| Fusarium head blight(FHB)is a serious plant disease in wheat,which is not only reduces the yield of wheat,but also threatens wheat quality and safety.Fusarium graminearum is the main fungal pathogen which can cause FHB and produce a virulence factor called DON.DON is harmful to both humans and mammals.Therefore,it is necessary to elucidate the pathogenicity of Fusarium graminearum as well as the mechanisms of pathogenesis and resistance.In eukaryotes,PI4 P is involved in many biological processesis,which located at different organelles,including trans-Golgi network(TGN),plasma membrane(PM)and endosomes.Type Ⅱ phosphatidylinositol 4-kinases(PI4Ks)plays a key role in phosphatidylinositol(PI)metabolism,which is involving regulating the production of PI4 P.The first step in the biosynthesis of PI lipids is catalyzed by phosphatidylinositol 4 kinases(PI4Ks).PI4Ks have been classified as type Ⅱ or type Ⅲ based on enzymatic properties.The research about type Ⅲ PI4 Ks are relatively extensive.Compared with type Ⅲ PI4 Ks,type ⅡPI4Ks are relatively less understood.In Saccharomyces cerevisiae and mammalian cells,typeⅢα and typeⅢβ are first identified and located in the Golgi apparatus and plasma membrane,respectively.Only typeⅡα PI4 K,Sc Lsb6,is identified and found to be located in the vacuolar membrane and plasma membrane.In mammalian cells,PI4KⅡα and PI4KⅡβ,were identified on the Golgi and endosomes.Since its molecular characterization,PI4KⅡα,which plays a role in post–trans-Golgi network(TGN)-to-endosome and TGN-to-late endosomal trafficking,has been known to be important in transport to the lysosome.However,it is remain not clear whether Sc Lsb6 is involved in membrane transport and the role of typeⅡ PI4 Ks in the mycelial growth and development,pathogenicity has not been investigated in Fusarium graminearum.In this study,the target gene Fg LSB6 is obtained by phylogenetic tree analysis and amino acid sequence alignment,which is homolog gene Sc LSB6 in Saccharomyces cerevisiae.It is revealed that Fg LSB6 has a conserved phosphatidylinositol kinase domain.To further explore the biological function of Fg Lsb6,a knockout fragment was constructed using Split-marker PCR technology,at least three gene knockout mutants strain ΔFglsb6 was obtained by PEG-mediated protoplast transformation.Based on the knockout mutants,a ΔFglsb6 /Fg LSB6 complement strain and ΔFglsb6/Fg LSB6-GFP transformed strains was further constructed.The main results obtained are as follows:(1)Experiments on the development and pathogenicity of Fusarium graminearum found that compared with the wild-type strain PH-1,both growth and pathogenicity of the ΔFglsb6mutant strain were inhibited.But there are no changes regarding sexual and asexual reproduction.(2)The ΔFglsb6 mutant strain is more sensitive to salt stress and cell wall integrity stress than the wild-type strain,but its tolerance to xidative stress is improved.(3)DON is an important virulence factor of Fusarium graminearum,and the toxin body is the main organelle for DON synthesis.The experimental results showed that the relative expression of TRI and DON production in ΔFglsb6 mutant strains were significantly lower than those of wild type,but the toxin body formation was normal.Therefore,Fg Lsb6 is related to the virulence of Fusarium graminearum.(4)In mammals,Type Ⅱ PI4 K regulates endosome transport by participating in the regulation of PI4 P production.The experimental results show that Fg Lsb6 is specifically localized to endosomes and vacuolar membranes,so Fg Lsb6 may affect the endocytic process of Fusarium graminearum.Finally,the experimental results showed that compared with the wild type PH-1,the cell endocytosis of ΔFglsb6 mutant strain was delayed at 10 min.We can conclude that Fg Lsb6 can reduce the pathogenicity of Fusarium graminearum by delaying the endocytosis pathway.To sum up,these results indicate that Fusarium graminearum Fg Lsb6 is essential for mycelial growth and development,pathogenicity and virulence. |
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