| Coniothyrium minitans is a highly specific parasitic fungus of the phytopathogenic fungi in the genus Sclerotinia.Because C.minitans is safe for animals and plants and is consistent with the growth environment required by Sclerotinia spp.,it has been developed as biological control agents to control sclerotinia disease commercially.In this article,the genome of C.minitans strain ZS-1was de novo sequenced and assembled;combinating with the interactional transcritptome of C.minitans and S.sclerotiorum,analysized the mycoparasitic mechanism.The main results are as follows:1.The C.minitans genome was assembled with size of 39.77 Mb,with 11,437 predicted transcriptional genes and 11,437 encoded proteins.Fungi genome phylogenetic analysis showed,C.minitans was most closely related to the Paraphaeosphaeria sporulosa.The main difference of the ITS sequence among two fungi was the exchange of nucleotide bases of C and T.Genomic component analysis showed that,the number and species distribution of snc RNA and t RNA in the fungus were similar to P.sporulosa.The number of sn RNAs was close to that of Trichoderma spp.,less than that of other fungi.However,the number of t RNA was less than that of Trichoderma spp.and most pathogenic fungi analyzed in the study.2.The carbohydrate activity enzymes(CAZymes),secered proteins,transporters and secondary metabolism biosynthesis gene clusters were analysized and compared between the genome of C.minitans and other 38 fungi.The CAZymes encoded by C.minitans play an important role in the degradation of the cell wall of S.sclerotiorum.The CAZymes encoded by genome of C.minitans and other fungi in the same order showed order specificity;and similar trend were presented of the classification of the plant cell wall degrading enzymes encoded by the fungi genome analysized,while the classification of the fungal cell wall degrading enzymes havn't shown order specificity.The number of 24 copper-dependent lysis polysaccharide monooxidase(AA9)encoded by C.minitans was significant expansion than that of 3 species of Trichoderma spp.(2 AA9 encoded in each of Trivi2,Triha1 and Triat2,respectively),2 species of Sclerotinia spp.(Sclsc2 and Sclbo1),and 3 species of Botrytis cinerea(10 AA9 encoded in each of B05.10,Bc DW1 and Bc T4,respectively).In addition,compared with the 3 species of Trichoderma spp.,the number of the CAZymes such as AA9,AA7,GH5,GH35,GH43,PL1,PL3 increased significantly in2020 C.minitans ZS-1,while the number of Trichoderma spp.in CAZymes such as GH18,GH55,GH64,GH71,GH75,GT69,GT70,PL20 extended significantly.The process of the life of C.minitans involves a large number of substances transport,and the transporters,especially the transporter proteins of MFS and ABC family,play a key role in the substance transport process of organisms.During the process of sporulation and mycoparasitism of C.minitans,showed that the molecular function of transporter activity(GO: 0005215)and the GO process of transporter(GO: 0006810)were significantly enriched(P?0.05).The number of Monocarboxylate transporters(MCT)was significantly expanded in the genome of C.minitans(16),P.sporulosa(18)and Trichoderma spp.(average of 23).The 7 of heavy metal transporters(HMTs)in the ABC transporter family of C.minitans was significantly larger than that of the other fungi(encoding 3-4)analyzed in the study.The secreted protein is a series of important proteins secreted to the extracellular environment and involved in the life cycle.1,171 signal peptide sequence proteins were predicted in the fungus and 948 of them were identified as extracellular secreted proteins,accounting for 8.29% of the total encoded proteins.And further studies showed that both of them had extracellular secretory properties.There were 114 predicted secreted protein genes that were significantly differentially expressed in the mycoparasitism stage of C.minitans.6 effector similar secreted protein-coding genes were cloned from the genome of C.minitans,and were expressed in S.sclerotiorum,respectively,resulted in the difference colony morphology of S.sclerotiorum,and the colony morphology was unstable.However,CDS with the signal peptide sequence removed showed no significant difference in the colony morphology expressed in S.sclerotiorum.The putative effector genes of C.minitans expressed in S.sclerotiorum influence the colony phenotype of the host,suggesting that the secreted protein of C.minitans could play an important role in the process of mycoparasitism.Polyketone compounds synthesized with decarboxylation of a series of polyketone synthases in fungi have significant antibacterial and antifungal activities;are the important components of fungal secondary metabolites.The number of 6 PKS gene clusters encoded in the genome of C.minitans was significantly