Analysis Of The Quantative Phosphoproteome And Functional Analysis Of PsMPK7 And Identification Of The PsMPK7-interacting Proteins In Phytophthora Sojae

Phytophthora sojae is one of the representative plant pathogen in oomycete,the soybean root rot it caused is one of the devastating diseases of global soybean production areas,and it will cause the loss of billions of dollars each year.Although the mycelium morphology of Phytophthora sojae is similar to many fungi,but it is evolutionarily fared to them,so often the pathogenic mechanism of Phytophthora sojae is very different with fungal pathogenesis,resulting in the majority of fungicides ineffective.Therefore,the primary task of researchers is to design new medicines in view of the unique pathogenic mechanisms.Because the genetic manipulation of Phytophthora is difficult,it is costly,inefficient and time-consuming to use traditional methods to study them.So we need the help of a high-throughput proteomic with transcriptome analysis to help us to find the key genes efficiently.In this study,we analyze the result of the quantitative phosphoproteomics of the stage of infection and sporangium in Psojae,we gained a lot of information about phosphorylated proteins.Based on the study,we found a unique PsMPK7 from large amounts of data,it can be phosphorylated in the stage of mycelium,infection and sporangium.To research the function of PsMPK7,we firstly corrected its gene modle,then made it silenced and studied its pathogenicity-related phenotypes.We found the gene not only involved in the virulence,the sexual reproduction and the response of the external stress,but also the activity of the extracellular laccase.Based on all of this,we used the yeast two-hybrid and affinity purification method to verify its interaction capabilities.The main contents are as follows:Large-scale quantitative phosphoproteomics study of infection and sporangium in P.sojae:In this study,we identified 4577 phosphorylation sites in the stage of infection and 3353 were identified in the stage of sporangium,compared to the previous qualitative phosphoproteomics which identified 1795 phosphorylation sites it has increased significantly.In the mixed sample of infected mycelium and mycelium,we identified 42 effectors(including 28 RxLRs,five NLPs,seven CRNs and two elicitins),14 ABC transporter proteins,20 transcription factors and 107 protein kinases were phosphorylated.In the mixed sample of sporulating mycelium and mycelium,we identified 16 effectors(one NLP and 15RxLR),9 ABC transporter proteins,15 transcription factors,47protein kinases were phosphorylated.After the analysis of the phosphorylated level in the mixed sample,we found some of the proteins' phosphorylated level has changed,some did not change,and the protein only in a stage of mixed samples by phosphorylation,can not be quantitative.This is the first report about quantitative phosphoproteomics in P.sojae,provides a basic data for the interaction between plant and oomycete.Functional analysis of PsMPK7 and identification of the PsMPK7-interacting proteins in phytophthora sojae:From the quantitative phosphoproteomics,we found an PsMPK7,it can be phosphorylated in both the stage of mycelium and infection.In the transcriptome.PsMPK7 is showed upregulated in the zoospore,cyst,and cyst germination stages.After the correction of the gene model,I have used the approach of gene silencing to make PsMPK7 silenced and study its function,and found the mutant of PsMPK7 affected oospore production,and the invasion of unwounded or wounded hypocotyls of susceptible soybean.We further found that the gene silencing also affects the laccase activity.In order to find which MEK or MEKs can phosphorylated MAPK7 playing an import role in signal transduction,we first used the yeast two-hybrid methods to validated four MEKs' and MAPK7 relationship and found that they are not interacted with each other.But that does not mean that they really having nothing to do with each other,the reasons to explain the result might be,Firstly,MEK and MAPK' interaction is instantaneous,which make the yeast two-hybrid system is not suitable for such a short transient interactions;Secondly,MAPK had no direct interaction relationship with MEK,in P.sojae may exist middle proteins involved in the interaction of the two;Thirdly,the proteins are imcomplete,it is also the possible reason that they do not interact with each other.We want to know which proteins can be regulated by PsMPK7.So we choosed the method of affinity purification to pull the proteins which can interact with the PsMPK7 in vivo of P.sojae.We inditified 113 proteins,including:protein kinases,14-3-3-like proteins,the biological significance of their interaction as well as one by one interaction verification remains to be further studied.