LAMP Method For Detection Of Plant Pathogenic Pythium Aphanidermatum/Ultimum And Functional Analysis Of Intrinsic Of Disorder Of RxLR Effectors In Phytophthora Sojae

Oomycetes are classified in the Mastigomycotina.Traditionally,the oomycetes were divided into four orders:Saprolegniales,Peronosporales,Lagenidiales and Leptomitales.Among Peronosporales,Pythium,Phytophthora and downy mildews are the main pathogens causing many destructiveplant diseases.The oomycete Pythium is a harmful genera of plant pathogen worldwide.Approximately more than 120 Pythium species have been identified,and most of them are soil-borne and saprophytic plant pathogens with broad host ranges.They usually attack young feeder roots and seedlings,and cause a variety of diseases including pre-and postemergence damping-off,seed rot,root rot and fruit rot.Pythium aphanidermatum is a pathogen with a wide host range.that causes damping-off and fruit or root rot in agronomic crops.It can infects the stem of corn,cucumber and tomato resultig in damping-off and root rot.Plants are most vulnerable to infection by P.aphanidermatum during germination and young stages.Pythium ultimum is one of the most important soilbome pathogen that mainly causes damping-off and root rot in a large number of plants included wheat,corn,soybean,potato,tomato.Conventional PCR and real-time PCR assays are two widely used techniques and have been used for specific diagnosis of more than twenty plant-pathogenic Pythium species.However,the above PCR methods require experienced technician and special equipments which are relatively expensive.Thus these methods may not be suitable in field and resource-limited laboratories.Loop-mediated isothermal amplification(LAMP)is a newly developped technique that amplifies target DNA with high specificity,rapidity and simplicity.To date,this technique has been successfully applied for the detection of fungi,bacteria,oomycetes and viruses.We successfully established a rapid and accurate loop-mediated isothermal amplification(LAMP)method for detecting P.aphanidermatum.The LAMP method was established,and was evaluated for specificity,sensitivity,and detection of inoculated plant tissues.One gene encoding Trypsin protease was specific for P.aphanidermatum,and was used as the target.The specificity test showed that positive results were only obtained from P.aphanidermatum isolates.The detection limit was 100 pg/?L,which was the same with Real-time PCR,and was 100 times higher than that of conventional PCR.In addition.this LAMP method was successfully applied to detect P.aphanidermatum DNA extracted from inoculated plant tissues.We successfully established a simple,rapid and accurate LAMP method for detecting P.aphanidermatum in the field.We developed a loop-mediated isothermal amplification(LAMP)method for rapid and accurate diagnosis of P.ultimum.Candidate targets were identified from P.ultimum genome using a comparative genomics apporach of other Pythium genomes.Among the pathogen isolates tested in this study,positive results were only obtained from P.ultimum isolates.The detection limit of the LAMP method was 1 pg/?L DNA,which was 104 times higher than that of conventional PCR.Moreover,this method was successfully applied to detect P.ultimum DNA extracted from infected plants.These results suggest that this LAMP method would be a useful tool for rapid,specific,and sensitive diagnosis of P.ultimum in the field.In this study,a large-scale prediction of intrinsic disorder and further feature descriptionwere carried out.These results extend our understanding ofRxLR effectors in protein structures,and open up new directions to explore novel mechanisms of oomycete RxLR effectors.Disorder affects RxLR effector activities.Intrinsic disorder is very common in nature and plays improtant biological functions.However,disorder in oomycete RxLR effectors has not been investigated previously and the roles are unknown.Our results of PONDR VL-XTdisorder analysis showedthat oomycete RxL Reffectors were significantly more disordered than other effectors andsecretome.The distribution of disorder content presented personal preference that RxLR-dEER regions were enriched in disordered residues,suggesting potential role of disorder in effector translocation.In contrast,the disorder content was depleted in the C-terminal regions,especially for W/Y/L motifs.We also found that around 42%of RxLR proteins were predicted to contain at least one a-helix-forming molecular recognition feature(a-MoRF),and most a-MoRFs were located in the C-terminal regions.Furthermore,both of the disorder delection mutants of PsAvh241 and PsAvh105 lost the cell death-inducing activity,indicating the potential important role of disorder structure in RxLR effector function.Overall,these results demonstrate that intrinsic disorder is a common characteristic of oomycete RxLR proteins,and we postulate that such structure feature may be important for effector translocation and function,which extends our understanding ofRxLR effectors in protein structures,and opens up new directions to explore novel mechanisms of oomycete RxLR effectors.