Location: Hard Winter Wheat Genetics Research
Project Number: 3020-21000-011-33-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2020
End Date: Aug 31, 2023
1. Determine localization of family 31 effectors in infested resistant and susceptible wheat seedlings. 2. Identify interaction targets of family 31 effectors in wheat using yeast two-hybrid system and immune-pulldown assays.
Larvae of the Hessian fly establish feeding sites at the base of wheat seedlings. In their saliva, they secrete small effector molecules to manipulate the host metabolism and suppress host defenses. We have identified a family (family 31) of putative effectors among the secreted salivary gland proteins of larvae. To study biochemical functions of candidate effector proteins, recombinant proteins will be produced using the pET (based on T7 polymerase promoter) bacterial expression system with a histidine-tag. Recombinant proteins will be purified and used to produce antibodies, which will be used to examine the effector location in infested wheat tissues via indirect immunostaining and confocal microscopy. Yeast two-hybrid libraries will be screened for wheat proteins that interact with putative effectors from Hessian fly. The purified recombinant proteins will also be used as a bait to identify interacting molecular targets from wheat. Specifically, individual effector proteins will be incubated with extracts of wheat tissues infested with Hessian fly larvae. If an effector protein interacts physically with a wheat target protein, the Hessian fly-wheat protein complex will be pulled down via the histidine-tag on the recombinant effector protein to solid beads. Interacting proteins from host plants will be identified by liquid chromatography/mass spectrometry.