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

豆科植物与根瘤菌共生的信号转导事件不仅涉及两种不同领域的生物，而且建立共生所需的通信发生在组织细胞层之间、植物器官之间，并且跨越时间，从6-12小时内第一次化学反应的诱导到接种后10天根瘤菌内固氮的建立。结果是生活在植物器官细胞内的分化细菌，由于植物提供碳骨架和低氧张力，细菌可以繁殖和固定氮。虽然在根瘤发育过程中对整个根的转录组分析扩大了我们对启动共生关系的基因的了解，但这些实验无法解决组织/细胞水平上事件的进展。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传导，有必要在特定时间点分析单个细胞的转录组，因为邻近细胞可能具有非常不同的转录组。这项资助的培训内容包括：对处于职业生涯中期的首席研究员（PI）进行基因组技术方面的培训，通过参与这项研究培训博士后学者和一名基因组学和信息学方面的研究生，在暑期工作坊中培训缺乏服务群体的研究生、博士后和PI，以及通过实地实验室体验，让缺乏服务和经济条件较差的中学生接触科学和高等教育。通过激光捕获显微解剖（LCM），然后使用RNAseq和文库的系统生物学分析，在与结节发展过程中的关键事件相关的特定时间点，测量参与结节形成的每种细胞类型的转录组。将解决以下问题：在给定的细胞类型中，在给定的时间，哪些基因相对于未接种的植物有差异表达？在一种细胞类型中，随着时间的推移，哪些基因与未接种的植物表现出保守的差异表达模式？是否存在多个基因的协同作用，表现出微小的表达变化，但有一个保守的模式？哪些基因网络包含已知的结节调节基因，因此，通过关联，这些基因控制什么？在这些早期时间点上，哪些基因在自我调节突变体中被错误调控，从而导致太多结节的发生？应用时间序列分析和系统生物学工具，将创建允许这些和其他查询的数据集。了解共生关系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关系的关键，并且需要在单个细胞水平上了解产生类似于根瘤的结构。这里提出的工作将提供关于在根瘤菌响应部分的特定细胞类型中哪些基因被差异调节的信息，关于在细胞类型中哪些基因被协调调节的信息，并结合从突变分析中发表的知识，将产生关于哪些组织中哪些协调调节的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系结瘤信号的认识，并为共生群落提供了数据挖掘资源。

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.