Interaction Machenism Research Of Stripe Rust Effect Protein Pst-1374 And Target Protein TaTrxm Deried From Wheat

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),Pst is a specific airborne fungus with the characteristics of easy infection and mutation.The emergence of new stripe rust fungus is a major cause of the large-scale outbreak of wheat stripe rust,causing a serious decline in wheat production.The traditional methods of prevention and control find the plants resistant to stripe rust by genetics to breed new wheat varieties resistant to the rust-like rust.However,due to the emergence of new races of pathogens,genetic methods cannot produce stable wheat varieties resistant to stripe rust.Stripe rust infects wheat by a specific infecting organ-haustorium.The haustorium is a highly specialized infecting organ formed by stripe rust in wheat during the process of infecting wheat.It forms a neck shape by the cell wall of the haustorium,the cell membrane,and the cell membrane of the host cell,making it a closed.The host cell is not a native structure.By secreting effector proteins to host cells,the haustorium changes the host’s defense response,thus absorbing nutrients and energy from the host and completing the wheat infection process.The effector protein is a small molecule protein produced by a pathogen that can change the structure and function of the host cell.It can interact with the macromolecule of the host cell to change the defense response of the host cell to achieve the purpose of infection.Pst-1374 contains 109 amino acids,including 6 cysteines,derived from Pst isolate PST-78.In previous studies,it was found that the transient expression of Pst-1374 in Ben’s cigarette inhibited BAX protein-induced cell necrosis,and that Pst-1374 also inhibited host effector-triggered immunity(ETI)response,which was manifested by the reduction of reactive oxygen species and necrosis area.Screening from the non-affinity cDNA library of stripe rust and wheat interaction and yeast double hybrid,Pull down identification,it was found that Pst-1374 can interact with wheat chloroplast thioredoxin TaTrxm and itself.Thioredoxin can untie disulfide bonds,Pst-1374 is rich in cysteine,and can interact with thioredoxin.Therefore,it is proposed that Pst-1374 interacts with TaTrxm to make the multimer open and exercise its function of interaction mode conjecture.In this experiment,biochemical and structural biological methods such as Gel Filtration Chromatography(GFC),thioredoxin activity measurement,Circular Dichroism(CD),Dynamic Light Scattering(DLS)were used to purify the effector protein Pst-1374 and the target protein TaTrxm in vitro using prokaryotic expression.The technique was used to determine the interaction mode between Pst-1374 and the target protein TaTrxm.The experimental results are as follows:1.The expression vectors of Pst-1374 and TaTrxm were successfully constructed,and Pst-1374 was purified by Ni column affinity chromatography and liquid chromatography.The expression and purification of TaTrxm were performed by amylose column to obtain electrophoresis-purified protein.2.The structure prediction shows that the overall structure of Pst-1374 is loose,only a small part of α-helical structure,most of which is irregularly curled;the α-helical structure of Pst-1374 can be induced by trifluoroethanol by CD.NaCl concentration can change the secondary structure of Pst-1374;DLS experiments show that Pst-1374 has two particle size molecules in solution.GFC experiments show that Pst-1374 is a multimer,and disulfide bonds participate in the formation of multimers.3.TaTrxm bovine insulin test proved its thioredoxin activity;TaTrxm and Pst-1374 reaction experiments proved that TaTrxm can degrade Pst-1374 by opening disulfide bond,which proves that Pst-1374 interacts with TaTrxm.The polymer opens to play function.From the above experimental results,we can prove that Pst-1374 is a multimer in solution through a disulfide bond.The TaTrxm can open the disulfide bond of Pst-1374 and depolymerize the polymer.These experiments results demonstrate that our proposed "Pst-1374 may form a multimer through a disulfide bond,and TaTrxm performs its function by depolymerizing Pst-1374 into a monomer by opening a disulfide bond" speculation.