Location: Temperate Tree Fruit and Vegetable Research
Project Number: 2092-21220-003-019-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2023
End Date: Jun 30, 2024
Objective:
Objective 1: Use a comparative genomics approach to identify pathogenicity-related genes and genomic elements in ‘Ca. P. trifolii’ BLTVA strain.
Objective 2: Use a targeted approach to profile the identified pathogenicity factors in BLTVA isolates identified in insect and plant specimens collected in the U.S. and Mexico.
Approach:
Objective 1. We will utilize several computational strategies to identify pathogenicity factors in the ‘Ca. P. trifolii’ BLTVA phytoplasma strain, including whole genome comparisons, phylogenetic inference, ortholog analysis, gene neighborhood analysis, and conservation analysis. Currently, 65 phytoplasma genomes are available in the NCBI GenBank, including the ‘Ca. P. trifolii’ BLTVA strain (GenBank Accession GCA_028595955.1), which is the only 16SrVI strain available, and numerous ‘Ca. P. asteris’ asters yellow strains that belong to the 16Sr1 phytoplasma group that is also associated with aster yellows disease of potato. We aim to identify the pathogenicity-related genes (toxins/effectors, secretion pathways, and other membrane proteins) or other genomic elements/islands that are present in BLTVA phytoplasma which induces potato purple top symptoms. We will also leverage state-of-the-art techniques such as deep learning-based AlphaFold2 modeling, protein domain analysis, structural comparisons, and profile analysis to gain insights into the potential functions of the genes we identify.
Objective 2. Following the successful identification of pathogenicity factors present in BLTVA phytoplasma, specific primers will be designed for use in targeted polymerase chain reaction (PCR) assays. Standard PCR using a high-fidelity, proof-reading polymerase will then be conducted using DNA previously isolated from symptomatic BLTVA phytoplasma-infected plant tissue and insect specimens collected in the U.S. and Mexico during the past decade. Amplified targets will be subjected to sequence analysis. Sequencing results will be further incorporated into comparative genomics and phylogenetic analyses (approaches in Objective 1) and used to assess if any spatial and temporal correlation exists between BLTVA samples, host plants or insects and the sequence of identified genes.
