良。 农杆菌介导的植物遗传转化已被用于许多植物物种的转化。 然而，许多植物物种和栽培种/品种对农杆菌介导的转化仍然高度排斥，部分原因是它们对农杆菌感染具有坏死防御反应。 作为一种非病原体，相关细菌Ensifer adhaerens不会引起强烈的植物防御反应。 Ensifer可以通过适当的遗传改变改变广泛的植物物种。 使用Ensifer作为农杆菌的替代物的限制是目前用于植物转化的最佳Ensifer菌株，E. adhaerens OV 14对通常用于操纵农杆菌的许多抗生素具有天然抗性。为了开发Ensifer介导的转化，该项目将首先删除负责卡那霉素抗性的Ensifer基因。一旦产生卡那霉素敏感性菌株，来自不同农杆菌菌株的各种毒力（vir）基因互补物将被引入其中，包括指导细胞分裂素合成的反式玉米素合酶（tzs）基因，其增强植物转化易感性，以及在不存在酚类化合物如乙酰双氢黄酮的情况下允许vir基因诱导的virG突变体（N54 D）等位基因。这些携带各种vir基因互补物的新型Ensifer菌株最初将与等同的农杆菌菌株在几种模式植物物种（包括拟南芥和烟草）上的毒力进行比较。最后，将最佳Ensifer菌株的转化效率与等同的农杆菌菌株在两种作物物种的各种栽培品种上进行比较：大豆（对AMT来说是高度不相容的）和马铃薯（其中许多品种对AMT的反应是坏死反应）该奖项反映了NSF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进行评估，被认为值得支持的搜索.

Agrobacterium-mediated plant genetic transformation (AMT) is the most commonly used technology for generating transgenic plants and introducing genome engineering reagents in plant basic research and in agricultural biotechnology. Although this technology was developed and used for plant improvement since the 1980s, Agrobacterium-mediated transformation has numerous limitations. Key among these is that Agrobacterium-mediated transformation is limited to specific plant species and/or varieties of particular plant species. Agrobacterium is a natural plant pathogen, and it therefore frequently elicits defense reactions from infected plants, causing tissue necrosis. Such necrotic tissue cannot be genetically transformed. Ensifer adhaerens is a bacterium related to Agrobacterium. When supplied with the relevant genetic elements it can, like Agrobacterium, genetically transform plants. Ensifer is not a pathogen and therefore fails to elicit necrotic plant defense responses. However, Ensifer-mediated plant genetic transformation remains inefficient and cumbersome to perform in the average plant biology laboratory. The work described in this project will develop an Ensifer-mediated plant genetic transformation system, greatly simplifying the use of Ensifer in the laboratory and improving the efficiency of Ensifer-mediated transformation. The resulting system will greatly extend the host range for plant transformation, allowing for the genetic improvement of a wider range of crop species than that now permitted by Agrobacterium-mediated transformation. Agrobacterium-mediated plant genetic transformation has been used for transformation of many plant species. However, numerous plant species and cultivars/varieties remain highly recalcitrant to Agrobacterium-mediated transformation, in part because they react with a necrotic defense response to Agrobacterium infection. As a non-pathogen, the related bacterium Ensifer adhaerens does not elicit a strong plant defense response. Ensifer can, with the appropriate genetic alterations, transform a wide spectrum of plant species. A limitation to using Ensifer as an alternative to Agrobacterium is that the best Ensifer strain currently used for plant transformation, E. adhaerens OV14, is naturally resistant to many antibiotics commonly used to manipulate Agrobacterium. To develop Ensifer-mediated transformation, this project will first delete Ensifer genes responsible for kanamycin resistance. Once a kanamycin-sensitive strain is created, various virulence (vir) gene complements from different Agrobacterium strains will be introduced into it, including the trans-zeatin synthase (tzs) gene that directs synthesis of cytokinins, that potentiates plant transformation susceptibility, and a virG mutant (N54D) allele that permits vir gene induction in the absence of phenolic compounds such as acetosyringone. These novel Ensifer strains harboring various vir gene complements will initially be compared to equivalent Agrobacterium strains for virulence on several model plant species, including Arabidopsis and tobacco. Finally, the transformation efficiency of the best Ensifer strains will be compared with equivalent Agrobacterium strains on various cultivars of two crop species: soybean (which is highly recalcitrant to AMT) and potato (many cultivars of which react to AMT with a necrotic response).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.