Location: Molecular Plant Pathology Laboratory2017 Annual Report
Objective 1: Develop novel plant virus-based expression vectors through the characterization of plant virus and viroid genomes. [NP 303, C2, PS2A] • Sub-objective 1.A. Characterize the genome expression strategies of plant viruses for vector development. • Sub-objective 1.B. Develop novel plant virus-based expression vectors utilizing modules derived from plant viruses, viroids, and plant genes. Objective 2: Identify changes in host gene expression and small RNA-mediated regulation associated with viroid and virus infection and spread as targets for disease management. [NP 303, C2, PS2A, PS2B] • Sub-objective 2.A. Perform a functional analysis of genes and proteins involved in protein phosphorylation pathways in virus and viroid infection to identify targets for disease control. • Sub-objective 2.B Identify the roles of viroid-specific small RNAs in plant disease. Objective 3: Develop strategies using plant viruses for the production of antimicrobials for the prevention, treatment, and control of plant and animal diseases. [NP 303, C2, PS2A] • Sub-objective 3.A. Evaluate novel functional proteins for control of plant diseases. • Sub-objective 3.B. Develop functionally active proteins in plants for treatment and control of animal diseases.
This project has two goals: reducing crop losses due to plant pathogens and developing novel compounds to promote growth, improve feed efficiency, and control diseases in farm animals without the use of antibiotics. Fundamental new knowledge of plant pathogen genomes and complex host-pathogen molecular interactions are required to develop novel strategies for disease control. In animals, there are increased challenges to controlling pathogens impacting food safety and infecting livestock and poultry, yet there is a conflicting need to reduce overused antibiotics. Therefore, there is a demand for antibiotic alternatives and novel vaccines, antimicrobials, diagnostic reagents, and therapeutic compounds with reduced cost and low risk to humans, animals and the environment. The unifying concept of this project is the development and use of plant viral-based vectors as tools for the expression of nucleic acids and proteins in plants as a means of studying plant/pathogen interactions, and to develop methodologies useful to control plant pathogens and animal pathogens. In Objective 1, we will study plant virus and viroid genomes (and genome expression strategies) and develop and modify novel plant virus-based vectors based on marafi- tobamo- and potexviruses, viroid genomes, and plant genes. The plant virus-based vectors will be utilized to gain fundamental knowledge of plant virus and viroid host interactions and as tools for expression of heterologous nucleic acids and proteins in plants for plant and animal disease control. In Objective 2, we will perform experiments to evaluate changes in plant host gene expression, and the role of small RNA-mediated regulation, in virus and viroid infection and to determine if phosphorylation signaling pathways play a role in virus and viroid pathogenesis by using protein interaction and gene editing tools. In Objective 3, we will design and express novel antimicrobial proteins in plants to protect against phytopathogenic bacteria and we will design and produce novel recombinant proteins and modified plant virus-like particles which retain functional activity and immunogenicity for control of animal pathogens.
We have made progress on all three objectives during the course of this project. For Objective 1, we developed modified full-length clones of Maize rayado fino virus and Pepper mild mottle virus that are being tested for infectivity and their ability to serve as plant virus-based vectors. For Objective 2, we analyzed changes in gene expression of key genes involved in fertility and fruit development in tomato plants infected with viroids. The fruits of viroid infected plants are small and have no commercial value and identification of interactions between viroid RNAs and their plant host contribute to our fundamental knowledge of viroid pathogenesis. For Objective 3, we constructed a plant codon-optimized triple-acting fusion gene encoding the enzymatically-active domains of three phage endolysins. The modified gene, when transiently expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT vector, produced the fusion protein that was examined for antimicrobial properties against two animal bacterial pathogens.