Studies On Rab5 Interaction Proteins In Magnaporthe Oryzae

Rab proteins are the GTP-ases with small molecular weight. They are members of the largest subfamily in Ras superfamily, composing of about 200 amino acids. Rab proteins in eukaryotes are composed of the conserved G-domain and highly variable N-terminal and C-terminal. Protein transporting intracellularly is accomplished by vescicle trafficking. Rab proteins play important role during the process of vesicle transport as molecular switches of membrane trafficking with specific interactions with its upstream regulators and downstream effectors. Rab5 is localized in early endosomes and responsible for regulation the fusion of vesicle eith early endosomes.It is shown that MoRab5 in M. oryzae may be involved in the regulation of infection disease in our previous studies. To study the interaction proteins and its regulatory network, we constructed the constitutively activated MoRab5-BD bait vector, and obtained MGG₀5480, MGG₁0315, MGG₀6684, MGG₀6683, MGG₀6884, MGG₀6587 and MGG₀3136 in total 7 genes may interaction with MoRab5 by yeast two-hybrid from M. oryzae cDNA library. The expression of MGG₀6587 gene was decreased significantly in MoRab5 deletion mutant by qRT-PCR detection. The expression of MGG₀6587 in conidia and appressoria was decreased significantly in MoRab5 deletion mutant. This further proved there is some interactions between MGG₀6587 and MoRab5.The pET-28a-MoRab5 fusion protein expression vector was then constructed to study MoRab5 interaction in vitro. pET-28a-MoRab5 fusion protein were induced purified and applied to pull-down its interaction proteins from the total proteins of M. oryzae. In total, 40 proteins were obtained through pull down and LC-MAS analysis possibly to interact with MoRab5. Six proteins are annotated as a mitochondrial F1 ATPase subunit alpha, a ATP synthase beta chain in mitochondrial, an elongation factor–alpha, an ADP-ribosylation factor and a trehalose 6-phosphate synthase subunit and actin, but others are hypothetical proteins in the M. oryzae protein database. When BLASTing these proteins in the yeast database, 9 homologous proteins were found in yeast which are demonstrated to interact with yeast Rab5 protein. The homologous proteins encoded by MGG₀0431 in yeast is an essential beta'-coat protein of the COPI coatomer, involved in ER-to-Golgi and Golgi-to-ER transport, suggesting MGG₀0431 is also a MoRab5 interacting protein in M. oryzae.In summary, we successfully found some MoRab5 interacting protein in this study. It provides a good foundation for further screening the effectors of MoRab5, and also for identifying the synergistic relationship between MoRab5 and its effectors during the signaling pathway. It is also helpful to better understand the molecular mechanism of MoRab5 in regulation of infection process of M. oryzae.