Regulatory Mechanism Of Magnaporthe Oryzae Pathogenesis By Tangeretin As A New Ferroptosis Inhibitor

Rice blast,caused by Magnaporthe oryzae,is one of the most serious diseases affecting rice yield.Elucidating the regulatory mechanism underlying this fungal pathogen’s growth,development and pathogenicity will provide the theoretical basis for the research and development of new sustainable prevention and control strageties against this disease.My study reveals that tangeretin could significantly delay M.oyrzae appressorium formation and block disease lesion formation on the infected rice leaves.To investigate the molecular mechanism underlying tangeretin’s effect on the rice blast,we carried out the detailed studies and obtained the results as stated in the follows.1.Tangeretin may act as an iron chelator and/or antioxidant to delay the appressorium formation,as indicated by the partial restore of appressorium formation by exogenous addition of a fungus-sourced siderophore or an oxidant menodione.Tangeretin was shown to suppress conidial death during appressorium formation.Treatment of M.oryzae conidia by an established ferroptosis inhibitor Liproxstatin-1（Lip-1）could similarly inhibit conidial death and leaf lesion formation,demonstrating that conidial ferroptosis is necessary for M.oryzae pathogenicity.By C11-BODIPY staining we visualized lipid peroxidation in conidium and found that it could be significantly reduced by tangeretin treatment.We further found that the strain over-expressing the NADPH oxidase NOX1 or NOX2 displayed reduced sensitivity towards tangeretin,suggesting that tangeretin may regulate conidial ferroptosis and thus reduce M.oryzae pathogenicity,by affecting Nox1 and Nox2.2.By comparative proteomics analysis we identified protein substrates of Gcn5-catalyzed acetylation.We found that Gcn5 may regulate autophagy induction at both epigenetic and post-translation level.Gcn5 also regulated other important aspects of fungal pathogenicity,including energy metabolism,stress response,cell toxicity and cell death.3.Exogenous addition of Fe Cl₃（ferroptosis inducer）could partially restore conidial death in the gcn5（35）,as well as its pathogenicity,indicating that Gcn5regulates conidial ferroptosis and thus positively regulate M.oryzae pathogenicity.It was interested to notice that Gcn5 may acetylate the key metabolic enzyme pyruvate kinase（PK）,and triggered its degradation via an autophagy-dependent manners.This process may contribute to ferroptosis induction.These results may partially reveal the molecular mechanism underlying ferroptosis regulation by Gcn5 and autophagy.The acetylation-mimicking PK mutant showed reduced sensitivity towards tangeretin treatment,suggesting that tangeretin may also target PK to regulate M.oryzae conidial ferroptosis and pathogenicity.Overall,this study showed that M.oryzae conidial ferroptosis may subject to regulation by Nox,Gcn5 and PK;tangeretin regulats M.oryzae pathogenicity likely by inhibiting conidial ferroptosis mediated by these proteins.Tangeretin has the potential to be developed as a new efficient fungicide for prevention and control of the rice blast disease.