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（TFs），已经实现了各种各样的细胞谱系转化-提供了对细胞分化的见解，并允许创建医学相关的细胞类型。更强的重编程植物细胞命运和诱导理想途径的能力可能对植物组织培养和合成生物学产生变革性影响。这个项目应用了一个新的框架来系统地评估异位TF在玉米中的表达，玉米是美国最重要的作物之一。机器人辅助分析将用于并行测试数千种tf对基因表达的影响。随后将进行有针对性的二次筛选，以评估植物组织中对选定的tf的生理反应。最终，这些实验将有助于更好地了解TF的功能和“重编程工具箱”，以帮助植物性状工程和改进植物育种技术的发展。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到本科院校讲述他们的职业生涯，这些院校中有很大比例的未被充分代表的群体。学生研究员将在演讲方面提供指导，在他们的研讨会期间将包括有关探索和导航科学事业的信息。该项目的科学目标是：(1)利用高通量原生质体分析方法测量对2000个克隆玉米TFs的转录反应；(2)建立解释和分析这些数据的方法，包括将tf与目标通路连接起来的方法，推断基因调控网络的方法，以及在分析高通量平板基因组学数据时更好地解释技术协变量的方法；(3)优先考虑并测试TFs诱导异位功能反应的能力，重点是组织再生-植物转化的关键步骤。数据和代码将可访问和免费使用，包括通过MaizeGDB基因登陆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，pi将举办一个关于植物高通量功能筛选的在线研讨会，帮助将该技术推广到社区。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。

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.