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Effectors are crucial for the interaction between endophytes and their host plants. However, limited attention has been paid to endophyte effectors, with only a few reports published. This work focuses on an effector of Fusarium lateritium, namely FlSp1 (Fusarium-lateritium-Secreted-Protein), a typical unknown secreted protein. The transcription of FlSp1 was up-regulated after 48 h following fungal inoculation in the host plant, i.e., tobacco. The inactivation of FlSp1 with the inhibition rate decreasing by 18% (p < 0.01) resulted in a remarkable increase in the tolerance of F. lateritium to oxidative stress. The transient expression of FlSp1 stimulated the accumulation of reactive oxygen species (ROS) without causing plant necrosis. In comparison with the wild type of F. lateritium (WT), the FlSp1 mutant of the F. lateritium plant (ΔFlSp1) reduced the ROS accumulation and weakened the plant immune response, which resulted in significantly higher colonization in the host plants. Meanwhile, the resistance of the ΔFlSp1 plant to the pathogenic Ralstonia solanacearum, which causes bacterial wilt, was increased. These results suggest that the novel secreted protein FlSp1 might act as an immune-triggering effector to limit fungal proliferation by stimulating the plant immune system through ROS accumulation and thus balance the interaction between the endophytic fungi and their host plants. [ABSTRACT FROM AUTHOR]