Function Analysis Of The Map Kinase PSSAK1Pathway And Myb Family In Phytophthora Sojae

Phytophthora sojae is a soil-borne plant pathogen that causes stem and rot rot of soybean, which are different to control. Each year in the most soybean-growing regions of the world, millions of dollars are lost. Therefore, slowing the assault of P. sojae pathogens has been being the primary task of researchers. However, Phytophthora is a genus of Chromalveolata of Eukaryota. The genetic distance between omycetes and fungal is so large that most fungicides show no control effect to plant diseases caused by Phytophthora spp. Therefore, Phytophthora spp. form the pathogenic mechanism which is different with these phytopathogenic fungi. Recently, after the draft genome sequences of P. sojae were reported, P. sojae has been developed as a model species for the Phytophthora spp..In this thesis, bioinformatics approaches as well as gene silencing for functional study provided an effect method to identify the downstream regulated gene of PsSAK1. Based on the analysis of PsSAK1-silenced line DGE (3’-tag digital gene expression) profiling, we identified a number of candidates that may be regulated by PsSAK1. And with the P. sojae gene silencing technique, we confirmed the biological function of PsMYB1, which is regulated by PsSAKl. The significance of this paper is that we found PsMYB1transcription factor functions downstream of MAP kinase PsSAKl and is required for zoospore development and P. sojae virulence. According to our current knowledge, this would be the first report on the downstream mechanism of MAPKs in plant pathogenic Oomycete.Identifing the genes related in growth, development and pathogenicity of Phytophthora sojae is the effective method to explain the mechanism of infection processes and control the plant disease. In previous study, we identified two MAPKs that were important in growth, development and infection processes by researching the phenotype of the silencing lines of MAPK. A challenging step in further understanding the MAPK pathway is the identification of downstream genes that are regulated by MAPK and related to pathogenicity. Thus, in this study, the3’-tag digital gene expression (DGE) profiling method were applied to sequence the global transcriptional sequence of PsMAPK-silenced mutants during different stages. The results showed that, The differentially expressed genes encode potential transcription factors, especially Myb, bZIP, zinc finger; potential cellular regulators, such as protein kinases; regulators of RNA synthesis; structural proteins, including putative glycoside hydrolases and proteins involved in cell wall biogenesis; and pathogenic factors, including proteins responsing to stress,and elicitin. Loss of PsSAK1lead to higher rate of TFs was detected. Meanwile, the malfunction of cell-wall integrity MAPK PsMPK1, resulted in the expression level of some elicition proteins and Avh proteins down-regulated. Based on the result of PsMPK1-silenced line could not suppress the HR of host, We therefore hypothesized that loss of PsMPKl, P. sojae could not response to various signals of infection processes, nor regulate elicition proteins or Avh proteins coding genes expression, and at least part of this response is functionally important for virulence of P. sojae.PsSAK1, a mitogen-activated protein (MAP) kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1remains unclear. In this study, the3’-tag digital gene expression (DGE) profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and1.5h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down) in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1decreased due to PsSAKl silencing. The transcriptional level of PsMYB1increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1results in three phenotypes:a) no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b) direct germination of sporangia, and c) afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1transcription factor functions downstream of MAP kinase PsSAK1and is required for zoospore development of P. sojae.Researching for the orthologs of PsMYB1have provided evidence for P. sojae may have a Myb superfamily. Using bioinformatics, annotation of the P. sojae genome database, Fungal Transcription Factor Database (FTFD), SMART (http://smart.embl-heidelberg.de), and manual evaluation of gene models,68Myb TFs with variable numbers of Myb DNA-binding domains were predicted. Most (47) of the proteins have a single Myb domain(PsMYB-like1-1～PsMYB-like1-47), whereas the remaining9(PsMYB-like2-1～PsMYB-like2-9),10(PsMYB-like3-1～PsMYB-like3-10),1(PsMYB-like4-1), and1(PsMYB-like5-1) proteins have two, three, four, and five Myb domains, respectively. The transcriptome of Myb family in P. sojae was profiled at10different developmental and infection stages based on a DGE protocol. Nearly all of the genes were detected in at least one stage. The Myb family was further clustered by distance threshold of0.85into seven groups based on their transcript patterns. Comparing the differential expression level of each in the three libraries of two MAPKs-silenced lines, we hypothesized that the Myb family may play key roles in the infection processes.PsMYB1plays an important role in the processes of growth, development and pathogenicity of P. sojae. Therefore, we hypothesized that the other candidates may be play key roles in the infection processes. To test this hypothesis, the transcript patterns in5asexual stages and5infection stages of genes, the expression level of genes in DGE profil of silenced-lines were analysed and double-stranded RNA mediated gene silencing were used in this chapter to identify the biological function of4candidates of Myb family. They were PsMYB-like2-2(Ps127211), PsMYB-like1-7(Ps131312), PsMYB-like1-39(Ps141522), and PsMYB-like3-6(Ps133941), which were all up-regulated in the infection stages. And the expression level of all showed markedly change at least in one library of three silenced-lines DGE profils. Among the4genes, we found one R1Myb transcription factor PsMYB-like1-39(Ps141522), is related to the release of zoospore from the vacuole at the beginning of sporangium releasing, and loss of this gene could not affect the pathogenicity of P. sojae.