Preliminary Study On Three-dimensional Structure Of Three Effectors From Haustorium Of Wheat Stripe Rust Fungus

Wheat stripe rust fungus,a serious fungal disease in the wheat production,whose haustorium is the only specialized infection structure.The fungus takes nutrients from the host by haustorium and delivers effector proteins directly into host cells in order to establish an intimate feeding relationship during infection.The whole immune process,the effector of wheat pathogen infection is very important.Therefore,it is very important to clarify the transport mechanism and mechanism of effectors.In this study,three effectors(pst-1374、pst-1178 and pst-8713)were cloned and expressed in E.coli BL21(DE3)cell.The target proteins were purified by Ni-NTA,HPLC and HiTrap Q column to electrophoretic purity.The three purified effectors were soluble in water buffer and suitable for the further structural studies.The feature structure of effector PST-1178 and PST-8713 was analyzed by CD and DLS.Finally,the solution structure of the effectors was analyzed by nuclear magnetic resonance(NMR)technique.(1)we had achieved three effectors of PST-1178 、PST-8713 and PST-1374,two of which have his tag on N-terminal.PST-1374 effector has no tag.We have constructed prokaryotic expression vectors,expression and purification.All the obtained proteins were identified by mass spectra and western bloting.It proved that they all were correct target proteins.(2)Conformation of PST-1178 and PST-8713 in different solution studied by means of circular Dichroism(CD)and dynamic light scattering(DLS),indicating that structure of PST-1178 changed easily,and usually transformed the folded conformation into random coil combined without target and PST-8713 has a little secondary structure.(3)The N-HSQC two-dimensional spectra of the effector PST-1178 at different salt concentrations were collected by nuclear magnetic resonance.We find different secondary structure.We had obtained the solution structure of PST-8713,consisted a little helix and random coil.The three-dimensional structure of PST-8713 was analyzed by software such as sparky,nmrpipe,talos,cyana and pymol by analysis the main chain spectrum and side chain spectrum data of NMR of effectors(4)Summary,The above experimental data can be inferred that the protein PST-1178 is Intrinsically Unstructured Proteins(IUP),and PST-8713 is a fusion-type Molten globule(conformational ensemble)in the absence of the target proteins.