| Activation of defense responses against Pathogen infeetion is usually regulated through modulation of a large set of defense genes at transcriptional level by specific transcription factors. Elicitors of systemic acquired resistance (SAR) in plants can trigger plant disease resistance in Arabidopsis. But the underlying molecular mechanism, how they switch on signal pathway and regulate plant defense responses to pathogen attack, remains unclear. Studies in this Ph.D project aim at determination of functions of transcription factor AtMYB44 in plants defense responses against pathogenic bacteria and mechanism of quercetin inducing defense responses against pathogen. We investigated the effect of methylation of AtMYB44 promotor on expression of AtMYB44 and responses against pathogen attack. The results here will provide basic clues to explore the mechanism of the transcription factor and flovoniods mediated plant defense and growth pathways and crosstalk with hormonal and non-hormonal signaling pathways.1. The production and identification of the atmyb44 transgenic ArabidopsisTranscription factor MYB plays the important role of regulation in response of plants stress. It can enhance disease resistance of plants. AtMYB44 belongs to the R2R3 MYB subgroup 22 transcription factor family in Arabidopsis (Arabidopsis thaliana). The atmyb44 knockout mutant line exhibited enhanced salt-induced expression of PP2C-encoding genes and reduced drought/salt stress tolerance compared to wild-type plants. Green peach aphid repellency and resistance to Pseudomonas syringae pv. tomato DC3000 (PstDC3000) are decreased in atmyb44, compared to these in wild-type plants. To analyze the role of transcription factor AtMYB44 in plants defense response, we constructed unit binary vector pCAMBI1301::44::6His with AtMYB44 gene and the vector was transformed into Agrobacterium tumefaciens strain EHA105.Then the recombinant unit was introduced into Arabidopsis Col-0. Transgenic homozygous Arabidopsis (35S-M), which can overexpress AtMYB44 under the regulation of 35S promotor, were obtained by screening with antibiotic resistance and identifying with molecular biology. Unit doublet vector pBI121::44P2000::GUS with 44P2000 gene was build.44P2000 gene is upriver 2000bp of AtMYB44 gene ATG. Then the recombinant unit was introduced into Arabidopsis Col-0. Transgenic homozygous Arabidopsis (44PGUS), which can express GUS protein under the regulation of 44P2000, were obtained by screening with antibiotic resistance and identifying with molecular biology. Whereafter, homozygous mutant atmyb44, produced by inseting T-DNA into promotor of AtMYB44, were obtained by screening with antibiotic resistance and identifying with molecular biology. A.tumefaciens EHA105 (pCAMBI1301: 44P2000::44CDS::GFP::His) was also build and the recombinant unit was introduced into Arabidopsis mutant atmyb44. Complementary Arabidopsis for AtMYB44(Catmyb44) gene were constructed. Homozygous Arabidopsis Catmyb44 was obtained by screening with antibiotic resistance and identifying with molecular biology.35S-M and 44GFP,44PGUS, Catmyb44, and atmyb44 will be applied in follow-up experiments as subject.2. Induced expression and subcellular localization of transcription factor AtMYB44Transcription factors are critical regulators of the changes in gene expression that drive developmental processes and environmental stress responses. Nuclear localization signal can enter transcription factor into nucleus and transcription factor can regulate expression of downstream genes which can determine organism phenotype. MYB is a DNA-binding transcription factor family. AtMYB44 belongs to the R2R3 MYB subgroup 22 transcription factor family in Arabidopsis(Arabidopsis thaliana). Expression of AtMYB44 gene can be enhanced by induction of abiotic stress including wound, cold stress, salt stress, and auxin, which can promote resistance response and development of plants. In this study, E.coli BL21 (pET30a::AtMYB44), which can express AtMYB44 gene, was constructed and antibody of AtMYB44 was prepared in rabbit by using strip of AtMYB44 PAGE electrophoresis. Subcellular localization of AtMYB44 was analyzed in onion epidermis by A.tumefaciens EHA105 (pCAMBI1301::44P2000::44CDS::GFP::His) and A.tumefaciens EHA105 (pBI121::44::GFP) and in Catmyb44 and 44GFP. The result shows that AtMYB44 is localized in nucleus and 44P2000 can activate downstream gene. GFP protein could generate more accumulation in Catmyb44 under the condition of induction of HrpNEa protein and ACC compared with EVP. The result of histochemical stain of 44PGUS showed that expression of AtMYB44 existed in each tissue of Arabidopsis, which was identified by RT-PCR of AtMYB44. Moreover, HrpNEa and quercetin could induce expression of Atmyb44 in each tissue of Arabidopsis. Expression of AtMYB44 gene was induced in 10 Catmyb44 lines and 535S-M lines by HrpNEa protein and quercetin, and optimal transgenic plants were screened. The results suggest that Expression of AtMYB44 gene can be induced by some biotic and abiotic stress. And AtMYB44 can function in plants.3. The roles of transcription factor AtMYB44 in defense resistance of Arabidopsis against Pseudomonas syringaeA common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. AtMYB44 belongs to the R2R3-MYB subgroup 22 transcription factor family in Arabidopsis (Arabidopsis thaliana). A large body of evidence suggests that plant R2R3 MYB transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of R2R3 MYB transcription factors in plant defense. We analyzed the role of the AtMYB44 transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae (PstDC3000). T-DNA insertion mutant atmyb44 for AtMYB44 increased growth of PstDC3000 and displayed increased disease symptom severity as compared to wild-type plants. The atmyb44 mutant plants also displayed reduced expression of the SA-regulated PR1 and PR2 genes after the pathogen infection. PstDC3000 could induce