Doxorubicin Inhibits Psd2 And Confers Broad-Spectrum Antifungal Activity

Fungal diseases occur worldwide and cause severe losses in agriculture.The environmentfriendly control of fungal diseases is critical for agriculture development and global food security.Magnaporthe oryzae is the causative fungus of devastating rice blast.M.oryzae produces abundant conidia,has a very short life-cycle,and rapidly develops fungicide resistance,so it is necessary to develop different strategies for rice blast control.In this study,we investigated the virulence factor in phosphatidylethanolamine(PE)biosynthesis,and analyzed functions of phosphatidylserine(PS)decarboxylase-encoding gene Mo PSD2 in M.oryzae.We found that the secondary metabolite produced by Streptomyces peucetiusdoxorubicin(DXR)inhibited the activity of Psd2 and showed a broad-spectrum antifungal activity to ten plant pathogenic fungi by suppressing growth,development and virulence.The main results are as follows:(1)Mo Psd2 plays important roles in fungal development and pathogenesis.The Mopsd2 mutant showed reduced vegetative growth,decreased conidiation,and abnormal morphology of conidia.In addition,the Mopsd2 mutant was hyper-sensitive to cell wall and reactive oxygen species(ROS)stressors,and delayed in the utilization of glycogen and lipid droplets.During plant infection,the Mopsd2 mutant induced the outbreak of ROS in host cells,which then manifested attenuated virulence.Lipidomics and thin layer chromatography(TLC)analyses showed that the content of PS was increased and the PE decreased in Mopsd2 mutants.The enzyme activity assays suggested that Mo Psd2 was involved in the de novo biosynthesis of PE in M.oryzae.Taken together,these results showed that Mo Psd2 was involved in the regulation of the growth and pathogenic process of M.oryzae,and played a role in the de novo synthesis of PE.(2)DXR,the inhibitor of Mo Psd2 suppresses plant infection of M.oryzae.The predicted protein structures and molecular docking showed that three predicted DXRinteracting residues,in the functionally conserved C2 domain of Mo Psd2,interacted with DXR.The phenotypes of resultant mutant alleles demonstrate that these key residues are important for Mo Psd2 function and the interaction of Mo Psd2 with DXR.RNA-seq and RT-q PCR assays showed that both DXR and Mo Psd2 regulated the signaling pathways such as phospholipid metabolism,signal transduction,protein phosphatases,and glycerolipid metabolism.(3)DXR shows broad-spectrum antifungal activity on plant pathogens.Chemical DXR inhibited vegetative growth,conidial germination,sexual production,and plant infection in ten phytopathogenic fungi including M.oryzae,consistent with the conserved Psd2 homologs in these pathogens.In addition,DXR efficiently reduced disease severity of rice blast and Fusarium head blight in the field.These results show the potential application of DXR as a fungicide for the control of multiple plant fungal diseases.In summary,our study demonstrates that Mo Psd2 is involved in de novo PE biosynthesis and contributes to the development and plant infection of M.oryzae and that DXR shows broad-spectrum antifungal activity as a fungicide candidate.