Functional Characterization Of The Phosphatidylsdrine Decarboxylase In Fusarium Graminearum

Fusarium head blight(FHB)is one of the main diseases that endanger wheat production areas around the world.FHB has the characteristics of rapid expansion and a wide range of harmful areas.Infected wheat will not only cause a large-scale reduction of production and severely reduce the quality of wheat production,then the toxins produced will also pose a serious threat to human and animal health.A variety of pathogenic bacteria can cause scab,but the dominant pathogen that causes scab in wheat is Fusarium graminearun.In recent years,lipids have received much attention in terms of virulence of microbial pathogens.Phosphatidylserine decarboxylase has been intensively studied in humans,mammals and even yeast,but there is relatively little research on the function of phosphatidylserine decarboxylase in filamentous fungi.In this study,two proteins expressing phosphatidylserine decarboxylase were identified in the whole genome of Fusarium graminearum,are FgPSD1 and FgPSD2.Analysis of the SMART domain revealed that both phosphatidylserine decarboxylase contained a C2 domain and a PS_decarboxylases domain.The phylogenetic tree results showed that FgPSD1 and FgPSD2 are closely related to the PSD1 and PSD2 genes of F.oxysporum and F.verticillioides,the phosphatidylserine decarboxylase gene is an orthologous gene of other Fusarium phosphatidylserine decarboxylase genes.In this paper,Split-marker PCR technology was used to construct FgPSD1 and FgPSD2 gene knockout fragments,and PEG-mediated protoplast transformation methods were used to obtain gene knockout mutants.By verifying more than 200FgPSD1 knockout transformants,no correct transformants were found,and FgPSD1 was determined to be a lethal gene.At the same time,four FgPSD2(?PSD2)geneknockout mutants were obtained after PCR verification and Southern Blot verification.On the basis of the ?PSD2 mutant,a ?PSD2-C recovery strain and a FgPSD2-GFP gene localization strain were further obtained.The results show that: compared to wild type,the ?PSD2 mutant grows slowly and the mycelium height is greatly reduced,conidia production is reduced,and germination is slow;compared to the wild type,the number of ?PSD2 mutant ascomycetes is not reduced but produce ascospores;compared with wild type,?PSD2 mutant greatly reduced pathogenicity,disease index decreased by 94.35%,meanwhile,DON toxin production only accounted for 15.6% of wild type,and important genes related to DON toxin synthesis TRI5,TRI6 And TRI10 expression levels decreased by 99.2%,96.0%,and83.3%;compared with the wild type,the cell wall integrity of the ?PSD2 mutant increased,but the resistance to osmotic capacity and oxygen stress was reduced,and it was more sensitive to oxygen stress;The mutant was ethanolamine auxotrophic,when1 mM Etn was added to the culture conditions,the phenotype of the ?PSD2 mutant could be partially restored;compared to the wild type,the phosphatidylserine content of the ?PSD2 mutant increased significantly,while phosphatidylethanolamine in addition,the ?PSD2 mutant has a lower lipid droplet accumulation and aggravated autophagy than the wild type;at the same time,we found that FgPSD1 is localized to mitochondria,while FgPSD2 is localized to Golgi apparatus.In summary,the results of these studies indicate that Fusarium graminearum FgPSD2 is crucial in the growth and development of hyphae,sexual and asexual reproduction,pathogenicity,stress response,lipid droplet accumulation and autophagy.