| Sclerotinia sclerotiorum (Lib.) De Bary, which caused sclerotinia stem rot disease, is a hugely destructive pathogen of many economically important crops, including oil crops and vegetables. Understanding of the S. sclerotiorum-resistant molecular mechanism will be greatly important theoretical significance for obtaining new transgenic disease-resistant crops. In this study, we identified a S. sclerotiorum-resistant MYB73 gene in Arabidopsis thaliana. We studied in detail by functional analysis of the MYB73 gene for its biological roles in plant defense responses against S. sclerotiorum and Pseudomonas syringae pv. tomato (Pst DC3000). Then we isolated the proteins that interact with MYB73 by yeast two-hybid system. These results were meaningful to perfect the plant defense signaling cascades and improve plant disease resistance. Main results in this paper are as follows:1. The myb73 mutant leaves appeared significantly larger disease lesions than wild-type (WT) leaves, and the transcript level of SsTUBULIN gene in the myb73 mutant was higher than that in WT plants, which showed that myb73 mutant was more susceptible to S. sclerotiorum than WT. In order to study the function of the MYB73 gene in disease resistance response, we constructed an overexpression vector 35S︰MYB73 and introduced it into WT plants using floral dip transformation protocol. Five 35S︰MYB73 transgenic lines (OE) were obtained. OE plants increased resistance to S. sclerotiorum compared with WT. The result demonstrates that the MYB73 gene is associated with S. sclerotiorum stress response.2. Disease phenotypes of the WT, myb73 and OE plants were analyzed after Pst DC3000 inoculation. The myb73 mutant enhanced resistance to Pst DC3000. OE plants increased susceptibility to Pst DC3000 compared with WT. After WT with Pst DC3000 inoculation, the expression of MYB73 gene was significantly down-regulated and the expression of the PR1, PR3, PR5 and PDF1.2 were up-regulated in WT plants by real-time PCR. These results demonstrate that the MYB73 is a negative regulator of Arabidopsis against Pst DC3000.3. RT-PCR analysis showed that the expression of PR1 and PDF1.2 gene were not changed in both of WT and myb73 plants, and in myb73 plants, the expression of SOD, POD and CAT were decreased and the expression of PAL and PPO were increased, which showed that the expression of the MYB73 didn't directly modulate that of the PR1 and PDF1.2 gene and was involved in the antioxidant response genes expression. The expression of MYB73 gene was reduced in the npr1,and the expression of NPR1 gene was also reduced in the myb73, which showed that the relationship between MYB73 and NPR1 was synergistic. The expression of the MYB73 gene was down-regulated in eds5,sid2 and jar1 mutant, and was induced in the NahG, sid2 and eds5 mutants by SA treatment and jar1 by JA treatment. These results showed that the expression of MYB73 gene required NahG, SID2, EDS5 and JAR1, and was modulated by SA and JA.4. The expression of the PR1 and PDF1.2 gene in WT and myb73 were similar through SA, JA and ACC treatment and S. sclerotiorum infection. However, the expression of the ICS1 gene was up-regulated 12 h after ACC treated in WT, and that was not increased 12 h after ACC treated in myb73, at the same time, that was not induced in WT after S. sclerotiorum inoculation, and that was reduced 1 h in myb73 after S. sclerotiorum inoculation.5. Inhibition of root elongation of the myb73 mutant was less severe compared with WT after JA treatment, and phenotype of myb73 was similar to WT after SA and ACC treatment. These results showed that the loss-of-function of the MYB73 gene affected to JA response.6. Through HPLC method, we found that the amount of SA was not altered in the myb73 mutant compared with WT plants, however, accumulation of SA in the myb73 mutant after inoculation with S. sclerotiorum was severely reduced relative to WT. Through ELISA method, the result showed the accumulation kinetics of the ABA in the WT and myb73 plants following S. sclerotiorum inoculation was similar to that of the SA. These results showed that the MYB73 gene is required for normal pathogen-induced SA and ABA accumulation.7. The full-length code domain sequence of MYB73 was amplified from Arabidopsis cDNA and was cloned into pAS1 vector. Transactivation analysis demonstrated that MYB73 was a transcriptional activator. Furthermore, deletion analysis of transactivation revealed that transactivation activity of the MYB73 protein was localized in a region at its C-terminal domain. MYB73 was used as bait to screen Arabidopsis cDNA library by yeast two-hybrid system. 15 putative positive colonies had been isolated and sequenced. RT-PCR result showed that F12F1.4 (gene NO. AT1G12080) expression was up-graduated in the myb73 mutant, indicating that the loss-of-function of the MYB73 gene affected the F12F1.4 expression. MYB73 interacted slightly with F12F1.4 by the yeast two-hybrid system.
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