Functional Analysis Of The Effector Proteins Pp18 And PpCys44/45 Secreted By Phytophthora Parasitica

Phytophthora parasitica has a broad host range,being capable of infecting plant species from more than 255 genera in 90 families.P.parasitica threatens natural ecosystem and crop production worldwide.P.parasitica belongs to oomycete,which is related to diatom and represents a distinct group of eukaryotic microorganisms distant from fungi.P.parasitica is unsensitive to most of the commonly used fungicides.The effectors secreted by oomycetes are important virulence factors,being evolved fast in interacting with host plants and overcoming genotype-specific resistance.Similar to other pathogens,P.paraisitica secretes an arsenal of effectors during infecting plant tissues to suppress plant immunity and promote pathogen colonization.Not only does the types and number of the effectors influence the pathogen virulence and occurrence of the disease,but also the pathogen population genetic structure in the field influences the occurrence and epidemic of the disease.Investigation of molecular mechanism of virulent effectors and a better understanding of P.parasitica evolution and population dynamics in the field will be essential for breeding resistant cultivars and developing disease control measures.In this research,in order to understand P.parasitica biology and pathology for developing efficient disease management strategies,we investigated this pathogen in two main aspects,effector function and population genetic variation.The main research includes:1)identification and functional analysis of effectors Pp18 and PpCys44/45,and preliminary functional analysis of elicitin-like gene 6C32;2)development of a new set of SSR markers for P.parasitica,and examined population genetic variation of P.parasitica isolated from tobacco fields in Chongqing.The main results obtained in this thesis are as follows.1.Identification of and functional analysis of virulent RXLR effector Pp18 of P.parasitica.Pp18 is highly conserved among different P.parasitica strains and highly upregulated during early stages of infection.Expression of Pp18 in N.benthamiana and A.thaliana consistently promoted P.parasitica colonization,and Pp18 suppressed significantly the cell death triggered by NIP(Necrosis-Inducing Protein secreted by P.sojae),indicating that Pp18 is a virulence factor and suppresses plant immune system.The P.parasitica transformants overexpressing Pp18 exhibited enhanced virulence during plant infection,suggesting its significant contribution to pathogen virulence.2.The RXLR effector Pp18 rendered plant more susceptible by targeting an ascorbate peroxidase.We identified an ascorbate peroxidase,MST35,as a host target of Pp18in N.benthamiana using Co IP-LC-MS/MS approach.Their interaction was confirmed with both Co IP and luciferase-complementary assays.Transient expression of fusion proteins in N.benthamiana showed that they were co-localized in vesicle-like structures.MST35 is an ascorbate peroxidase,which is involved in the clearance of ROS(H₂O₂).MST35-silenced plants,generated by VIGS in N.benthamiana,showed enhanced susceptibility,suggesting that MST35 is a positive regulator of plant immunity and Pp18 may suppress plant immunity by interfering its biological function.3.Two cysteine protease genes from P.parasitica,PpCys44 and PpCys45,induce cell death in various Nicotiana species and function as virulence factors.We performed preliminarily bioinformatics analysis of the cysteine protease genes in P.parasitica.The results showed that all the predicted secreted cysteine proteases are conserved among different P.parasitica strains and most members were upregulated during plant infection.We identified three members,PpCys44,PpCys45 and PpCys69,that could induce cell death in various Nicotiana species,with PpCys44 and PpCys45 encoding the identical amino acid sequence.By creating enzymatic activity-deficient mutants and performing transient expression and silencing in N.benthamiana,we showed that cell death triggered by PpCys44/45 is dependent on its enzymatic activity and host component NPK1.By using CRISPR/Cas9 system,we successfully generated genome edited knockout mutants simultaneously for both PpCys44 and PpCys45 in P.parasitica.Further infection assays showed their decreased virulence,indicating that PpCys44/45 are pathogen virulence factors.4.The elicitin-like gene 6C32 of P.parasitica induces SGT1-and HSP90-dependent cell death on various Nicotiana species.6C32 is highly upregulated during plant infection and its encoded mature protein sequence is highly conserved among different P.parasitica strains.Functional analysis of truncated 6C32 constructs showed that both signal peptide and elicitin domain are required for its cell death triggering activity.The cell death triggered by6C32 is dependent on the host components SGT1 and HSP90 and could be suppressed by P.infestans RXLR effector Avr3a.5.The P.parasitica populations from Chongqing tobacco fields are from a clonal linage sensitive to metalaxyl.We performed bioinformatics analysis for SSR sequences using publicly available P.parasitica genome sequences,and developed a new set of SSR markers for P.parasitica population genetic analysis.Systematic sampling of P.parasitica from diseased tobacco plants in different regions of Chongqing,China,was performed for two consecutive years.We further analyzed P.parasitica populations isolated from tobacco fields in Chongqing using the developed SSR markers.The results showed very low level of genetic variations among different populations.Mating type analysis showed that A2 is dominant and no A1 or self-fertile isolates were detected.All examined P.parasitica isolates that represent different multilocus genotypes were sensitive to the fungicide metalaxyl.