Analysis Of Secreted Proteomics Between Isolates Of Ustilago Esculenta From Jiaobai And Huijiao Plants

The interaction between biotrophic Ustilago esculenta and Zizania latifolia causes the swollen structures on upper parts of plants,which call as the Jiaobai.The Jiaobai is an important aquatic vegetable in China.The fungus survives in the form of mycelium within the host plants for long time and transfers to a new plant by plant asexual propagule,establishing endotrophic relationship.Sometimes,the black teliospore sori produce in a swollen structure on a plant,which is called as the Huijiao.Biotrophic and endophytic relationshipes established between Ustilago esculenta and the host are unique in the Ustilaginomycetes.The establishment of biotrophic relationship involves a large number of molecular interaction mechanisms between fungus and host.Although its genome sequencing has finished,the function of a large number of genes still is unclear.Especially,fungal secreted proteins are an important component in study on interaction mechanisms.In this study,proteomic sequencing and bioinformatics analysis were conducted for revealing the differences of isolates of U.esculenta from Jiaobai and Huijiao plants,providing important information for understanding the molecular mechanism of interaction between U.esculenta and its host.1.Secretory protein sequencing.Basidiospore production was induced from the Jiaobai ane Huijiao samples containing teliospore sori,they were cultured in Czapek Dox media and secreted proteins were obtained,respectively.The secreted proteins were separated by 3K protein separation columns(ZipTip C18),less than 3K proteins were used directly as sequemcing and more than 3K proteins were sequenced after digestion with enzymes.Two datasets obtained by protein mass spectrometer from the Jiaobai ane Huijiao samples were mapped with four genomes U.esculenta 4tube from Jiaobai,U.esculenta H3 from Huijiao,U.maydis 521 and Sporisorium reiliana SRS1_H2-8.The results showed that in JB samples,602,600,402 and 407 gene-coding proteins in more than 3K samples were predicted from 4tube、H3、521 and SRS1_H2-8 genomes,respectively,and 345、345、281 and 294 in less than 3K samples were from 4tube、H3、521 and SRS1_H2-8 genomes,respectively.Similarly,in Huijiao samples,183,182,133 and 142 in more than 3K samples and 690,569,505 and 920 in less than 3K samples were predicted from 4tube、H3、521 and SRS1_H2-8 genomes,respectively.After merging the same genes from less than and more than 3K samples in per samples,821、816、637 and 658 gene-coding proteins using dataset from JB samples were obtained from from 4tube、H3、521 and SRS1_H2-8 genomes,respectively,and 691、689、619 and 1018 gene-coding proteins using dataset from HJ samples were obtained.2.Analysis of secretory proteomeAnalysis of homologous proteins showed that 97 gene-coding proteins from dataset of JB samples shared in 4tube、H3、521 and SRS1_H2-8 genomes and 163、118、377、128 proteins were isolates-specific in 4tube、H3、521 and SRS1_H2-8 genomes,respectively.Similarly,64 gene-coding proteins from dataset of HJ samples shared 4tube、H3、521 and SRS1_H2-8 genomes and 173,110,326,501 proteins were isolates-specific in 4tube、H3、521 and SRS1_H2-8 genomes,respectively.3.Prediction of secretory protein functionSignal peptide prediction.The signal peptide prediction of secretory proteins showed that 133 signal peptide-containing proteins were found from 821 secretory proteins from 4tube genome,and 98 signal peptide-containing proteins were found from 689 secretory proteins from H3 genome.Gene function annotation.Functional annotation of secreted proteins showed that secretory proteins from 4tuber genome contained 274 annotated proteins,407 hypothetical proteins and 140 uncharacterized proteins.Similarly,secretory proteins from H3 genome contained 250 annotated proteins,320 hypothetical proteins and 114 uncharacterized proteins.4.Analysis of carbohydrate active enzymesAnalysis of carbohydrate active enzymes showed that 32 CAZyme families were identified in secretory proteome from 4tuber genome.Among them,glycoside hydrolases(GHs)were grouped into 11 families(34.38%)based on amino acid sequence in the CAZy database,glycosyltransferases(GTs)into nine families(28.13%),Carbohydrate esterases(CEs)into seven families(21.88%)and Carbohydrate-binding modules(CBMs)into five families(15.63%).Similarly,The 84 CAZyme families were identified in secretory proteome from H3 genome.Among them,GHs were grouped into 41 families(48.81%),CEs)into 13 families(15.48%)and CBMs into 11 families(13.10%).No PLs were found in the CAZyme families from 4tube and H3 genomes.