Botrytis cinerea,the causative agent of gray mold rot,is an aggressive fungal pathogen that infects more than 200 plant species,particularly almost all vegetable and fruit crops,and annually causes $10 billion to $100 billion in losses worldwide.Actually,this fungal pathogen is more destructive on flesh fruits,such as grape,straw berry,peach,pear,apple,kiwifruit and so on,resulting in a serious decay during postharvest periods.Reactive oxygen species(ROS)play an important role in all fungus-plant interactions,mostly as signaling components.The specific role of ROS in pathogen defense is still unclear.Small GTPase of the Rho subfamily has been proved to be an important role in the ROS generation by NADPH oxidases in animal,plant,and fungi.Here,we found that deletion of the rho3 from B.cinerea significantly suppressed vegetative growth and conidiation,effectively reduced appressorium formation and decreased virulence.Moreover,we observed that mitochondria were the main source of intracellular ROS in B.cinerea by dual staining with 2,7-dichlorodihydrofluorescein diacetate and MitoTracker orange.The △rho3 mutant showed less accumulation of ROS in the hyphae tips compared to the WT strain of B.cinerea.Based on the yeast two-hybrid assay,we confirmed that Rho3 protein could interact with NoxA,NoxB and NoxR in vitro,but Rho1 and Rho2 did not interact with them,suggesting that Rho3 may be involved in regulation of function of Nox complex.These results reveals the biological function of small GTPase Rho3 contributed to virulence of B.cinerea via mediating growth,conidiation and ROS metabolism.The finding leads to a better understanding of the important role of small GTPase Rho3 in virulence of fungal pathogens.