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The effector proteins secreted by a pathogen not only promote the virulence and infection of the pathogen but also trigger plant defense response. <i>Lasiodiplodia theobromae</i> secretes many effectors that modulate and hijack grape processes to colonize host cells, but the underlying mechanisms remain unclear. Herein, we report LtGAPR1, which has been proven to be a secreted protein. In our study, LtGAPR1 played a negative role in virulence. By co-immunoprecipitation, 23 kDa oxygen-evolving enhancer 2 (NbPsbQ2) was identified as a host target of LtGAPR1. The overexpression of NbPsbQ2 in <i>Nicotiana benthamiana</i> reduced susceptibility to <i>L. theobromae</i>, and the silencing of NbPsbQ2 enhanced <i>L. theobromae</i> infection. LtGAPR1 and NbPsbQ2 were confirmed to interact with each other. Transiently, expressed LtGAPR1 activated reactive oxygen species (ROS) production in <i>N. benthamiana</i> leaves. However, in NbPsbQ2-silenced leaves, ROS production was impaired. Overall, our report revealed that LtGAPR1 promotes ROS accumulation by interacting with NbPsbQ2, thereby triggering plant defenses that negatively regulate infection.