Development of a CRISPR/Cas9-mediated transformation procedure for the wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most destructive diseases of wheat worldwide. Although the Z. tritici genome encodes hundreds of predicted effector proteins, functional characterization through the use of genome-editing techniques has been limited due to low homologous recombination efficiency and extensive effector redundancy. In this study, we established and evaluated a CRISPR/Cas9-based genome editing procedure for targeted effector gene disruption in Z. tritici using in vitro-assembled Cas9-sgRNA ribonucleoprotein (RNP) complexes combined with short (60 bp) homologous donor DNA flanks. Using this approach, we successfully generated knockout mutants for a selected candidate effector gene, the Hce2 domain-containing effector Mycgr3107904. Virulence assays on the susceptible wheat cultivar Taichung 29 revealed that two independent{Delta} Mycgr3107904 mutants exhibited a pronounced delay in symptom development compared to the wild-type strain IPO323, with disease onset and progression delayed by approximately 4-5 days. While mutant strains ultimately followed a similar disease trajectory, wild-type-infected leaves displayed extensive necrosis and pycnidia formation at earlier time points, indicating a significant reduction in virulence upon loss of Mycgr3107904. Together, our results demonstrate the feasibility of CRISPR/Cas9-mediated effector gene knockout in Z. tritici and provide functional evidence that Mycgr3107904 contributes to timely disease progression. This work advances genome editing tools for Z. tritici and facilitates systematic dissection of effector functions underlying fungal virulence.