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Functional Analysis Of Autophagy-related Genes In Fusaium Graminearum

Posted on:2020-03-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:W Y LvFull Text:PDF
GTID:1360330575496034Subject:Plant pathology
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Autophagy is a subcellular degradation pathway,highly conserved in eukaryotes,which maintains intracellular homeostasis and helps cells overcome environmental stresses through the formation of autophagosomes to degrade macromolecular proteins and cell organelles.So far,38 ATGs have been identified and elucidated in Saccharomyces cerevisiae.However,in Fusarium graminearum,only FgATG8 and FgATG15 have been reported,while the biological properties and functions of other ATGs have not been studied.In this paper,we identified 28 ATGs by BLASTP analysis,and preliminary illustrated their functions in vegetative growth,asexual and sexual reproduction and pathogenicity in F.graminearum.Besides,we focused on the roles of FgATGl,FgATG5 and FgATG20 in autophagy pathway.The main results as follows:1.28 ATG genes were identified in the genome of.graminearum by BLASTP analysis using S.cerevisiae functional annotations as guides.And we generated targeted gene deletion mutants of 28 ATGs,which were confirmed by PCR and Southern blot.Based on differences in growing patterns,the 28 ATG mutants could be divided into two groups.In Group 1,colonies showed a statistically significant difference from the wild-type strain PH-1 in radial growth under nutrient-rich conditions(PDA plates).In the mutants of Group 2,no significant difference in growth rate was observed compared to the wild-type strain.Colonies of most ATG mutants in the two groups showed significantly decreased development of aerial mycelium compared to PH-1.These results indicate that autophagy is necessary for proper vegetative growth in F.graminearum.Moreover,we found that autophagy is important for asexual/sexual sporulation and is required for full virulence,and is involved in DON biosynthesis in F.graminearum.2.Introduction of FgATG1 and FgATG5 into Mag;aporthe oryzae AMoatgl and AMoatg5 complements their phenotypic defects respectively.AFgatgl and AFgatg5 mutants completely failed to form any perithecia after two-week self-fertilization.Besides,when strains were cultured in MM-N liquid medium in the presence of 2 mM PMSF for 4 h,autophagic bodies were observed clearly in the vacuoles of the wild-type strain PH-1,while no autophagic bodies or a few autophagosome-like structures were seen in the vacuoles of ?Fgatg1 and ?Fgatg5 mutants.Furthermore,GFP-FgAtg8 in hyphal cells of ?Fgatg1 and AFgatg5 mutants was defective in fusion with vacuoles when autophagy was induced.And GFP-FgAtg8 proteolysis was impaired in AFgatg1 and AFgatg5 mutants.Taken together,we conclude that the autophagy pathway is impaired in the ?Fgatg1 and ?Fgatg5 mutants and that this process is critical for fungal pathogenesis.By analysis of point mutations,we found that the morphology of colonies of FgATG1K53A,FgATG1D213A,and FgATG1T228A was consistent with ?Fgatg1 mutants.The result indicates that K53,D213 and T228 are three key amino acid sites for the FgAtg1 protein.3.Among the 28 ATG mutants,colony morphology of AFgatg20 and AFgatg24 on PDA plates is different from other mutants.Meanwhile,colony morphology between two mutants is very similar,and Pfam based domain prediction analysis revealed that FgAtg20 and FgAtg24 contain the same domains.Therefore,we focus on FgAtg20,whose biological characters and functions are emphasically studied.(1)?Fgatg20 mutants showed slow growth,rare aerial hyphae,almost no conidia production and reduced pathogenicity,but normally produced perithecia,asci and ascospores.(2)After loss the PX domain of FgAtg20,the growth phenotypes and pathogenicity of FgAtg20?PX-C strains were similar to ?Fgatg20 mutants.(3)The subcellular localization showed that FgAtg20-GFP dispersed throughout the cytoplasm and were localized in the punctate structures close to vacuoles,which colocalized with RFP-FgAtg8,(4)Through transmission electron microscopy,we found that there were no autophagic bodies in the hyphae cells of AFgatg20 mutants.(5)In terms of the Cvt pathway,FgApel-RFP signals dispersed throughout the cytoplasm,which different from the punctate structures in hyphal cells of the wild-type PH-1.Bedides,the mature of FgApe1 precursor was impaired in the AFgatg20 mutants.(6)In terms of mitophagy,western blot analysis confirmed that the degradation of Porin in the AFgatg20 mutants,a mitophagic marker protein,was consistent with PH-1.As well as,confocal microscope showed that MitoFluor-Red stained mitochondria colocalized with CMAC-strained vacuoles in the hyphal cells of both PH-1 and AFgatg20.(7)In terms of pexophagy,the degradation of Pex14 marking peroxisome in the AFgatg20 mutants was suppressed.(8)Y2H experiments confirmed that FgAtg24,FgAtgl7 and FgAtgl 1 interact with FgAtg20 respectively,and FgAtgl interacts with FgAtg17 and FgAtg24,but not FgAtg20 and FgAtgl 1.From the above results,we concluded that FgATG20 is involved in the vegetative growth,asexual reproduction and pathogenic process in F.graminearum,and PX domain is necessary for FgAtg20 protein.In the process of autophagy,FgATG20 is indispensable for the formation of autophagic bodies and involved in general autophagy and pexophagy,but is unnecessary for mitophagy in F.graminearum.FgAtg20 forms a multimer with FgAtgl,FgAtg1 1,FgAtg17 and FgAtg24,co-involved in the autophagy process of F.graminearum.This study suggests that autophagy is involved in the vegetative growth,asexual and sexual reproduction,DON biosynthesis and pathogenicity,and autophagy related proteins interacts with each other and co-regulate the process of autophagy in F.graminearum.In F.graminearum,in addition to autophagy process regulated by the TOR pathway,FGSG 05304(FgHLTF1)is also regulated by this pathway.FgHLTFl is identified as a transcriptional factor at the downstream of TOR pathway.The biological function analysis showed that expression levels of FGSG 05304(FgHLTF1)were significantly decreased by 5.9 folds in the AFgppgl mutant.Then its molecular biological functions were preliminarily analyzed.We found that FgHltfl is located in the nuclei,and ?AFghltfl mutants showed slower vegetative growth,less pigment,no perithecia and significantly reduced pathogenicity compared to the wild type PH-1.However,the DON production and conidiation of the mutants showed no significant difference compared to PH-1.Besides,on the CM plates with 1M Sorbitol,KC1 and NaCl,the growth of ?AFghltfl mutants was remarkably inhibited.Meanwhile,FgHogl could not transfer into the nuclei from the cytoplasm in order to response the osmotic stress after 2 h of 0.7 M NaCl treatment.These results indicated that FgHLTF1 plays important roles in vegetative growth,sexual reproduction and pathogenicity in F.graminearum,and also is involved in the HOG signaling pathway,but it is not required for DON production and asexual reproduction.
Keywords/Search Tags:Fusarium Head Blight, Fusarium graminearum, autophagy, FgATG20, pathogenicity
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