Study On Functionalmechanism Of Two RxLR Effectors From Phytophthora Sojae

Phytophthora sojae is an oomycete,which is a soil-borne plant pathogen causes stem and root rot of soybean,leading to a ?2 billion losses worldwide annually.As an aggressive hemibitroph pathogen,Phytophthora sojae has an initial phase of biotrophic growth require living host cells to support their obligate parasitism followed by a transition to necrotrophy.During the long-term interaction process between Phytophthora sojae and host plant,Phytophthora sojae deliver kinds of extracellular effectors into the plant cell to help itself promote infection,while plant generates resistance by resistance genes recognizing the effectors.So understanding the mechanism of plant disease resistance interfered buy effects,and clearing pathogenic ways and the target site in the host plant,can effectively provide new ideas and cirection of disease prevention and control strategies,and be of important significance to design and improve disease resistance.P.sojae secrets a large number of cytoplasmic effectors to modulate multiple signaling pathways in the host cells and promote P.sojae pathogenicity.The transcriptional level of GmPirin significantly up-regulated in the process of P.Sojae entrance host plant.After functional analysis of Avrlk and another RxLR effector Avh262,we found that Avrlk and Avh262 inhibit HR triggered by Avh241,INF1,Avh238 and Bax.It can promote P.sojae infection of the host plant.To illustrate the molecular mechanisms of the interaction between host plants and Avrlk/Avh262,screening and identifying effector in vivo targets is required.Firstly,To identify the protein interacting with Avrlk The experiment is to screening the target of Avrlk in the soybean library using yeast two-hybrid assays.We take Avrlk as bait protein and to screen soybean cDNA library by yeast two-hybrid assayas prey to identify the protein interacting with Avrlk.Finally we get got 200 positive clones.Among them,we found a very interesting protein GmPirin.The transcriptional level of GmPirin significantly up-regulated in the process of programmed cell death.overexpression ofGmPirin leads to enhance the infecting capability of P.capsicei we use yeast two-hybrid and Pull-down to verify the interaction between Avrlk and GmPirin.By constructing different mutant struncates,we found 21-128 and 222-280 amino acids of Avrlk are the critical region that responsible for the critical region in the interaction is consist of 21-128 and 222-280 amino acids of Avrlk.Co-expression of Avrlk or GmPirin with Bax in Nicotiana benthemiana can suppress plant HR triggered by Bax,indicating that the GmPirin might take part in the plant immune response,provided that GmPirin could possibly be the host target manipulating by Avrlk to suppress plant immune responses during,in order to achieve the promotion of P.sojae infection of host plants.Secondly,We showed that the RxLR effector PsAvh262 from P.sojae,which is highly expressed during early stages of infection.Using in plant co-immunoprecipitation(Co-IP)followed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),plant redundant Endoplasmic Reticulum(ER)-luminal Binding immunoglobulin Proteins(BiPs)from N.benthemiana warw identified as host target of Avh262.we futher confirmed the interaction between Avh262 and GmBiP1 by BiFC assay.