| Tomato gray mold disease caused by Botrytis cinerea is a serious disease of tomato production. Clonostachys rosea is an antagonistic microorganism to Botrytis cinerea. To investigate the mechanism of C. rosea induced tomatoes’ resistance, four treatments, including B. cinerea treatment (treatment1), C. rosea treatment (treatment2), C. rosea and B. cinerea treatment (treatment3), C. rosea+B. cinerea (treatment4) and water (control) on tomato leaves were carried out. In this study, we found that the microorganisms lead to changes in the activities of seven enzymes of defense (PAL, PPO, POD, SOD, POX and LOX) and second messengers (NO and H2O2). Compared to the control, all treatments induced higher levels of all defence enzymes and increased NO and H2O2levels. We detected high level of two proteins in leaves treated with C. rosea and inoculated with B. cinerea (treatment4): LEXYL237(β-xylosidase) abbreviated as "Lexyl2" and ATP synthase CF1alpha subunit (unit α in the ATP synthase CF1) abbreviated as "atpA". The expression levels of atpA gene in treatment4(tomato leaves treated with C. rosea and inoculated with B. cinerea) was higher than that the expression levels of Lexyl2gene. To determine the functions of atpA protein in mediating defenses, it was cloned in tobacco plant by Agrobacterium-mediated leaf disc transformation. The explants were grown on selective media and then transferred to jars and pots respectively. Molecular and genetic analyses such as Q-PCR, PCR, RT-PCR, Southern Blot, Sequence Analysis, Northern Blot and Anatomy were performed with transgenic plants. Southern Blot, Sequence Analysis, Northern Blot and RT-PCR they confirmed the expression of the atpA gene in tobacco transgenic plants. Change on the activity of four defense enzymes (PAL, PPO, POD and SOD) and second messengers (NO, H2O2, O2-) were examined with the tobacco leaves from treatment A (Transgenic plants treated with B. cinerea), treatment B (Transgenic plants treated with distilled water) and treatment C (Non-transgenic plants treated with B. cinerea). Comparing to the treatment B, treatment C, treatment A induced the highest level of PAL, PPO, POD and SOD in tobacco leaves, also the increased levels ofNO,H2O2, and O2-. Especially in treatment A, the physiological index showed significantly high level. The expression of the pathogenesis related proteins PR1, important signals related to plants’ defense response, were profiled in treatment A and treatment C. The results showed that the expressions of this protein up-regulated in treatments A and B, especially treatment A. Subsequently we performed the expression of Ca2+-ATPase andCa2Mg2ATPase. The results showed that the expressions of these two proteins up-regulated in treatment A and B, especially treatment A. Similar results were observed for chlorophyll content.Finally, some analysis of hypersensitivity reactions were performed with putative transgenic plants treated with B. cinerea and non-transgenic plants treated with B. cinerea. We found that the putative transgenic plants treated with B. cinerea developed a late and reduced HR while non-transgenic plants treated with B. cinerea developed an accelerated and high HR. These results indicated that C. rosea could be a potential candidate for plant vaccine preparation to combat gray mold in tomato and plants putative transgenic tobacco plants which carry atpA transgene can be used in the study for the related diseases’ resistant genes in tomato. |
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