Nitrogen makes up over 70% of the air around us but is unavailable to living organisms to use to make proteins and DNA until it is reduced ("fixed"). Legume plants set up a symbiosis with bacteria that can fix nitrogen, supplying carbon from photosynthesis to the bacteria while the bacteria fix nitrogen from the air for the plant. The bacteria live inside the plant in special root structures called nodules. Legumes provide 33% of human nutrition in the world, and our research is designed to understand nodule development and the plant control of nodulation to benefit agricultural production, both in legumes and other plants. We use molecular genetic tools 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 the long distance communication pathway between the roots and shoots. 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. All plants can sense nitrogen needs and balance those needs with carbon resources; in legumes the perception and processing of these signals influences nodule number. Discovering how the plant regulates nodule number will also help determine how the plant senses the environment around and within it, “makes decisions” based on the information and acts on those decisions.
Co-localization of MtCORYNE (35S-CRN-YFP; Green) and
the plasma membrane (35S-AtPIP2A-mCherry; Red).
We have three project areas in the lab:
We have identified multiple mutants that make too many nodules and are currently cloning 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 used an immunoprecipitation approach to identifying SUNN interacting partners. A putative epigenetic silencer of SUNN, termed lss, is another mutant in the lab that has led to experiments investigating genome organization, while our root-controlled nodule number mutant rdn1 encodes an enzyme that modifies a signal peptide. We have other mutants that make too many nodules awaiting characterization, and several suppressors of the sunn mutation. We order the genes into a pathway using grafting experiments, hormone measurements, expression analysis and epistasis analysis. The components of our model include the plant hormones auxin, cytokinin and ethylene.
Some of the genes we have identified by forward genetics have been discovered in Arabidopsis, where the signaling partner molecules are better known. We use information about what those genes do in Arabidopsis to identify the corresponding M. truncatula genes and mutate them by CRISPR technology to see if they affect nodulation. We are also characterizing mutants in these genes from the Tnt1 insertion collection at the Nobel Research Institute as part of an NSF funded project.
The signal transduction events in the legume-bacterial symbiosis involves two organisms from different kingdoms and the communication required to establish the symbiosis occurs between cells layers in tissues, between organs in the plant, and across time. Signaling starts with the induction of the first chemical responses within 6-12 hours an continues past 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. We use laser capture microdissection (LCM) followed by RNAseq and a systems biology analysis of the data. This is allowing 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?
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).