Establishment Of The Haustorium Extraction System Of Wheat Powdery Mildew Fungus Bgt And Functional Verification Of Five Secreted Effective Proteins

Powdery mildew fungus of wheat is a kind of specific living nutritional parasite,which is a specialized wheat type of Brumeria graminis.In the process of compatibility and interaction between powdery mildew and host wheat,once the pathogen invades the host epidermal cells,it will form a highly specialized infection structure,namely haustorium.Powdery mildew haustorium is not only a place to absorb nutrients and exchange energy from the host,but also participates in the biosynthesis of pathogens to varying degrees,induces the expression of effector genes in the haustorium of powdery mildew,identifies disease-resistant genes of host plants and inhibits defense responses of plants.In this study,susceptible wheat cultivar L958 was used as experimental material to establish a method for isolation and extraction of wheat powdery mildew fungus haustorium.The wheat powdery mildew fungus haustorium with complete morphology and high RNA concentration was successfully isolated.In addition,based on the transcriptome sequencing results of wheat powdery mildew,candidate effector genes were obtained by bioinformatics method,and the secretory function of selected effector genes was studied by yeast secretion system.Results were showed as follow:1.High concentration and complete morphology of powdery mildew haustorium were isolated from wheat leaves inoculated with powdery mildew by column chromatography and gradient centrifugation.However,by comparing the number of haustoria isolated and the total RNA concentration of haustoria detected,it was concluded that the number of haustoria obtained by gradient centrifugation was about 1.89 x 10~5in 20 g fresh wheat leaves,and the total RNA concentration of haustoria was 324 ng/?L,The number of haustoria was about2.19×10~5and the total RNA concentration of haustoria was 725.8 ng/?L.It is proved that chromatography column method and gradient centrifugation method are two suitable methods for isolation and extraction of wheat powdery mildew fungus haustorium,and chromatography column method is better than gradient centrifugation method.2.On the basis of genomic study of powdery mildew in wheat,11 candidate genes of powdery mildew in wheat were predicted by signalP and TMHMM software,and the transmembrane region of effector protein was analyzed.The 11 candidate genes were secretory effector genes.The protein sequences of these candidate effector genes may contain a signal peptide sequence,and the probability of signal peptide is between 90%and 100%.Meanwhile,this signal peptide prompts them to target the endometrial system and export it to extracellular space.3.Total RNA of conidia,hyphae and haustorium were extracted respectively.Five effector-secreting genes were screened out from 11 candidate effector genes of powdery mildew fungus by RT-PCR and qRT-PCR.They were TID_seq6013,TID_seq1173,TID_seq4971,TID_seq2965 and TID_seq4396.4.The secretory function of five candidate effector genes highly expressed in powdery mildew haustorium was verified by yeast secretory system.The insertion of yeast pSUC2empty vector into the predictive signal peptide sequences of the five effector proteins of wheat powdery mildew can make yeast strain YTK12 grow well on CMD-W and YPRAA media,which indicates that the effector proteins encoded by these five effector genes are typical secreted proteins.