Genomics Of Monilinia Spp. And The Functions Of MfOfd1 Gene In Monilinia Fructicola

Monilinia species are the important Ascomycota pathogens in peach production,which cause blossom blight,twigs canker and brown rot of fruits on peach plants,with the severe yield losses during both field production and post-harvest processing.It can not only infect stone fruits,such as peach,plum,apricot,but also damage apple,pear and other pome fruits.Therefore,the occurrence of brown rot disease is one of the main factors that restrict the yield and quality of fruit production.In this study,Monilinia spp.the casual agents of brown rot were used to explore the pathogenic mechanism based on the combination of genomics,transcriptomicse,and molecular biological technology.The main results were as follows:The genomes of M.fructicola,M.mumecola and M.yunnanensis,the three main pathogens causing brown rot of peach in China,were sequenced,assembled and functionally annotated.The M.fructicola genome was assembled into 163 scaffolds?>2kb;N50,2.2 Mb?with a total size of 45.40 Mb,in which the longest sequence length was4.19 Mb.The M.mumecola genome was assembled into 144 scaffolds?>2 kb;N50,1.0Mb?with a total size of 40.18Mb,in which the longest sequence length was 2.42 Mb.The M.yunnanensis genome was assembled into 146 scaffolds?>2 kb;N50,1.01 Mb?with a total size of 44.74 Mb,in which the longest sequence length was 3.36 Mb.M.fructicola,M.mumecola and M.yunnanensis were predicted to have 10636?secreted protein,872?,11077?secreted protein,908?and 10948?secreted protein,886?protein coding genes,as well as 336,232 and 229 transfer ribonucleic acids?tRNAs?,respectively.M.fructicola had most tRNAs than other species.The samples of M.fructicola isolate Bmpc7 at 1 h,3 h,6 h,12 h,24 h and spore germination?sg?stage after inoculation were collected to perform RNA-Sequencing.Specific differentially expressed genes were identified for different infection stages.One hour post inoculation?hpi?was an important period of recognition and interaction between pathogen and host,1548 differentially expressed genes were identified,among them 773 genes were up-regulated and 775 genes were down-regulated.Compared with the other early stages of infection?3 hpi,6 hpi and sg?,188 specifically expressed genes were detected at 1 hpi,in which 100 genes were up-regulated and 88 genes were down-regulated.In addition,the Gene Ontology?GO?function enrichment analysis of this stage showed that the down-regulated genes were mainly enriched in the redox-regulated processes,both in biological process and molecular function category.When pathogens infect plants,hosts accumulate large amount of reactive oxygen species?ROS?,the expression of redox-related enzymes encoded genes are inhibited in pathogens,and the detoxification ability of ROS are weakened,resulting in slow development of the diseases at initial stage of infection.The MfOfd1 gene was significantly up-regulated at 1 hpi,thus was selected for further study.It contained an intron of 61 bp,the length of coding sequence?CDS?was2037 bp,encoding 678 amino acids.The gene was knocked out by combining CRISPR/Cas9 with homologous recombination,and the sporulation of knockout transformants were reduced by 53%to 83%.The knockout transformants were more sensitive to H₂O₂ and showed decreased virulence.Besides,the transformants were more sensitive to exogenous osmotic stress,such as glycerol,D-sorbitol and NaCl,and increased sensitivity to dicarboximides fungicides?iprodione and dimethachlon?.In a word,these results indicated that MfOfd1 gene palys an important role in M.fructicola sporulation,oxidative response,osmotic pressure signal transduction pathway and pathogenic process.