Functional Analysis Of Phytophthora Sojae Apoplastic Effector PsAEPl And Structural Analysis Of Phytophthora Parasitica Molecular Pattern CBEL

Phytophthora belongs to the oomyceta of antler flagellate biota.There are more than 180 species of Phytophthora.Phytophthora is similar to fungi in morphological characteristics,but is significantly different from fungi in evolutionary relationship and pathogenic mechanism.Phytophthora infests thousands of plants,including almost all dicotyledons.The devastating diseases caused by Phytophthora causing economic losses to tens of billion dollars in agricultural production every year,such as soybeans,potatoes,vegetables and trees.It is difficult to control Phytophthora because of its fast onset in the field,rapid epidemic,drug resistance and strong adaptability to host.Taking the pathogenic effectors of Phytophthora as a breakthrough point and resolving the interaction between Phytophthora and plants from the molecular level,which can provide theoretical guidance for the improvement of crop disease resistance.Phytophthora genome encodes hundreds of effectors.The effectors of Phytophthora are divided into intracellular effectors and extracellular effectors,according to the subcellular localization.In recent years,the function of a large number of intracellular effects of Phytophthora has been revealed,however relatively little is known about the function of extracellular effectors.Phytophthora sojae apoplastic effector PsAEPl mediates the uptake of extracellular aldose and is be recognized by plants:The apoplastic fluid of soybean leaves which infected by Phytophthora sojae could trigger cell death in Nicotiana benthamiana.Protease K treatment destroy the cell death triggered by apoplastic fluid,which indicated there are proteins elictor exist in the infected apoplastic fluid.32 proteins from P.sojae containing signal peptide were identified by LC-MS.One of the 32 proteins is PsAEP1 which encodes a secreted aldose 1-epimerase.PsAEP1 is recognized solely when secreted/infiltrated into the apoplast and triggers cell death as well as multiple canonical MAMP-related immune responses in N.benthamiana.PsAEP1 is well conserved among Phytophthora species,and the homologues cloned from P.parasitica and P.infestans could also trigger cell death in N.benthamiana.Furthermore,PsAEPl elicits ROS burst in multiple plant species including Arabidopsis,potato,pepper,eggplant and tomato.These data illustrated that the recognition of Phytophthora apoplastic PsAEP1 is a conserved mechanism evolved in different plant species.Meanwhile,PsAEP1 mediates the mutarotation of extracellular aldose from α-anomer to β-anomer,which are essential for the sugar uptake of P.sojae.This study revealed a novel function of microbial effectors,highlighting the significance of extracellular sugar uptake for Phytophthora infection.As a counteract,plants evolved membrane-localized receptor complex to recognize this key effector for sugar conversion to activate plant immunity.PERA-BAK1 receptor complex participates in the recognition of PsAEPl and mediates the basal resistance to Phytophthora in N.benthamiana:The recognition of PsAEP1 depends on the cell membrane receptor BAK1,but not SOBIR1.And the cell death triggered by PsAEP1 could be suppressed by RXLR effectors of Phytophthora.The LRR receptor of N.benthamiana was screened for the recognition of PsAEP1.LRR-RLP PERA was identified and PsAEP1 triggered cell death and immune responses were significantly suppressed in PERA silencing or knockout plants.The immune responses triggered by flg22,INF1 and XEG1 were also impaired in PERA silencing or knockout plants.Further analysis revealed that PERA interacts with BAK1 and the protein accumulation of BAK1 was positively regulated by PERA.Pathogen inoculation experiments of Phytophthora sojae and Phytophthora parasitica showed that PERA-BAK1 receptor complex was essential for the basal resistance of N.benthamiana.The results confirmed the central role of BAK1 mediated immune response in the resistance to Phytophthora.The finding of PERA regulating the protein accumulation level of BAK1,provided theoretical guidance for the exploitation of the resistance resources.Crystal structure and function analysis of the molecular pattern CBEL from Phytophthora parasitica:CBEL reported as a molecular pattern identified from the cell wall of Phytophthora parasitica.The N-terminal encodes a signal peptide,followed with a cellulose binding domain(CBM1)and functional uncleared domain(PAN)in tandem.CBEL is quite conserved in Phytophthora species.And this combination of domains is unique to Phytophthora,which implies that CBEL plays a distinctive role in the growth or infection of Phytophthora.Intended to reveal the biological function of CBEL,we obtained and analyzed the structure firstly.CBEL full length and truncated CBA(CBM1a+PAN1a)protein were expressed by Pichia pastoris.The purified protein was cultured and screened for crystals.Data were collected after X-ray diffraction.The crystal structure with resolution of 1.22 ? was obtained after phase determination by selenium substitution.Sugar staining experiment showed that O-glycosylation occurred in the linker region of CBEL,which enhanced the binding ability of CBEL to cellulose.It was necessary for CBEL to trigger necrosis in N.benthamiana.Therefore,CBAL(CBA+linker)functional complete truncated mutant was constructed.According to the structural information of CBA,the point mutation combined with cellulose binding experiment confirmed the key amino acids of cellulose binding were phenylalanine at the seventh position and tyrosine at the thirty fourth position.The mutant protein still triggered the burst of reactive oxygen species and lost the function of inducing necrosis in N.benthamiana.The structure of PAN was found to be similar to that of SML2,an apoplastic effector of Toxoplasma gondii.SML2 reported that it recognized host cells by binding galactose.Then we mutated the key sites of CBAL corresponding to the sites of SML2 binding galactose,mutation of aspartic acid at position 51,aspartic acid at position 56,tyrosine at position 94 and valine at position 109 could still trigger the burst of reactive oxygen species in N.benthamiana,while could not trigger necrosis any more.Thus,we supposed that the PAN domain of CBEL is responsible for the binding of carbohydrate.In conclusion,crystal structure of CBEL truncated mutant was determined and analyzed.Combining with biological experiments,results showed that the function of CBEL to trigger necrosis depends on the binding of cellulose and carbohydrate.However,the specific function of CBEL on the growth or toxicity of Phytophthora required more biochemical and genetic experiments.In this study,we identified several apoplastic effectors from the apoplastic fluid of soybean leaves after Phytophthora infection.One of them is an aldose 1-epimerase PsAEP1,PsAEP1 mediates Phytophthora sugar uptake by mutarotation extracellular aldose,and can be recognized by many Solanaceae plants or Arabidopsis thaliana to trigger PTI.The cell death triggered by PsAEP1 was used as a probe to screen receptors.LRR-RLP PERA(PAMP Elicitation Reduced A)was identified.PERA interacts with BAK1 without PAMPs treatment and positively regulates BAK1 accumulation in planta.PERA-BAK1 complex contributes to basal resistance of N.benthamiana.In addition,we also analyzed the crystal structure of CBEL truncated mutant,which has been reported as a molecular pattern of Phytophthora.And revealed the functions of different domains of CBEL according to the structure.In conclusion,our study focused on the functions of three apoplastic effectors from Phytophthora,which provides new theoretical basis supports for revealing the interaction between Phytophthora and plant in apoplast and controlling diseases caused by Phytophthora.