| Fusarium head blight(FHB)caused by Fusarium graminearum can lead to the reduction of wheat yield and quality deterioration.The deoxynivalenol(DON)toxin produced by the pathogen will seriously harm the health of human and animals.Therefore,the prevention and control of FHB and DON toxin contamination are of great significance to ensure food quality and food safety.In the absence of wheat germ plasm resources with high resistance to FHB,an in-depth understanding of the gene functions and metabolic regulatory pathways of DON synthesis by pathogenic bacteria is needed in order to develop antimicrobial agents for controlling FHB.So far,the biosynthetic pathway of DON toxin has been clear in Fusarium graminearum.The regulatory mechanism is not fully understood.In this study,we focus on the mechanism of DON toxin regulation by Fusarium graminearum stress response protein NST1,and explore the gene targets of fungicide action by using the response characteristics of NST1 to antagonistic bacteria.The main results include the following:(1)The conserved domain and phylogenetic analysis of FgNST1 showed that FgNST1 contained NST1(Negative for salt tolerance)domain.FgNST1 was the most similar to the stress response protein of Fusarium culmorum.The effects of FgNST1 on DON biosynthesis and Tri gene expression were analysed.The results showed that the deletion of FgNST1 gene resulted in a significant reduction in DON toxin synthesis and Tri gene expression.Under Na Cl stress,the synthesis of DON toxin in the wild-type and ?FgNST1 mutant was significantly reduced.The DON produced by ?FgNST1 mutant was significantly lower than that in wild-type,and only the expression of transcription factor Tri6 gene was down-regulated.The results indicate that FgNST1 could be respond to salt stress and play a role in regulating DON biosynthesis.(2)In order to reveal the mechanism of FgNST1 regulating DON biosynthesis,the differentially expressed genes regulated by FgNST1 were analyzed by transcriptome.There were 452 up-regulated genes and 1002 down-regulated genes in the differentially expressed genes regulated by FgNST1 in the normal growth condition.480 and 776 genes were significantly up-and down-regulated under Na Cl stress.277 genes were regulated by FgNST1 under both conditions.Through GO and KEGG enrichment analysis,the differentially expressed genes related to DON synthesis regulation under FgNST1 regulation were screened,including salt stress related genes,antioxidant enzyme genes,amino acid metabolism genes,MAPK related genes and so on.(3)The effects of FgNST1 on genes related to salt stress,antioxidation,glycerol metabolism and proline metabolism were further analyzed.The results showed that the deletion of FgNST1 gene regulated the gene expression.The salt-tolerant genes were significantly down-regulated,antioxidant enzyme genes,glycerol kinase genes and proline synthesis genes were significantly up-regulated.FgNST1 showed different regulation modes under Na Cl stress.This indicated that FgNST1 could regulate the DON synthesis by influencing a variety of stress response-related proteins.(4)The effect of FgNST1 on Fg MGV1 in MAPK pathway was analyzed.The results showed that Fg MGV1 gene was almost not expressed in ?FgNST1 mutants in the lack of Na Cl stress.Under Na Cl stress,the expression of Fg MGV1 gene was activated.Yeast two-hybrid analysis shows that FgNST1 can interact with Fg MGV1.The results indicate that FgNST1 could cooperate with Fg MGV1 to regulate DON synthesis.(5)The function and expression of two acetaldehyde dehydrogenase genes were analyzed.The conidiation of ?FGSG-05831 and ?FGSG-02296 mutants were reduced.The sensitivity to osmotic pressure and Congo red were increased.The sensitivity to oxidative stress,pathogenicity and DON synthesis were all significantly decreased.It indicates that acetaldehyde dehydrogenase plays an important role in growth,stress-response and DON toxin synthesis.The expression of FGSG-05831 and FGSG-02296 in ?FgNST1 mutants was significantly down-regulated,suggesting that FgNST1 regulates the DON synthesis through regulating the expression of acetaldehyde dehydrogenase.(6)In order to verify whether FgNST1 gene can be used as a target of antagonistic substances,the response of FgNST1 to antagonistic bacteria was analyzed.The results showed that antagonistic substances had a higher inhibition rate on the mycelium growth of wild-type than in FgNST1 mutants under a certain concentration.The antagonistic substances had a significant inhibitory effect on DON synthesis.FgNST1 could be a potential target of fungicides.In summary,FgNST1 regulates DON biosynthesis by regulating gene expression and metabolic pathways,which play an important role in the prevention and control of Fusarium head blight and DON pollution.This study can provide a theoretical basis and technical parameters for the systematically elucidation of the regulation mechanism of DON toxin biosynthesis and the development of new fungicide. |
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