Fusarium head blight (FHB) caused by Fusarium graminearum (Fg) is an important disease of wheat and barley, resulting in significant yield loss and reduced grain quality due to mycotoxin contamination.  Most commonly grown barley cultivars in the U.S. are susceptible to Fg infection.  The mechanisms of FHB resistance in barley are not well defined.  Genetic engineering of barley with transgenes including those that confer FHB resistance and the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-gene editing constructs can improve barley FHB resistance and aid our studies for the underlying molecular mechanisms of FHB resistance.  To genetically engineer barley, we have developed an optimized plant tissue culture protocol to regenerate multiple barley plantlets from a single scutellum explant for cultivars of the two-rowed Conlon, Genesis and the six-rowed Morex.  Many transgenic barley plants have been produced via both gene gun and Agrobacterium-mediated transformation.  We have also developed our own CRISPR-gene editing platform to knock-out (KO) several host genes involved in conditioning F. graminearum susceptibility.  Our studies with the model plant Arabidopsis have shown that CRISPR-mediated knock-outs of the At2OGO (DMR6) gene encoding a putative 2-oxoglutarate Fe(II)-dependent oxygenase and the AtEIN2 (ethylene insensitive 2) gene result in an augmented Fusarium resistance.  Another potential FHB susceptibility gene identified in Arabidopsis is AtHSK encoding homoserine kinase.  We have constructed both transient and integrating CRISPR vectors to KO barley Hv2OGO, HvEIN2 and HvHSK.  We have also constructed CRISPR vectors to disrupt the promoter for barley HvUGT encoding the UDP gluconosyltransferase in order to study the dynamic role of HvUGT in FHB resistance in different barley cultivars.  We have obtained CRISPR-edited Hv2OGO Conlon mutant plants and found that the mutations induced by our CRISPR-editing vector are inherited into the T₂ generation.  Additionally, we have successfully applied the oligo-directed mutation method to enhance the CRISPR-gene editing efficiency and the online ICE (Inference of CRISPR Edits) analysis to efficiently screen for barley mutants.

ACKNOWLEDGEMENT AND DISCLAIMER

This material is based upon work partially supported by the U.S. Department of Agriculture, under Agreement No. 59-0206-0-170. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.