Analysis And Identification Of Partners Of Protease MAP From Rice Blast Fungus

Rice blast caused by Magnaporthe oryzae is the most important fungi diseasethat affects rice production and leads to enormous economic losses. In the previousstudy, mitochondrial AAA+protease in M. oryzae (MoMAP) was proved to beinvolved in the growth of infectious hyphae and regulation of pathogenicity. MAP iswidespread among organisms and plays a major role in cell cycle and differentiation,stress response and respiration regulation and so on. MAP was well studied but itspathogenicitiy mechanism and regulation network was not known. To further elicit therelationship between MoMAP and pathogenicity, we performed the followingresearch and finally set up MoMAP’s regulatory network. This work will provideimportant information for understand the molecular mechanisms of rice blast fungusinfection hyphae and host-pathogen pathogenic process, and will provide a scientificbasis for the effective prevention and control of rice blast fungus.1. Purification of MoMAP. The full length open reading frame (3360bp) ofMoMAP predicting a1120amino acid polypeptide was cloned by RT-PCR. Thededuced amino acid sequence of MoMAP (122kDa, pI5.45) displayed high homologyto AAA+protease family from other organisms. MoMAP was inserted to pETexpression vector and induced by IPTG in E. coli. The purification of recombinantMoMAP from E. coli was performed by Ni-NTA Agarose chromatography method.SDS-PAGE analysis of recombinant MoMAP revealed a molecular weight ofapproximately122kDa.2. Identification of MoMAP’s binding proteins. The purified MoMAP was usedto identify the binding protein by His-tag pull down and Tandem Affinity Purification(TAP). Totally,18proteins was extracted and identified by Maldi-TOF MS, whichincluded enoyl-CoA hydratase、ceramide glucosyltransferase、leucyl-tRNA synthetaseand so on. In addition, these two methods identified the same interaction protein: enoyl-CoA hydratase.3. Yeast two hybrid of MoMAP and binding proteins. To further prove therelationship between MoMAP and the proteins. All of the proteins identified frompull-down method was cloned and inserted into bait vector in yeast two hybrid system,which used MoMAP as a pray. The results show that enoyl CoA hydratase、C3HCzinc finger domain-containing protein、Zinc finger,C2H2type etc can bind toMoMAP.In conclusion, we purified MoMAP from E. coli and reveal the networkregulated by MoMAP by using two pull down methods. The net work was furtherconfirmed by yeast two hybrid. The result showed that MoMAP play important rolesin fatty acid metabolism、metal transporter、transcription、protein synthesis、sulphurmetabolism、lon channels and cell death. MoMAP and interacting proteins by manybiological pathways co-mediated regulation of the expansion of infection hyphae of M.oryzae and regulation of pathogenic.