Fusarium head blight (FHB; scab) is a devastating disease in barley and wheat caused by the same pathogen. While significant progress has been made in understanding and improving host resistance in wheat with molecular cloning of the major QTL Fhb1 and Fhb7, similar research with barley has lagged behind due to the lack of highly resistant genotypes, which makes it difficult to effectively control FHB and DON contamination. Thus, there is an urgent need for a breakthrough in gene discovery and germplasm development to achieve higher levels of FHB resistance and a greater capacity to detoxify DON in barley using transformative approaches.

The use of wheat genes to breed barley FHB resistance is the road not taken because of strong reproductive barriers. Considering that Fhb7 detoxifies DON, we hypothesize that Fhb7 can also greatly contribute to FHB resistance in barley. Taking advantage of our ongoing work on Fhb7 and CRISPR-based genome editing, we propose to continue our effort with an overall goal to transfer Fhb7 to barley through CRISPR-mediated targeted gene insertion as a proof of concept. The proposed research includes three objectives:

1)      Generate transgenic barley expressing both CRISPR/Cas9 and Fhb7 donor.

2)      Evaluate the Fhb7 function in transgenic barley.

3)      Screen the transgenic plants for targeted Fhb7 insertion events.

Supported by the USWBSI-TRSC program, we are establishing a CRISPR-mediated targeted gene insertion system in barley: developed an all-in-one construct to express CRISPR/Cas9 and the Fhb7 donor DNA and 39 transgenic plants by Agrobacterium mediation. Detached leaf assay analysis of the transgenic plants together with the non-transgenic control showed that Fhb7 functions in barley in suppressing the growth of Fusarium graminearum. While screening the T1 population for targeted insertion events, we have transformed thousands of embryos of Gold Promise (GP) and Excelsior Gold (EG; an elite two-row barley cultivar from the Cornell University) embryos by Biolistic-bombardment of an optimized CRIPSR/Cas construct together with phosphorylated and phosphorothioate linkage-protected PCR product of the Fhb7 gene. After optimizing the parameters, both EG and GP transformation starts to regenerate transgenic plants.

Results from the proposed research will have a positive impact on barley production and the (malting, feed, and food) industry, benefiting barley growers and end-users.