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

Clemson University, Clemson SC

Investigators

Abstract

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.