Location: Hard Winter Wheat Genetics Research
Project Number: 3020-21000-012-015-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2022
End Date: Aug 31, 2024
Objective:
The objective of this research is to determine the biochemical functions of Hessian fly effectors and their interacting proteins in wheat plants.
Approach:
Hessian fly is an important insect pest of wheat. The insect can be controlled through plant host resistance, but the insect population typically overcomes new resistance genes within a few years after deployment. To develop strategies for more effective, durable resistance, the molecular foundations of the plant-insect interactions need to be better understood.
Hessian fly larvae are thought to inject secreted salivary gland protein (SSGP) effectors into wheat seedlings to help obtain nutrients and dampen the host resistance response. The largest group of SSGPs is known as family 31. Several genes encoding putative Hessian fly effector family 31 members have been cloned previously. We will use the pEAQ-HT-DEST1 protein transient expression vector system to screen for candidate effectors that can trigger measurable plant responses. We will use changes in the gene expression levels of Mds-1 and the lipid transfer protein gene BT008922 as biomarkers to measure plant responses using RT-qPCR. Mds-1 is upregulated >100 fold in compatible interactions, but there is no change in incompatible interactions. The lipid transfer gene is upregulated >50 fold in incompatible interactions, but downregulated >50 fold in compatible interactions.
Next, we will generate recombinant proteins using the cloned genes that have shown a measurable plant response. The recombinant proteins will be used as antigens to produce polyclonal antibodies. The antibodies will be used for western blots or immunostaining to detect the presence of the Hessian fly proteins in infested wheat tissues.
The same antibodies will be used for immunoprecipitation to identify interacting targets of Hessian fly family 31 effectors in wheat tissues. A His-tag will be added to each recombinant protein to facilitate protein purification. The His-tag may be also used to immobilize proteins to a solid support (such as resin) for identifying interacting targets in wheat. To identify interacting targets, Hessian fly-infested and non-infested wheat extracts will be incubated with immobilized family 31 proteins. Non-interacting proteins or other molecules will be washed off with buffers. The molecules bound to recombinant family 31 proteins will be eluted and will be identified via liquid chromatography/mass spectrometry quadrupole time of flight (LC-Q-TOF) instrumentation.
Lastly, a candidate for the broadly effective resistance gene Hdic has been identified. The cognate effector for Hdic is predicted to interact with important plant defense networks. To identify the effector recognized by Hdic, we first need to verify the sequence of Hdic. The genome of the wheat line WGRC42 will be sequenced via the PacBio platform. The region that contains Hdic will be compared between wild-type WGRC42 and its gene-edited, susceptible mutants.
