Location: Molecular Plant Pathology Laboratory
2022 Annual Report
Objectives
Objective 1: Develop new plant virus-based expression technologies and diagnostics through the characterization of plant virus and viroid biology. [NP 303, C1, PS1A, PS1B]
Sub-objective 1A: Characterize the genomes and genome expression strategies of plant viruses and viroids for vector development (Non-hypothesis driven)
Sub-objective 1B: Develop novel plant virus-based expression vectors utilizing modules derived from plant viruses, viroids, and plant genes. (Non-hypothesis driven)
OBJECTIVE 2: Identify changes in host gene expression and small RNA-mediated regulation associated with viroid and virus infection and disease development as potential targets for disease management [NP 303, C2, PS2A]
Sub-objective 2A: Functional analysis of genes and proteins involved in transcription and protein phosphorylation pathways in virus and viroid infections.
Sub-objective 2B Identify the roles of viroid-specific small RNAs and host factors in plant disease.
OBJECTIVE 3: Develop new practical strategies for the production in plants of proteins and nucleic acids for prevention, treatment, and control of plant and animal diseases [NP 303, C2, PS2A; NP301, C2, PS2A]
Sub-objective 3A: Evaluate novel functional proteins and nucleic acids for control of plant diseases (Non-hypothesis driven)
Sub-objective 3B: Express functionally active proteins in plants for treatment and control of animal diseases. (Non-hypothesis driven)
Approach
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 characterize the genomes and genome expression strategies of plant viruses and viroid genomes and develop and modify novel plant virus-based vectors based on modules derived from 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.
Progress Report
The unifying concept of this project is development and use of plant virus-based expression vectors as tools for expression of nucleic acids and heterologous proteins in plants. 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. It builds on progress and accomplishments made in the previous project, 8042-22000-295-00D entitled “Development of Novel Control Strategies for Diseases Caused by Cellular and Sub-cellular Pathogens”. In Objective 2, we used an omics approach that integrates root and leaf transcriptomic data, gene regulatory network analysis, and identification of affected biological processes revealed that specific BHLH, MYB, and ERF transcription factors regulate genes involved in molecular mechanisms underlying critical signaling pathways associated with viroid infection of tomato. Functional enrichment of regulons shows that BHLH-MTRs are linked to metabolism and plant defense, while MYB-MTRs are involved in signaling regulation and hormone-related processes. Members of the BHLH-TF family have a potential specific role as microproteins involved in the post-translational regulation of hormone signaling events. For the severe viroid variant, ERF-MTRs were characteristic, while ZNF-TF, tf3a-TF, BZIP-TFs, and NAC-TF act as unique MTRs. Altogether, our results lay a foundation for further research on the PSTVd and host genome interaction, providing evidence for identifying potential key genes that influence symptoms during development in tomato plants. In Objective 3, one of the novel antimicrobial proteins we are producing in plants as an alternative to antibiotics is the glycosyl hydrolase bacteriophage endolysin (PlyCP41). PlyCP41 lyses the bacterial cell wall of Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, that is the third leading cause of human foodborne bacterial disease and necrotic enteritis in poultry and is controlled using antibiotics. We expressed an Escherichia coli codon-optimized gene encoding the modified PlyCP41p expressed in Nicotiana benthamiana plants using a series of pepino mosaic virus (PepMV)-based transient expression vectors, developed in the previous project. In our recent studies. PlyCP41p displayed the highest accumulation of ~2.5% total soluble protein in one of the virus vectors. Plant sap containing the protein lysed C. perfringens poultry strains Cp39 and Cp509 in a plate lysis assay. Optimal expression of PlyCP41p was achieved at 7 days post-infection. Experiments are underway to determine the most appropriate formulation of plant material containing CP41 for oral delivery to chicks to control C. perfringens in a poultry model.
Accomplishments
