Functional Relationship Between MoRab5 A And MoRab5B And Their Downstream Effectors Screening In Magnaporthe Oryzae

The rice blast fungus Magnaporthe oryzae can infect various important crops,especially rice,and it is also an important model organism for studying biology of filamentous fungi.Rab GTPases belong to Ras superfamily,and act as molecular switches to regulate intracellular tracfficking process such as vesicle formation,transport and fusion.There are three human Rab5 homolog,Ypt51,Ypt52 and Ypt53 in Saccharomyces cerevisae,which are involved in early endocytic pathway.MoRab5A and MoRab5B in M.oryzae are homologous protein of Ypt51 and Ypt52.Point mutation and RNAi mutants of and showed similar defects in growth,conidiation and pathogenicity on barley and rice in previous study.In order to know if MoRab5A and MoRab5B possess redundant function,we overexpressed them in wild types strain Guy 11 separately,and at the same time,we overexpressed MoRAB5A in MoRab5B RNAi mutant and MoRAB5B in MoRab5A RNAi mutant respectively.The results showed that overexpression of MoRAB5B led to significant defects in growth,conidiation and pathogenicity on rice and barley,while overexpression of MoRAB5A did not result in obvious defects.Overexpression of MoRAB5A in MoRab5B RNAi mutants could not restore growth,conidiation and pathogenicity deficiencies,and vice versa.All the results suggested that MoRab5A has not functional overlapping with MoRab5B in these aspects.To screening the downstream effectors of MoRab5A and MoRab5B,we conducted transcriptome sequencing using their dominant negative(DN)and constitutively active(CA)strains.There were 507 genes upregulated in MoRab5A CA but downregulated in MoRab5A DN,292 genes upregulated in MoRab5B CA but downregulated in MoRab5B DN.Among these genes,there are 47 genes upregulated both in MoRab5A CA and MoRab5B CA but downregulated both in MoRab5A DN and MoRab5B DN simultaneously.Their expression pattern may suggest they are possibly the downstream effectors of MoRab5A and MoRab5B.Furthermore,some genes shared the same expression patterns between MoRab5A CA and MoRab5B CA,or between MoRab5A DN and MoRab5B DN,indicating MoRab5A and MoRab5B may regulate some similar gene network.Most of the differentially expressed genes above are involved in cell development,metabolism and intracellular trafficking process through GO(gene ontology)analysis,and some of them are involved in unique cellualr process belonged to MoRab5A or MoRab5B.These results demonstrated that MoRab5A and MoRab5B have both functional similarities and differences in regulation of gene network and the cellular processes.This provides a good theoretical bases for understanding the functional mechanism of MoRab5A and MoRab5B and screening their downstream effectors.