Lipopeptide Surfactin Induces Fusarium Graminearum Apoptosis Studies

Fusarium head blight is one of the most common diseases of wheat,which seriously affects wheat yield and even endangers human and animal health.Fusarium graminearum is the main pathogenic species of Fusarium head blight,and effectively inhibiting the growth of Fusarium graminearum is the key to controlling Fusarium head blight.Surfactin is a cyclic lipopeptide synthesized by Bacillus nonribosomals with excellent surface activity and antibacterial activity,but its antifungal activity is less reported.In this study,the induction effect of Surfactin on the early,middle and late apoptosis characteristics of F.graminearum hyphae was specifically studied,and the related mechanisms of ROS-mediated,Metacaspase-dependent and MAPK signaling pathway were further analyzed.The main findings are as follows:1.Surfactin can induce apoptosis in F.graminearum hyphae.DCFH-DA fluorescence staining showed that compared with the blank control group,the number of hyphae with green fluorescence in the Surfactin treatment group was significantly increased,and the green fluorescence intensity was significantly enhanced（p<0.001）,which was 6.96 times higher than that in the blank group,indicating positive ROS accumulation;JC-1 fluorescence staining showed that the number of hyphae with green fluorescence in the Surfactin treatment group was significantly increased,and the green-red fluorescence ratio was significantly increased（p<0.001）,which was 4.89 times higher than that in the blank group,indicating a positive decrease in mitochondrial membrane potential;the expression levels of Metacaspase gene FGSG₀9204 and FGSG₁2913 in the Surfactin treatment group were significantly increased（p<0.01）,which were 1.43 times and2.07 times higher than that of the blank control group,respectively;at the same time,Caspase enzyme biopsy showed that the activities of caspase 9 and caspase 3 in the Surfactin-treated group were significantly increased（p<0.001）,which were 4.15-fold and 1.40-fold higher than those in the blank control group,indicating positive Metacaspase enzyme activation,and Fluo-3 AM fluorescence staining showed that the number of hyphae with green fluorescence in the Surfactin treatment group was significantly increased,and the green fluorescence intensity was significantly enhanced（p<0.001）,2.92-fold higher than that of the blank group,indicating positive intracellular Ca2+ overload;Hoechst 33342/PI double-staining assay showed that there was no hyphae showing red fluorescence in the Surfactin treatment group,the number of hyphae showing blue fluorescence increased significantly,and the blue fluorescence intensity was significantly enhanced（p<0.001）,4.2-fold higher than in the blank group,indicating positive chromatin concentration.In summary,Surfactin can lead to early apoptosis characteristics such as intracellular ROS accumulation and mitochondrial membrane potential decline in F.graminearum hyphae,mid-stage apoptosis characteristics such as intracellular Ca2+ overload,Metacaspase enzyme activation,and late apoptosis characteristics such as chromatin posity,that is,Surfactin can induce apoptosis of F.graminearum hyphae.2.Surfactin’s induction of apoptosis in F.graminearum hyphae is ROS-mediated and Metacaspase-dependent.NAC（ROS scavenger）pretreatment significantly reduced the intracellular ROS accumulation（p<0.001）of F.graminearum hyphae,and the apoptosis features such as decreased mitochondrial membrane potential,elevated Metacaspase enzyme activity and chromatin concentration were significantly weakened or inhibited by Surfactin,suggesting that Surfactin-induced apoptosis of F.graminearum hyphae was mediated by ROS.Pretreatment with Z-VAD-FMK（Caspase Broad-spectrum Inhibitor）significantly reduced the increase in Metacaspase enzyme activity of F.graminearum hyphae（p<0.001）caused by Surfactin,while apoptosis features such as mitochondrial membrane potential decrease,ROS accumulation and chromatin concentration caused by Surfactin were also significantly weakened or inhibited,suggesting that Surfactin-induced apoptosis of F.graminearum hyphae depended on Metacaspase activation.3.Surfactin induces apoptosis in F.graminearum hyphae involved by the Mgv1Gmpk1 MAPK signaling pathwayQuantitative real-time PCR detection showed that the expression levels of Mgv1 and Gmpk1 genes in F.graminearum hyphae in the Surfactin-treated group were significantly increased（p<0.001）,which were 6.84-fold and 1.76-fold higher than those in the blank control group,respectively,while Hog1 was not significantly changed.ERK inhibitors significantly inhibited the upregulation of Mgv1 and Gmpk1 gene expression（p<0.001）in F.graminearum hyphae-induced by Surfactin,reducing them by 55% and 32%,respectively,while there was no significant change in JNK inhibitor and p38 inhibitor pretreatment（p>0.05）.At the same time,ERK inhibitor and JNK inhibitor pretreatment could significantly reduce the apoptotic characteristics of F.graminearum hyphae intracellular ROS accumulation,decreased mitochondrial membrane potential,and elevated Metacaspase enzyme activity caused by Surfactin,while p38 inhibitors had no obvious effect（p>0.05）.It was shown that apoptosis of F.graminearum hyphae caused by Surfactin was conducted by Mgv1 and Gmpk1 MAPK signaling pathways.