Location: Crop Production and Pest Control Research
Project Number: 5020-21000-001-006-A
Project Type: Cooperative Agreement
Start Date: Aug 22, 2025
End Date: Aug 21, 2026
Objective:
(i) Transcriptomics and proteomics of wheat EMS mutant to identify candidate differentially expressed genes/proteins
(ii) In silico identification, validation, and subcellular location of effectors from wheat insect pests (Hessian fly and aphids)
(iii) Subcellular localization of insect effector(s) and defense/susceptibility protein(s) within the insect and host plant
(iv) Validation of resistance and susceptibility factors via heterologous expression in Nicotiana benthamina and subcellular localization of protein interaction dynamics
Approach:
Total RNA and proteins will be extracted from uninfested and infested-wheat EMS mutant and wild type wheat samples collected over a time course. The samples will be used for RNA and protein sequencing. Bioinformatics will be used to identify differentially expressed genes and proteins. Using previously generated RNA-sequencing data from insect samples over a time course, in silico prediction will be undertaken to identify insect effectors. The candidate insect effectors and plant proteins identified from sequencing data will be validated using quantitative real-time PCR and western blot analysis. The coding sequence of the candidate plant (resistance/susceptibility) and insect effector proteins will be cloned into expression vectors. Subcellular localization of the candidate insect and plant proteins will be done using cryostat sectioning and immunolocalization on sectioned tissue with target-specific antibodies. The protein-protein interaction and subcellular localization of the protein interaction dynamics will be performed using heterologous transient expression in Nicotiana benthamiana via split luciferase assay and quantitative imaging.