LF-RPA Detection Of Phytophthora And The Virulence Function Study Of Phytophthora Novel Effector

Oomycetes,the eukaryotes in the subdivision of Stramenopiles,are phylogenetically related to diatoms and brown algae but distanced from fungi.The genus Phytophthora belongs to oomycete,which includes many important plant pathogens.These pathogens cause destructive diseases on economic crops and pose a serious risk to global food security.In order to control of Phytophthora diseases,on the one hand,a rapid detection technique for Phytophthora is established to develop the monitoring for early plant disease,on the other hand,the pathogenic mechanism of Phytophthora is analyzed in plant-pathogen interaction to help to make control strategy.The traditional plant pathogen detection method of isolation,culture,and pathogenicity testing is time consuming and requires professional training.Many molecular detection methods based on conventional polymerase chain reaction(PCR)have high efficiency,specificity and sensitivity.However,these techniques require expensive materials and equipment that may not be available in resource-poor regions.Therefore,the development of a convenient pathogen detection method is important for sustainable agricultural production.To successfully colonize plants,Phytophthora secretes effectors to manipulate plant immunity and peoduces external phosphatidylinositol 3-phosphate(PI3P)to mediate effectors translocation into plant cells and/or to increase their stability in planta.In our previous studies,PI3 P produced by Phytophthora sojae binding effectors is important for infection.Knocking out PI3K1/2 which contribute to PI3 P production significantly reduces the pathogenicity on soybean indicating that PI3 P is essential for the full virulence of P.sojae.FYVE is the PI3P-specific binding domain widely existing in eukaryotes.Some novel strategies have been found for disease control by engineering plants to secrete PI3 Pbinding proteins,including either competitive binding PI3 P or secretion of PI3 Pmetabolizing enzyme to block effector entry,targeting of anti-microbial proteins to the hyphal surface enhancing resistance of plants.However,the studies of FYVE domaincontaining proteins of Phytophthora remain largely unknown.In this study,we used the ras-related protein gene Ypt1 to develop the LF-RPA assay for the sensitive visual detection of Phytophthora,which successfully detected field potato late blight leaf samples and strawberry Phytophthora rot disease fruit samples.Bioinformatics analysis indicated that the sequence of PsFYVE1 encoded a FYVE domain-containing protein was available to be used as a target for detecting P.sojae.Functional analysis showed that PsFYVE1 was also a novel effector protein containing a N-terminal signal peptide.PsFYVE1 could target toRZ-1A/1B/1C,which are the splicing factors of plant alternative splicing complex.PsFYVE1 interferes with the alternative splicing and expression of immunityrelated genes to suppress plant immunity by affecting the assembly of spliceosome comple at the level of RNA regulation.The contents are as follows:Establishment of the rapid LF-RPA method for detection Phytophthora: Lateral flow strip-recombinase polymerase amplification(LF-RPA)is a novel isothermal DNA amplification technology.LF-RPA assay has high sensitivity,specificity and operability,and therefore has been widely used for rapid detection pathogens of mammalian and plants.The nucleotide sequence of the Ypt1 gene varies sufficiently among Phytophthora species,and therefore acts as the target gene for LF-RPA detection of P.infestans and P.cactorum.We designed different LF-RPA primers in the specific gene regions of Ypt1 and screened the primers had highest sensitivity and specificity.Then,we determined the optimal conditions for RPA reactions,and the results showed that RPA reaction performed in a wide range of temperatures from 25℃ to 45℃ and needed no less than 10 minutes.Compared with detection technology of conventional polymerase chain reaction,the LF-RPA has higher efficiency and sensitivity,whtich successfully detects sample with fg/μL level within 30 min.The LF-RPA assay combined with the simplified PEG-Na OH method of DNA extraction were used to detect field potato leaf samples and naturally diseased strawberry samples.In conclusion,a novel method enables detection at temperatures from 25 to 45°C within 40 min with no specialized equipment.Moreover,this detection technology can also be applied to develop the monitoring system for early plant disease.Because the Ypt1 targets are unavailable to distinguish closely related species with their relatives,more targets should be found to improve the molecular detection technology for Phytophthora.Bioinformatics analysis indicated that the sequences of PsFYVE1 are conserved among 25 different pathogenic