Localization And Biochemical Analysis Of Rab Proteins In Magnaporthe Oryzae

Rice blast disease is one of the most serious and recurrent problems affecting rice production worldwide. The disease is caused by the pathogenic fungus Magnaporthe oryzae (M. oryzae), which may serve as a model for understanding the growth and pathogenicity of filamentous fungi. The infection process of M. oryzae is complex, including conidia germination, appressorium formation and differentiation, colonization and expansion in the host tissues, and involves membrane transport, signal transduction, protein transport and other complex activities. It is shown that endocytosis is important for the pathogenicity of M. oryzae.Rab GTPases are molecular switches that regulate intracellular membrane trafficking on endocytic, exocytic and recycling pathways. Each Rab targets to a specific organelle and controls multiple steps of vesicular transport by alternating between GTP-bound "on" conformation and GDP-bound "off conformation with the assistance of upstream regulators and downstream effectors. Among the 11 putative Rabs identified from the M. oryzae genome database (MoRabs),6 MoRabs were cloned and expressed in mammalian cells, and their intracellular localization was determined, including MoRab1, MoRab8, MoRab11, MoYpt7, MoRab5A and MoRab5B. Considering the requirement of MoRab5B for the infection and pathogenicity of M. oryzae and the lethality of MoRab5A knockout mutants, we analyzed in-depth the biochemical and functional characteristics of MoRab5A and MoRab5B. Both MoRab5A and MoRab5B can co-localize with human Rab5 (hRab5) on early endosomes in the cell. Likewise, MoRab1, MoRab8 and MoRab11 can co-localize with their human Rab conterparts (hRabs), respectively, to the ER, exocytic vesicles and recycling vesicles, indicating that they are indeed corresponding Rab orthologs. In contrast MoYpt7 showed dispersed distribution in the cytoplasm, possibly due to limited post-translational isoprenylation and targeting to the late endosomal membrane.Furthermore, like mammalian Rab5, MoRab5A and MoRab5B can bind to several Rab5 effectors in a GTP-dependent manner, including EEA1, Rabenosyn-5 and Rabaptin-5. However, MoRab5A shows distinct binding characteristics in the sense that both the wild-type and the GTP hydrolysis-defective constitutively active mutant (CA) bind the effectors equally well in GST pull-down assays, suggesting that MoRab5A is defective in GTP hydrolysis and mostly in the GTP-bound conformation in the cell. Indeed, GTP hydrolysis assays indicate that MoRab5A GTPase activity is dramatically lower than MoRab5B and hRab5 and is insensitive to RabGAP5 stimulation. We have further identified a Pro residue in the switch I region largely responsible for the distinct MoRab5A properties by characterization of MoRab5A and MoRab5B chimeras and mutagenesis. The differences between MoRab5A and MoRab5B extend to their functions in the cell. Although they both target to early endosomes, only MoRab5B closely resembles human Rab5 in promoting early endosome fusion, stimulating fluid phase endocytosis and negatively regulating the Nerve Growth Factor (NGF)-induced neurite outgrowth. In contrast, MoRab5A correlates with another related early endosomal Rab, Rab22, in terms of the presence of the switch I Pro residue and the blocked GTPase activity. Our data thus identify MoRab5B as the Rab5 ortholog and suggest that MoRab5A specializes to perform a non-redundant function in endosomal sorting.In summary, MoRab5B and MoRab5A represent the functional orthologs of Rab5 and another member of the Rab5 subfamily, respectively, in M. orzyae during the evolution from the last eukaryotic common ancestor (LECA) to mammals. MoRabs may organize the transport of proteins and membranes on endocytic and exocytic pathways and play important roles in the development and pathogenicity of M.oryzae.