Location: Crop Production and Pest Control Research
Project Number: 5020-21220-014-020-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Sep 15, 2022
End Date: Sep 14, 2026
Objective:
The overall objective of this project is to evaluate the roles of Fusarium effector proteases on pathogenesis in wheat and barley. Two of the specific aims are:
1. Identify host proteins targeted by the Fusarium graminearum proteases using enzyme-catalyzed proximity-dependent labeling.
2. Generate and test decoy substrates for activation of a photosystem biogenesis regulator (PBR1.c) in a model plant, Nicotiana benthamiana, and wheat and barley protoplasts.
Approach:
Specific Aim 1. To identify host proteins targeted by the translocated F. graminearum proteases, we will generate lipase-tagged derivatives of the F. graminearum proteases and transiently express these constructs in N. benthamiana leaves and wheat protoplasts using previously established protocols. The biotinylated proteins will then be captured using streptavidin-based affinity purification and identified using mass spectrometry.
Specific Aim 2. Once we have identified a preferred cleavage site sequence for the F. graminearum proteases, we will generate ‘decoy’ proteins in wheat and barley. We will then transiently co-express the F. graminearum protease and the modified protein constructs in N. benthamiana and evaluate induction of cell death. In parallel, we will also co-express the protein derivatives and the corresponding F. graminearum protease along with a reporter of cell viability in either wheat or barley leaf protoplasts to test whether cleavage of the decoy protein will activate endogenous PBR1.c, using a luciferase assay.
