Functional Analysis Of The Peroxisome Proteins FgPEX4 And FgPEX22 Of Fusarium Graminearum

Fusarium head blight(FHB)is an economically devastating disease in the world.FHB was discovered in England in the nineteenth century for the first time,the frequency of disease outbreaks in Asia,Europe,North America and South America occurrence gradually increases.FHB seriously affects the yield and quality of wheat.The main pathogen of FHB is F.graminearum.Peroxisomes are indispensable organelles that exist in eukaryotic organisms and involving a variety of physiological and biochemical processes Proteins involved in peroxisomal biogenesis are named peroxins,which are encoded by PEX.To date,over 30 peroxins have been identifed in eukaryotic cells.Relationship and function of PEX4 and PEX22 in phytopathogenic fungus are still unknown.The relationship and function of F.graminearum PEX4 and PEEX22 were analyzed systematically in this study,the main results were summarized as follows:(1)Homologous protein of Saccharomyces cerevisiae PEX4 and Colletotrichum orbiculare PEX22 were identified from the F.graminearum genome.SMART-PFAM analysis revealed that FgPEX4 protein contains a UBCc domain,which is a characteristic region of the protein,in addition,FgPEX4 protein also contains a PEX22 interaction region.FgPEX22 contains a transmembrane domain and a possible PEX4 interaction region.(2)To study the interaction between FgPEX4 and FgPEX22 in F.graminearum.yeast twohybrid and immunocoprecipitation were used in this research,results showed that the two proteins could interact each other directly.(3)To understand the biological functions of FgPEX4 and FgPEX22 in F.graminearum,we generated single gene knockouts mutants ?PEX4,?PEX22 and double gene knockouts mutant ?PEX4/22 by Split-marker PCR and protoplast transformation.Complementary strain ?PEX4-C and ?PEX22-C were generated based on ?PEX4 and ?PEX22.To understand the relationship between the two proteins further,a fusion expression vector of FgPEX4-GFP was constructed and transferred into wild-type strains and ?PEX22 respectively.The results showed that the deficiency of FgPEX22 can result in abnormal subcellular localization of FgPEX4,FgPEX22 functions as the rivet of FgPEX4.(4)The related phenotypic changes of all mutants were further studied: in comparison with the wild type strain,colony diameter of ?PEX4 and ?PEX22 were reduced significantly and FgPEX22 have no effect on colony diameter.All mutant showed a distinct colony morphology with shorter aerial hyphae and unregular edges.FgPEX4 and FgPEX22 also regulate asexual and sexual reproduction of F.graminearum: in comparison with the wild type strain,spore production of the mutants was reduced and the morphologies was abnormal,including the excretion of intracellular content and the absence of nuclei.Abnormal spores could not germinate normally;In comparison with the wild type strain,number of perithecium formation in ?PEX4,?PEX22 and ?PEX4/22 was reduced.The phenotype of ?PEX4-C and ?PEX22-C was restored to the level of the wild type strain.(5)Pathogenicity assays were conducted to determine the effect of FgPEX4 and FgPEX22 on flowering wheat heads and corn silks by artificial inoculation.We found that ?PEX4,?PEX22 and ?PEX4/22 were defective in pathogenicity.Date of qRT-PCR indicated that FgPEX4 and FgPEX22 were involved in the regulation of TRI expression.(6)In comparison with the wild type strain,?PEX4??PEX22 and ?PEX4/22 exhibited increased sensitivity to cell wall-damaging agents and cell wall-degrading enzymes.In addition,oxidative stress capacity of ?PEX4??PEX22 and ?PEX4/22 decreased significantly.The mutants couldn't get rid of the reactive oxygen in time,relative growth rate of ?PEX4,?PEX22 and ?PEX4/22 were decreased on medium containing hydrogen dioxide.(7)Transmission electron microscopy and lipid droplet staining were used to determine the effect of FgPEX4 and FgPEX22 on organelles in F.graminearum.We found that in all mutants,phenomenon of cell breakage was observed,the volume and number of lipid droplets increased,peroxisomes and Woronin body were absent.Further,take advantage of subcellular localization of organelle genes,we found that the number of peroxisomes decreased significantly,and the Woronin body was absent.(8)TMT-based proteomics analysis was applied to wild type strain PH-1 and ?PEX4,The results showed that proteins related to amino acid biosynthesis,protein translation,translational elongation were down-regulated,and it may explain why ?PEX4 displayed defects in growth,sexual and asexual reproduction.Proteins related to oxidation–reduction reactions,biosynthesis and degradation of fatty acid were up-regulated,and it may explain why ?PEX4 displayed changes in sensitivity to oxidative stress and utilization of fatty acids.These data can explain the molecular mechanism of the phenotype changes of F.graminearum after FgPEX4 gene deletion.