Subcellular Location Of Carabrone In Gaeumannomyces Graminis Var. Tritici And Its Role In Induced Cell Death

Carabrone,a sesquiterpene lactone compound,is widely distributed in many plant species of Compositae Carpesium.The results of previous researchs indicated its potential as a new fungicide.However,the antifungal mechanism of carabrone still remains unclear.Subcellular location technology is one of the important methods to reveal the action mechanism of a fungicide.In this study,three techniques of fluorescent tracer,immunofluorescence and immune colloidal gold were used to investigate the target organelle of carabrone using Gaeumannomyces graminis var.tritici（Ggt）as the tested fungus.Simultaneously,carabrone induced oxidative stress and cellular apoptosis of Ggt were also measured.The main results and conclusions of the thesis are as follows:1.A fluorescent conjugate（TTY）analogous of carbrone was designed and synthesized to explore the subcellular location of carabrone in Ggt using MitoTrackerR Green FM（a mitochondrial dye）and RedDot?（a commercial nuclear probe）as the co-locating probes.TTY and MitoTrackerR Green FM overlapped perfectly with a pearson’s colocalization coefficient of 0.83 whereas no overlay was observed between TTY and RedDot?.These results strongly supports the mitochondrial localization of carabrone.2.A polyclonal antibody TPAbs-4 specific to carabrone was prepared to determine the subcellular location of carabrone in Ggt using a mitochondrial dye of MitoTrackerR Red CMXRos as the co-locating probe.Excellent colocalization was observed between the secondary antibody Anti-Mouse IgG（whole molecule）-FITC and the MitoTrackerR Red CMXRos,with the Pearson’s co-localization coefficiency being 0.86.These results declared that carabrone is predominantly present in the mitochondria.3.F2B4,a monoclonal antibody specific to carabrone was also prepared with a good sensitivity,the IC50 value of which was as low as 2.05 ng/mL.It also exhibited high specificities of a cross reaction rate of 1.8%with carabrol,and no obvious cross reaction withγ-phenyl-α-methylene-γ-butyrolactone.The immuno-gold localization study revealed that the gold particles extensively distributed on the cytomembrane after 30-min exposure to the carabrone.Subsequently,they moved to the cytoplasm and entered into mitochondria after 1 h incubation.When the time extended to 2 h or more longer,the gold particles were selectively present in the mitochondria and no gold particles were observed in other subcellular structures,indicating that mitochondria are the action area of carabrone in Ggt.4.The role of carabrone on the induction of Ggt oxidative stress was detected and the results shew that ROS burst emerged in the mycelium after 3-h incubation with carabrone and that the mitochondrial membrane potential was reduced after 6-h incubation.Moreover,carabrone could inhibit the activities of SOD,CAT and GR,but activate the activity of GSH-PX leading to a remarkable decreasion of GSH level in Ggt.That is,carabrone exerts oxidative stress on Ggt through activating ROS production and concurrently inhibiting their elimination.Notably,GR was most sensitive to carabrone with a IC₅₀ value lower than 3.125μg/mL.This inhibition can be explained by the reaction of cysteine residues in the GR active center with carabrone.Taken together,GR may be a potential target of carabrone.5.carabrone induced cell apoptosis of Ggt was also conformed.phosphatidylserine eversion was observed in mycelium treated with carabrone for 2 h.DNA damage was detected by TUNEL analysis after 6 h carabrone treatment,which was exacerbated with extension of the exposure time.Meanwhile,the expression levels of Ggmet1 and Ggmet2were also upregulated obviously.All these results indicate the role of carabrone on induced cell apoptosis of Ggt.In summary,carabrone selectively acted on mitochondria and subsequently induce the oxidative stress and apoptosis of Ggt.Taking previous study results into consideration,we can speculate the mechanism of action of carabrone against Ggt as follows:carabrone will accumulate in the mitochondria specifically after entering into Ggt and then affects the functions of complex III,which can block the electron transport process of the respiratory chain and affect its energy generation;Meanwhile,carabrone can also inhibit the activities of antioxidases including GR,leading to successive linkage effects of ROS accumulation,high mitochondrial membrane permeability and cell apoptosis.