expression of AtMYB44 in Arabidopsis. Overexpression Arabidopsis 35S-M-1, and-4 for AtMYB44 reduced growth of PstDC3000 and displayed decreased disease symptom severity as compared to wild-type plants. The 35S-M (1,4) plants also displayed increased expression of the SA-regulated PR1 and PR2 genes after the pathogen infection. Expression of AtMYB44 has obvious up-regulation by induction of PstDC3000. However, after complementary Arabidopsis Catmyb44-2,-7, and-9 for AtMYB44 were inoculated by PstDC3000, Catmyb44-2,-7, and-9 had the same disease symptom severity and expression of PR1 and PR1 as compared to wild-type plants. Analysis of cellular defense response against PstDC3000 in WT,35S-M, and Catmyb44 and expression of AtMYB44 under the condition of environmental stress (SA, MeJA, ET, ABA, HrpNEa, and quercetin) showed that stress-induced AtMYB44 functioned as a positive regulator of defense responses to PstDC3000. Analysis of PstDC3000-induced AtMYB44 in the defense signaling mutants NahG and nprl-1 further indicated that this gene is positively regulated by the salicylic acid (SA) signaling pathway. AtMYB44 was a transcriptional activator and was able to activate the expression of genes involved in plant defense.4. Quercetin-induced H2O2 mediates the pathogen resistance against Psedomonas syringae pv. tomato DC3000 in Arabidopsis thalianaQuercetin has been previously demonstrated to function as important antioxidant in human disease therapy. However, some reports show it is also prooxidant and can provoke hydrogen peroxide (H2O2) under certain conditions. In the current study, we demonstrated that quercetin induced the production of H2O2 and enhanced Arabidopsis thaliana (Arabidopsis) resistance against the virulent strain Pseudomonas syringae pv. tomato DC3000 (PstDC3000). When Arabidopsis was pretreated with quercetin before PstDC3000 inoculation, the leaves exhibited several defense responses, including H2O2 burst, callose deposition, hypersensitive cell death, and the expression of defense response genes PR1 (pathogenesis-related1) and PAL1(Phe ammonia-lyasel) expression. These responses were consistent with the enhancement of plant resistance to PstDC3000, which had significantly decreased population in the quercetin-pretreated plants than control plants. When we applied catalase and PstDC3000 together on the Arabidopsis pretreated with quercetin, the defense responses and other relevant symptoms vanished. Application of quercetin or inoculation of PstDC3000 on leaves of Arabidopsis could only induce limited defense responses in cellular or molecular level. In the further study, we treated the Arabidopsis mutants with quercetin or/and PstDC3000. These similar defense responses also occurred in the Arabidopsis mutant jar1, ein2 and abi1-2, while disappeared in the Arabidopsis mutant NahG and nprl. These studies illustrate that quercetin enhance Arabidopsis resistance against PstDC3000 infection by quercetin-mediated H2O2 elevation and depend SA and NPR1.5. The effect of DNA methylation of atmyb44 promotor on resistance defense response of ArabidopsisDNA cytosine methylation is important for many epigenetic processes including X chromosome inactivation, genomic imprinting, epigenetic changes during carcinogenesis, and silencing of transposons, of specific genes during development and of certain transgenes. To adapt plants to environment stress, gene expression in plants is controlled by epigenetics. Inheritable character of organism is not fully determined by DNA. Under the condition of bioctic and obiotic environment stress, transcription factor plays an important role on Inheritable character of organism, and expression of transcription factor is controlled by DNA genetic modification. In this study, expression of transcription factor AtMYB44 is analyzed in Arabidopsis treated by ET, ABA, MeJA, SA, HrpNEa, quercetin, PstDC3000, and E. carotovora subsp.carotovora (Ecc). And methylation of CG island in 44P2000.The result shows that different treatment leads to the discrimination of level of CG-island methylation.5-aza-2’deoxycytidine (5-aza) can remove methylation in DNA. Wild type Arabidopsis Col-0, mutant atmyb44, and complementary transgenic Arabidopsis Catmyb44 were treated by 5-aza. Then, expression of AtMYB44 was detected by RT-PCR. It was initially determined that 5-aza cold promoter expression of AtMYB44 gene in Arabidopsis. GUS protein from transgenic Arabidopsis P44GUS treated by 5-aza was detected qualitatively or quantitatively. It was found that 5-aza had an effect on the role of AtMYB44 promotor. After plants treated by 5-aza were inoculated by PstDC3000, Disease symptom development and expression of PR1 and PAL suggest that 5-aza has relation with resistance response against disease and plays a role in expression of AtMYB44. These results suggest that outside environmental stress can have an effect on the number of CG-island methylation in the upper stream promoter of AtMYB44 and the expression of AtMYB44 in different level provoked by methylation of CG island promotes expression of resistance and defense genes. In fine, plants possess resistance against pathogen.Conclusive remarksData obtained from studies described above have provided us the further understanding on mechanisms and functions of pathogen defense in plants by transgenic plant and exogenous quercetin. Firstly, transcription factor AtMYB44 can be induced by biotic and abiotic stress in plants defense responses against pathogen attack. It is found that AtMYB44 functions as positive regulation against PstDC3000 by SA mediated signaling pathway. Secondly, Quercetin can induce expression of AtMYB44 in Arabidopsis and enhance resistance against PstDC3000 by the level of H2O2. The mechanism of defense response against pathogen depends on SA and NPR1. Thirdly, expression of AtMYB44 is regulated by up-stream promotor methylation of AtMYB44 gene in biotic and abiotic stress. |
PDF Full Text Request