| Fusarium Head Blight(FHB)caused by Fusarium graminearum infection of wheat is one of the most destructive fungal diseases in wheat production.Ascospores produced by sexual reproduction of F.graminearum,as the primary infection source,play a significant role in the occurrence of FHB.The key to control the primary infection source is to block the sexual reproduction of F.graminearum.Therefore,it is of great theoretical significance to reveal the molecular regulation mechanism of F.graminearum sexual reproduction for prevention and control of FHB.Autophagy is a highly conserved subcellular degradation pathway in eukaryotic cells,which plays an important role in maintaining intracellular homeostasis and helping cells to survive adversity.In plant pathogenic fungi,autophagy has been studied mainly on its functions in vegetative growth,asexual sporulation and plant infection,while there is still a lack of in-depth and systematic research on the sexual reproduction of fungi.In the early stage,24 genes related to autophagy were knocked out in F.graminearum,and phenotypes such as colony growth,conidial production and virulence of these mutants were preliminarily observed.In this study,we systematically observed the phenotypes of 24 autophagy gene deletion mutants during sexual development,including the formation of perithecium,development and eruption of ascospores.Subsequently,we identified the specific stagnant period of Fgatg8 during sexual development by fluorescence microscopy and scanning electron microscopy,and speculated the function of Fgatg8 in sexual reproduction by hybridizations between Fgatg8 and PH-1 or Fgmat1-1-1.We detected the autophagy levels of F.graminearum during different stages of sexual development by Western-Blot,and observed the expression of GFP-FGAtg8 in the wall of perithecium,paraphysis,asci and ascospores.Finally,we detected the function of FgATG8 in late sexual reproduction of F.graminearum by tandem repeated induction of specific silencing of gene at the sexual stage.The main results of this paper are as follows:1.Sexual reproduction phenotype identification revealed that 12 of 24 autophagy related gene mutants of F.graminearum had severe sexual defects,and these 12 genes were all key genes in autophagy pathway.The frequency of mycelial fusion of the 12 mutants with severe sexual defects was significantly lower than that of the wild type.2.After staining with WGA488,we found that Fgatg8 mutant could not form spheroidal perithecium precursor.Scanning electron microscopy results showed that the sexual development of Fgatg8 stagnated in the initial stage of sexual reproduction.Sexual hybridization results showed that Fgatg8 could be fertile as a male parent,but could produce a large number of empty perithecium as a female parent either with wild PH-1 or Fgmat1-1-1.The core autophagy gene deletion mutants as female parent hybridized with Fgmat1-1-1 could produce small perithecium,and they could also produce extremely small perithecium when cultivated on wheat straw.3.Western blot results showed that GFP-FgAtg8 was completely degraded in the aerial hyphae of F.graminearum and in the samples after 4,6 and 8 days of sexual induction.Fluorescence microscopy results showed that GFP-FgAtg8 was highly expressed in the wall of perithecium and ascospores,but not in the young asci and paraphysis.4.During sexual development,the specific silencing of FgATG8 may not affect the development of asci and ascospores,nor the accumulation of glycogen and lipid droplets in ascospores. |
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