Biological Function Of Chitinase PbChia1 In Plasmodiophora Brassicae

Clubroot caused by the soil-living,obligate biotrophic protist Plasmodiophora brassicae Woronin is the most devastating diseases of cruciferous crops worldwide.Clubroot development is characterized by the formation of club-shaped galls on roots of infected plants,interrupted absorption of water and nutrients,caused to show wilting,stunting,yellowing and premature senescence disease symptoms,results in a 10%-15%reduction in yields.In 2015,genome sequence provided an opportunity to further explore the pathogenic mechanism of P.brassicae.The resting spore wall of P.brassicae has chitin,while animals and plants do not.Chitin metabolization-related enzymes can be potential targets for green pesticides.A large number of genes related to chitin metabolism were found in the P.brassicae genome,but the process of chitin synthesis and hydrolysis has not been reported.In this study,the chitinase gene of P.brassicae was explored.The main findings are as follows:P.brassicae has chitin.the results of fluorescence WGA488 in situstaining showed that chitin can be detected in the entire life cycle of P.brassicae,indicating that the metabolisms of chitin played an important role in the development of P.brassicae.The crudely chitin extracted from resting spore was characterized by Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD)and thermogravimetric analysis(TGA).Chitin in resting spore of P.brassicae were identified as α-chitin,the content of chitin is about 2.17%;commercial chitinase can directly degrade resting spore of P.brassicae.These results indicated that P.brassicae contained chitin,suggesting that chitin and chitin metabolism-related enzymes can be used as targets for control clubroot.Pb Chia1 is a functional chitinase in P.brassicae.genome-wide analysis showed 17 chitinase genes in P.brassicae,with 16 chitinases in the GH18 family,and 1 chitinases in the GH19 family,7 chitinases contained signal peptide and were predicted secretory protein;different life stage transcriptome data of P.brassicae showed that these 17 chitinases may functional differentiation;phylogenetic tree analysis showed that 13 chitinases were closely related to plant-derived chitinases and only 2 chitinases were closely related to fungal-derived chitinases.Through LC-MS/MS method,15 chitinases were screened from clubroot galls of rapeseed.14 were derived from the host and 1 was derived from P.brassicae,the gene number was Plas B＿10400,named Pb Chia1;Pb Chia1 gene was 1203 bp,encoded 400 amino acids,and contained a signal peptide and GH18(Glyco＿18 domain)domain.Using yeast system and tobacco transient expression system,confirmed that Pb Chia1 protein had secretory function.Purified Pb Chia1 protein could bind to chitin and showed chitinase activity in vitro,the activity of chitinase was the highest at 37 ℃.Pb Chia1 could degrade resting spore of P.brassicae in vitro and reduce the germination rate of resting spore of P.brassicae.Used Pb Chia1 to treat with rapaseed-infected by P.brassicae,the content of clubroot was significantly reduced,the development of P.brassicae was delayed,the clubroot symptom was relieved,and the biocontrol efficiency was 61.29%.Overexpression of Pb Chia1 in Arabidopsis thaliana exhibits strong resistance to P.brassicae.In order to understand the biological founction of Pb Chia1,we acquired 3overexpression of Pb Chia1 lines in A.thaliana,L2,L3 and L5 transgenic lines and control plant showed no significant difference in the growth phenotypes,such as root hair growth,leaf size and seeds yield;the colonization rate of P.brassicae in root hair cells or root cortex cells could reduce at 60%～75% compared transgenic lines with control plant,transgenic lines could significantly relieve the severity of clubroot symptoms and delay the life cycle of P.brassicae.The biocontrol efficiency of transgenic lines could be reach to 62.3%,the survival rate was increased 2.4 times and the yield was increased 100 percent.Transgenic lines also showed resistance to other pathogens,such as biotrophic bacteria Pst.DC3000,necrotrophic fungi Sclerotinia sclerotiorum 1980 and Rhizoctonia solani.Take together,these results suggested that Pb Chia1 has a broad application prospect.Research on the mechanism of disease resistance in transgenic lines: the chitinase activity of transgenic lines was significantly increased compared with Col-0 plants,and the expression level of Pb Chia1 was positively correlated with the level of disease resistance.Treated plants with PAMPs flg22 and chitin,which could induce innate immune responses in plants,transgenic lines significantly enhanced the ROS burst,MAPKs the phosphorylation activation and immunity-related genes expression.All these results suggested that overexpression of Pb Chia1 in plant would enhance plant immunity,which provided theoretical support for Pb Chia1 application.Two strains of Trichoderma with biocontrol potential for clubroot were screened and identified.We screened and identified two strains(Hz36,Trichoderma guizhouense;Hk37,T.koningiopsis)of Trichoderma from the gall of clubroot in rapeseed fields with biocontrol potential for clubroot.The fermentation broth of Hz36 could significantly inhibit the germination of resting spore of P.brassicae and promote the seed germination and root growth of rapeseed.The biocontrol efficiency of Hz36 strain on clubroot for rapeseed and A.thaliana was 44.29% and 52.18%,respectively.The q PCR results revealed that strain Hz36 treatment could significantly reduce the content of P.brassicae in root cells,and paraffin section analysis revealed that it could delay the development of P.brassicae.Strain Hk37 showed similar effects to strain Hz36,whose biocontrol efficiency of clubroot could reach 57.30% in rapeseed and 68.01% in A.thaliana.These results indicate that strains Hz36 and Hk37 have the potential for the biocontrol of clubroot.In conclusion,this was the first to study the chitinase gene Pb Chia1 of P.brassicae,and analyze its biological function and disease resistance phenotype of transgenic lines.Two biocontrol strains were obtained and their phenotypes against clubroot were determined,which enriched the microbial resource pool for clubroot biocontrol.The research results provided new research ideas for clubroot biocontrol.