TUBP1 protein lead to mitochondria-mediated apoptotic cell death in Verticillium dahliae.

Verticillium wilt, caused by Verticillium dahliae, is a cancer of cotton which affects cotton quality and yield in China. In our previous study, a novel anti-Verticillium dahliae protein TUBP1 was obtained from Bacillus axarquiensis. Then, we have systematically studied the anti-V. dahliae activity and the pore formation action of TUBP1 protein on V. dahliae membrane. In present study, we provide detailed whether TUBP1 protein induced mitochondrial damaged and mitochondria-mediated apoptotic cell death in V. dahliae. In V. dahliae cells exposed to the TUBP1 protein, the mitochondrial dehydrogenases, F0F1-ATPase, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) activities were reduced and reactive oxygen species (ROS), which is a major cause of apoptosis, were increased. The results demonstrated that mitochondria dysfunction and ROS-induced oxidative stress caused the release of apoptotic factors. The following cellular changes, which are characteristic of apoptosis, were measured including mitochondrial membrane potential (MMP), Cytochrome c (Cyt C) release, metacaspase activation, phosphatidylserine (PS) exposure, and DNA condensation and fragmentation. The results showed that an important feature of apoptosis, MMP, was caused by ROS. Significantly, cyt c was released, which is a factor in metacaspase activity after treatment with the TUBP1 protein. Number of stained cells with activated intracellular metacaspases exposed to TUBP1 protein was increased in a concentration-dependent manner. We also showed that in the early and late stages of apoptosis, the effects of the TUBP1 protein were mediated by PS and DNA fragmentation and condensation in the plasma membrane, respectively. There turned out that the TUBP1 protein led to mitochondria-mediated apoptotic cell death in V.dahliae. The results of this investigation indicated that TUBP1 stain or protein is a potent candidate against V.dahliae infections in crop species.