Location: Molecular Plant Pathology Laboratory
2024 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. In support of Objective 1, leaf extracts from clover displaying yellow mosaic were processed for high-throughput sequencing. Two distinct isolates each of the potexviruses, clover yellow mosaic virus (ClYMV-Iso1 and Iso2) and white clover mosaic virus (WClMV-Iso1 and Iso2), were identified as being present in a mixed infection. To generate plant virus-based vectors for legumes, full-length copies of the RNA genomes of ClYMV-Iso1 and ClYMV-Iso 2 were generated and are being engineered into bacterial plasmids for use as infectious transient expression vectors for experiments described in Objective 3. Yield losses caused by viroids can reach 17-64% depending on the viroid strain and plant crop species worldwide. Viroid systemic infection in tomato is commonly associated with the development of severe symptoms, including stunting, leaf epinasty and distortion, veinal chlorosis, reduction of flower size, flower abortion, and reduced size and numbers of fruits. Mexico represents a geographical landscape of viroids that has been linked to their origin and comprises considerable biodiversity. The biological features of viroid species endemic to Mexico in tomato, citrus, and avocado were reported, including a broad host range and possible long-distance, seed, or insect mediated transmission, to yield a better understanding of the biology of viroid diseases and future programs on movement and spread control to avoid economic losses in agricultural industries.
In support of Objective 1 and 2, we identified potential host genome targets of vd-siRNAs of seed-transmitted pospiviroids tomato planta macho viroid (TPMVd) and potato spindle tuber viroid (PSTVd), in TPMVd-infected tomato plants. A subset of 43,000 vdsRNA reads were mapped against the tomato genome to identify potential host targets. An average of 65 tomato genome targets with at least 10x coverage were identified. Current work focuses on experimental validation of target cleavage sites and gene expression of predicted vdsiRNA targets in infected tomato by RT-qPCR and evaluation of RNA-Seq datasets. In our study of viroid-host interactions, we developed gene knockout lines in tomato (cv. Moneymaker) using CRISPR/cas9/RNA-guided editing to target two single-copy genes: ViRP1 (viroid RNA binding protein 1) and PKV (protein kinase viroid induced). Plants generated for each edited gene consisted of homozygous and heterozygous lines for the expected deletions, and small indels. Phenotypes of the T0 and T1 plants for ViRP1 were like wild type, while PKV T0 plants had small fruits and few or no viable seeds. Current work focuses on the heritability of the mutations, phenotypes of stable, homozygous lines, and subsequent effects of the mutations on viroid infectivity and pathogenesis when challenged with PSTVd and TPMVd.
In support of 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 (Cp). We previously expressed an Escherichia coli codon-optimized gene encoding the modified PlyCP41p in Nicotiana benthamiana plants using a series of virus-based transient expression vectors. 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. In a NIFA-funded team effort between USDA-ARS researchers in Beltsville, Maryland, and Peoria, Illinois, and the University of Maryland Eastern Shore, Princess Anne, Maryland, we demonstrated that recombinant PlyCP41 as either a purified enzyme (expressed in E. coli and purified via nickel chromatography) or as a part of intact Tg organisms [Tg yeast or Tg plants] can kill Cp in vitro or ex vivo in chicken gut fluids. When utilized in an in vitro assay, the modified yeast containing PlyCP41 eliminated 99.997% (P<0.0001) of C. perfringens. Similar results were observed during ex vivo studies with PlyCP41 eliminating 90-99.9% of the C. perfringens in all three regions of the gut that were tested. The next step is to test the Tg plants and Tg yeast for the ability to reduce Cp in live chickens. 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.
In 2024, progress was made in support of Objective 1 with the engineering of an infectious clone of clover yellow mosaic virus with the intent of developing this virus into a vector to produce antimicrobial proteins in legumes for use in animal feed. Additionally, molecular protocols were developed to detect tomato brown rugose fruit virus, an emerging and devasting virus of tomato and pepper, in seed to prevent its dispersion to growers.
In support of Objective 2, fifteen T0 lines of ViRP1 CRISPR-edited plants consisted of homozygous, heterozygous, and biallelic lines for the expected deletions, and small indels. Phenotypes of the T0, T1, T2 and T3 ViRP1-edited plants were not phenotypically different from wild type, unedited plants. Characterization of mutation events in T0 plants and T1/T2 segregating progenies homozygous for potential mutant alleles revealed size differences in the PCR amplified products. Finally, the edited plants displayed various disease index scores where some lines displayed suppression of viroid infection. The edits in the ViRP1 bromodomain may result in a nonfunctional protein that is unable to interact with the viroid RNA molecule and facilitate transport into the nucleus.
