A Method To In Vivo Detect Redox State In Plants During Virus Infection

Plant virus, one of the important pathogen groups, can cause many serious plant disease and loss of crops. Cucumber mosaic virus (CMV) is one of the most common, widely distributed and harmful plant viruses, can induce mosaic, necrosis and blight in a variety of vegetables. Understanding pathogenesis of CMV and resistance mechanism of host would be of great significance to prevent the viral infection.After infected, host would induce several defense mechanisms such as hypersensitive response (HR) and system acquired resistance(SAR) to prevent the spread of virus. During the development of these mechanism, oxidative burst may be the first step to prevent viral spread, which would generate lots of Reactive Oxygen Species (ROS) to transmit signals, or to kill the pathogen immediately. How to in vivo measure the variation of ROS during viral infection (i.e. the variation of redox state in cells) is a difficult problem. To overcome the destructive, irreversible and interference disadvantages of conventional methods to detect ROS we adopt a mutant of Green Fluorescent Protein(GFP) to in vivo detect the redox state occurring in hosts during virus infection. By substitution of surface-exposed residues on the GFP with cysteines in S147and Q204to form disulfide bonds, the roGFP show rapid and reversible ratiometric changes in their fluorescence in relation to ambient redox status.Thus we can use the405/488ratio to detect the dynamic range of redox status. The ratio increase in oxidation state and decrease in reduction state.This thesis reviewed the progress of the role of ROS in plant resistance mechanism and the role of GFP and roGFP in biology, constructed roGFP vectors that target chloroplast and mitochondrion. We used PEG-mediated transfection to verify the function of roGFP and Agrobacterium-mediated transformation to in vivo measure the dynamic redox changes during cucumber mosaic virus (CMV) infection.The results showed that vector K13-roGFP could be expressed in nucleus and cytoplasm. The vectors with Fd61could be expressed in chloroplast,vectors with TR-BMY could be expressed both in nucleus and chloroplast, and vectors with P-ATPase could be expressed in nucleus. In the Agrobacterium-mediated transformation, the data showed that26hours post injection,the fluorescence of roGFP could be detected and the fluorescence intensity maximizing after48h post injection. In the control test, the healthy tobacco had a stronger fluorescence intensity than CMV-infected tobacco in 488nm, and had a weaker fluorescence intensity in405nm.That is, contrast to healthy tobacco, the cytoplasm in CMV-infected tobacco tend to be more oxidized. But further research is needed.In summary our word uses a method to detect ROS dynamically in plant in vivo, and preliminary detect ROS in plant during CMV-infection, trying to provide a basis for the further research of pathogenesis.