less than that of the related fungi P.sporulosa(16)and all other phytopathogenic fungi(12-16)and biocontrol fungi(12-20),but more than that of the genome of B.cinerea Bc T4.3.The whole growth stages could be roughly divided into 5 stages of conidium(Cop),conidium germination(Spg),the hypha growth period(Myp),later mycelium extend stage(Lmy)and maturation of conidium(Com).Based on RNA-seq analysis,the dynamic gene expression of the 5 stages were conducted in this article.During conidial germination,the KEGG pathways of protein translation,oxidative phosphorylation and amino acid related enzymes,cytoskeletion proteins,energy metabolism and glutathione metabolism were significantly enriched(P?0.05).Signaling and cellular processes,Nitrogen metabolism,tyrosine metabolism,exosomes,carbohydrate metabolism and transporters in the KEGG pathways were significantly enriched(P?0.05)in the mycelium growth stage.In the sporeproducing stage,carbohydrate metabolism,biosynthesis of other secondary metabolites,starch and sugar metabolism,transporters,and pyruvates,amino sugars and nucleotide sugar metabolism pathways were significantly enriched(P?0.05)to participate in the production of conidia of C.minitans.4.In order to better understand the mycoparasitism of C.minitans,RNA-seq data at three time-points of interaction between C.minitans and S.sclerotiorum were analyzed.At 4 h and 12 h,622 and 875 genes were significantly up-regulated and 443 genes of them were commonly up-regulated.In the process of recognizing the presence of S.sclerotiorum,many related genes were upregulated in C.minitans participate in the mycoparasitism process.Cellular communication(GO: 0007154),biological regulation(GO: 0065007),transporter(GO: 0006180),substrate localization(GO: 0051179),heterocycles and organics biosynthesis were significantly enriched in the early 4 h and 12 h of mycoparasitism(P?0.05).Based on the biological processes and molecular functions can be predicted that: C.minitans could recognise the host S.sclerotiorum through the cellular exchange process(0007154),improve the related genes expression of organic cyclic compound(GO: 0097159),heterocyclic organic compounds binding(GO: 1901363),smaller molecules binding(GO: 0036094),drug binding(GO: 0008144),protein binding(GO: 0005515)and ion binding(GO: 0043167),as well as the related genes of transporter activity(GO: 0005215).And the expression of related genes involved hydrolase activity(GO: 0016787)was significantly enriched at the interaction stage of 12 h(P?0.05).On the other hand,S.sclerotiorum presented a related response during the mycoparasitism,such as oxidation-reduction process(GO: 0055114),aromatic amino acid biosynthesis process(GO: 0009073)and chorismate biosynthetic pathway(GO: 0009423),and oxidoreductase active(GO: 0016491),response to stress and heat(GO: 0006950,GO:2020 0009408),choline dehydrogenase activity(GO: 0008812),and carbon-nitrogen ligase activity with glutamine as amido-N-donor(GO: 0016884)in molecular function related genes expression were significantly inhibited.The expression of shikimate pathway and the biosynthesis of phenylalanine involving genes was significantly suppressed in S.sclerotiorum induced by the mycoparasite C.minitans.5.The expression of gene Ss NEP2,encoding a necrotic protein in S.sclerotiorum,was significantly up-regulated in the interaction of S.sclerotiorum and C.minitans,and the gene Ss NEP2 silencing mutants of S.sclerotiorum were got by ATMT.C.minitans produced less conidia when grow on medium amended with fermatation broth of the Ss NEP2-silencing transformants.Silencing the gene could improve its resistance to mycoparasitism of C.minitans.And the extract of the silenced sclerotia could promote conidia germination of C.minitans.While lower parasitic ability of C.minitans was observed on the hyphae and sclerotia of Ss NEP2-silencing mutants than on those of wild-type S.sclerotiorum,and less condia production of C.minitans growing on the hyphae of Ss NEP2-silencing transformants than on that of the wild-type strain.The gene expression of inhibition in shikimate pathway and induction of Ss NEP2 in S.sclerotiorum induced by C.minitans,indated all of them could be one of reason of the C.minitans mycoparasitism.The shikimate and Ss NEP2 were conjectured has an effect the mycoparasitic process of C.minitans.In a conclusion,the mycoparasitism is a complex and a two-way process between C.minitans and S.sclerotiorum.The whole genome sequencing and analysis of C.minitans in this study provided a platform for the further study of the biological characteristics of the fungus on growth,development and parasitics,and provided a foundation for genetic resources and secondary metabolites resources. |
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