Research Project: Molecular Identification, Characterization, and Biology of Foreign and Emerging Viral and Bacterial Plant Pathogens

Location: Foreign Disease-Weed Science Research

2020 Annual Report

Objectives
Objective 1: Develop broad range nucleic acid, antibody and metabolomics based diagnostics for vectored plant pathogens. [NP303, C1, PS1] 1-A. Develop E-probe Diagnostic Nucleic acid Assay (EDNA) diagnostics for the detection of plant pathogen vectors. 1-B. Develop massively parallel sequencing based diagnostic for the detection of bacterial pathogens in vectors. 1-C. Develop immunodiagnostic reagents for specific and sensitive detection of Rathayibacter toxicus and tunicamycin toxin in plant products. 1-D. Collect and characterize foreign and emerging bacterial plant pathogens. Objective 2: Assess the effects of host metabolism and environmental factors on transmission, biology and evolution of threatening and emerging insect-transmitted plant pathogens. [NP303, C2, PS2C] 2-A. Assess the effects of vernalization on Plum pox virus adaptation to new hosts. 2-B. Determine the effects of Plum pox virus infection on host plant metabolomics. 2-C. Xylella fastidiosa subsp. pauca comparative genomes and proteomes. 2-D. Transmission of Xylella fastidiosa subsp. pauca CoDiRO by glassy-winged sharpshooter. Objective 3: Identify genes and proteins required for infection, toxin production and pathogenicity of foreign bacterial plant pathogens. [NP303, C2, PS2A] 3-A. Control of toxin production in Rathayibacter toxicus. 3-B. Rathayibacter toxicus gall transcriptome and proteome. Objective 4: Identify factors in the retention and transmission of insect-transmitted viruses and develop strategies to disrupt this transmission, with an initial focus on the cotton leafroll dwarf virus. [NP303, C2, PS2A, PS2B; C3, PS3B]

Approach
Metagenomics based detection of pathogens and vectors will utilize E-probe Diagnostic Nucleic acid Assay (EDNA) diagnostics. E-probes will be developed for vectors and vectored bacterial pathogens, and tested on metagenomes from controlled simulated insect traps, then extended to test assay success on real world samples. Immunoassays for Rathayibacter toxicus will be developed by the identification of soluble, high abundance, extracellular and/or secreted pathogen proteins as potential diagnostic targets followed by production of monoclonal antibodies. Obtain cultures of target bacteria from major international collections, foreign collaborators, and by traveling abroad. Accessions will be cloned, checked for authenticity using biochemical tests and added to the FDWSRU International Collection of Phytopathogenic Bacteria. Effects of vernalization on Plum pox virus (PPV) biology will be assessed using parallel lines of PPV in peaches, one undergoing artificial vernalization and the other without undergoing vernalization. PPV effects on the metabolome of peaches will be assessed using standard methods, testing PPV positive symptomatic and non-symptomatic trees and comparing the results to metabolomic profiles from healthy and Prunus necrotic ringspot infected trees. The genomes of multiple Xylella fastidosa subsp. pauca isolates will be sequenced, and comparative genomics will be used to assess potential host range and pathogenicity factors. The transmission of the olive strain will be tested using glassy-winged sharpshooter biotypes from the U.S. The genes responsible for toxin production in Rathyaibacter toxicus will be confirmed by gene knockouts, and the regulation and control of these genes will be studied using transcriptomics and proteomics.

Progress Report
The goals of Objective 1 are to develop novel diagnostic techniques and reagents for vectored plant pathogens. The departure of the unit plant virologist and EDNA specialist has left a critical vacancy in our ability to proceed with the planned EDNA goals (Subobj 1B). Under Subobjective 1C, we generated monoclonal antibodies that specifically detect the USDA-APHIS Plant Pathogen Select Agent Rathayibacter toxicus, which allow for the rapid, sensitive detection of the pathogen in immunoassays on in seed and environmental samples from surveys of the U.S. Pacific northwest. We are currently conducting diagnostic immunoassays on R. toxicus -infested seed samples collected in Australia in FY 2019. We have isolated additional new strains of R. toxicus from Australian annual ryegrass seed samples and novel strains of Rathayibacter from grass seed from the U.S. Pacific Northwest and from Maryland (Subobj 1D). Objective 2 focuses on characterizing two vectored pathogens: plum pox virus (PPV) and the bacterium Xylella fastidiosa. The PPV vernalization projects continue to be somewhat delayed by a powdery mildew epidemic in our containment greenhouse. The lack of treatment options during the FY2019 shutdown allowed this epidemic to get out of control and has set back progress. Metabolomic samples for PPV infected and control sets have been collected, extracted, and sent to our collaborator for analysis. qRT-PCR analysis of PPV titers and amplicon sequencing is in process. We had changed the control virus to tomato ringspot virus (ToRSV); however, this may need to be changed again as the collaborator we had identified with a source of ToRSV no longer has infected budwood to share with us. Under Subobj 2C, proteomic analysis of sequenced Xylella fastidiosa genomes has not progressed as we are awaiting results from our proteomics collaborator on samples shipped two years ago. The USDA-APHIS listed select agent Rathayibacter toxicus is the focus of Objective 3. In related work, we are currently performing a comparative secretome analysis of toxigenic and atoxigenic Rathayibacter species, in order to identify bacterial surface-associated and secreted proteins may influence nematode attachment, in planta disease development, and microbial competition/defense. Our project has a new Objective 4 this fiscal year that accompanies new money focused on cotton blue disease caused by the cotton leafroll dwarf virus. This work awaits the arrival of our new virology SY that we anticipate will be on board by the end of calendar 2020.

Accomplishments

Review Publications
Tancos, M.A., Sechler, A.J., Davis, E.W., Chang, J.H., Schroeder, B.K., Murray, T.D., Rogers, E.E. 2020. The identification and conservation of tunicaminyluracil-related biosynthetic gene clusters in several Rathayibacter species collected from Australia, Africa, Eurasia and North America. Frontiers in Microbiology. 10:2914. https://doi.org/10.3389/fmicb.2019.02914.
Tian, B., Gildow, F.E., Stone, A.L., Sherman, D.J., Damsteegt, V.D., Schneider, W.L. 2019. Aphid vectors impose a major bottleneck on Soybean dwarf virus populations for horizontal transmission in soybean. Phytopathology Research. 1:29. https://doi.org/10.1186/s42483-019-0037-3.
Collum, T.D., Stone, A.L., Sherman, D.J., Rogers, E.E., Dardick, C.D., Culver, J.N. 2019. Translating ribosome affinity purification profiling of plum pox virus (PPV) infected leaf tissues in Prunus domestica L reveals post-dormancy spatial coordination of defense responses in phloem tissues. Molecular Plant-Microbe Interactions. 33(1) 66-77. https://doi.org/10.1094/MPMI-06-19-0152-FI.
Luster, D.G., Mcmahon, M.B., Carter, M.L., Sechler, A.J., Rogers, E.E., Schroeder, B.K., Murray, T.D. 2020. Immunoreagents for development of a diagnostic assay specific for Rathayibacter toxicus. Food and Agricultural Immunology. 31(1):231-242. https://doi.org/10.1080/09540105.2020.1714554.
Kappagantu, M., Collum, T.D., Dardick, C.D., Culver, J.N. 2020. Viral hacks of the plant vasculature: the role of phloem alterations in systemic virus infection. Annual Review of Virology. 7:10.1-10.20. https://doi.org/10.1146/annurev-virology-010320-072410.