Functional Analysis Of Siderochrome-iron Transporter Like Protein CmSIT1 In Conithyrium Minitans

Coniothyrium minitans produces antifungal substances and mycoparasitizes the hypha and sclerotia of Sclerotinia sclerotiorum which make it an important fungus for biological control of sclerotinia disease.Using Agrobacterium tumefaciens-mediated transformation technique,a T-DNA insertional library was constructed,from which more than 20 mutants distinct from the wildtype strain ZS-1 were obtained.In this research,a mutant ZS-1TN1812 was studied in detail.ZS-1TN1812 showed abnormal growth phenotype and could inhibit the growth of S.sclerotiorum significantly when dual-cultured on a PDA plate.When spraying the filtrate of ZS-1TN1812 on the leaves of rapeseed,S.sclerotiorum infection was significantly inhibited,suggesting that the antifungal substances produced by this mutant were effective on rapeseed leaves.The thermo-tolerant antifungal substances could specifically suppress the growth of S.sclerotiorum,but could not significantly suppress the growth of another fungus,Colletotrichum higginsianum.However,C.higginsianum was more sensitive to proteinous antibiotics than S.sclerotiorum.The T-DNA insertion in ZS-1TN1812 activated the expression of CmSIT1.Southern blot analysis showed that only one insertion event in mutant ZS-1TN1812.Overexpression of CmSIT1 led to the same phenotypes with ZS-1TN1812.The full length of CmSIT1 is 2510 bp with seven exons,and the full length of c DNA is 2052 bp coding for a putative protein with 683 amino acidic residues(Gen Bank Acc.No.MF447899).Blastp searching the NCBI database showed that CmSIT1 had a conserved domain(aa124-aa490)of the Major Facilitator Superfamily(MFS)transporter(cd06174).This putative protein may have 13 transmembrane domains predicted with TMHMM Serverv.2.0.The three dimensional(3D)model and phylogenetic analysis t proved that CmSIT1 is most likely to be a MFS transporter for iron uptake and CmSIT1 encodes a putative siderophore-mediated iron transporter.In order to verify the functions of CmSIT1,the Quantichrom TM Iron Assay Kit(Bioassay Systems,USA)was used to determine the total iron concentrations in the mycelium of C.minitans.ZS-1TN1812 produced hypha with higher iron levels than stain ZS-1 which indicated that stain ZS-1TN1812 had stronger ability to uptake iron ion.When growing on 2.0 m M Fe Cl3-amended PDA,the iron levels in both ZS-1 and ZS-1TN1812 were significantly increased,and growth inhibition of stain ZS-1 was observed.Colony morphologys of these two strains were similar under 2.0m M Fe Cl3-amended situation.These results suggested the abnormal phenotype of ZS-1TN1812 was relevant to high accumulation of iron in hyphae.In order to study the effect of CmSIT1 on the interactions between C.minitans and other fungi,S.sclerotiorum and C.higginsianum,stain ZS-1 was co-cultured with other fungi,and then the expression of CmSIT1 was determined with q RT-PCR.The results showed that when contacting S.sclerotiorum,the CmSIT1 was significantly expressed at 2 days post-inoculation(dpi),and the expression was declined at 4 dpi to 5 dpi.This result suggested that CmSIT1 may play a role during early stage of parasitizing,such as competing for iron with its host,S.sclerotiorum.Therefore,activated expression of CmSIT1 leads to the enhanced antifungal ability,and CmSIT1 is a potential gene for improving the biological control ability of C.minitans.In order to clarify the molecular mecanisms in ZS-1TN1812,the 48 hpi data of ZS-1TN1812 and ZS-1 was analyzed on the transcription level.Based on the differentially expressed genes(DEGs),the categories of GO and Pathway enrichment were related to very important biological processes,such as carbohydrate metabolism,lipid metabolism,transport,immune system.The differential expressed genes mainly involved in carbohydrates metabolism,lipid metabolism and other basic metabolism,indicating that CmSIT1 is an important gene in C.minitans.The overexpression affects the most basic life activities of C.minitans,such as growing and conidiation.C.minitans is reported to produce macrosphelide A,while there are no differences on macrosphelide A production.Therefore metabolites of ZS-1TN1812 were analyzed.The types of substances identified in ZS-1 samples were ketones,alkanes,olefins,esters and fatty acids,etc.,the material types of ZS-1TN1812 samples were alcohols,terpenoids(sesquiterpene and Single-chain terpenes),alkanes,olefins,esters,fatty acids and alkaloids.Alkanes,olefins and alcohols involved in carbohydrate metabolism;ketones,terpenes,esters and fatty acids involved in lipid metabolism;alkaloids are secondary metabolites.By comparing substances and metabolic pathways,they were corresponding to each other.The major presumptive compounds can be grouped into the corresponding pathway.(1)Carbohydrate metabolism.In the metabolomics corresponding compounds were cetane,n-octadecane,docosane,heptadecane,mannitol,sorbitol,D-mannitol;(2)lipid metabolism.Biosynthesis of fatty acids and unsaturated fatty acids(linoleic acid,oleic acid,ergosterol,amylol),glycerophospholipid metabolism,ether lipid metabolism(butyl-isobutyl-o-phthalate,o-benzene(2-ethylhexyl)phthalate,dibutyl phthalate),the metabolism of polyketone compounds(cyclopentyl ethanone)and terpenoids(antelenol,squalene);(3)Secondary metabolites included 4-methyl-2,6-quinoline diol,2-(2-benzothiazolylthio)ethanol and other alkaloids.And the interaction between the metabolic node and the transcriptome data,such as the analysis of sugars and hydrogen peroxide,suggested that the changes of the relevant genes and enzymes were also partial reasons to ZS-1TN1812 phenotype.In conclusion,overexpression of CmSIT1 could affect the growth,sporulation and mycoparasitization of C.minitans,led to stronger antifungal activity.Siderophore-mediated iron transport(SIT)has substrate specificity and can be used to genetically modify.Antimicrobial agents for biological control of sclerotinia can be made.Transcriptional analysis indicated that CmSIT1 had a significant effect on C.minitans,so CmSIT1 could be an important gene.Metabolomics group could be verified with the results of transcripts,indicating that the results are reliable.The genetic function of C.minitans is quite complex,and the change of one gene may lead to a lot of genetic changes.These genes regulated and maintened life activities together.