Performing Department
(N/A)
Non Technical Summary
Increased crop plant production is necessary to feed a growing global population and support renewable resources. Novel crop resistance to nematodes and other biotic attackers is required to meet these demands. Xylan, a plant cell wall component, plays a central role in plant integrity and health. The high amount of xylan biomass incorporated into grass cell walls highlights the importance of this polysaccharide, as U.S. grass crops provide many staple foods and renewable resources. Xylan influences cell wall structure and flexibility, ultimately contributing to stored plant biomass, mechanical structures, and resilience.Discoveries from basic research in plant biology can inform engineering efforts and industrial collaborations. Insights from interactions between rice plants and root-knot nematode (RKN) pests have highlighted a host-parasite arms race at the cell wall interface. Endoparasitic RKNs are devastating pests that respond to the plant host's xylan barrier by secreting xylanases, xylan-degrading enzymes. To counteract this, rice protects xylan via proteases with inhibitory activity on xylanases. This multidisciplinary project complements an evolutionary systems biology framework with biochemistry, cell biology, and genetics to gain a deeper understanding of xylan properties, which can inform efforts to improve plant health and engineering of high-impact agricultural products, thereby contributing to global food and resource security. To characterize these molecular interactions and determine regulation of xylan-related genes, I will use varying molecular biology techniques with computational analyses. Exploration of xylan metabolism and hydrolysis could enhance plant products, such as biofuels, and plant quality traits, biomechanics, and resilience to stressors.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
15%
Developmental
10%
Goals / Objectives
Major GoalsThe goal of this project is to define the molecular interplay between xylan (hemicellulose, a polysaccharide) and cell wall-related proteins in interactions between rice plants and nematode pests. By investigating this molecular interaction surrounding xylan, this project can translate discoveries for two major outcomes: 1) crop plant resistance to nematodes and 2) tools to engineer xylan deposition and cell wall structure. This work has the potential for sustainable agricultural intensification via crop protection and value-added innovation of xylan for its dynamic roles in biofuel production, biomechanics, and crop plant resilience to stressors.ObjectivesSurvey genes and regulators of rice xylan deposition during nematode infection to advance genomic engineering of xylan metabolism.Map spatial and temporal xylan fluctuations in root cell walls during infection.Test rice xylan synthase function during infection.Build gene regulatory networks of rice responses to nematode infection.Define key regulators in xylan synthesis during rice responses to nematodes.Characterize nematode xylanase hydrolytic activity to find tools for xylan catabolism.Define nematode xylanase expression patterns.Test xylanase hydrolytic activity with enzymatic assays.Silence xylanases in nematodes to determine their role in infection.Test the effectivity of rice xylanase-inhibiting proteases (XIPs) to expand our toolkit for engineering cell wall structure.Model protein-protein interactions between rice XIPs and nematode xylanases.Test rice XIP function during nematode parasitism.Test inhibitory activity of rice XIPs on nematode xylanases with enzymatic assays.Confirm XIP-xylanase interactions in vitro and in planta.
Project Methods
Project MethodsBroadly, this project incorporates molecular biology techniques, bioinformatics analyses, and nematode infection assays to define the xylan-based molecular interactions occurring between rice plants and nematode pests at the cell wall interface. This project will be conducted using histochemistry, microscopy, biochemistry, molecular genetics, in vitro, and computational tools and methods.To profile xylan fluctuations in rice roots across nematode infection cycles I can purchase commercial antibodies to stain for xylan and nematodes and quantify xylan composition during infection with fluorescent microscopy and associated software.Enzymatic assays will be performed to determine the function of nematode xylanases and rice xylanase-inhibiting proteins. I will generate recombinant proteins and incubate proteins with purified xylan. I will use mass spectrometry to determine the enzymatic activity and quantify xylo-oligo fragmentation patterns.Transgenic rice mutant lines for overexpression, reporter constructs, and targeted mutagenesis will be generated using established plant transformation protocols for rice. These lines will be used in nematode infection experiments in the greenhouse and in vitro.Using sterile technique, infection experiments will be accomplished on media to functionally test the rice mutant lines and quantify xylan fluctuations. In these experiments I will include treatments with xylanase-silenced nematodes induced by dsRNA.Using programming in R I can clean, analyze and visualize my data, build gene regulatory networks, and run statistical analyses and models. The Geneious Platform allows me to manage my genetic material and data (e.g., sequences, primers, plasmid maps).EffortsThis project seeks to focus efforts on mentorship of undergraduates through laboratory instruction, training, and agricultural science development. Information will be disseminated through science communication, outreach, and education to my local community. Through extension work I can deliver genetic markers and plant lines for rice breeders in the U.S. and globally.?EvaluationI will evaluate the progress of the project through dedicated milestones. Major milestones will include presentations of data at conferences/symposia and finished manuscripts (a minimum of two will be produced). Experimental milestones will include the completion of each sub-objective (e.g., generating transgenic rice mutant lines, successful enzymatic assays, successful imaging of cell walls stained for xylan). Accountability of milestones and progress will be evaluated during my annual reviews and during meetings with my mentors.