Bioinformatical Analysis Of Functional Domains In Oomycetes Cytoplasmic Effectors And The Transcriptome Analysis Of Arabidopsis Ago4-2 Mutant

Oomycete is a type of unique eukaryotic microorganism in evolutionary history.Although it is similar with the fungi in nutrient absoption and morphology,the evolutionary relationship is more close with algae.Oomycete contains a large number of animal and plant pathogens.During infection,the oomycetes pathogenes can secrete a large number of RxLR(Arg-x-Leu-Arg)and CRN(Crinkle and necrosis)effectors to inhibit or interfere with the host plants immune responses,leading to promote its infection and colonization.Meanwhile,the host plants also regulate the expression of a series of denfense genes to resist the invasion of the pathogens,among which mediated by Argonaute(AGO)protein is an indispensable regulation component.Therefore,it's very important to indentify and analyse the domain types of RxLR and CRN effectors in oomycetes.Furthermore,the research on transcriptional regulation of AGO is also meaningful to understand the process of pathogens interact with host plant.This thesis regard effectors and Arabidopsis ago4-2 mutant as research objects,using the bioinformatics methods to predict and analyse their biological functions.The main results are below:1)The identification of RxLR and CRN effectors in oomycetes,and the analysis of thier DNA-binding domain:The motif structure of RxLR and CRN effectors shows that RxLR-dEER and FLAK-HVLVVVP conservative elements are existed in RxLR and CRN effectors respectively.According to those features,This study has also predicted 1811 RxLR and 1120 CRN effectors in 9 oomycete species respectively.The Pfam domain annotation shows that many DNA-binding doamin among those effectors,so we use three methods(GO annotation/PDB 3D structure/bioinformatics software)to verify.What's more,we select PsCRN115 effectors to generate transgenic Nicotiana benthamiana plants.According to the analysis of DGE tag profiling,we find the reasons of effectors mediates disease resistance and abiotic stress tolerance in transgenic plant.And also analyse the evolution of HNH(His-Asn-His)domain family and infer the CRN's pathogenicy keeps path with its evolution tendency.2)Analyse the Small RNA data of Arabidopsis ago4-2 mutant to inoculation with Pc35 and V991:We identify and predict the miRNAs from the genome of Arabidopsis and pathogenic oomycetes.We can directly calculate the expression of miRNAs in mirBase,and predict the novel miRNAs according to its secondary harpin structure and its minimum free-energy.The result shows that many novel miRNAs in our samples,we select the differentially expressed miRNAs and predict their target genes.The results shows that those miRNAs can target genes not only in Arabidopsis genome but also in pathogenic oomycetes.The GO annotation manifests that the target genes in Arabidopsis are involved in the pathway of signal transduction,plant hormones and transcription factors.etc,however,the target genes in pathogenic oomycetes are those PcCRN167 and PcAvh10/15 effectors.Due to the mutual interference of miRNAs between host plant and pathogens,the normal plant innate immune defense responses and the pathogenicity of pathogens might be highly affected.3)Analyse the transcriptome data of Arabidopsis ago4-2 mutant to inoculation with Pc35 and V991:Using the high-throughput sequencing to acquire the transcript profiling of Arabidopsis Col-0 and ago4-2 mutant to inoculation with P.capsici andV.dahlia for 10h,and obtain the RNA-seqs data of control samples inoculation with sterile water respectively.After the analysis of transcriptome data,we map clean reads to the Arabidopsis genome.After significant difference and gene expression pattern analysis,we find the degree of whole differently expressed genes in ago4-Pc/m are higher than ago4-V991/m.Furthemore,the GO annotation of those differently expressed genes manifests that the most of genes are related to the resistant pathway in Arabidopsis.Base on the mapman pathway cluster software,we find the resistant and biotic stress tolerance pathway are highly different between ago4-Pc/m and ago4-V991/m,and those differences are likely to be the reason of the Arabidopsis ago4-2 mutant are more resistance to Pc-35 and more susceptible to V991.