| Septin is a highly conserved family of GTP-binding proteins and is considered to be the fourth cytoskeleton protein after microtubules,micro filaments and intermediate fibers.Septin is widely present in all eukaryotes except plants.In plant pathogenic fungi,such as Magnaporthe oryzae,Botrytis cinerea,Fusarium graminerarum,it is involved in the growth and development of pathogens,morphogenesis related to disease,stress response and secondary metabolism,etc.However,it has not been reported in Setosphaeria turcica.Therefore,in this study,bioinformatics technology was used to identify Septin gene family within the scope of S.turcica genome.Then bio informatics analyses including gene structure,physical and chemical properties and transmembrane region structure of the Septins were conducted.The gene knockout mutant of StSep4 was obtained by using gene knockout technology.The differences in growth,development and pathogenicity betweenΔStSep4 and wild-type(WT)strains were compared.The main results were as follows:1.Through homology search in genomic database of S.turcica,six candidate Septins were obtained.They were named as StSep1(JGI accession No.162710),StSep2(JGI accession No.163440),StSep3(JGI accession No.93639),StSep4(JGI accession No.110191),StSep5(JGI accession No.165795),and StSep6(JGI accession No.99177),respectively.2.Bio informatics method was used to analyze the structural characteristics and domains of 6 Septin genes,and online alignment was performed in NCBI-CDD(Conserved Domain Database)database.The results showed that these 6 Septin genes,randomly distributed in the genome of S.turcica.Among them,four typical Septins named by StSep1,StSep1,StSep3 and StSep4 contained G1,G3,and G4 motifs.StSep5 and StSep6 were mainly members of the P-loop-N TPase domain,which were used to bind theβ-γ phosphate portion of nucleotides(ATP/GTP)and Mg2+.The gene expression pattern of Septin was analyzed by RT-qPCR.StSepl and StSep4 were actively expressed during the germ tube formation and appressorium formation stage,respectively,and the expression of StSep2,StSep3 and StSep4 was up-regulated during the invasive hypha formation stage.It was found that only the expression of StSep4 was significantly up-regulated during the formation of appressorium and invading nail.It was preliminarily determined that StSep4 is the main Septin that regulates the formation of appressorium and invading nail.3.In order to further clarify the relationship between Septin and the infection structure and pathogenicity of the pathogen.The small molecule drug FCF(Forchlorfenuron)against the Septin cytoskeleton was used to preliminarily study the function of Septin.The results showed that the growth of the colony of S.turcica treated by FCF was inhibited,the aerial hyphae formed dense clusters,and the distance between hyphal septa increased.After 1 mM FCF treatment,the spore production of the pathogen was only 0.2×104/mL,the development of its appressorium was delayed,and the formation rate of infecting hyphae was weakened by 21%.4.According to the principle of homologous recombination,StSep4 gene knockout vector was constructed using plasmid pBS.Transformation of recombinant plasmids into protoplasts of S.turcica.After glufosinate ammonium(Bar)-resistant screening,sometrans-formants were obtained followed by identification with Bar sequence amplification and RT-PCR.Two strains of StSep4 gene knockout mutants ΔStSep4-1 andΔStSep4-2 were obtained.5.The deletion of StSep4 gene affected the growth,development and pathogenicity of the pathogen.On the PDA medium,Both ΔStSep4-1 and ΔStSep4-2 showed slower colony growth,less aerial hyphae and denser than the wild-type 01-23 strain.The mycelium morphology of the mutants showed different degrees of deformity.The wild-type strain was smooth and linear,while the mycelium of the mutant grew with greater curvature,and part or even the whole mycelium was wavy and don’t produce conidia.Stsep4 gene knockout delayed the process of appressorium induction from hyphae by the mutant,significantly reduced the appressorium formation rate,and the appressorium swelled abnormally,and the infection nail formation rate was significantly reduced.Compared with the wild-type strain,the ΔStSep4 mutant has a weakened ability to penetrate cellophane and formed smaller lesions on corn leaves,indicating that the StSep4 knockout mutant has a reduced pathogenicity.6.Deletion of the StSep4 gene reduces the tolerance of the bacteria to hyperosmotic stress.Under the treatment of 0.5M sorbitol and 0.5M NaCl,the ΔStSep4 mutants are more sensitive to hyperosmotic stress than wild type.The size and distribution of lipid droplets in mycelial cells are closely related to the regulation ability of osmotic stress.Histochemical staining of appressorium and hyp ha with saturated Oil red O(Oil-Red)showed that the lipid droplets(LDs)in ΔStSep4 mutants was enlarged,large and small lipid droplets coexisted,and the distribution was uneven.7.The StSep4 gene is involved in the formation of bacterial cell walls and the correct localization of chitin.Under the treatment conditions of 100μg/mL Congo Red(CR),50μg/mL CFW and 0.01%SDS,compared with WT,the tolerance of the mutant was significantly reduced,The chitin content in hyphae of the two mutant strains decreased by 41.23%and 41.85%,respectively.In order to verify the relationship between chitin content and StSep4,we tested the transcription level of its synthase encoding gene.The results showed that the transcription level of the chitin synthetase encoding gene was down-regulated inΔStSep4-1 and ΔStSep4-2 mutant strains,and the expression of StCHS3 in the ΔStSep4 mutant was significantly down-regulated(P<0.01),which was consistent with the changing trend of chitin content.CFW staining of mycelial cells found that the fluorescence signal of the mycelium tip of WT strain was stronger,indicating that chitin had a dominant aggregation phenomenon at the tip of the hyphae to provide for cell wall synthesis.In the ΔStSep4-1 and ΔStSep4-2 mutants,the fluorescent signal was evenly distributed in the hyphae,and only the signal at the hyphae septum was slightly stronger,the dominant aggregation of chitin in the mutants disappeared at the end of the hyphae,indicating that the deletion of StSep4 gene may affect the transport of chitin synthase to the end of the hyphae,thereby causing the aggregation of chitin in the cell wall to be blocked,which is one of the reasons for the slowdown of the growth of the mycelium.8.The deletion of StSep4 gene affects the nucleoplasmic synchrony.We co-stained wild-type and ΔStSep4 mutants with PI(nucleus specific dye)and CFW to observe the distribution of nuclei in the hyphae.It was found that the nuclei in the wild-type strain were evenly distributed along the hyphae,and clustered nuclei were observed in ΔStSep4,and the number was more than that of the wild-type strain.This further indicates the importance of StSep4 in the sclerotomy of S.turcica.9.Yeast two-hybrid verification of core Septins interaction.Connect the StSepl and StSep2,StSep3,and StSep4 genes to pGADT7 and pGBKT7,respectively,and transform the yeast strain AH109.Detect the growth of the colony on the SD-Ade-His-Leu-Trp plate to verify the interaction between StSepl,StSep2,StSep3 and StSep4.The results showed that there were interaction between StSepl.StSep2,StSep3,and StSep4,suggesting that Septin might play a role as a complex in S.turcicaIn summary,this study confirmed that Septin has a regulatory effect on the polar growth of S.turcica,especially the formation of invasive nails,and the four core Septins of the pathogen were obtained.Among them,the expression of StSep4 was significantly up-regulated during appressorium and invasion nail formation.It was preliminarily determined that StSep4 is the main Septin that regulates the formation of pathogenic appressorium and invasion nail.The ΔStSep4 knockout mutant was constructed to clarify the regulation effect of StSep4 on the polar growth,cytokinesis,and the development of infection structure of the mycelium of S.turcica.The results lay the foundation for the discovery of new pathogen-based disease prevention and control strategies. |
