The Molecular Mechanism Of Ethylene Suppressed MAPK-WRKY33 Immune Signaling Pathway Induced By The Apple Ring Rot Pathogenenic Fungi Botryosphaeria Dothidea

The apple crop disease caused by the pathogenic fungi Botryosphaeria dothidea seriously affects the yield and quality of apple fruit,causing huge economic losses and restricting the development of apple industry.At present,there is no effective method for the prevention and treatment of the disease.It is of great significance to study the immune signal pathways of apple ring rot disease,to explore the genes that control the resistance of the disease and to cultivate new varieties of apple disease resistance.The immune response pathway(PTI)induced by the pathogen-associated molecular pattern(PAMPs)is activated when the apple is infected with Botryosphaeria dothidea.The interaction between plants and pathogens usually stimulates the changes of hormone levels.Therefore,the mechanism of apple and pathogen Interaction enriches apple's own immune regulatory network.At present,there are few studies on the mechanism of interaction between the apple and the fungi.Apple fruit infected with the fungi are accompanied by the biosynthesis of ethylene.And the role of ethylene in the process of interaction and the specific regulatory mechanism between the two is unsolved.In this study,we used the apple immature fruit and callus infected with Bd and the exogenous ethylene treatment as the test materials to study the function and regulation mechanism of ethylene using genetic and biochemical methods.First,it clarified the effect of ethylene on the interaction process of two:ethylene as a virulence factor specially enhanced the susceptibility of apple ring rot pathogen Bd.Second,we analyzed the molecular mechanism of MAPK-WRKY33 immune signaling pathway through the widespread MAPK cascade in plants.The results are as follows:1.The ethylene negatively regulated the resistance of apple to the fungus,the regulation varies with different pathogens.Ethylene,as an immune factor,weakened spot expansion of the gray mold and apple canker.However,in contrast to the above two,ethylene accelerated the spot expansion of apple ring rot,and the same in apple fruit and callus,indicating that the effect had no organization specificity.Therefore the interaction between apple and the fungus is unique.2.At present,the study of apple ring rot and MAPK cascade has not been reported.In this study,the results showed that ethylene inhibited MAPK activity during the infection of Bd,and Vm was the opposite of it.3.MAPK activity will alter the expression levels of the target genes downstream of MAPK cascades.The results showed that the expression of MdWRKY33-1/2/3,MdPR4/5/8 and MdChiB-1 exhibited a decreasing trend with the increase of ethylene concentration,while for MdWRKY40,MdERF3/6,MdChiB-2 and MdPGl,it is disproportionate to the dose of ethylene,indicated that ethylene specifically reduced the transcriptional level of MdWRKY33-1/2/3,MdPR4/5/8 and MdChiB-1.4.The result indicateed that ethylene mainly affected MPK3/6 activity by western blot analysis.We constructed the over-expression vector of MdMPK3/6,and obtained the transgenic callus of MdMPK3/6-OE.Compared with wild type,MdMPK3/6 over-expression callus infected with Bd significantly increased the expression of MdPR4/5/8 and MdWRKY33-1.The results showed that exogenous ethylene inhibited the activity of MdMPK3 and inhibited the phosphorylation of MdWRKY33-1 by MPK3,for MdMPK6,its background expression level is too high,the role of ethylene is minimal.Although the evidence of ethylene affecting MdMPK6 activity was not found,the results of the pull down assay confirmed that ethylene supressed the interaction of MdMPK6 with MdWRKY33-1 during the infection of Bd.In order to verify the specificity of ethylene to apple ring rot disease,transgenic plants were infected with Vm and treated with exogenous ethylene,the result indicated that ethylene enhanced the activity of MdMPK3 and its phosphorylation of MdWRKY33-1.5.In this study,ethylene specifically inhibited the expression of MdWRKY33 induced by Bd,MdChiB-1 and MdACS5A were up-regulated after overexpression of MdWRKY33-1,while MdPR4/5/8 was not significant Changes,suggesting that MdChiB-1 and MdACS5A may be regulated by MdWRKY33-1.The results of EMSA assay showed that MdWRKY33-1 could specifically bind to W-box in MdChiB-1 and MdACS5A promoters,and the binding domain was located in the first WRKY domain of MdWRKY33-1 protein.There is a sequence near the W-box of the MdChiB-1 promoter:AAACT,another binding site for the MdWRKY33-1 and MdChiB-1 promoters.MdWRKY33-1 differs from the DNA-protein complexes formed by the two sites,suggesting that it may regulate the expression of MdChiB-1 by for wing homologous or heterodimer.The results of ChIP assay showed that ethylene inhibited the binding of MdWRKY33-1 to the MdChiB-1 promoter.The promoter sequence of MdChiB-1 was constructed into the transient expression vector containing the luciferase gene,and was co-transfected into apple callus protoplasts with MdWRKY33-1-HA tag to detect luciferase activity.The results showed that the activity of luciferase was slightly improved after co-expression of MdChiB-1 pro-LUC and MdWRKY33-1-2HA,which was about 2.73 times higher than control(MER),but enhanced after co-expression with AtMKK7-2HA,which was 10.5 times to the control,not AtMKK5.Western blot result showed that AtMKK7 overexpression significantly promoted the activity of MAPK in apple protoplasts,while AtMKK5 did not.It suggested that the transcriptional activity of MdChiB-1 may require MAPK activation.6.Previous studies have shown that the release of ethylene during the pathogen infection is achieved by the induction of MdACS5A and MdACS5B.The apple callus of MdWRKY33-1 over-expressed,inhibited the infection of Vm.However,the inhibition effect on Bd is not as significant as Vm.MdWRKY33-1 can regulate MdChiB-1 and combine with MdACS5A promoter,and ethylene supressed the resistance of apple to Bd,Therefore,it is speculated that the phenotype of Bd may be related to MdACS5A.7.More interestingly,western blot results of MdMPK6 showed that there was always a significant band shift at the target position of MdMPK6 during the infection of Bd and ethylene inhibited the shift.The pull down assay indicated that ethylene inhibited the interaction between MdMPK6 and AtMKK7ac.In order to verify the relationship between the interaction and the band shift,the modification of MdMPK6 protein was identified.The results showed that the presence of ethylene changed the phosphorylation sites of the protein:the infection of Bd caused the phosphorylation of 236 T site and ethylene,which resulted in the change of phosphorylation site from 236 T to 231 T.The specific mechanisms remain to be investigated.