Screening Of Dimorphic Switch Inhibitors In Sporisorium Scitamineum And Its Mechanism

The morphological changes of pathogenic fungi are critical to their path-ogenicity.The transition from the single cell yeast form to the infective filamentous one is crucial for Sporisorium scitamineum.The dimorphic switch of fungi is that fungi senses environmental signal factors through its signal transduction system and transfers it to cells,causing a specific functional gene expression process.Ustilago is a typical dimorphism fungus,while its haploid is a non-pathogenic yeast-like,mating leads to the formation of a dikaryon filament,dikaryon filament can infect the host and cause disease.Screening from the natural product with a new type of active compound that inhibits the dimorphic switch of S.scitamineum,it is important to clarify the target and mec hanism of the compound to clarify the signal transduction pathway of dimorphic switch in S.scitamineum.In order to solve this scientific problem,we screened from one hundreds of compounds available in the laboratory,including organic synthesis sources and natural extractionsources,found that a new class of phenolic compounds has a significant inhibitory effecton the dimorphic switch,thus greatly reducing its pathogenicity.The mechanism of the inhibitory activity of these compounds was preliminarily determined by transcriptone sequencing and analysis,real-time fluorescence quantitative analysis,and target gene knockout complementation.Listed below are the key research findings.1.The compounds with inhibitory effect on the dimorphic switch of S.scitamineum were screened by 24-well plate method.Among the more than 100 compounds,a class of phenolic compounds has been screened to significantly inhibit the dimorphic switch of S.scitamineum,with a MEC between 0.9 and 42 ?m.Wherein the compound PB-1 has the best activity(MEC value is 0.92 mM).The compounds had no effect on the growth of S.scitamineum at the lowest effective concentration.In addition,the compound PB-1 also inhibited the teliospores germination of S.scitamineum.2.Transcriptome analysis of MAT-1 Ss17 treated with PB-1 for 4h,8h and 12 h,was carried out with the corresponding control group without PB-1 treatment.The results showed that most of the differentially expressed genes were eniched in amino acid metabolism,especially tyrosine metabolism and histidine metabolism.The real-time fluorescence quantitative PCR analysis was carried out to determine the pra1,prf1,b E and mfa1 gene expression.In combination with transcriptome analysis,the effect of the compound PB-1 was confirmed in the c AMP-PKA signal pathway.3.The full-length sequences of two mating genes MAT-1 and MAT-2 Ss16 were obtained by homologous gene cloning technique.MAT-1 and MAT-2 Ss16 were both 1346 bp in length.Phylogenetic analysis was carried out on the Ss16 gene sequence with Ustilago maydis,Sporisorium reilianum,Ustilago bromivora,Kalmanozyma brasiliensis,Pseudozyma hubeiensis,Ustilago hordei,Moesziomyces antarcticus,Melanopsichium pennsylvanicum.The results showed that Ss16 gene was homologous to S.reilianum and Moesziomyces antarcticus in the same branch,and with U.maydis,U.brom ivora and U.hordei is relatively distant,but with a large evolutionary cluster.4.MAT-2 Ss16 of S.scitamineum was deleted by homologus recombination mediated by Agrobacterium tumefaciens.Through the mating test,the results show that the deletion of Ss16 gene influenced the mycelialization of S.scitamineum.The expression of pra2,prf2 and mfa2 in the mutant of Ss16 gene was significantly decreased by RT-q PCR analysis.The pde1,pde2 and Ubc1 were up-regulated.These results indicate that the Ss16 gene is associated with c AMP-PKA signal pathway.The above results indicate that PB-1 has the potential of being developed into a new type of biocontrol agent.Without the inhibitory the growth of S.scitamineum,PB-1 may affect the normal expression of mating genes,pra1,prf1,mfa2 and b E,to further affect switch from a non-pathogenic yeast-like form to polarized pathogenic filament,and then ultimately reduce its pathogenic ability to achieve effective control effect.