Research in My Lab

photo, Nodulation in wild type M. truncatula. Atmospheric nitrogen makes up over 70% of the air around us, but is unavailable to living organisms until it is reduced ("fixed") by certain bacteria. Legume plants set up a symbiosis with some of these bacteria, supplying carbon from photosynthesis to the bacteria while the bacteria fix nitrogen from the air for the plant inside special plant root structures called nodules. Since legumes provide 33% of human nutrition in the world, we wish to understand nodule development and the plant control of nodulation to benefit agricultural production, both in legumes and other plants. My lab focuses on molecular genetics in the legume model system Medicago truncatula. Our goal is to identify the plant genes, hormones and environmental signals involved in nodule number regulation and construct a signal transduction pathway for this process. These signals are part of a long distance communication pathway between the roots and shoots and many of the genes involved plant control of nodulation are genes involved in general plant growth and development, making our findings applicable to all plants.

Forward and Reverse Genetics Approach

We have identified multiple mutants that make too many nodules and are currently funded by the NSF to clone the genes and construct the pathway. For example, our sunn mutant, a mutation in a leucine-rich-repeat receptor kinase, makes 7-10-fold more nodules than wild type plants. Grafting experiments with sunn mutants have shown that the signal regulating nodule number occurs in the shoot and we are currently undertaking an immunoprecipitation approach to identifying SUNN interacting partners. A putative epigenetic silencer of SUNN, termed lss, is another mutant in the lab that has lead to experiments investigating genome organization, while a recently cloned root controlled nodule number mutant rdn1 encodes a protein involved in glycosylation in the Golgi. We have other mutants that make too many nodules awaiting characterization, and several suppressors of the sunn mutation. We have begun to order the genes into a pathway regulating nodule development based on grafting experiments, hormone measurements, expression and epistasis analysis.

The components of our model include the plant hormones auxin, cytokinin and ethylene. In addition, the plant can sense its nitrogen needs and balance those needs with its carbon resources; perception and processing of these signals also influence nodule number. Thus discovering how the plant regulates nodule number will also help determine how the plant "thinks."

photo, M. truncatula photo, CRN and PM

Co-localization of MtCORYNE (35S-CRN-YFP; Green) and the plasma membrane (35S-AtPIP2A-mCherry; Red).

Video of RDN1roothair
MtRDN1-GFP localized in tiny moving organelles in the cytoplasm of M. truncatula root hair cell


Genomics Approach

The signal transduction events in the legume-rhizobial symbiosis not only involve two organisms of different kingdoms, a bacterium and a plant, but the 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. Each organism influences the development of the other, and the end result is differentiated bacteria living inside the cells of a plant organ that allows the bacteria to reproduce and fix nitrogen by providing carbon skeletons and a low oxygen tension. While transcriptome profiling of whole roots at one or two ‘snapshot’ time points 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, where much sub-organ gene expression signals are likely diluted by the complex root tissue mixture.

With funding from the National Science Foundation, we (the Frugoli lab and the Feltus lab) are measuring the transcriptome for each cell type involved in nodule formation at specific time points tied to critical events during the progression of nodule development by using laser capture microdissection (LCM) followed by RNAseq and a systems biology analysis of the libraries. This will allow us to answer questions such as: 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, we will create datasets that will allow for these and other queries.



National Science Foundation

Summer Workshop

We will offer a week-long summer workshop at Clemson University June 11-16, 2017 for a small group of graduate students, postdocs and principle investigators to learn LCM and RNASeq.

Application Details for the workshop and travel support awards at our website:
Spatial and Temporal Resolution of mRNA Profiles During Early Nodule Development

I have a professional interest in research ethics, specifically the encouragement and transmission of ethical research practices (often called "best practices") to graduate students, and the definition of these practices. To that end, I interact frequently with Clemson's Rutland Institute for Ethics and Clemson's Office of Research Integrity and am currently serving as Associate Chair of the Department of Genetics and Biochemistry.

Support:

This research has been supported by the US Department of Agriculture, Clemson University, the National Science Foundation, and awards to undergraduates from Pfizer, the American Society for Plant Biology, and the Howard Hughes Medical Institute (through the SC LIFE program).