Functional Analysis Of The Peroxisome Proteins FgPEX2and FgPEX12 Of Fusarium Graminearum

Fusarium head blight?FHB?is a wheat disease with a worldwide prevalence,caused by Fusarium graminearum,the frequency of disease shows an upward trend year by year in our country.FHB not only damages potentially high-yielding crops within a few weeks of harvest,but also produces trichothecene mycotoxins,and mycotoxin-contaminated grains are unsafe for human and animal consumption or malting.Peroxisomes are ubiquitous in eukaryotic cells and are involved in all kinds of biochemical phenomena,which contain more than 50 kinds of enzymes.Peroxisomal gene?PEX?is the general name of a class of genes encoding peroxin-related proteins.Thus far,more than 30 different PEX genes are known,most of which are conserved among eukaryotic.In this study,bioinformatics method was used to get the FgPEX2?FGSG₀0724?and FgPEX12?FGSG₀9756?genes in genome of PH-1.The fusion cassette containing the gene-flanking sequences by split-marker PCR.And we got the ?PEX2,?PEX12 mutants and ?PEX2-C,?PEX12-C complementary strains by using the polyethylene glycol-mediated fungal transformation and homologous recombination.Studying the phenotypic changes of FgPEX2 and FgPEX12 gene knockout mutants further can explain the function of them,which will lay a foundation for further analysis of the molecular mechanism of F.graminearum and also provide a theoretical basis for the control of FHB.The main results as follows:?1?Compared with the WT strain,colony diameter of ?PEX2 and ?PEX12 were reduced significantly and the aerial hyphae of mutants in germ tubes were shorter.The phenotypes of ?PEX2-C and ?PEX12-C were basically restored to the level of the PH-1.?2?Compared to the WT strain,conidiation in the ?PEX2 and ?PEX12 mutants was reduced and mutants conidia displayed an abnormal morphology,in that they burst and the contents oozed out.The number of perithecium formation in ?PEX2 and ?PEX12 mutants was reduced.The phenotypes of ?PEX2-C and ?PEX12-C were basically restored to the level of the PH-1.?3?Virulence determination results found that ?PEX2 and ?PEX12 mutants on flowering wheat heads and corn silks in virulence were significantly reduced.The phenotypes of ?PEX2-C and ?PEX12-C were basically restored to the level of the PH-1.DON toxin production measurement results showed that ?PEX2 and ?PEX12 mutants produced DON toxin ability were significantly reduced than that of the PH-1.Date of qRT-PCR showed that Tri5?Tri6 and Tri10 genes were significantly downregulated in the ?PEX2 and ?PEX12 mutants in comparison with the WT strain.?4??PEX2 and ?PEX12 mutants are more susceptible to the disruption of CWI,the expression of chitin synthase FgCHS7 quantity is also significantly reduced.In addition,mycelia of ? PEX2 and ?PEX12 mutants were well digested and released abundant protoplasts.The phenotypes of ?PEX2-C and ?PEX12-C were basically restored to the level of the PH-1.?5?ROS production was observed via NBT staining in mutants and WT strains.The staining assay revealed that ROS abundantly accumulated in the ?PEX2 and ?PEX12 mutants.The utilization of fatty acid was decelerated in ?PEX2 and ?PEX12 mutants.The phenotypes of ?PEX2-C and ?PEX12-C were basically restored to the level of the PH-1.?6?Transmission electron microscopy showed that the quantity and volume of lipid droplet increased in ?PEX2 and ?PEX12 mutants than that of the PH-1.The accumulation of lipid droplets in ?PEX2 and ?PEX12 mutants was demonstrated by lipid droplet staining.Furthermore,fluorescence microscopic analysis of intracellular localization of GFP-PMP70 and GFP-HEX1 revealed that deletion of FgPEX2 and FgPEX12 does not affect peroxisomes;however the Woronin bodies were absent in ?PEX2 and ?PEX12 mutants.These results show that FgPEX2 and FgPEX12 are required for growth,conidiation,virulence,cell wall integrity,and fatty acid metabolism in F.graminearum.