| The control of viral disease in plants has become an importance-area of research in the current-phytopathology. Biological activity, physiological achieve and inhibitory mechanistic actions of Gu188 [2-cyano-3-(methylthio)-3-(2-nitrophenylamino)acrylate], synthesized by Center for Research and Development of Fine Chemicals of Guizhou University are useful for further development of this kind of antivirus chemicals.1,Biological activities against TMVPrevention effects of GU188 on inhibition of TMV infecting tobacco were either higher than or equal to that of the control chemical, Ningnanmycin. Controlling effects of GU188 on common tobacco had reached 58.33% and higher than that on Nicotiana glutinosa. Compared with the result of the isolated inactivation experiment, blight inhibition rate of tested chemicals in living tobacco leaves was remarkably higher. The blight inhibition rates for preventive are curative effects reached up to 30.04% and 53.31% respectively. The results also showed that 250 mgL-1 of Bingduxing WP30% had preventive and curative effects, similar to those of 250 mgL-1 of GU188 and control (Ningnanmycin).2,Analysis of biochemical activitiesActivities of defense enzymes: Activities of several defense enzymes in the tobacco plant treated with the tested compounds were studied. When inoculated after being induced by the compound, the activity peak value of the POD in tobacco leaf appeared at the same time as the CK after treatment with GU188 activity peak value was found to be the highest. When treated with the compounds after inoculation, the activity of POD in the tobacco leaves treated with D2 and CK1 reached the peak value in the beginning. The PAL activity in the compound-induced tobacco leaf reached the highest level 6d after inoculation similar to the control treated tobacco leaf with the D2 treated are assuring the highest value. The PAL activity in the tobacco leaf treated with the tested compound after inoculation equally reached the highest level 6 days after being treated and was much higher than that of CK, which reached the peak value 2 days after inoculation. When inoculated after being induced by the tested compounds, the appearance of PPO activity peak in the tobacco leaf was delayed. From 4th day onward after being inoculated, the PPO activities of all the tested compounds were found to be remarkably higher than that of CK. When treated with the tested compounds after being inoculated, the PPO activities of all the treatments were significantly higher than that of CK and the peak value appeared earlier than that of CK. When induced by the tested compounds, except for D2's, CAT activity initially reached the highest level 0 day after inoculation, other CAT activities of CK achieved the peak value 8 days after inoculation. At the same time, the D treatment could cause some changes in the protein expression spectrum in the tobacco leaf inoculated with TMV.Stimulatory of resistance of tobacco: A better antivirus compound should not only inactivate action of virus but should also be able to stimulate ability of host's antivirus mechanism to inhibit virus' replication, spread and syndrome. In this study the compound D2 (250 mg·L-1 of GU188), showed better preventive and curative effects, could change the profile of protein expression after TMV infection. Most of the new protein bands the appeared should related to various PR proteins, especially the members of the group of PR-2a, PR-2b, PR-3 and members of the group of PR-1, PR-4, PR-5b. GU188 could also change the profile of protein expression in common tobacco in which there was no background of N gene after TMV infection. A decreasing trend was noticed with 24 h after treatment of GU188 reached the lowest point and 48 h resuming to the level before treatment. 3 bands appeared at common tobacco might be the acidic members of PR-1 and PR-4 group. The phenomenon is of enormous significance.3,Analysis of mechanism action of tested compound at the molecular levelIn order to study the mechanistic aspects pertaining to GU188 in gene expression for host plant leaves were detected by the analysis of microarrays based on Arabidopsis thaliana genome sequence. The results showed that the 260 genes in tobacco leaves inoculated by TMV alone (treatment A) were induced to exhibit significant expression changes, including 180 up-regulated genes and 80 down-regulated genes. There were 175 genes in tobacco leaves treated by inoculation of TMV and spray of GU188 at same time (treatment B) to display effective expression, which consisted of 55 up-regulated genes and 120 down-regulated genes. 83 genes in leaves treated by the compound but without inoculation of TMV (treatment C) were induced to exhibit effective expression, including 10 up-regulated genes and 73 down-regulated genes. Remarkable phenomena of the gene expression differences in 3 treatments were observed: almost entire up-regulated genes by TMV were down-regulated by the usage of the tested chemical, while most all of the down-regulated genes by TMV were found to be up-regulated by GU188. Similar results were obtained in treatment C except for the face that fewer genes were expressed as compared with that in treatment B. Analysis of gene functions showed that TMV-infection up-regulated expression of the genes in relation to the host nucleoproteins, synthesis of RNAs and proteins, and the protein degradation pathways mediated by ubiquitin and SUMO; TMV disturbed the normal gene expression associated with phytohormones metabolism in its host, down-regulated expression of the genes related to photosynthesis, respiration, construction of cell wall and cytoskeleton, normal anti-stress pathways such as heat shock protein and active oxygen scavenger protein; gene expression of main components in calcium signal pathway was altered sharply due to TMV infection; the cDNA microarray also identified the up-regulated genes of DNA demethylation, miRNA, HDACs, which demonstrated that TMV might regulate the host gene expression at various levels.To sum up, this paper explained that role of GU188 in improving tobacco resistance against TMV infection was probably achieved by counteraction or correction of abnormal gene expression in the host leaves induced by TMV. The key mechanism underlined that inhibition of GU188 against TMV reproduction was down-regulated the expression of genes relation to RNA synthesis in leaf cells because the RNA synthesis of TMV completely depends on the host's machine of RNA synthesis. And the judgment of the results also indicated that curing effects of tested chemical was probably better than its' preventing effect by judgment at gene transcription level. In addition, out of the 260 significant expression genes, there were 57 whose function could not be ascertained. We believe that with continuous of these gene functions, a clearer profile of gene expression induced by TMV infection in tobacco leaf would be achieved.
|
PDF Full Text Request