The Regulation Of Sensitivity Of FgAglZ And FgGlc3 To Phenamacril In Fusarium Graminearum

Wheat head blight is a worldwide epidemic disease caused by Fusarium graminearum,which seriously reduces the yield and quality of wheat.Phenamacril is a new fungicide developed by our country which can control wheat head blight.It is known that F.graminearum can easily develop resistance to phenamacril under indoor conditions,and the main reason is the point mutation of Myosin5 amino acid.Current studies have found that there are other proteins or pathways in F.graminearum that have regulatory effects on the drug sensitivity of phenamacril.For example,β2-tub、FGSG＿06149、FGSG＿03716、Fgspt3、Fgspt8、Fg Aps B、Fg Fim、Myosin2B、Fa Smy1、Fg EB1 and Fg Tfm I are all involved in the regulation of the drug sensitivity of phenamacril.Therefore,we speculate that there is a regulatory network of F.graminearum susceptibility to phenamacril.Based on this,the mass spectrometric results of Myosin5,the target of phenamacril.Two proteins potentially interacting with Myosin5,Fg Agl Z（Adventurous gliding protein）and Fg Glc3（1,4-alpha-glucan branching enzyme）,were obtained.The purpose of this study was to study the drug sensitivity regulation of Fg Agl Z and Fg Glc3 to phenamacril and the effect on the survival fitness of F.graminearum.So we did the following research:1.Agl Z is a gliding protein in Fusarium langsethiae and a fibrous curled protein in Myxococcus aureus,which regulates gliding movement.Fg Agl Z,2645 bp in length,encodes 774 amino acids and has 1 intron and 2 exons.In addition,protein domain analysis showed that Agl Z belonged to the Smc family.Phylogenetic analysis showed that Agl Z was conserved in Fusarium,and Fg Agl Z and Fusarium austroamericanum Agl Z had the highest similarity in protein sequences.The protein sequences of Fusarium graminearum Fg Agl Z and Botrytis cinerea Agl Z and Sclerotinia sclerotiorum Agl Z were relatively low in similarity.In Fusarium graminis,the knockout and replicant of Fg Agl Z were constructed using the wild-type sensitive strain PH-1 and the highly resistant strain YP-1 as the starting strains,respectively.The results showed that:After Fg Agl Z knockout,the drug sensitivity of the knockout with PH-1 as the starting strain to phenamacril had no significant change,and the drug sensitivity of the knockout with YP-1 as the starting strain to phenamacril increased significantly,specifically,the EC₅₀of phenamacril onΔFg Agl Z-32 andΔFg Agl Z-37 with YP-1 as the starting strain was 82.8403±4.3694μg/m L and78.6605±3.8196μg/m L,respectively.The EC₅₀was 113.7125±5.5779μg/m L for YP-1 and103.8056±4.6606μg/m L for the replicant,which was recovered to the level of YP-1.butΔFg Agl Z had no change in drug sensitivity to penazolol,prothiazole,fluazolimide,etc.Fg Agl Z did not interact with Myosin5,but the transcription level of Myosin5 in the knockout body was decreased by adding 80μg/m L phenamacril.Fg Agl Z is localized in the phenamacril;At the same time,the growth rate ofΔFg Agl Z on PDA board was lower than that of its parent strain,and the top branches of hyphae were reduced.In terms of asexual reproduction,the conidia production capacity ofΔFg Agl Z decreased,the conidia morphologic curvature decreased,and the proportion of the number of conidia septum in1-3 increased.In terms of sexual reproduction,ΔFg Agl Z lost the ability to produce ascomyte shell;Under external stress,the inhibition rate ofΔFg Agl Z on 0.7M Na Cl and0.7M KCl increased significantly.In terms of pathogenicity,the pathogenicity ofΔFg Agl Z to wheat decreased.In conclusion,Fg Agl Z knockout of Fusarium graminis not only reduces the resistance to phenamacril,but also regulates the growth,reproduction,stress,toxicity and pathogenicity of the strain.2.Glc3,glycogen branching enzyme,regulates glycogen accumulation and branching level in Saccharomyces cerevisae.Fg Glc3 has a total length of 3046 bp,encoding 707amino acids,and has 5 introns and 6 exons.In addition,protein domain analysis showed that Fg Glc3 contains three domains,namely CBM＿48,alpha-amylase and alpha-amylace＿c.Phylogenetic analysis showed that the protein sequences of Fg Glc3 and Fg Glc3 of Fusarium pseudograminearum had the highest similarity.The protein sequence similarity between F.graminearum Fg GLC3 and Candida albicans GLC3 was the lowest.we constructed deletion mutantΔFg Glc3 and its complementation to determine whether Fg Glc3 is involved in the regulation of the sensitivity of phenamacril and to verify the biological function ofΔFg Glc3 and explore the role of Fg Glc3 in the growth and development process of F.graminearum.The results showed that phenamacril EC₅₀of theΔFg Glc3 obtained from sensitive strain PH-1 and high-resistant strain YP-1 was significantly increased.The EC₅₀of phenamacri against p H-1 starting strainΔFg Glc3-83andΔFg Glc3-185 were 0.3432±0.02284μg/m L and 0.3325±0.03445μg/m L,respectively.Significantly higher than the EC₅₀value of 0.2255±0.02885μg/m L for PH-1and the EC₅₀value of 0.2199±0.01906μg/m L for the replicant.The EC₅₀values of cydene ester for YP-1 starting strainsΔFg Glc3-47 andΔFg Glc3-54 were 196.7009±3.9631μg/m L and 204.3119±4.5792μg/m L,respectively.EC₅₀122.9003±4.9702μg/m L was significantly higher than that of YP-1.However,there was no interaction between Fg Glc3 and Myosin5,and the expression of Myosin5 in theΔFg Glc3 did not significantly change with the addition of phenamacril.At the same time,compared with those of the parent strains,the mycelial growth rate of Fg Glc3 deletion mutant was slowed down,and branches of the mycelial tip increased.In terms of sexual reproduction,ascospores cannot be produced normally;sensitivity to SDS,Cogon Red and Mg Cl₂significantly increased in external stress.In terms of pathogenicity,there was no significant change in pathogenicity,and the ability to produce DON toxin increased.In conclusion,Fg Glc3 is involved in the regulation of the drug sensitivity to phenamacril,which may be related to the influence on the growth,reproduction,stress and toxicity of the strains.