Analysis Of Disease Resistance Mechanism Of SDR6 Gene In Arabidopsis Thaliana

Botrytis cinerea was an important phytopathogenic fungus caused gray mold in many kinds of plants.It has a wide range of host plants and often caused sever economic losses to agricultural production.Exploring related genes of plants against Botrytis cinerea and making clearly the molecular mechanisms of their resistance to the disease will lay a foundation for elucidating the molecular mechanism of the interaction between plants and Botrytis cinerea and control of the gray mold.In the previous study,the anti-Botrytis cinerea associated gene SDR6 was obtained from Arabidopsis thaliana and it was clarified that the SDR6 gene was involved in the resistance of Arabidopsis thaliana to B.cinerea and Pst DC3000(Pseudomonas syringae pv.tomato DC3000).This study analyzed the relationship between SDR6 gene with SA and JA signaling pathways,tested the mechanism of SDR6 gene regulating Arabidopsis resistance to Botrytis cinerea and Pst DC3000,identified candidates for SDR6 by yeast two-hybrid technology and two-molecule fluorescence complementary technology.The results provide the basis for further elucidating the molecular mechanism of Arabidopsis SDR6 gene regulation in Arabidopsis disease resistance.1.RT-qPCR was used to detect the expression of SDR6 gene in SA,BTH,ACC and MeJA treated Arabidopsis wild-type Col-0 and SA,JA/ET signaling pathway mutants,and to analyze the relationship with SDR6 gene and SA,JA/ET signaling pathway.The results showed that SA,BTH,and MeJA treatment had an effect on SDR6 gene expression in wild-type,and SDR6 gene in SA signal pathway mutants eds5,sid2,and NahG showed significantly higher expression levels than wild-type after SA treatment.After the mutant eds5 was treated with SA and BTH,the expression level of SDR6 gene was significantly higher than in the wild type.After the mutant ein2 was treated with ACC for 3 h or mutant eto1 was treated with ACC for 12 h,the expression level of SDR6 gene was significantly higher than in the wild type.MeJA treated for 24 h,the expression level of SDR6 gene in mutant jar1 was significantly higher than that in wild type.It indicates that the expression level of SDR6 is regulated by SA,JA/ET signaling pathway.2.The research detected Arabidopsis wild type Col-0,mutant sdr6,revertant mutant CE,mutants NahG,eds5,jar1 and double mutants such as sdr6/NahG,sdr6/eds5 and sdr6/jar1 against Botrytis cinerea resistance.The result showed that all the wild-type Col-0,mutant sdr6,and revertant mutant CE of Arabidopsis showed obvious disease-resistance symptoms,while other mutants showed obvious symptoms of susceptibility.The lesion area of double mutants sdr6/NahG,sdr6/eds5 and sdr6/jar1 were significantly larger than their respective single mutants sdr6,NahG,eds5,jar1.Using the trypan blue and DAB staining methods,the mutants were inoculated with leaves for histopathology,and the BcACTIN gene was analyzed at the same time.The results were consistent with the symptoms of the mutants,indicating that the mutant sdr6 was sensitive to Botrytis cinerea.The susceptibility of double mutations sdr6/NahG,sdr6/eds5 and sdr6/jar1 to Botrytis cinerea was higher than that of their respective single mutants,indicating that SDR6 gene is positively regulating the resistance of Arabidopsis thaliana to Botrytis cinerea.RT-qPCR was used to detect the expression of disease resistance-related genes ICS1,JAR1,PDF1.2 and PR1 after inoculation with Botrytis cinerea.The result showed that the expression levels of ICS1,JAR1 and PR1 genes in mutant sdr6 were significantly lower than those in Arabidopsis wild type Col-0 and revertant mutant CE.The expression level of PDF1.2 in mutant sdr6 was significantly higher than that in Arabidopsis wild-type Col-0 and revertant mutant CE.It was shown that SDR6 gene affects the resistance of Arabidopsis thaliana to Botrytis cinerea by affecting the expression of ICS1,JAR1,PR1 and PDF1.2 genes.3.The research detected Arabidopsis wild type Col-0,mutant sdr6,revertant mutant CE,mutants NahG,eds5,jar1 and double mutants sdr6/NahG,sdr6/eds5 and sdr6/jar1 against Pst DC3000 resistance.The results showed that the mutant sdr6 showed resistance to Pst DC3000 compared to Arabidopsis wild-type Col-0 and revertant mutant CE.The mutants NahG,eds5,jar1 were sensitive to Pst DC3000,and the double mutants sdr6/NahG,sdr6/eds5 and sdr6/jar1 were more sensitive to Pst DC3000 than the corresponding single mutants.Histopathological examination and detection of pathogenic bacteria in inoculated leaves were consistent with the symptoms of the mutant inoculation.It was shown that the SDR6 gene negatively regulates Arabidopsis resistance to Pst DC3000.RT-qPCR was used to detect the expression of disease resistance-related genes ICS1,JAR1,PDF1.2 and PR1 after Pst DC3000 inoculation.The results showed that the expression levels of ICS1,JAR1 and PR1 genes in mutant sdr6 were significantly higher than that in Arabidopsis wild type Col-0 and revertant mutant CE.The expression level of PDF1.2 in mutant sdr6 was significantly lower than that in Arabidopsis wild-type Col-0 and revertant mutant CE.It was shown that SDR6 geneaffects Arabidopsis resistance to Pst DC3000 by affecting the expression of ICS1,JAR1,PDF1.2 and PR1 genes.4.Identification of candidate interacting proteins of SDR6 using yeast two-hybrid technology.As a result,it was found that co-transformed AD-SDR6+BD-AT1G06050,BD-SDR6+AD-AT1G06050,AD-SDR6+BD-AT1G21400,BD-SDR6+AD-AT1G21400,AD-SDR6+BD-AT2G19480,BD-SDR6+AD-AT2G19480 the yeast colonies combined in the three-null media(-Leu/-Trp/-His and-Leu/-Trp/-His/3-AT)and the four-null medium(-Leu/-Both Trp/-His/-Ade)were able to grow normally,indicating that SDR6 and AT1G06050,AT1G21400,and AT2G1948 can directly interact in yeast cells.Using the transient expression technology of GFP in tobacco cells,subcellular localization of AT1G06050,AT1G21400 and AT2G19480 genes was performed.The results showed that AT1G06050 was localized in the nucleus,AT1G21400 was localized in the cell membrane,and AT2G19480 was located in the cell membrane.Identification of candidate interacting proteins for SDR6 using dual-molecular fluorescence complementary techniques.As a result,it was found that SDR6-nYFP and AT1G06050-cYFP,SDR6-cYFP and AT1G06050-nYFP,SDR6-nYFP and AT1G21400-cYFP,SDR6-cYFP and AT1G21400-nYFP,SDR6-nYFP and AT2G19480-cYFP,SDR6-cYFP and AT2G19480-nYFP were co-injected.Fluorescence signal appeared in tobacco leaves of combination,but no fluorescent signal was detected in other negative controls.This shows that SDR6 and AT1G06050,AT1G21400 and AT2G19480 can directly interact with each other in tobacco cells.