| Summary: | Fusarium head blight (FHB) caused by <i>Fusarium graminearum</i> is a significant disease among cereal crops. In <i>F. graminearum</i>, biosynthesis of leucine, which is a branched chain amino acid, is achieved by converting α-isopropylmalate to β-isopropylmalate catalyzed by isopropylmalate isomerase encoded by <i>LEU1</i>. Considering the potential for targeting this pathway by fungicides, we characterized the gene <i>FgLEU1</i> (FGSG-09589) in the <i>Fusarium graminearum</i> genome using bioinformatics methods. For functional characterization, we constructed a deletion mutant of <i>FgLEU1</i> (Δ<i>LEU1</i>) through homologous recombination. Compared with the wild-type strain PH-1, Δ<i>LEU1</i> showed slower colony growth and fewer aerial mycelia. Leucine addition was needed to ensure proper mutant growth. Further, Δ<i>LEU1</i> showed decreased conidial production and germination rates, and could not produce ascospores. Moreover, Δ<i>LEU1</i> showed complete loss of pathogenicity and reduced ability to produce deoxynivalenol (DON) and aurofusarin. Upstream and downstream genes of <i>FgLEU1</i> were significantly upregulated in Δ<i>LEU1</i>. Contrary to previous reports, the deletion mutant was more resistant to osmotic stress and cell wall-damaging agents than the wild-type. Taken together, FgLEU1 plays a crucial role in leucine synthesis, aerial mycelial growth, sexual and asexual reproduction, pathogenicity, virulence, and pigmentation in <i>Fusarium graminearum</i>, indicating its potential as a target for novel antifungal agents.
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