Effect Of SlMLO2 And SlMLO4 Gene Over-expression On Tomato Powdery Mildew Resistance

The harm of tomato powdery mildew to its quality and yield is becoming more and more serious.The mutation frequency of powdery mildew is high,which can easily cause the loss of disease resistance gene function.And the loss of the function of the susceptible factor leads to broad-spectrum resistance,As a susceptible factor,MLO has important research significance in disease resistance breeding.In this study,four types of T3 generation transgenic lines SLMLO2 and SLMLO4 were respectively transferred to susceptible variety Moneymaker(MM)and disease resistant material 62579 as materials.4 transgenic positive lines of T3 generation were identified after transgenic positive plant detection.After inoculation with powdery mildew bacteria,the expression profile of target gene,the hypersensitivity necrosis(HR)was observed by trypan-blue staining,the accumulation of reactive oxygen species,the activity of protective enzymes and the content of malondialdehyde were detected by DAB staining,and the expression profile of pathogenesis-related protein genes was analyzed.The influence of SLMLO2 and SLMLO4 gene overexpression on the disease resistance of tomato powdery mildew was studied.The results were as follows:1.Both TPS(T2-MM)and TP18(T2-62579)amplified a band of about 1.5 KB,which was consistent with the full length of SLMLO2 gene.Both TP7(T4-MM)and TP 17(T4-62579)amplified a 1.6 KB band,which was the same as the full length SLMLO4 gene.However,wild-type tomato WT-MM and WT-62579 showed no bands after amplification,indicating that T2-MM and T2-62579 were positive line in the T3 generation of SLMLO2 gene transfer to MM and 62579,respectively,and T4-MM and T4-62579 were positive plants in the T3 generation of SLMLO4 gene transfer to MM and 62579,respectively.2.After After being infected with powdery mildew,The relative expression level of SLMLO2 in WT-MM increased first and then decreased,and reached the peak at 6h after inoculation.At 0h-12h after inoculation,the relative expression level of T2-MM was significantly higher than that of wild-type WT-MM,and the highest at 12h after inoculation,which was 2.92 times of that of wild-type.The relative expression level of SLMLO2 in WT-62579 peaked at 12h after inoculation.At 0h and 12h after inoculation,the relative expression level of T2-62579 was significantly higher than that of wild-type WT-62579,reaching 2.37 and 2.52 times,respectively.Moreover,the relative expression level of SLMLO2 was the highest at 12h after inoculation.At 0h and 12h after inoculation,the relative expression levels in T2-MM and T2-62579 were higher than those in the wild type.These results indicated that T2-MM and T2-62579 were more susceptible,suggesting that SLMLO2 might be the susceptible gene.3.After After being infected with powdery mildew,The relative expression of SLMLO4 in both WT-MM and T4-MM increased first and then decreased,and the relative expression of SLMLO4 was the highest at 12h after inoculation.At 0h-12h after inoculation,the relative expression of T4-MM was significantly higher than that of wild-type WT-MM,reaching 2.74,4.02 and 1.19 times respectively.The relative expression level was significantly lower than that of wild-type wt-MM.The expression peak of WT-62579 was 24h after inoculation.At 0h and 12h after inoculation,the relative expression level of T4-62579 was significantly higher than that of wild-type WT-62579,up to 3.05 and 1.85 times,respectively,and the highest at 12h after inoculation.At 6h and 12h after inoculation,the relative expression levels of WT-MM and T4-MM were significantly higher than those of wild-type.At 0h and 12h after inoculation,the relative expression levels in T4-MM and T4-62579 were higher than those in the wild type respectively.These results indicated that T4-MM and T4-62579 were more susceptible,suggesting that SLMLO4 might be the susceptible gene.4.At 6h after inoculation,T2-MM and T4-MM were the first to appear HR site,and within 6h-72h,HR site showed a gradually increasing trend.The HR sites of T2-62579 and T4-62579 began to appear at 12h,and the HR sites reached the maximum at 72h after inoculation,with little change compared with the wild type.The HR sites of T2-MM and T4-MM were more than those of T2-62579 and T4-62579,respectively,from 12h to 72h after inoculation.The T2-MM and T4-MM of tomato infected with powdery mildew were more affected by powdery mildew,indicating that they were more susceptible to powdery mildew,that is,SLMLO2 and SLMLO4 may be associated with susceptible powdery mildew.5.After being infected by powdery millet,the accumulation of H2O2 increased sharply,and the maximum number of H2O2 producing sites in T2-MM,T2-62579,T4-MM and T4-62579 reached 36.67,18.67,35.33 and 22.33,respectively,36h after inoculation.The accumulation of H2O2 in T2-MM and T4-MM at each time period after inoculation was greater than that in T2-62579 and T4-62579,respectively,indicating that T2-MM and T4-MM had weaker disease resistance to powdery mildew fungus than T2-62579 and T4-62579,that is,SLMLO2 and SLMLO4 may be susceptible related genes.6.The activities of SOD,POD and CAT and the content of MDA in T2-MM were the highest at 12h,36h,0h and 0h after inoculation,which were 1.50,0.92,1.23 and 2.97 times of those in wild type,respectively.The activities of SOD,POD and CAT and the content of MDA in T2-62579 were the highest at 12h,36h,0h and 36h after inoculation,which were 1.67,0.95,0.88 and 2.11 times of those in the wild type,respectively.The activities of SOD,POD and CAT of T4-MM peaked at 12h,which were 1.29,1.85 and 1.43 times of those of wild type,respectively.The highest content of MDA appeared at 24h,which was 2.75 times that of the wild type.The activities of SOD and POD of T4-62579 peaked at 12h,which were 1.81 and 1.08 times of those of wild type,respectively.The activity of CAT and the content of MDA peaked at 24h,which were 1.36 and 1.56 times of that of wild type,respectively.This indicated that the expression of protective enzymes was up-regulated after being infected by powdery mildew,thus protecting the plants,while the increase of MDA content indicated that the plants were damaged by powdery mildew fungus.It is inferred that SLMLO2 and SLMLO4 genes were more susceptible to powdery mildew.7.The relative expression levels of pathogenesis-related protein genes Glu,chi3,PR1B1,NPR1,EDS1 and PAD4 of T2-MM,T2-62579 and T4-62579 showed a peak at 12h after inoculation.The relative expression levels of pathogenesis-related protein genes Glu,PR1B1,NPR1,EDS1 and PAD4 of T4-MM peaked at 12h after inoculation,and the relative expression levels of chi3 peaked at 6h after inoculation.The relative expression levels of T2-MM,T2-62579,T4-MM and T4-62579 were higher than those of the wild type respectively.These results indicated that the expression of these pathogenesis-related protein genes was stimulated after being infected by powdery mildew fungus,and the expression of these pathogenesis-related protein genes all indicated that the plant was invaded by powdery mildew fungus.Therefore,SLMLO2 and SLMLO4 were involved in the response to stress of powdery mildew fungus,and were susceptible genes.In conclusion,SLMLO2 and SLMLO4 are susceptible genes,and the results provide a theoretical basis for broad spectrum resistance breeding of tomato powdery mildew and resistance mechanism.