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ARS Home » Pacific West Area » Pullman, Washington » Grain Legume Genetics Physiology Research » Research » Research Project #444928

Research Project: Plant Defense Mechanisms Against Sclerotinia Effector PINE1

Location: Grain Legume Genetics Physiology Research

Project Number: 2090-21000-034-037-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 30, 2023
End Date: Sep 29, 2028

The Objectives are to investigate pathogenicity mechanisms of grain legume pathogens and to enhance plant resistance to fungal pathogens through modifying existing plant defense proiteins.

A. Approaches for plant defense proteins: A1. Clone and heterogonously express chickpea polygalacturonase-inhibiting proteins. A2. Characterize chickpea polygalacturonase-inhibiting proteins at molecularly and functionally. A3. Determine physical interactions of chickpea PGIPs with fungal polygalacturonases and SsPINE1 using yeast two-hybrid, BiFC and co-immuno precipitation techniques. A4. Conduct site-directed mutagenesis on the chickpea PGIPs at the specific interaction sites. A5. Test the reduced interactions of the mutant variants of PGIPs with the effector protein SsPINE1. A6. Transform model tobacco plants with the PGIP mutant variants that shoed reduced interactions with SsPINE1. A7 Verify the enhanced plant resistance to Sclerotinia white mold in transformed mutant lines A8. Prepare official reports and publish the research findings in peer-reviewed journals. B. Approaches for pathogenicity effectors: B1. Specifically delete the SsE3 gene in Sclerotinia sclerotiorum using the split-marker technique and confirm the deletion in the mutants using specific PCR and southern hybridization. B2 Characterize the functions of SsE3 by testing the SsE3-deletion mutants B3 Determine SsE3 is a secreted protein from Sclerotinia sclerotiorum. B4. Clone and heterogeneously express the effector gene SsE3 in expression yeast. B5. Screen Arabidopsis thaliana mRNAs that code for proteins that could interact with SsE3 using yeast two hybrid technique. B6. Determine and confirm the interactions between SsE3 and the plant protein using three independent techniques yeast two hybrid assay, BiFC and CO-IPs B7. Develop mutants of Arabidopsis thaliana with SsE3 and confirm the integration of SsE3 in the Arabidopsis thaliana genome. B8. Characterize the phenotypes of the transgenic lines. B9. Prepare official reports and publish the research findings in peer-reviewed journals.