Screenings Of Proteins Interacting With Pik-H4/Avr-Pik By IP-MS And Primary Study Of Their Functions

Rice blast,caused by ascomycotina filamentous fungus Magnaporthe grisea,is a worldwide fungal disease that seriously threatens the world’s food security.The interaction of rice-Magnaporthe oryzae is known to follow a classical gene-for-gene system.By screen-ing of disease resistance genes and Avr gene interaction protein separately,we can explore the signal pathway and show the interaction mechanism between host and pathogen.Pik-H4is composed of Pik₁-H4 and Pik₂-H4,which shows high resistance to the physiological race of Magnaporthe grisea in Guangdong.The Pik₁-H4 protein contains the NBS-LRR domain and the Pik₂-H4 contains the NB-ARC domain,its play an important role in the plant’s imm-une system.As the physiological traits of rice blast fungus are susceptible to genetic variat-ion,it is necessary to clear the mechanism of disease resistance to solve the problem funda-mentally,which provides a new way for disease breeding.In this study,We screened Pik-H4 and Avr-Pik interacting proteins mainly by immunop-recipitation techniques based on the protoplast transient expression system.The mainresults are as follows:1.In this study,we planted rice seeds in 1/2 MS medium about 8-10 days,to obtained rice protoplasts by digesting rice leaf sheath with cellulase,transformed transient vector into cells by PEG4000,the target protein will be highly expressed at 14-16 h.Through experimen-ts,the best time to digest is 4-6 h and the most suitable time for transforme is 14-16 h.After the dark culture cells also have a high vitality by FAD dyeing.The concentration of protein was up to 10 mg/ml by BCA method,which was in accordance with the requirements ofCo-immunoprecipitation.2.By fusion of the target protein with GFP transforme to protoplasts.Pik₁-H4 wasmainly located in the endoplasmic reticulum,Pik₂-H4 was mainly located in the plastid and Avr-Pik was mainly located in the cell membrane by laser laser scanning confocal microsco-pe.The Avr-Pik protein is localized on a rice cell membrane,it is presumed that the protein may be first accumulated on the cell membrane that is transported by some form to the host cell as an exciton to trigger a series of reactions.From the subcellular localization resultspresumption,Pik₁-H4 may be mainly involved in the recognition of Avr-Pik protein and signal transmission,Pik₂-H4 mainl-y play a role in changing the energy transmission and regulation of downstream disease caused hypersensitive reaction.3.In this study,We were sequenced by MALDI-TOF gel band mass spectrometry,Pik₁-H4 gained 4 interaction protein,and Pik₂-H4 obtained 6 interaction protein.Indicating that Pik-H4 and lectin-related proteins have a strong interaction.It was also found thatPik-H4 and lectin proteins had strong interaction.Sequencing by QE solution,Pik₁-H4 gaine-d 45 interaction protein,and Pik₂-H4 obtained 57 interaction protein,Avr-Pik gained 36 inte-raction protein.4.Through the bioinformatics analysis of DAVID online database,it was found that the interacting proteins were divided into four types of biosynthesis,energy transfer,transcripti-on regulation,membrane related protein.In the analysis of the GO cluster,the molecularfunctions mainly focused on the catalytic properties,and the biological pathways mainly focused on the metabolic process.In the cytological components,the interacting proteins were mainly involved in the cell composition,mainly related to nucleic acid binding and transport-related proteins.GO cluster analysis results are similar to those of DAVID datab-ase,and most proteins are related to energy transport and nucleic acid binding.It is presum-ed that screening has a great research value with transcription factor-related proteins and energy transfer proteins associated with Pik₂-H4.