Research On Drug-sensitive Related Genes Of Fusarium Graminearum And Its Regulation

Fusarium graminearum not only causes Fusarium head blight(FHB)to reduce production,but also produces toxins that contaminate the grain and threaten human and animal health.Therefore,it is a primary task for us to study the theory and technology of safe,efficient and sustainable control of FHB.For a long time,the prevention and control of FHB at domestic and abroad mainly rely on chemical control,and the benzimidazole fungicide carbendazim has been mainly used in China.However,with the prolongation of the medication time and the increase of the dosage,the F.graminearum has formed a resistance population to benzimidazole fungicides in nature,resulting in the failure of chemical control in some areas.Studies have shown that point mutations in the carbendazim receptor β2-tubulin of Fusarium spp.are responsible for pathogen resistance.β2-tubulin is a structural and functional regulatory protein in Fusarium,which has an interaction with various proteins.In this paper,the interaction between FgASK1 andβ2-tubulin and the sensitivity regulatory mechanism of FgASKl to tubulin inhibitors were studied.Besides,the behavior of dsRNA nucleic acid pesticides to control FHB in pathogenic fungi and host plants was explored,which provide scientific basis for revealing the toxicology mechanism of the fungicide and its efficient application technology.We first used the yeast interaction protein library(Uniprot)to screen for FgASK1 protein,which is known as a subunit of the DASH protein complex that plays an important role in chromosome separation during cell division.This study found that this protein interacts with Fgβ2-tub,and the absence of FgASK1 causes different changes in the sensitivity of F.graminearum wild-type strains and Fgβ2-tub resistant point mutants to carbendazim.The sensitivity to other types of fungicides remains unchanged.The sensitivity to carbendazim of wild-type strains,Y50C and E198K FgASK1-deficient mutants was increased by 32.2%,42.5%and 40.8%,respectively,while the resistance level of F167Y lacked FgASK1 to carbendazim remains unchanged.It indicated that FgASK1 has a significant regulating effect on the sensitivity to benzimidazole drug by interacting with Fgβ2-tub,and positions 50 and 198 of Fgβ2-tub may be the sites binding benzimidazole drugs or FgASK1,while the residue 167 has a lower affinity to FgASK1.Compared with that of wild-type strain,the interaction strength of Fgβ2-tub of Y50C-2 and E198K mutants and FgASK1 in vitro was reduced by 44.2%and 48.9%,respectively.The interaction strength of Fgβ2-tub of F167Y and FgASK1 decreased by only 2.3%.Co-localization observations using cells with the double-fluorescence-labeled proteins revealed that the interaction between FgASK1 and Fgβ2-tub to form a complex was very short,and separation occurred after about 2 seconds.We found that the sexual reproduction of F.graminearum reduced significantly when the fungus absents FgASK1.Meanwhile,the growth,asexual reproduction and the division of the nucleus in conidial period of F.graminearum,weakened remarkably for the FgASK1 deletion mutants.What’s more,Y50C-2 and E198K Fgβ2-tub mutants also showed the same phenotype,which suggested that part of the function of FgASK1 depends on the interaction with Fgβ2-tub,but some functions are not related to its interaction with Fgβ2-tub.Myo5-8 dsRNA,derived from a fungicide target myosin-5 based on the principle of RNA interference,is a bioactive nucleic acid against F.graminearum.By spraying Myo5-8 dsRNA,Myo5-8 dsRNA induces the gene silencing of the fungicide target(Spray-induced gene silencing,SIGS)to control FHB,but the mechanism of SIGS is still unclear.This study found that after spraying Myo5-8 dsRNA on wheat leaves,Myo5-8 dsRNA can be more effectively absorbed by the leaves through the wounds and enter the vascular bundle for redistribution.The Myo5-8 dsRNA entering plant cells can be transformed into a large amount of siRNA through the processing of wheat leaf tissue cells and the siRNA secondary amplification machinery.However,Myo5-8 dsRNA penetrating into cells could not induce RNAi secondary amplification machinery in F.graminearum.The siRNAs in fungal cells were all primary siRNAs generated by the cleavage of exogenous dsRNA and the expression of genes related to amplification was not induced,which resulted in lower concentration of the dsRNA and shorter persistent period of the dsRNA.This study revealed the behavior of Myo5-8 dsRNA in pathogenic fungi and plants,which will benefits us to optimize of Myo5-8 dsRNA formulation and its application technology.