Molecular Mechanisms Of Phytophthora Sojae PsGPA1 In Regulation Of Zoospore Chemotaxis To Soybean

The detection of plant-specific molecules is critical for microbes to recognize and subsequently colonize potential hosts.Phytophthora sojae is the soybean pathogen responsible for the soybean root rot disease that causes around＄1-2 billion in losses per year and is a threat to soybean production all over the world.Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen P.sojae.Previously,in work exploring the mechanism of P.sojae chemotaxis,we performed a series of studies using gene silencing.First,we found that the heterotrimeric G-protein a subunit PsGPA1 controlled zoospore behavior,including chemotaxis to the isoflavone daidzein.However,there is hardly any knowledge about the mechanisms underlying the G-protein signaling events in Phytophthora.In this thesis,affinity purification and gene silencing method are used to explore molecular mechanisms of PsGPA1 in regulation of zoospore chemotaxis to soybean.In the present study,we identified several putative PsGPA1-interacting proteins,including PsHint1,and the function of PsHint1 was investigated based by the RNAi strategy.Screening and indentification of PsGPA1-interacting proteins in P.sojae:Chemotaxis of zoospores to isoflavones released by soybean has been observed in P.sojae and is thought to be important for root pathogens to localize host.We previously identified a G-protein a subunit encoded by PsGPA1 that regulates the chemotaxis and pathogenicity of P.sojae.To elucidate the function of PsGPA1 and identify PsGPA1-interacting proteins,we generated P.sojae transformants expressing 3×FLAG-tagged PsGPA1.The protein,isolated from sporulating hyphae of the transformants,was next purified by admixing total protein lysates with anti-FLAG M2 beads,and the bead-bound proteins were analyzed by MS(Mass spectrometry).Several proteins,such as Histidine triad nucleotide-binding protein 1(PsHintl),Acetyl-CoA synthetase(PsACS1)and Glutaredoxin(PsGrx1)co-precipitated with 3×FLAG-tagged PsGPA1.Glutathione S-transferase(GST)-PsGPA1 and 6×His-taged putative Ga-binding proteins were expressed(separately)in Escherichia coli and purified;a pull-down experiment followed.Physical interactions of PsGPAl with PsHint1 or PsACS1 were verified by this method.PsHint1,associated with the G-protein alpha subunit PsGPA1,is required for the chemotaxis and pathogenicity of P.sojae:We previously identified a G-protein a subunit encoded by PsGPAl that regulates the chemotaxis and pathogenicity of P.sojae.In the present study,we used affinity purification to identify PsGPA1-interacting proteins,including PsHint1,a HIT domain-containing protein orthologous to human HINT1.For testing protein interaction in vivo,we established a dual expression system.We constructed a vector expression PsHint1-6×HA and PsGPA1-3×FLAG fusion proteins and did the transformation.Using co-IP,we validated the association between PsHintl and PsGPA1.We also used in vitro GST pull-down assay to confirm that PsGPA1 interacts with PsHint1.The protein interacted with both the GTP-and GDP-bound forms of PsGPA1.An analysis of gene-silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein.During interaction with a susceptible soybean cultivar,PsHint1-silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants.In addition,the transformants displayed defective cyst germination,forming abnormal germ tubes that were highly branched and exhibited apical swelling.These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1,it participates in a Ga-independent pathway involved in the pathogenicity of P.sojae.Analysis of downstream signaling pathways regulated by pathogenicity related gene PsHint1:G-proteinαsubunit encoded by PsGPAl regulates the chemotaxis and pathogenicity of P.sojae.We identified a PsGPA1-interacting proteins,PsHintl,using affinity purification.The PsHint1-silenced transformants were hypersensitive to exogenous oxidative stress.During interaction with a susceptible soybean cultivar Hefeng 47,PsHint1-silenced transformants displayed significantly reduced infectious hyphal extension.DAB staining revealed the PsHint1-silenced transformants lost the ability on detoxification of the ROS at the infection sites.Inhibition of the plant NADPH oxidase by DPI prevented host-derived H2O2 accumulation and partially restored infectious hyphal growth of the PsHint1-silenced transformants in soybean cells.In addition,transcriptional patterns of P.sojae effectors were altered in PsHint1-silenced transformants.We analyzed of the RNA-Seq(Quantification)results about the PaHint1-silenced transformant and the wild-type strain P6497 at the stage of 3 hpi,and the results from RNA-seq had a well linear correlation with the results from qRT-PCR.In the RNA-seq results,most of the elicitin-like genes were down-regulated in the PsHint1-silenced transformant.Meanwhile,the transcription of many transcription factors also displayed changed expression level in the transformant.These results suggest that PsHint1 functions as a pathogenicity gene that might regulates the transcription of effectors,and compromises ROS-mediated plant defense.We also used affinity purification to identify PsHintl-interacting proteins.Heat shock 70 kDa protein,AGC protein kinase,importin alpha and several translation related proteins were involved in the MS results.Screening and fuctional analysis of potential PsGPA1 effectors in P.sojae:Many extracellular signals are detected by cell surface receptors and further transmitted inside the cell by G proteins,which serve as molecular switches.We previously identified a G-protein a subunit encoded by PsGPA1 that regulates the chemotaxis and pathogenicity of P.sojae.Among many proteins identified by affinity purification,some function as potential Ga-interacting proteins,such as PsHintl and PsACS1,although proteins identified so far are not sufficient to explain divergent functions of PsGPA1.For this reason,an improved way of affinity purification for better understanding of the Ga molecular mechanism is sorely needed.We generated P.sojae transformants expressing 3×FLAG-tagged PsGPA1 and isolated protein from sporulating hyphae of the transformants.We loading the lysates with GTPγS,a non-hydrolyzable analogues of GTP,which leading to the activation of the Ga subunits.From the MS results,many potential targets were identified,such as two protein kinases(PsSNF1 and PsYPK2),a serine/threonine-protein phosphatase(PsPPEF)and a Rab2 family GTPase(RABBI).Physical interactions of PsGPA1 with each candidate proteins were verified by GST pull-down.Transcription profiling of target genes showed that these four genes were all up-regulated in infection stages,indicating potential roles in pathogenicity.Three of the target proteins,PsSNF1,PsPPEF and PsYPK2,were identified in quantitative phosphoproteomics.In the present study,we used affinity purification to identify PsGPA1-interacting proteins,and then an improved way was carried out for better understanding of the Ga molecular mechanism.This method represents a powerful tool for native protein complex purification and characterization,and can easily be modified to purify other native protein complexes in Phytophthora.We identified Ga-downstream signaling pathway in Phytophthora.PsHint1,one of the binding proteins we screened,not only regulates chemotaxis by interacting with PsGPA1,it participates in a Ga-independent pathway involved in the pathogenicity of P.sojae.These results will help in unraveling the signaling networks that underlie the chemotaxis and pathogenicity of Phytophthora pathogens.