Screening And Identification Of Interactive Proteins Of E3 Ubiquitin Ligase Subunit Mogrr1 Using Yeast Two Hybrid In Magnaporthe Oryzae

Rice blast disease,caused by Magnaporthe oryzae,leads to significant losses to the global rice production.As one of the most extensively distributed and harmful disease in the rice of the world,this disease is also known as "rice cancer" with occurring throughout the growth period and scattering symptoms across various parts of rice.The availability of the genome sequences of M.oryzae provided a theoretical basis for further exploring the pathogenesis of M.oryzae and investigating relevant virulence genes,which lays a foundation for the scientific control of rice blast disease.The ubiquitin proteasome pathway is extensively involved in a series of physiological processes of eukaryote,such as cell cycle,genetic expression,transcription regulation,signal transmission,immunization and so on,through the combined effects of ubiquitin protein and three ubiquitin enzymes.Among the three ubiquitin enzymes,E3 ubiquitin ligase assumes recognizing the target protein and then connecting it to the ubiquitin protein,and it plays an important role in the ubiquitination process.Through the preliminary work in laboratory,it was found that E3 ubiquitin protein subunit Mogrr1 was an important factor in regulating the development and pathogenicity of M.oryzae.For further analyze biological function of Mogrrl in M.oryzae,the yeast two hybrid method is used in this study to screen the cDNA library of M oryzae using Mogrr1 protein as a bait.In total 28 proteins that may interact with Mogrr1 were preliminarily obtained from cDNA library of M.oryzae,and then co-transformation was further conducted for those preliminarily screened proteins via the yeast two hybrid method.The results indicated that Mogrr1 had a strong interaction with MoPP2Ac（MGG₀6099）and Mougal（MGG₀1662）,but a weak interaction with Moppel（MGG₀3911）and MoPP2Ab（MGG₀5637）.Moreover,the prey vector of MoPP2Ac and the bait vector of Moppel,MoPP2Ab,Mouga1 were respectively co-transformed into yeast AH 109.As revealed by the results,MoPP2Ac also had an interaction with Moppel,MoPP2Ab,Mougal.Furthermore,the F-box domain and LRR domain of Mogrr1 were deleted severally in this study.Based on the test through yeast two hybrid,it was discovered that Mogrr1 did not interact with MoPP2Ac and Moppel when F-box domain was deleted;while the interaction between Mogrrl and MoPP2Ac/Moppe1 was not affected by the deletion of LRR domain.To sum up,four proteins that interacted with Mogrr1,including MoPP2Ac,Moppel,MoPP2Ab,Mougal,were acquired through the yeast two hybrid method in this study;and the co-transformation revealed there was interaction between MoPP2Ac and Moppel,MoPP2Ab,Mouga1.Meanwhile,the study clearly indicated that F-box was the key domain for affecting the interaction between Mogrrl and MoPP2Ac,Moppe1.Thus the study laid a solid foundation for screening and identifying the interacting protein of Mogrr1 subsequently and exploring the molecular mechanism of Mogrr1.