Studies On Transcriptome Of Infection Process,Long Non-coding RNA Identification,and Species-specific Molecular Detection Of Ustilaginoidea Virens

Rice false smut is one of the most important panicle diseases caused by Ustilaginoidea virens.It can not only cause yield losses,but also pollute grains with toxins produced by U.virens,which seriously threatens food security and food safety.In this study,based on observation of infection process on Wanxian 98 by U.virens,samples of each infection stage were sequenced using RNA-seq.Differently expressed genes(DEGs)and lnc RNAs were identified and analyzed in the infection process.Functional studies were conducted on the gene Uv Ces A that related to development of rice false smut ball and the lnc RNA UvlncNAT-MFS that related to growth of U.virens.In addition,a comparative genome method was developed for the mining of species-specific detection targets of U.virens,and six sets of nested PCR primers were designed for the detection of U.virens.The main results of the study are described as follows:(1)The infection process of U.virens was divided into five stages(Stage 1-5)based on the result of microscopic examination.Samples of Stage 1,Stage 2,Stage 3,and Stage 4 were sequenced using RNA-seq.Compared the RNA-seq data of Stage 2,Stage 3,and Stage 4 with the initial period of infection at Stage 1,1031,1965,and 1972 up-regulated genes and 1216,1666,and1755 down-regulated genes in the each stage were identified,separately.The q RT-PCR results of selected pathogenetic genes of U.virens,including MAPK genes,G-protein subunit genes,pectinase genes,cutinase genes,and cellulase genes,indicated high correlation with RNA-seq data.GO and KEGG enrichment of DEGs in the four stages showed that DEGs were mainly enriched in carbohydrates metabolism and transport activity,which indicated that carbohydrates metabolism was an important factor of the spikelet infection in U.virens.Based on the RNA-seq data of four infection stages,1724 lnc RNAs were identified,including 1084 linc RNs,566 lnc NATs,51 inc RNAs,and 23 sense transcripts.GO enrichment of differentially expressed linc RNAs and lnc NATs of four infection stages indicated that they were mainly enriched in transporter and transport activity.Expression analysis of seven selected transport related lnc NATs and their target genes in the sense strand using q RT-PCR,that demonstrated the positive relationship of expression between each lnc NAT and its target genes.(2)A cellulose synthase gene UvCesA,which belongs to glycosyl transferase 2 family gene,was selected for functional studies.Uv Ces A,containing three exons and two introns with 2567 bp,codes for a protein with 786 aa which contains a conserved CESA?Cel A?like domain.Functional analysis based on knockout and complementation of Uv Ces A in U.virens indicated that it was involved in regulation of vegetative growth,conidiation,response of high osmotic,cell membrane and oxidative stresses,utilization of glucose and lactose,synthesis of cell wall,and pathogenicity of U.virens.Subcellular localization analysis of Uv Ces A showed that it was mainly distributed in cytoplasm and vacuoles.(3)UvlncNAT-MFS and its sense transcript Uv MFS,which was annotated as a putative MFS transporter,have a 452 bp overlapping region in the 3' region.FISH of Uvlnc NATMFS showed it was located in cytoplasm.The silencing and overexpression of Uvlnc NATMFS and Uv MFS showed positive relationship of expression between UvlncNAT-MFS and Uv MFS,and they were involved in the regulation of growth,conidiation,and the response of high osmotic,cell membrane,and oxidative stresses.RNase protection assay indicated that UvlncNAT-MFS could form an RNA duplex with Uv MFS.This study expanded knowledge of the regulatory mechanism of lnc RNA in filamentous fungi.(4)Based on the annotated genome of U.virens,a comparative genome method was carried out for U.virens-specific target mining.Ninety-six candidate protein sequences of 8231 annotated proteins in U.virens were found using BLASTP through first round homology screening against a local database comprised of 46 genomes,with second round comparison to the Gen Bank NR database to further identify sequences unique to U.virens.Among 96 remaining candidate sequences,20 of them were randomly selected for nested PCR primer designing.Genome DNA of 97 U.virens strains collected from eleven provinces of China were used to test the stability of 20 nested PCR primer sets.Nineteen of the twenty primer sets could detect the targets in all genome DNA of 97 strains.The rest 19 primer sets were tested for specificity and sensitivity,using genome DNA of some closely related fungal species of U.virens,different plant pathogens in the fields,different rice species,and serially diluted DNA of U.virens.Eventually six sets of nested PCR primers were developed.PCR assays with these six sets nested PCR primers were also proceeded with field samples and artificial inoculation samples.The results showed that all the six sets of nested PCR primers could be used to detect U.virens in different rice tissues or even in the field,which is an essential tool for prediction of rice false smut.