Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch] is the fungal pathogen that causes Fusarium head blight (FHB), which is the most devastating disease in cereal crops worldwide. FHB significantly reduces yield and grain quality under favorable environmental conditions. Understanding the molecular mechanism underlying the virulence of the pathogen is critical for designing novel strategies to manage the disease. In this study, we aimed to develop a CRISPR/Cas9-mediated genome editing protocol for efficiently knocking out genes in F. graminearum. The Tri5 gene required for DON production in the fungus was chosen as the target and two sites of the gene were selected for designing single guide RNAs (sgRNAs) for the genome editing experiments. The hygromycin resistance gene (HygR) was amplified by PCR using primer pairs containing 50 bp or 60 bp sequences flanking the target sites of Tri5 and used as donor DNA.  PEG-mediated transformation of F. graminearum protoplasts was conducted using the donor DNA with or without the Cas9/sgRNA complex added. The results showed that more transformants with the target gene disrupted were generated with the donor DNA combined with the Cas9/sgRNA complex than with the donor DNA only, suggesting that the Cas9/sgRNA complex increased the gene knockout efficiency. Our experiments also indicated that more gene knockout mutants were generated with the donor DNAs containing the 60 bp flanking sequences than those with the 50 bp flanking sequences. The Cas9/sgRNA-mediated gene knock strategy developed in this study will be helpful in advancing functional genomics studies in F. graminearum.

ACKNOWLEDGEMENT AND DISCLAIMER

This material is based upon work partially supported by North Dakota Wheat Commission and the U.S. Department of Agriculture under Agreement No. 59-0206-2-162 (USWBSI). 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.