TRTech-PGR: Reprogramming the Plant Cell with Transcription Factors

在生物医学中，通过异位表达转录因子(TF)实现了各种各样的细胞谱系转换--提供了对细胞分化的洞察，并允许创建与医学相关的细胞类型。更强的重新编程植物细胞命运和诱导理想途径的能力可能会对植物组织培养和合成生物学产生革命性的影响。这个项目应用了一个新的框架来系统地评估在玉米中异位表达转铁蛋白可以实现什么，玉米是美国最重要的作物之一。机器人辅助化验将被用来测试数千种转录因子对基因表达的平行影响。随后将进行有针对性的二级筛选，以评估植物组织对选定的TFS的生理反应。最终，这些实验将有助于更好地理解转铁蛋白的功能，并为植物性状工程和改良植物育种技术的发展提供一个“重新编程工具箱”。为了将研究和教育联系起来，将设立一个奖学金，派遣研究生和博士后讲述他们在本科院校的职业生涯，这些院校中代表不足的群体比例很高。这个项目的科学目标是：(1)使用高通量原生质体分析来测量对2000个克隆的玉米转录因子的转录响应；(2)建立解释和分析这些数据的方法，包括将转录因子连接到靶途径，推断基因调控网络，并在分析高通量平板基因组数据时更好地考虑技术协变量；以及(3)确定和测试转录因子的异位诱导功能反应的能力，重点是组织再生--植物转化中的关键步骤。数据和代码将变得可访问和免费使用，包括通过MaizeGDB基因登录页面上的数据报告和图形可视化。质粒将通过拟南芥生物资源中心(ABRC)分发。最后，PIS将举办一个关于工厂高通量功能筛选的在线研讨会，帮助将这项技术提供给社区。该奖项反映了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。

A wide variety of cell lineage transformations has been achieved in biomedicine by ectopically expressing transcription factors (TFs) – providing insight into cell differentiation and allowing for the creation of medically-relevant cell types. A greater ability to reprogram plant cell fate and induce desirable pathways could have transformative impacts on plant tissue culture and synthetic biology. This project applies a new framework to systematically assess what can be accomplished with ectopic TF expression in maize, one of the most important US crops. Robotics-assisted assays will be used to test the effect of thousands of TFs on gene expression in parallel. This will be followed with targeted secondary screens to evaluate the physiological response to selected TFs in plant tissues. Ultimately, these experiments will contribute a greater understanding of TF function and a “reprogramming toolbox” to aid in the engineering of plant traits and development of improved plant breeding technologies. To connect research and education, a fellowship will be established to send graduate students and post-docs to speak about their careers at undergraduate institutions with a high proportion of underrepresented groups. Student fellows will be provided mentorship on speaking, and during their seminars will include information about exploring and navigating scientific careers.The scientific aims of this project are to: (1) measure the transcriptional response to a collection of 2000 cloned maize TFs using high-throughput protoplast assays; (2) establish methods to interpret and analyze these data, including approaches to connect TFs to target pathways, infer gene regulatory networks, and better account for technical covariates when analyzing high-throughput plate-based genomics data; and, (3) prioritize and test TFs for the ability to induce functional responses ectopically, with an emphasis on tissue regeneration – a crucial step in plant transformation. Data and code will be made accessible and free to use, including through data reports and graphical visualization on the gene landing pages of MaizeGDB. Plasmids will be distributed through the Arabidopsis Biological Resource Center (ABRC). Finally, the PIs will host an online workshop on high-throughput functional screening in plants, helping to make the technique available to the community.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.