MCA-PGR: Spatial and Temporal Resolution of mRNA Profiles During Early Nodule Development

豆科植物-根瘤菌共生中的信号转导事件不仅涉及不同界别的两种生物，而且建立共生所需的通讯发生在组织中的细胞层之间、植物器官之间，以及从接种后6-12小时内诱导第一次化学反应到根瘤内建立固氮的一段时间。其结果是，分化的细菌生活在植物器官的细胞内，由于植物提供碳骨架和低氧分压，使细菌能够繁殖和固定氮。虽然在根瘤发育过程中对整个根的转录组图谱扩展了我们关于启动共生的基因的知识，但这样的实验无法在组织/细胞水平上解决事件的进展。为了以连贯的方式理解这些细胞之间在空间和时间上发生的信号，有必要分析单个细胞在特定时间点的转录组，因为相邻的细胞可能具有非常不同的转录组。该补助金的培训部分包括对首席研究员(PI)职业生涯中期的基因组技术进行培训，通过参与这项研究对博士后学者和研究生进行基因组学和信息学方面的培训，在暑期工作坊培训来自服务不足群体的研究生、博士后和PI，以及通过实地考察实验室体验让服务不足和经济困难的中学生接触科学和高等教育。将在与结核发育进程中的关键事件有关的特定时间点，通过使用激光捕获显微解剖(LCM)以及随后的RNAseq和对文库的系统生物学分析，测量参与结核形成的每种细胞类型的转录体。以下问题将被解决：在给定的时间，与未接种的植物相比，在给定的细胞类型中，哪些基因有差异表达？在一种细胞类型中，哪些基因表现出与未接种植物随着时间的推移差异表达的保守模式？表达有微小变化但模式保守的多个基因是否存在协同效应？哪些基因网络包含已知的根瘤调控基因，因此，通过关联，这些基因控制什么？在这些早期时间点，哪些基因在自律突变体中被错误调控，从而导致太多的结节形成？应用时间序列分析和系统生物学工具，将创建允许进行这些和其他查询的数据集。了解共生建立过程中的基因调控是能够在其他植物中重建固氮共生体的关键，而在单个细胞水平上的了解是产生类似于根瘤的结构所必需的。这里提出的工作将提供关于根瘤菌在根的特定细胞类型中被差异调控的基因的信息，关于在细胞类型中被协调调控的基因的信息，并与来自突变分析的已发表的知识相结合，将产生关于在哪些组织中的哪些协调调控基因组对早期根瘤发育至关重要的信息。最终的结果将是一个时空模型，极大地扩展了我们对根瘤信号的了解，并为共生群落提供了一个数据挖掘资源。

The signal transduction events in the legume-rhizobial symbiosis not only involve two organisms of different kingdoms, but communication required to establish the symbiosis occurs between cells layers in tissues, between organs in the plant, and across time, from the induction of the first chemical responses within 6-12 hours to the establishment of nitrogen fixation in the nodules 10 days after inoculation. The result is differentiated bacteria living inside the cells of a plant organ that allows the bacteria to reproduce and fix nitrogen due to the provision of carbon skeletons and a low oxygen tension by the plant. While transcriptome profiling of whole roots during nodule development has expanded our knowledge of what genes are involved in initiating symbiosis, such experiments are unable to resolve the progression of events at the tissue/cellular level. In order to understand the signaling occurring between these cells in space and time in a coherent manner, it is necessary to analyze the transcriptome of the individual cells at specific points in time, as neighboring cells may have very different transcriptomes. The training components of this grant include training mid-career of the principal investigator (PI) in genomic techniques, training postdoctoral scholars and a graduate student in genomics and informatics through their participation in this research, training graduate students, postdocs and PIs from underserved groups in the summer workshop, and exposing underserved and economically disadvantaged middle school students to science and higher education through field trip lab experiences. Transcriptomes will be measured for each cell type involved in nodule formation at specific time points tied to critical events during the progression of nodule development by use of laser capture microdissection (LCM) followed by RNAseq and a systems biology analysis of the libraries. The following questions will be addressed: What genes are differentially expressed relative to uninoculated plants in a given cell type at a given time? What genes show conserved patterns of differential expression from uninoculated plants over time within a cell type? Is there a synergistic effect of multiple genes showing small changes in expression but a conserved pattern? Which gene networks contain known nodule regulatory genes and therefore, by association, what do these genes control? What genes are misregulated in autoregulatory mutants at these early time points that allow too many nodules to develop? Applying time series analysis and systems biology tools, datasets will be created that will allow for these and other queries. Understanding gene regulation during the establishment of symbiosis is key to being able to recreate nitrogen-fixing symbioses in other plants, and understanding at the level of individual cells is required to generate a structure similar to a nodule. The work proposed here will provide information about what genes are being differentially regulated in a specific cell type in the part of the root responding to rhizobia, about what genes are coordinately regulated within a cell type, and combined with published knowledge from mutant analyses, will yield information about which groups of coordinately regulated genes in which tissues are critical to early nodule development. The end result will be a spatiotemporal model greatly expanding our knowledge of nodulation signaling in roots and a data mining resource for the symbiosis community.