Location: Molecular Plant Pathology Laboratory
2019 Annual Report
Objectives
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.
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 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.
Progress Report
The project is continuing on track for meeting goals for fiscal year 2019. We have made progress on all three objectives during this project. In Objective 1, we developed and modified full-length clones of Maize rayado fino virus, Pepper mild mottle virus, cymbidium mosaic virus that are being tested for infectivity and their ability to serve as plant virus-based vectors. To date, only the cymbidium mosaic virus full-length clone has exhibited low levels of infectivity. Modifications are being made on the marafivirus and tobamovirus full-length clones to enhance infectivity. Tobacco rattle virus-based vector constructs were engineered to express the antisense RNAs for two tomato genes that are involved in viroid pathogenesis for use in gene silencing assays in tomato. Full-length clones of pepino mosaic virus (PMV) were generated and were shown to replicate in inoculated leaves and express the GFP protein; work is ongoing to mutate the movement protein to generate virus that can systemically infect inoculated plants, including tomato.
In Objective 2, we analyzed changes in gene expression of key genes, including basic helix loop helix transcription factors, involved in fertility and fruit development in tomato plants infected with pospiviroids. 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. We have identified three genes that are key for viroid infectivity and pathogenesis and designed and constructed guide RNAs to edit the genes using CRISPR technology to verify their role in disease and plant development. As no naturally occurring resistance is available for plant breeding, gene editing approaches offer more promise. We developed a rapid diagnostic assay for the detection of tomato apical stunt viroid (TASVd) based on isothermal reverse-transcription-recombinase polymerase amplification. TASVd is not present in the U.S. and is seed-transmitted, therefore the need for a rapid and sensitive assay to test for the viroid in seeds and propagation materials pre- and post-entry. We also developed real-time RT-PCR-specific assays for detection of coconut cadang-cadang, tomato apical stunt, pear blister canker, and coconut tinangaja viroids for the Animal and Plant Health Inspection Service (APHIS) Cooperate Agricultural Pest Survey Program (CAPS) program and used these assays to test plant materials to verify presence/absence of the viroids for collaborators in the CAPS program.
In Objective 3, we constructed a plant codon-optimized triple-acting fusion gene (TFnt) encoding the enzymatically-active domains of two bacteriophage endolysins and the mature version of lysostaphin for control of Staphylococcus aureus. The modified gene, when transiently expressed in Nicotiana benthamiana plants using the non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT vector, produced 0.12 g/g fresh weight tissue of TFnt; purified TFnt was preferentially active against the gram-positive S. aureus. Therefore, the combination of codon optimization and transient expression facilitated production of a chimeric phage endolysin in plants. Experiments initiated with ARS researchers in the Animal Biosciences and Biotechnology Laboratory, Beltsville, Maryland resulted in the production of biologically active phage endolysins specific for Clostridium spp. in plants. Several endolysins were evaluated using a plant virus-based expression system, with resulting varying levels of protein expression. Further experimentation revealed that one endolysin, CP41, retained high levels of biological activity in plant sap and future plans are to formulate plant tissue expressing CP41 for feeding studies in chicks. Bacilliform and isometric plant virus-like particles, based on prunus necrotic ringspot virus and maize rayado fino virus, respectively, were engineered to display cationic antimicrobial peptides on their surface. Our results reveal that the antimicrobial peptides can be incorporated into the particle without compromising particle integrity, as revealed by electron microscopy, and experiments to determine the antimicrobial activities of the displayed peptides revealed moderate biological activity against gram-negative bacteria.
Accomplishments
1. Method to produce biologically active Clostridium perfringens phage endolysins in plant sap. Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and causes necrotic enteritis, which can lead to significant levels of mortality and lost productivity in poultry. The disease is commonly controlled using antibiotics in drinking water or feed, widespread use of which may lead to development of resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglyans in the bacterial cell wall are potential replacements for the antibiotics. ARS researchers in Beltsville, Maryland, demonstrated the expression in plants of the soluble, functional bacteriophage CP41 endolysin using a plant virus-based expression system. The endolysin retained activity against C. perfringens in crude plant sap. The incorporation of the lysin in plant cells eliminates the need to purify the protein for addition to animal feed as an effective antimicrobial agent against C. perfringens. These results are of value to animal production scientists who are developing alternatives to antibiotics to control disease in poultry and livestock.
Review Publications
Xu, L., Wang, J., Chen, X., Zhu, D., Wei, H., Hammond, R., Liu, Q. 2019. Molecular characterization and phylogenetic analysis of hop stunt viroid isolates from sweet cherry in China. European Journal of Plant Pathology. https://doi.org/10.1007/s10658-019-01693-3.
Kovalskaya, N.Y., Herndon, E., Foster Frey, J.A., Donovan, D.M., Hammond, R. 2019. Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus. AIMS Microbiology. https://doi.org/10.3934/microbiol.2019.2.158.
