Screening And Identification Of Effectors Of Plamodiophora Brassicae Woron

Plasmodiophora brassicae causes clubroot,a major disease of cruciferous crops worldwide.The pathogen can quickly infect the susceptible varieties of cruciferous plants and cause significant damage to crop production.After the P.brassicae infects the host,the secreted effectors can regulate the plant's immune response and ultimately help the pathogen to infect the host.The screening and identification of P.brassicae effectors can reveal the interaction mechanism between Chinese cabbage and P.brassicae,which is important for explaining the disease resistance mechanism of Chinese cabbage and cultivation of resistant cultivars.Based on previous studies on the P.brassicae genome,In this study,We searched the genome of nine P.brassicae isolates for potential secreted proteins by bioinformatics software.And Ortho MCL was used to analyze the gene family of these secreted proteins.There are totally 30 candidate effectors including candidate effectors selected by Effector P 2.0 and candidate effectors with typical motif.These candidate effectors that could suppress or induce cell death were screened through an Agrobacterium mediated plant virus transient expression system.in the end,the expression levels of 30 effectors were analyzed by q RTPCR.The main results obtained are as follows:1)In previous studies,the P.brassicae isolate e3 genome from genomic DNA were sequenced?Arne Schwelm et al.2015?.However,Our research group sequenced eight P.brassicae isolates genome from genomic DNA of resting spores,Pb1,Pb3,Pb4,Pb9,Pb10,Pb11,Pb14,ems,respectively,Pac Bio data was spliced and assembled using HGAP.We searched the genome of nine P.brassicae isolates for potential secreted proteins which might act as effectors,using Signal P 4.1 server,TMHMM server v.2.0 and Target 1.1 server.The results show that predicted secreted proteins of Pbe3 is 503,and Pb1?757?,Pb3?752?,Pb4?754?,Pb9?665?,Pb10?780?,Pb11?761?,Pb14?650?,ems?755?.2)Furthermore,to gain insight into the protein families diversity of various P.brassicae isolates by Ortho MCL analysis.The results displays among nine P.brassicae isolates,228 protein families were identical,114 protein families only in Pbe3,23 in Pb1,7 in Pb4,51 in Pb9,18 in Pb10,4 in Pb11,17 in Pb14,26 in ems,respectively,but there is no Pb3-specific protein families3)Effector P 2.0 the machine learning classifier for fungal effector prediction.Although P.brassicae is distinct from fungi,prediction of effectors of P.brassicae by Effector P 2.0 can be used as a reference.Twenty-five candidate effectors were detected in 228 protein families by effector P2.0 analysis.In addition,we analysis the domains of Pb4 secretome based on PFAM and MEME,we ultimately screened12 proteins contain domain,such as,RXLR,LXAR,CRN,KAZAL,Chitin,LRRs and Cysteine-rich secretory protein family.It's reported that twenty-six P.brassicae secreted proteins were identified as effectors were expressed in primary infection?Chen et al.,2019?.Four genes of the thirty-seven candidate effectors of were found to be identical by Blast P.and three genes can't cloned,thus,Thirty candidate effector proteins were selected for the following studies.4)30 candidate effectors that could suppress or induce cell death were screened through an Agrobacterium mediated plant virus transient expression system.Only effector Pb4₁08104 was found to be capable of inducing cell death,and the other 29 effectors could suppress mouse Bcl-2-associated X protein?BAX?-induced cell death.Besides,To further detect the accumulation of H2O2 and O2-in the leaves after injection by DAB/NBT staining,Thus,we found that the accumulation of H2O2 and O2-of on the tobacco leaves of Pb4₁08104,and the other 29 effectors inhibit BAX-induced cell death were relatively small compared to negative control.5)To identify the expression period,we analyzed the expression of 30 genes during resting spores,germinating spores,mature spores period by q RT-PCR.It was found that expression of Pb4₁04106 was highest during resting spores,3 genes?Pb4₁05970,Pb4₁06019,Pb4₁02521?was highly expressed during germinating spores period,and remaining genes was expressed to the highest level during mature spores periods.