In support of Objective 3, collaborative studies with researchers in Peoria, Illinois, and the University of Maryland Eastern Shore, Princess Anne, Maryland, we demonstrated the anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) suspended in PBS via mixing experiments with cultured Cp and transgenic yeast slurries followed by serial dilution plating and colony counts on TSC (Cp indicator) plates. The transgenic yeast containing PlyCP41 eliminated 99.997% (P<0.01) of the cultured Cp. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous Cp content in fluids from three different gastrointestinal (GIT) regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast slurries eliminated 90 to 99.99% of the endogenous Cp (P<0.01). These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is highly effective at reducing the endogenous Cp in the presence of gastrointestinal fluids of broiler chickens. Future studies will measure the anti-Cp effect in vivo by administering transgenic yeast to broiler chickens in the feed and to optimize production of PlyCP41 in plant cells. New studies have begun to utilize advanced computational tools to design modular soluble and thermostable phage endolysins to control animal bacterial pathogens, with the goal of delivery in formulated feed additives. To date, seven candidate proteins have been designed, expressed in E. coli, and purifed to test in vitro against multiple poultry isolates of Clostridium perfringens, in addition to other bacterial pathogens of animals.
Accomplishments
1. Developed optimized molecular methods for routine detection of tomato brown rugose fruit virus in tomato and pepper seeds to reduce dispersion. Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus is stable and mechanically transmitted, through both standard horticultural practices and contaminated seeds, serving as the primary modes of transmission. In collaborative research with scientists from Irapuato, Mexico, ARS researchers in Beltsville, Maryland, examined the relevance of emergent diseases associated with potential seed dispersion of ToBRFV Mexican strains to agronomically important Solanaceous hosts, focusing on a comparison of molecular biology techniques for rapid and sensitive virus detection in seed lots. We developed optimized extraction and amplification protocols to achieve optimal sensitivity and accuracy of ToBRFV detection for routine detection of in seeds lots and plants, reducing the risk of dispersion. The results of this study will be of value to the vegetable seed industry, growers, regulatory agencies, and researchers worldwide.
2. Demonstrated that a recombinant phage endolysin produced yeast is active in killing poultry stains of Clostridium perfringens. Necrotic enteritis (NE) is a prevalent and often fatal gastrointestinal disease in poultry caused by the bacterium Clostridium perfringens. NE not only impacts the health and welfare of chickens, but global losses have been estimated to cost the poultry industry $6 billion annually. Antibiotics were commonly used for the treatment and prevention of NE. However, alternative antimicrobial solutions are necessary to address rising concerns about antibiotic resistance.In collaboration with USDA-ARS researchers in Beltsville, Maryland, Peoria, Illinois, and the University of Maryland Eastern Shore, Princess Anne, Maryland, a novel approach was developed to control Clostridium perfringens using an enzyme, called phage endolysin PlyCp41, that has the unique ability to selectively target and destroy the harmful bacterium. The enzyme, when produced in yeast, was able to eliminate up to 99.99% of the harmful bacteria in laboratory testing and experiments using the contents from chicken intestines. These promising results suggests that incorporating this modified yeast, and in the future, modified plant material into chicken feed could serve as an effective strategy to control this devastating disease, reducing dependency on antibiotics.
Review Publications
Zamora-Macorra, E.J., Avina-Padilla, K., Hammond, R., Ochoa-Martinez, D.L. 2023. Comparison of molecular protocols to detect Tomato brown rugose fruit virus in solanaceae hosts. Mexican Journal of Phytopathology. 41(4):2. https://doi.org/10.18781/R.MEX.FIT.2023-5.
Barnas, M.R., Attuquayefio, W.D., Donovan, D.M., Skory, C.D., Hammond, R.W., Siragusa, G.R., Timmons, J.R. 2024. Yeast expressing a phage endolysin reduces endogenous Clostridium perfringens ex vivo in 21-day-old broiler chicken intestinal fluids. Avian Diseases. 68(2):129-133. https://doi.org/10.1637/aviandiseases-D-23-00088.
Abrahamian, P., Mollov, D.S., Hammond, R., Rivera, Y. 2023. Coding-complete genome sequence of a novel isolate of papaya virus E in tomato. Microbiology Resource Announcements. Article. e00344-23. https://doi.org/10.1128/MRA.00344-23.
