Study On The Antifungal Molecular Target Of Carabrone

Carabrone is a sesquiterpene lactone compound of carabane type,which is widely distributed in the plants of Carpesium L.and its related genera.Since our research group reported the antifungal activity of the compound,a lot of research work has been done on its antifungal spectrum,pot activity and mechanism of action.In previous research,it was found that carabrone had good protective and therapeutic effects on wheat take-all,wheat powdery mildew and cucumber anthracnose.Subcellular localization experiments confirmed that it acted on the mitochondria of the test stain and significantly affected the mitochondrial respiratory chain,but its target protein was still unclear.Based on this,in this study,Gaeumannomyces tritici was used as the test strain,and the results of time-series transcriptome analysis were used as the starting point.The suspected target proteins of carabrone were screened by ABPP（Activity-based protein profiling）,NAD⁺/NADH ratio detection and RNAi.The physiological and biochemical indicators and overexpression techniques were used to verify the target of carabrone,in order to clarify the antifungal target of carabrone.The main results are as follows:1.The effect of carabrone on the transcriptional level of the test strain was analyzed by time-series transcriptome.The results showed that the gene expression level of the test strain was significantly affected by the treatment of different time.At 1 h,2 h and 4 h,1178,2377and 2823 differentially expressed genes（DEGs）were identified,respectively.All differentially expressed genes were clustered into four gene clusters.Among them,the down-regulated expression trend gene cluster 1 and cluster 2 were mainly involved in translation,TCA cycle,pyruvate metabolism and oxidative phosphorylation processes,while the up-regulated expression gene cluster 3 and cluster 4 were mainly involved in cofactor biosynthesis,redox balance,autophagy and other biological processes.Further analysis confirmed that carabrone promoted the expression of NAD metabolism-related genes,induced the destruction of cell redox homeostasis and the occurrence of mitophagy.Taken together,these results demonstrated that carabrone significantly affected the transcriptional levels of energy metabolism and NAD metabolism-related processes,and induced autophagy in G.tritici.2.ABPP was used to identify the binding proteins of carabrone.The results showed that CAR-Y could stably label the 30 k Da,50 k Da,58 k Da and 75 k Da proteins of the test strain in vitro and in vivo,and was mainly labeled in mitochondria.After magnetic bead enrichment and LC-MS/MS detection,56 suspected target proteins of carabrone were finally determined,and a large number of them were related to NAD⁺and NADH.Therefore,it is speculated that the antifungal mechanism of carabrone is related to NAD.3.The NAD⁺/NADH ratio of the test strain was detected.The results showed that:（1）Carabrone could significantly reduce the NAD⁺/NADH ratio of the test strain in a dose-dependent and time-dependent manner;（2）The NAD⁺orthogonal pathway can significantly reduce the sensitivity of the test strain to carabrone;（3）Pyruvate could significantly reduce the sensitivity of the test strain to carabrone and increase the NAD⁺/NADH ratio after carabrone treatment.The above results indicate that the target of carabrone is related to the conversion of NAD⁺and NADH.4.The silencing mutants of NAD-related suspected target proteins were constructed,and the sensitivity of the silencing mutants to carabrone was determined.The results showed that the complex I subunit mutantsΔnuo49,Δndufv1 and the ATP synthase subunit mutantΔatp3 were significantly increased in the sensitivity to carabrone.The fumarate hydratase silencing mutantΔfh had no significant change in the sensitivity to carabrone,while the D-lactate dehydrogenase silencing mutantΔdld2 was significantly reduced in the sensitivity to carabrone.It is speculated that carabrone has a significant effect on mitochondrial respiratory chain,and Nuo49,NDUFV1 and ATP3 may be the target proteins of carabrone.5.The effects of carabrone on the physiological and biochemical indexes of mitochondrial respiratory chain were determined.The results showed that carabrone could significantly reduce the ATP level of the test strain and induce the production of superoxide anion.At the same time,FCCP treatment significantly increased the antibacterial activity of carabrone and maintained a decrease in the ratio of NAD⁺/NADH,while it reduced the antifungal activity of oligomycin and increased the ratio of NAD⁺/NADH.The above results showed that carabrone had a significant effect on mitochondrial respiratory chain,but the mechanism of action was different from that of oligomycin.6.The overexpression strains of OE-ndi1,OE-nuo49,OE-ndufv1 and OE-nuo78 were constructed,and the sensitivity of overexpression strains to carabrone and the change of complex I enzyme activity were detected.The results showed that:（1）The sensitivity of S.cerevisiae ndi1（rotenone-insensitive NADH oxidase）to rotenone and carabrone was significantly reduced,and the ratio of NAD⁺/NADH was increased.（2）The activity of complex I could be significantly reduced by carabrone.The inhibition rate of 2μM carabrone was 74%,and the IC₅₀ value was 309.03 n M.（3）The sensitivity of OE-nuo49 overexpressing strain to carabrone was significantly reduced.The above results indicate that mitochondrial respiratory chain complex I is one of the targets of carabrone,and its direction action site may be Nuo49 subunit.In summary,carabrone targets the mitochondrial respiratory chain complex I of the test strain and reduces the NAD⁺/NADH ratio.Combined with the previous research results,the mechanism of action of carabrone is as follows:carabrone quickly accumulates in mitochondria after entering G.tritici,and acts on mitochondrial respiratory chain complex I,thereby destroying the cell NAD⁺/NADH ratio,resulting in an imbalance of redox homeostasis,a large amount of ROS is accumulated,and ATP synthesis is blocked;at the same time,under the stimulation of complex I inhibition,mitophagy may be induced by ROS and AMPK pathway,but under the continuous action of carabrone,excessive mitophagy may be caused,leading to apoptosis,and ultimately leading to the death of test strain.