strains of P.sojae from different area,and varies sufficiently among Phytophthora species.PsFYVE1 contains an N-terminal signal peptide and is up-regulated in the infection process suggesting that it is not only available to be used as a target for detecting P.sojae,but also could involves in pathogenicity.PsFYVE1 is a novel virulent effector from Phytophthora sojae: In this study,FYVE domain-containing proteins were systematically identified and analyzed in oomycete and other organisms.We observed that all the oomycete genomes contained much larger numbers of FYVE proteins compared with other eukaryotes.Analysis of gene expression profiling and domain architectures,we identified a potential novel effector,termed PsFYVE1,in 116 FYVE proteins of P.sojae.PsFYVE1 encodes a FYVE protein that contains an N-terminal signal peptide,one classic FYVE domain,and two oomycete-specific FIST domains.The RNA-seq data verified that PsFYVE1 was up-regulated in the infection process,suggesting that PsFYVE1 might associate with pathogenicity of P.sojae.Here,we demonstrated that PsFYVE1 carried a functional secretory signal peptide and bound to PI3 P in a FYVE domaindependent manner.We generated three PsFYVE1-silenced transformants by using a gene silencing technology,and the PsFYVE1-silenced transformants are less virulent on soybean hypocotyl compared to control.Furthermore,transient expression of PsFYVE1 in N.benthamiana can promote Phytophthora infection and suppress both flg22-and chitintriggered ROS burst,which demonstrate that PsFYVE1 plays an important role during infection.PsFYVE1 targetsRZ-1 proteins which are positive regulators of plant immunity:To further elucidate PsFYVE1 virulence function,we performed immunoprecipitation combined with LC-MS/MS assay and identified the RNA-binding proteins NbRZ-1A/1B/1C in N.benthamiana targeted by PsFYVE1.We demonstrated that PsFYVE1 interacted with both NbRZ-1A/1B/1C and GmRZ-1A/1B/1C by co-immunoprecipitation and luciferase complementation assay.To further explore the biological function ofRZ-1A/1B/1C,we focused on the GmRZ-1A and NbRZ-1A which have the highest expression levels at infection stage.A total of four PsFYVE1 mutants are used to pinpoint that the FIST＿N and FIST＿C domains are essential for interaction of PsFYVE1 —RZ-1A and virulence function of PsFYVE1.When transient silencing of NbRZ-1A/1B/1C in N.benthamiana respectively promote Phytophthora infection and suppress ROS accumulation.These results indicate that NbRZ-1A/1B/1C are positive regulators in plant immunity against Phytophthora and are the target proteins related of PsFYVE1 virulence function.Transient expression of GmRZ-1A in the NbRZ-1A-silenced N.benthamiana leaves can recover the immunity phenotype,suggesting that NbRZ-1A and GmRZ-1A act as orthologous proteins and maintain an identical positive regulatory role in plant immunity.Besides,the C-terminus deletion mutants of GmRZ-1A and NbRZ-1A that fail to bind PsFYVE1 or suppress Phytophthora infection,suggesting that the glycine-rich region at the C-terminus of GmRZ-1A/NbRZ-1A are the key regions for interaction and positive immune regulation.By observing the subcellular localization of deletion mutants of GmRZ-1A and NbRZ-1A,it was determined that the nuclear speckles localization of GmRZ-1A and NbRZ-1A were also in a C-terminusdependent manner.PsFYVE1 regulates alternative splicing of the immune-related gene to suppress plant immunity by interfering association of NbRZ-1A and the splicing factor: In order to further study the biochemical function of NbRZ-1A,immunoprecipitation and LC-MS/MS analyses found that NbRZ-1A associated with alternative splicing factor Nb GRP7 in N.benthamiana.Co-expression of PsFYVE1,NbRZ-1A and Nb GRP7 reveal that PsFYVE1 impacts of the association between NbRZ-1A and Nb GRP7,but not protein stability of NbRZ-1A.These results suggest that PsFYVE1 affects either the complex of splicing factors or the assembly of spliceosome comple to regulate plant immunity at the RNA level.To test this hypothesis,RNA-seq and subsequent experimental analysis demonstrated that PsFYVE1 and NbRZ-1A not only modulated pre-m RNA alternative splicing but also co-regulated transcription of immunity-related genes.Collectively,we successfully established rapid LF-RPA assays for the diagnosis of diseases caused by Phytophthora.The PsFYVE1 gene was mined as a target for detecting P.sojae,meanwhile identified as a virulence effector.The sequence of PsFYVE1 contains a functional secretory signal peptide,a conserved FYVE domain binding to PI3 P,and the Cterminal FIST domain interacting with target proteinRZ-1A.These studies indicate that PsFYVE1 interferes with the association ofRZ-1A and splicing factor to mediate pre-m RNA alternative splicing and gene transcription and subvert plant immunity at the level of RNA regulation.