Functional Characterization Of The Virulent Protein PcAvh2 Of Phytophthora Capsici And Establishment Of LAMP Detection Method For Phytopythium Helicoides

Most of crop diseases caused by oomycete infection are devastating soil-borne diseases.Due to the lack of effective detection means and control measures,oomycete diseases often cause serious economic losses worldwide.At present,application of chemical oomycides is the major control measure,but this strategy has lots of limitations.Resistance to oomycides are often observed in oomycete populations,which increases the cost of disease management.A thorough understanding of the pathogenic mechanisms of oomycetes is conducive to the development of new disease management technologies,which is of great significance for safeguarding agricultural production.It is widely believed that Phytophthora capsici secretes virulent effector proteins into plant cells to subvert plant immunity.However,the action mechanism underlying this presumption is unclear.To clarify this,the following studies were carried out in this thesis:(1)Effector protein screening.One virulent RxLR effector protein PcAvh2 was identified from P.capsici through bioinformatics,real time quantitative RT-PCR and gene transient expression.(2)Localization analysis of PcAvh2.The functional activity of the signal peptide of PcAvh2 was verified by yeast sucrase secretion assay.It was found to be localized in plant nucleus and cytoplasm using laser confocal microscope.(3)Gene function analysis.Using CRISPR/Cas9 technique to knock out PcAvh2 from P.capsici,it was found that the virulence of P.capsici on host plants was significantly compromised.At the same time,the resistance of PcAvh2 transgenic Nicotiana benthamiana to P.capsici was significantly decreased.(4)Interaction protein analysis.The interaction proteins of PcAvh2 in N.benthamiana were identified by immunoprecipitation mass spectrum.The interaction between each of three candidate targets,i.e.histone H4,multi-protein bridging factor and an unknown protein and PcAvh2 were confirmed by bimolecular fluorescence complementation and GST pull down analyses.These results laid a foundation for further analysis of the function and action mechanism of the virulent protein PcAvh2,and also provided important data for understanding the pathogenesis of P.capsici.(5)A loop-mediated isothermal amplification(LAMP)detection method was established for the newly identified oomycete phytopathogen Phytopythium helicoides,which causes stem rot and leaf blight in citrus.The sensitivity and specificity of LAMP assay,and field simulation detection were carried out.This work provides a rapid detection tool for monitoring and early warning of Ph.helicoides.The thesis determined the subcellular localization,virulence function and plant interaction proteins of P.capsici PcAvh2,and established the LAMP assay for detection of the causal agent of citrus stem root and leaf blight.The results provided not only important data for elucidating the pathogenesis mechanism of P.capsici,but also a tool for monitoring and early warning of citrus stem rot and leaf blight occurrence.