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Zn₂Cys₆ transcription factors are unique to fungi and have been reported to be involved in different regulatory functions. Here, we characterized EBR1 (enhanced branching 1), a novel Zn₂Cys₆ transcription factor of Fusarium graminearum. Knocking out EBR1 in F. graminearum PH-1 caused reduction of both radial growth and virulence. The conidia of knock-out strain PH-1Δebr1 germinated faster than those of wild-type PH-1, but the conidiation of the mutant was significantly reduced. Detailed analysis showed that the reduced radial growth might be due to reduced apical dominance of the hyphal tip, leading to increased hyphal branching. Inoculation assays on wheat heads with a green fluorescent protein (GFP)-labeled PH-1Δebr1 mutant showed that it was unable to penetrate the rachis of the spikelets. Protein fusion with GFP showed that EBR1 is localized in the nucleus of both conidia and hyphae. Knocking …