The Molecular Mechanism Of CAMP-PKA Pathway Signaling Regulates Mycelium Polar Growth Of Setosphaeria Turcica

Maize is one of the three major food crops in China,which occupies an important position in the agricultural production of our country,but it is deeply affected by Setosphaeria turcica.Setosphaeria turcica is a typical filamentous fungus which causes maize macrophyte disease,Its pathogenicity is regulated by the signal transduction pathway of cAMP.PKA,the second messenger of the signaling pathway,is a tetromer composed of four subunits,including two regulatory subunits R and two catalytic subunits C.The main purpose of this study is to identify the main functions of the structural genes in the PKA signaling pathway and their interactions with transcription factors in the pathogenic process of maize macrophytes,and systematically describe the signaling process of this signaling pathway.On the basis of the StpkaC2 deletion mutant obtained in the laboratory.the transcription factor Stflo8 deletion mutant downstream of the PKA signaling pathway was obtained through gene recombination and filamentous fungi transformation,and the gene function was analyzed through phenotypic differences.The interaction between the genes was verified by yeast two-hybrid system,imolecular fluorescence complementation,electro mobility shift assay and other test methods.The function of downstream gene StRab1 was determined by specific inhibitor test.The main results are as follows:1.Verify the interaction between StEFG1 and StpkaC2 through BiFC.Build pSPYCE(M)-StpkaC2,pSPYCE(M)-StEFG1,pSPYNE(R)-StpkaC2,pSPYNE(R)-StEFG1 four carrier recombination,two combination into agrobacterium GV3101 feeling states,injecting recombinant strains to leaf age of 15 d young in tobacco leaves,in the dark environment training in confocal microscope after 2 d fluorescence in tobacco leaves,in an injection of strains of tobacco leaf as a negative control.In the tobacco leaves of the injection combination of pSPYCE(M)-StpkuC2::pSPYNE(R)-StEFG1,the fluorescence signal of GFP could be seen,while no fluorescence signal was detected in the negative control,indicating that StpkaC2 and StEFG1 had an interaction relationship and could conduct signal transduction.2.Gene with protein ID 162018 was obtained by qPCR detection of ?SpkaC1 and ?SpkaC2,which were differentially expressed with WT strain and were related to mycelium growth.Sequence analysis showed that the gene encoding product had the highest homology with small G protein Rabl,which was named StRabl.Its expression level in strain ?SpkaC1 was 13 times that of strain WT,and higher than strain ASpkaC1,which was consistent with the previous transcriptome data.3.Verify the interaction between StEFG1 and StRab1 promoter by(EMSA).Lab early received StpkaC2 gene deletion mutants,analysis of mutant and wild type transcriptome data,to obtain a gene expression differences related to the growth of the mycelium and StRab1,will be StEFG1 prokaryotic expression,using IPTG inducing expression,active protein,extraction and purification of selection on StRab1 promoter sequences StEFG1 protein sequences of anchor anchor point before and after 30 bp as a probe,with bovine serum albumin(BSA)as the negative control,fumble incubate conditions,set different protein concentrations and DNA probe temperature,The non-denatured gel was used to separate the probe and the probe was stained with ethidium bromide.30 bp for the length of the probe,the probe to incubate StEFG1 protein,80 bp to 200 bp in the denatured gel between three main belt,showed that protein and probe to probe the migration rate of change,and as a negative control of BSA didn't change migration rate of probe,suggests StEFG1 the combination of protein and probe is the specificity.4.qPCR test was used to detect StRab1 gene expression in different stages of the infection process of maize macula.The relative expression levels of ?-tubulin gene were 0.24,1.14,1.35 and 4.51 respectively at spore stage,bud tube stage,appressorium stage and invasive nail stage.The expression level of StRab1 was the highest at the stage of invasion,indicating that StRab1 may play an important regulatory role in the growth process of hypha polarity.5.The function of StRab1 was verified by inhibitor test.Lab early received StpkaC2 gene deletion mutants,analysis of mutant and wild type transcriptome data,to obtain a gene expression differences related to the growth of the mycelium and StRab1,for clear appearance of mutant and wild type phenotypic differences,including chitin synthesis and distribution and the change of spore production ability,etc.,it's caused by the high expression of StRab1,design the specific inhibitors of Rabl test,after treated with inhibitors,deletion mutants of spore production ability get certain recovery,Confocal microscopy was used to observe the distribution of chitin in wild-type strain treated with inhibitor.6.The interaction between Stflo8 and StpkaC1,Stflo8 and StEFG1 was verified by yeast two-hybrid test.pGADT7-StpkaC1/StEFG1/Stflo8,pGBKT1-StpkaC1/StEFG1/Stflo8 vector,pGADT7/pGBKT7-Stflo8 and pGADT7/pGBKT7-StpkaC1,pGADT7/pGADT7/pGBKT7-StEFG1 recombinant plasmids were constructed.A total of 7 combinations were used to co-transform each group of plasmids into yeast AH109 competent cells and to select monoclones of resistant inverters.They were cultured on SD/-leu-trp and SD/-ade-his-leu-trp plates,and observed after 3 days.It was found that all the inverters could grow normally on the SD/-leu-trp plates,but only pGADT7-StpkaC1 and pGBKT7-Stflo8,pGADT7-Stflo8 and pGBKT7-StEFG1 could grow on the SD/-ade-his-leu-trp plates.This indicates that there is an interaction between protein Stflo8 and proteins StpkaC1 and StEFG1,and this interaction is specific.7.Created the Stflo8 deletion mutant and analyzed the function of the gene.Successful built Stflo8 knock out the carrier,using PEG mediated transformation of filamentous fungi,successful knockout mutant,comparing gene deletion mutant and wild type of phenotypic differences,analysis the function of the gene,knock out that gene,germs spore production quantity is almost zero,wild type in hydrophobic medium culture 36 h can form into nail.mutant without this feature,and knock out that gene after the germ of melanin synthesis ability is affected.8.Expression of Stflo8 protein was induced by prokaryotic induction,and proteins interacting with the gene downstream were fished by pull-down technique.Analysis results,will through the pull-down fish for protein mass spectrometry analysis,obtained more than 240 proteins in the ncbi compare analysis,clear its function and gene families,statistics found that 6.67%were glycoside hydrolase(glycoside hydrolase),4.44%of glycosyl hydrolase(glycosyltransfersae),2.22%carbohydrates esterase(carbohydrate esterase).the rest of the protein has not yet been marked its function.In this paper,the signaling network of the cAMP signaling pathway was mainly combed to clarify the interaction between genes in this pathway,and the functions of important genes in the pathogenic process of bacteria were analyzed,so as to provide certain reference information for inhibiting the pathogenicity of bacteria.