The Preliminary Study On The Inhibitory Effect Of Histone H3K27me3 Inhibitor GSK126 Which Targets On The Methyltransferase E(z) In Phytophthora Infestans

Phytophthora infestans is a devastating disease in agricultural production,it can cause potato late blight,leading to necrosis of stems and leaves and decay of tubers.In the mid-19th century,the outbreak of potato late blight in Ireland led to the Great Irish Famine.At present,the occurrence of potato late blight in China is very common,causing huge economic losses to agricultural production every year.Although the application of potatoes that have resistance genes can alleviate the potato late blight,the new physiological races of P.infestans in the field lead to the loss of resistance of plants to pathogens.Therefore,chemical control is still an important measure for the prevention and control of potato late blight in agricultural production.Histone modification is a kind of epigenetic modification and H3K27me3 modification is widespread in many organisms and has important biological functions.H3K27me3 is a modification associated with gene transcriptional repression that alters the structure of chromatin,making it more compact and inhibiting gene expression.H3K27me3 modification is highly conserved in fungi,plants,animals and humans,affecting many biological processes,including gene expression,cell growth and differentiation and tumorigenesis.Studies in Arabidopsis thaliana and Drosophila melanogaster have confirmed that the PRC2 complex is could methylate the histone H3K27 and the methyltransferase E（z）of the complex is a catalytical subunit.GSK126 is a potent,highly selective and S-adenosyl-methionine competitive H3K27me3 inhibitor that inhibits the E（z）methyltransferase activity.The inhibitory effect of GSK126 against Phytophthora has not been studied so far.Therefore,the following studies were conducted to explore the inhibitory effect of GSK126 against Phytophthora and the influence of GSK126 on the pesticide effect of propamocarb.Detection of H3K27me3 modification and sequence analysis of E（z）homologous genes of P.infestans.First,we detected the H3K27me3 modification in P.infestans and P.sojae by Western blot.Then we found five E（z）homologous genes in P.infestans and seven E（z）homologous genes in P.sojae.By constructing the phylogenetic tree,it was found that P.infestans PITG₀2096,P.sojae Ps₁44554 and Drosophila Dm_E（z）were closely related and contained a conserved SET domain.In addition,by comparing the sequences of the E（z）homologous protein of six P.infestans races,we found that the protein sequences of PITG₁0068,PITG₁2169,PITG₀2096,PITG₁3837 and PITG₁3838 is highly conserved within the SET domain.This study demonstrated that E（z）homologous genes can be expressed in P.infestans,suggesting that they may be involved in the regulation of histone H3K27me3 in P.infestans.Sensitivity of P.infestans and P.sojae to GSK126.Because GSK126 is able to target the E（z）homologous protein EZH2 in the human PRC2 complex,it also has a significant inhibitory effect on the growth of some tumor cell lines and H3K27me3.E（z）homologous genes are also present in P.infestans and P.sojae,so we speculated that GSK126 may be able to influence the biological processes of P.infestans and P.sojae by targeting E（z）proteins.So we measured the sensitivity of P.infestans and P.sojae to GSK126 by growth rate method.The EC50 value of the growth inhibition rate of the P.infestans T30-4 strain was 177.9 μM,and the EC50 value of the growth inhibition rate of the P.sojae P6497 strain was 107.6μM.We further demonstrated that 100 μM GSK126 has a strong inhibitory effect on the histone H3K27me3 modification level of P.infestans and P.sojae by Western blot.At the same time,we simulated the structure of the Phytophthora E（z）homologous protein on the SWISS-MODEL website.The E（z）homologous protein Ps₁17600 of P.sojae can find the protein structure template with GSK126 inhibitor and we simulated the structure of Ps₁17600.There are four amino acid residues that may interact with GSK126,these residues are highly conserved in the E（z）homologous protein sequences of the P.infestans T30-4 strain.The inhibitory effect of GSK126 and propamocarb against the P.infestans.Propamocarb is a carbamate fungicide with local systemic action.It can inhibite the biosynthesis of phospholipids and fatty acids of membrane.It has good control effect on oomycete diseases such as potato late blight,pepper blight and tobacco black shank.Currently,it is widely used in the prevention and control of oomycete diseases.Considering that GSK126 can change the epigenetic state of Phytophthora genome by influencing H3K27me3,it is speculated that this compound will affect the sensitivity of Phytophthora to propamocarb.Therefore,this study intends to determine whether there is synergism of GSK126 and propamocarb.We first determined the sensitivity of P.infestans to propamocarb by growth rate method.The EC50 value of the inhibitory rate of P.infestans T30-4 was 10.17μg/mL.Further,we used GSK126 with a final concentration of 93.69μg/mL to mix with propamocarb and determine the inhibition rate on the mycelium growth of P.infestans.According to the Horsfall method,we calculated the toxicity ratio by the inhibition rate.We found that mixture of GSK126 and propamocarb has a synergistic effect against P.infestans.In the future,we will conduct further study on this phenomenon and explore the synergistic effect of epigenetic inhibitors and chemical pesticides,which will provide new ideas for disease prevention and control.