NSF Postdoctoral Fellowship in Biology FY 2019: The Effects of Tomato Domestication on the Circadian Clock and its Interaction with Resistance to Phytophthora infestans

本行动资助NSF国家植物基因组计划2019财年生物学博士后研究奖学金。该奖学金支持奖学金获得者在主办实验室的研究和培训计划，该奖学金获得者还提出了扩大生物学参与的计划。本·曼斯菲尔德奖学金的研究和培训计划的标题是“番茄驯化对生物钟的影响及其与抗疫霉菌的相互作用”。该奖学金的主办机构是唐纳德·丹福斯植物科学中心，赞助科学家是丽贝卡·巴特博士。西红柿是世界上第二大蔬菜作物，它的果实可以生吃，也可以在汤和酱汁等加工产品中食用。西红柿富含重要的维生素、矿物质以及具有潜在抗癌和其他健康功能的抗氧化剂。番茄的原产地是南美洲的赤道国家。因此，在数百万年的时间里，番茄植物进化出了一个适应赤道周围白天长度的生物钟。然而，在过去的几千年里，随着人类驯化西红柿，他们也带着西红柿的种子四处迁徙，把西红柿培育成高纬度地区更成功的作物。通过这个过程，番茄的生物钟被改变，以适应地球上这些地区新的长日。通过驯化改变作物生物钟的过程并不是番茄独有的，而且可能相当普遍；然而，由于其在调节植物如何保护自己免受疾病和昆虫侵害方面的重要作用，改变的生物钟可能因此增加植物对疾病的易感性。这项研究旨在以番茄为模型，更好地理解作物驯化是如何影响生物钟的，以及这反过来又可能如何影响植物抵御疾病的先天能力。这项研究的首要社会目标是在基本层面上提高作物防御能力，帮助种植者减少投入，为不断增长的人口生产更便宜、可持续的食物。该项目的培训也将进一步提高研究员在植物遗传学、基因组学和植物-病原体相互作用方面的技能。更广泛的影响包括指导对生物信息学、植物病害和作物改良有兴趣的高中生和本科生。这项研究将探索生物钟的改变是否影响了栽培番茄对感染的反应，特别是对引起番茄和马铃薯晚疫病的病原菌疫霉的反应。首先，将使用自动摄像系统筛选野生和驯化番茄品系的昼夜节律特征。然后将使用高通量生物测定来筛选这些品系是否在不同时间对感染P. infestans表现出不同的反应（“门控”反应）。时钟节律改变的转基因番茄株系也将被培育和筛选，以测试时钟基因的错误调控对感染门控反应的影响。在门控反应中表现出最强差异的两个品系将用于72小时的基因表达研究，其中叶片将在黎明和黄昏接种病原体，每4小时采样一次进行RNA测序。然后，加权基因共表达网络将用于比较野生和驯化番茄的转录反应，并确定网络共表达模块中心的枢纽基因，这些基因在品系之间存在差异。最后，为了绘制导致野生和驯化番茄差异的基因座，将对分离群体进行表型分析，并使用散装分离方法绘制数量性状基因座。候选基因将使用基于crispr的诱变技术进行验证。本研究中产生的所有数据将通过在线web应用程序以及公共存储库（如NCBI的Short Read Archive （SRA）、Dryad （https://datadryad.org）和Sol Genomics Network (SGN; https://solgenomics.net).This）进行访问，该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

This action funds an NSF National Plant Genome Initiative Postdoctoral Research Fellowship in Biology for FY 2019. The fellowship supports a research and training plan in a host laboratory for the Fellow who also presents a plan to broaden participation in biology. The title of the research and training plan for this fellowship to Ben Mansfeld is "The Effects of Tomato Domestication on the Circadian Clock and its Interaction with Resistance to Phytophthora infestans". The host institution for the fellowship is the Donald Danforth Plant Science Center and the sponsoring scientist is Dr. Rebecca Bart.Tomato is the second most important vegetable crop in the world, the fruit of which is eaten fresh or consumed in several processed products such as soups and sauces. Tomatoes are rich with important vitamins, minerals as well as antioxidants with potential anti-cancer and other healthful capabilities. The origin of the tomato plant is in equatorial countries in South America. As such, over millions of years, tomato plants have evolved a biological clock adapted to the day length around the equator. However, as humans domesticated tomatoes over the last few thousand years, they also traveled with the seeds, breeding tomatoes to be more successful crop plants in high latitude regions. Through this process, the tomato biological clock was altered to fit the new long days in these regions of the planet. The process of altering a crop's biological clock through domestication is not unique to tomato and may be quite common; however, because of its important role in regulating how the plant protects itself from disease and insects, an altered biological clock may thus increase a plant's susceptibility to disease. This research aims to use tomato as a model to better understand how crop domestication affected the biological clock and how this, in turn, may have affected the plant's innate ability to defend itself from disease. The overarching societal goal for this research is to contribute to increased crop defenses at a basic level, helping growers reduce their inputs and produce cheaper, sustainable food for a growing population. Training though this project will also further the Fellow's skills in plant genetics, genomics and plant-pathogen interactions. Broader impacts include mentoring high school and undergraduate students interested in learning more about bioinformatics, plant disease and crop improvement.This research will explore the hypothesis that alterations to the biological clock have affected cultivated tomato responses to infection, specifically by Phytophthora infestans, the pathogen that causes tomato and potato late blight. First, a collection of wild and domesticated tomato lines will be screened for their circadian rhythm traits, using an automatic camera system. A high-throughput bioassay will be then be used to screen if these lines show a different response to infection by P. infestans, at different times (a "gated" response). Transgenic tomato lines with altered clock rhythms will also be generated and screened to test the effects of mis-regulation of clock genes on the gated response to infection. The two lines showing strongest difference in gated response will be used in a 72-hour gene expression study, in which leaves will be inoculated with the pathogen at dawn and dusk and sampled every 4 hours for RNA sequencing. Weighted gene co-expression networks will then be used to compare the transcriptional responses of wild and domesticated tomato and identify hub genes at the center of network co-expression modules that differ between lines. Finally, to map loci contributing to differences in wild and domesticated tomato, a segregating population will be phenotyped and quantitative trait loci mapped using a bulk segregant approach. Candidate genes will be validated using CRISPR-based mutagenesis. All the data generated in this research will be made accessible through an online web application as well as through public repositories such as the NCBI's Short Read Archive (SRA), Dryad (https://datadryad.org), and the Sol Genomics Network (SGN; https://solgenomics.net).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.