Location: Hard Winter Wheat Genetics Research
Project Number: 3020-21000-010-33-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2016
End Date: Aug 31, 2019
1) Produce recombinant protein encoded putative effector genes from the Hessian fly. 2) Identify target proteins that interact with Hessian fly effector poteins.
1). Selected effector genes from the Hessian fly genome will be cloned into pT7expression vector. In this expression system, the putative effector protein will be expressed as a fusion protein with a His Tag, which will be used for purification of the recombinant protein. The expression constructs will be delivered into BL-21 (Rosette)-DE3 bacterial strain, which allows to expression eukaryotic protein at high levels. The recombinant proteins will be purified to homogeneity for functional assays and for antibody production. 2). Purified recombinant Hessian fly effectors will be incubated with wheat protein extracts infested with Hessian fly. The proteins from wheat that interact with Hessian fly effectors will be monitored and purified with HPLC columns (gel filtration or ion exchange). The interactive complexes will be further analysis through SDS/PAGE, western blots, and LC/MS/MS to identify and characterize host proteins.