BRC-BIO: MediCARGO- Decoding peptide perception during Medicago-Sinorhizobium symbiosis using CRISPR-cas9 As a Reverse Genetics tool

Bioactive small molecules play an important role in optimizing agricultural systems, allowing plants to tolerate abiotic stresses like high temperature and biotic stresses like disease-causing pathogens, while enabling interactions with beneficial microbes. As our planet is faced with uncertain environmental challenges, research seeking to understand roles of novel, plant-based small molecules can provide much needed solutions.

Small signaling peptides, or peptide hormones, are genome-encoded bioactive molecules that plants naturally deploy as signals to mediate physiological responses to their environment, such as the absence of essential macronutrients, including nitrogen. This project will use the model legume, Medicago truncatula, which is capable of interacting with beneficial bacteria to help acquire nitrogen from the environment in the absence of added nitrogenous fertilizers.

This ability is mediated by detection of peptides in the environment, although the mechanism is poorly understood. Understanding this process in more detail could provide an avenue to the potential use of chemically synthesized peptides as alternatives to nitrogen-based fertilizers, which can be harmful to the environment. Thus, the outcomes of this work could provide an affordable, non-GMO route to regulate plant growth.

The project will also have broad impact by providing career development opportunities for a new faculty member at a Minority-Serving Institution; mentoring and research training for graduate and undergraduate students; and outreach to farmers, producers, and the general public on factors influencing agricultural sustainability.

The objective of this project is to understand how legumes perceive peptides during root nodule symbiosis by genetically dissecting gene redundancy in the receptor class most commonly associated with peptide binding, namely, Leucine-Rich Repeat Receptor Like Kinases (LRR-RLKs). Molecular mechanisms of plant peptide signaling, and perception are poorly understood.

Several peptide hormones have been identified in legumes; however, their cognate receptors remain elusive. The project will use reverse genetics tools, primarily CRISPR-cas9 mediated gene editing, and make use of the pre-existing Tnt1 insertion mutant collection in the model legum, M. truncatula, to develop an in-house lrr-rlk mutant collection. Another objective of the project will be to develop a working protocol to identify peptide-receptor pairs using co-immunoprecipitation followed by mass spectrometry.

The development and curation of mutant lines in M. truncatula lrr-rlks will serve as a vital resource for the legume research community, facilitating investigation of peptide hormone function not only in root nodule symbiosis and nitrogen uptake, but also in interactions with arbuscular mycorrhizal fungi, and responses to macronutrient deficiency. In the long term, these resources will provide fundamental knowledge for understanding effects of putative agrochemicals in control of plant-microbe interactions.

This work using the model legume M. truncatula will have a profound impact on the state of knowledge in this nascent field and uncover roles of symbiotically expressed receptors exclusively present in legumes.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.