The Study Of The Molecular Mechanism Of Dry Mycelium Of Penicillium Chrysogenum Induced Resistance In Arabidopsis And The Function Of Gene AT1G13520

Dry mycelium of Penicillium chrysogenum,DMP means the water extract of dry mycelium of Penicillium chrysogenum,a biological control agent,can protect plant from disease by inducing resistance and activating system acquired resistance in plant.DMP has been widely used on the crops such as rice,tobacco,Panax pseudoginseng,and tomatoes in the field.The area that DMP has been used in the field has reached more than 3000 km~2in the past 5 years and the control effection of DMP is better than most of other bio-control agents.Nevertheless,the mechanism of DMP induced SAR remains unknown.In this paper,we examined the variation of transcriptome in Arabidopsis thaliana(L.)Heynh under the treatment of water,jasmonic acid(JA),DMP or salicylic acid(SA)respectively to reveal the signaling pathway induced by DMP,and analyzed the major signaling pathways induced by DMP,then compared the differences of the differentially expressed genes(DEGs)between the plant treated with SA and JA.Furthermore,we verified the variation of genes involved in some pathways by using Quantitative Real-time PCR(q RT-PCR).On the other hand,genes with unknown function were annotated and predicted by online database.Knockout and overexpression Arabidopsis mutants were acquired by using CRISPR/Cas9 and over-expression system.Function of the gene was explored by the phenotypes of the mutants.The main results are listed as following:(1)RNA-Seq based transcriptome analysisArabidopsis under different four treatments including DMP,SA,JA and water were using for m RNA sequencing.23,153,931,24,526,880,22,357,353 and 22,357,353reads were detected in DMP,SA,JA and water library.After assembling the high quality sequencing reads into unigenes,gene expression levels were evaluated according to transcript levels calculated in FPKM from transcriptome data with 19577,19802,19746 and 19742 genes expressed in treatment water,JA,DMP and SA,respectively.We identified 1018,445 and 668 DEGs with at least 2-fold change after JA,DMP,and SA treatment compared with the water control(CK),respectively.The DEGs were annotated against the GO(Gene Ontology),COG(Cluster of Orthologous Groups of proteins)and KEGG(Kyoto Encyclopedia of Genes and Genomes)database.Enrichment in different functions are basically the same in the ratio of DMP vs CK,SA vs CK and JA vs CK by GO and COG analysis.The involved pathways of different groups show the result that the DEGs after DMP treatment were significantly enriched in three metabolism pathways shared by SA treatment and one additional pathway shared by JA treatment.Key DEGs,including PR1,EIN3 and FRK1,in the SA,JA/ethylene(ET)and pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI)pathways for inducing resistance were significantly regulated.These result shows that the genes expression in Arabidopsis treated by DMP was similar with SA treatment.And the DMP treatment can induce the crosstalk of the signaling pathway in Arabidopsis.(2)Verification of the pathway induced by DMP in ArabidopsisThe genes involved in SA,JA/ET and PTI were further confirmed by q RT-PCR in Arabidopsis treated with DMP.EDS5 and NIMIN1 are upstream regulatory genes,after DMP treatment this two genes were up-regulated at 24 h and 48 h.The positive regulator NPR1,NPR3 and NPR4 were consistently up-regulated at 24 h.Genes involved in the downstream of each pathway,including PR1,PR2 and PR5 were significant enhanced at 24-72h.TGA1-7 were further analyzed using q RT-PCR,and the results showed that all TGAs,and particularly TGA1,3 and 7,were up-regulated.We also detected the JA-and ET-responsive genes from 0-72 h and found that the expression levels of EIN2,ERF1 and PDF1.2 were enhanced by DMP,while the expression of MYC2 and VSP2 were decreased.It means that DMP can induce the expression of genes involved in SA and JA signaling pathway in Arabidopsis.Furthermore,SA accumulated significantly after DMP treatment and SID2 and FRK1involved in PTI pathway also showed increased expression,which means DMP induced genes expression in SAR via PTI.It has been the first time proved that the DMP-induced resistance in Arabidopsis is regulated by PAMP-triggered immunity activated SA signaling pathways and also have a crosstalk with JA/ET pathway.Our results provide guidance for further identification of the activators in DMP.And it also provides more molecular theory supporting the application of DMP in fields.(3)Gene function prediction and exploration of AT1G13520By transcriptomic data analysis,we inclined to screen out gene which can be induced by different elicitor,but the function is still unclear.In particular,one unigene AT1G13520 was screened out.Experiments showed that AT1G13520 responds to different treatments and stresses.The expression of this gene was increased after SA,ABA and DMP treatment in Arabidopsis and was down regulated after JA treatment.This indicated that this gene may play a role in signaling transduction.At the same time,AT1G13520 has different response patterns to cold and drought treatments.By DNA cloning and sequencing,we found that one nucleotide is different from the information in NCBI.The protein of AT1G13520 was expressed using prokaryotic expression system for the preparation of antibodies.Additionally,the online prediction showed that AT1G13520 function was related to SA mediated signaling pathway.Through structural analysis,the structure is similar to a toxin protein.According to previous yeast two-hybrid results,the function was related to energy metabolism.These results support a reference for the gene function identification.In order to explore the function of the AT1G13520 gene,we built knockout mutants and overexpression mutants in Arabidopsis by using CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats/CRISPR associated systems 9)system and overexpression technology,respectively.Knockout mutants can prematurely terminate the translation of AT1G13520.q RT-PCR results showed that the m RNA level of AT1G13520 in the knockout mutant was significantly lower than the control.In addition,q RT-PCR result showed the genes expression in overexpression mutants was slightly higher than the control.Phenotype shows that rosette leaves of knockout mutants were significantly larger than wildtype control,and the rosette leaves of overexpression mutants showed slightly smaller than control group.It means AT1G13520 could regulate the growth of Arabidopsis.Moreover,the time of flowering was advanced in knockout mutants.In addition,comparing with the wildtype plants,expression of SID2,WRKY70 and PR1,regulation and response genes involved in SA signaling pathway,were all significantly repressed in knockout mutants.On the other side,when treated the knockout mutant with SA,endogenous SA is not enriched.These results showed that AT1G13520 can affect the growth of Arabidopsis.Furthermore,AT1G13520 may activate SA signaling pathway by regulating the upstream genes and it is consistent with the prediction result.