Screening And Mechanism Of Novel Inhibitors Against Phytophthora Infestans

Phytophthora infestans（P.infestans）is an oomycete causing potato late blight.The disease is responsible for the devastating Irish Potato Famine occurred during1845-1852.Until now,late blight is still the most severe threat to potato industry,being responsible for significant economic losses worldwide.Chemicals are widely used to control pathogenic oomycetes.However,their applications are challenged by several issues including environment pollutions,food safety,human health,and emergence of chemical resistance in pathogen populations.Thus,the development of effective,safe and eco-friendly alternatives is an urgent task for controlling P.infestans.The TOR（Target of Rapamycin）protein is highly conserved in eukaryotes cell and its mutation can cause cell death.However,it is still unknown whether the TOR protein exists in P.infestans or the TOR protein is a new fungicide target to control this pathogen.Targeting on TOR protein could be a novel strategy to control P.infestans.To verify this hypothesis,the following studies were conducted:1)The bioinformatics analysis of P.infestans genome was performed to determine if the TOR protein existed in pathogen.The analysis showed that the TOR protein might exist in P.infestans and it was highly conversed.2)To confirm the result of bioinformatics,TOR inhibitors were used to treat P.infestans.It was shown that AZD8055(chemical formula:C₂₅H₃₁N₅O₄,CAS:1009298-09-2)significantly inhibited mycelial growth,reduced sporulation quantity,suppressed spore germination and pathogenicity of P.infestans.3)The integrated analyses of the phosphoproteome and transcriptome were used to explore the mechanism of the effect of AZD8055（AZD for short）on P.infestans.The analysis showed that AZD might target on PiTOR protein to suppress the phosphorylation of PiS6K and PiRPS6,leading to the disturbance of the biological function on gene expression and phosphorylation modification.4)RNAi was tested to comfirm the functions of PiTOR.The results showed that the synthetic RNAi was effective on inhibiting the pathogen.It confirmed that PiTOR protein was a vaild target to control this pathogen.5)According to above studies,we hypothesized that there may be some natural products that also target on PiTOR protein to control P.infestans.Here,melatonin（chemical formula:C13N2H16O2,CAS:73-31-4）was chosen as one of the nature products to investigate its effect on P.infestans.The result showed that melatonin significantly attenuated the potato late blight via inhibiting mycelial growth,changing cell ultrastructure and reducing stress tolerance of P.infestans.Notably,synergistic anti-fungal effects of melatonin with fungicides on P.infestans suggest that melatonin could reduce the dose levels and enhance the efficacy of fungicide against potato late blight.6)The integrated analyses of the phosphoproteome and transcriptome suggested that melatonin might target on PiTOR protein to suppress the phosphorylation of PiS6K and PiRPS6,leading to the suppression of the protein metabolic processes.Moreover,the differentially expressed genes/phosphoproteins associated with stress tolerance,fungicide resistance,and virulence were also dysregulated by melatonin.In summary,this study reveals that the PiTOR protein of P.infestans might be a new fungicide target of P.infestans for the first time,and it may be useful to explore the PiTOR-based approaches for the late blight control.It is also the first time to reveal that the inhibitory effects of melatonin on P.infestans and explain the mechanisms of melatonin on plant pathogens.These findings provide a potential new biocontrol approach using a natural product to prevent potato late blight.