Effect Of Carabrone On Respiratory Chain In Gaeumannomyces Tritici

Take-all,caused by Gaeumannomyces tritici,is one of the most important wheat root diseases worldwide,as it results in serious yield losses.Carabrone,a botanical bicyclic sesquiterpenic lactone isolated from Carpesium macrocephalum,which is widely distributed in Asia and Europe,is a promising fungicidal agent that is environmentally friendly and can effectively control G.tritici.However,the mechanism of action of carabrone against G.tritici remains largely unclear.In our previous study,we identified the mitochondria as the subcellular location of carabrone by confocal microscopic detection of a fluorescently-labeled molecule,and carabrone has a significant effect on biological oxidation.With G.tritici as the target organism,this study has confirmed the antifungal activity of carabrone against G.tritici and completed the histopathology observation on the strain after treatment by carabrone.Then with antimycin A as a positive control,we performed the research on effects on mitochondrial respiratory chain by carabrone.Finally,we explored the functional analysis of genes GgCytc₁,GgCytb,and GgIsp of the mitochondrial respiratory chain cytochrome bc₁ complex in G.tritici by RNA silencing and overexpression as a possible target of carabrone.Following results were obtained:1.The activity of carabrone against G.tritici was confirmed.The potentiation of the effect of carabrone in G.tritici by salicylhydroxamic acid?SHAM?was measured as well.Carabrone resulted in significant inhibition of G.tritici with EC₅₀ value of 40.30?g/mL.For mixture of carabrone and SHAM?1:3?,the value of CTC was 146.1,which is significantly greater than 120,indicated that the mixture of carabrone and SHAM against G.tritici showed a synergistic effect,and carabrone has a significant inhibition on mitochondrial respiratory chain in G.tritici.2.The activity of mitochondrial respiratory chain complexes I,II,III,IV,V,I+III,II+III and citrate synthase?CS?was measured,and quantitative real-time polymerase chain reaction?qRT-PCR?analysis was performed when treated with carabrone.The activity of respiratory chain complexes I-V were inhibited,especially complex III,I+III and II+III,and the activity of CS did not change significantly.Compared with the control,the activity of complex III?30,60 and 87%,respectively?,complex I+III?26,45 and 76%,respectively?and complex II+III?50%,60%and 85%?decreased significantly following treatment with carabrone at EC₃₀,EC₅₀ and EC₇₀.The activity of complexes III,I+III and II+III in G.tritici were approximately negatively correlated with the treatment of carabrone in concentration and time.The gene GgCyc of respiratory chain complex III in G.tritici was highly sensitive to carabron by qRT-PCR.These results declared that respiratory chain complex III in G.tritici is highly sensitive to carabrone.3.An optimised polyethylene glycol?PEG?-mediated protoplast transformation system for G.tritici was established,and the genes of hygromycin phosphotransferase were transformed to G.tritici.The final optimized conditions were determined to be:1 mL of 1.4%lysing enzyme reacting with 0.035 g of hyphae at 31^?C for 2.2 h at 90 rpm.The yield of protoplast was 9.83×10⁷ protoplasts/mL,and the protoplast vitality was high,reaching96.27%under the optimized conditions.Under the optimal conditions,the genes of hygromycin phosphotransferase were successfully inserted into the genome of G.tritici,with the highest transformation frequency?46–54 transformants/?g DNA?.The establishment of protoplast transformation laid a foundation for the construction of mutants'bank of G.tritici.4.The genes GgCytc₁,GgCytb,and GgIsp of the mitochondrial respiratory chain complex III in G.tritici were silenced and overexpressed,and mutants were successfully obtained.The growth rate,colony morphology and sensitivity to carabrone and H₂O₂ of the wild type strain and mutants were determined.The results showed that the silencing mutants?GgCytc1??GgCytb and?GgIsp has less sensitive to H₂O₂,and that the hyphae were thin with hyphal branching reducing compared with the wild-type strain and the overexpression mutants.The silencing mutant?GgIsp is less sensitive to carabrone with EC₅₀ value of 156.18?g/mL,and the overexpression mutant OEIsp is significantly sensitive to carabrone with EC₅₀value of 25.88?g/mL.The results declared that GgIsp is sensitive to carabrone and the subunit Rieske is a potential target of carabrone.5.The activity of peroxidase,laccase,and mitochondrial respiratory chain complex III,melanin concentration,pathogenicity,and the rates of mitochondrial respiration in the wild type strain and the mutants were assessed.?1?Compared with the wild-type strain and overexpression mutants,the production of melanin in the strain treated with carabrone?100?g/mL?,silencing mutants?GgCytc₁,?GgCytb,?GgIsp,and carabrone-resistant isolate24-HN-1?Laboratory preservation?were significantly increased?50-100%?.?2?The activities of laccase were significantly inhibited in?GgCytc₁,?GgCytb,?GgIsp and 24-HN-1?47,37,41,and 44%?,which was consistent with pathogenicity.The activity of peroxidase was significantly increased in wild type strain and the mutants,except for?GgIsp,when treated with carabrone(EC₈₀).?3?The activities of respiratory chain complex III of wild type strain and mutants were significantly decreased,except for?GgIsp and 24-HN-1,when treated with carabrone(0,EC₂₀,EC₈₀)in vitro and in vivo.?4?Following treatment with antimycin A?20?g/mL?,the rates of mitochondrial respiration of the?GgCytb had no signicant change,and other strains were significantly inhibited.The rates of mitochondrial respiration of the wild-type strain,?GgCytc₁,?GgCytb,24-HN-1,OECytc₁,OECytb,and OEIsp were significantly inhibited following treatment with carabrone at EC₈₀?approximately 81,77,79,57,72,73,and 72%,respectively?,while?GgIsp had no effect compared with the control.The results declared that?GgIsp is insensitive to carabrone and the subunit Rieske is a potential target of carabrone.6.With 3D structure of Saccharomyces cerevisiae respiratory chain complex III cytochrome c₁?Cytc1?,cytochrome b?Cytb?and Rieske?ISP?subunit as template,homologous modeling of Cytc1 and ISP subunit from G.tritici,and Cytb subunit from Neurospora crassa were conducted through the Swiss Model software.Based on the reliable homologous model,active binding cavities of Cytc1,Cytb and ISP subunit were predicted.Then,the carabrone were docked flexibly with the binding cavities in Cytc1,Cytb and ISP respectively,and eight amino acid residues?Tyr200?Arg194?Gly47?Gln43?Leu225?Arg171?Gln166 and Tyr230?were the important binding sites to combine carabrone.In conclusion,this study proposed the mechanism of carabrone against G.tritici as following:carabrone enriched in the mitochondria,which affected the respiratory chain complex III,leading to electrons were unable to transfer,then lots of ROS were generated by respiratory chain complex III.More ROS would be accumulated in mitochondrial,and a series of oxidative stresses were produced.Finally,apoptosis factor was released in mitochondria,causing cell death of G.tritici.