Biochemical And Molecular Characterization Of A Putative Ferulic Acid Esterase In Magnaporthe Grisea

Plant cell walls are pivotal battleground between microbial pathogens and their plant hosts. Micrbial pathogens secrete an array of cell-wall degrading enzymes (CWDEs) to deploymerize the noncellulose polysaccharides of primary cell walls to facilitate colonization of the host tissue. To better understand their role in the pathogenesis, a putative feruloyl esterase from Magnaporthe grisea was characterized.Multiple alignment shows that the protein eccoded by gene MGG₀1403.5 in M.grisea (named MgFAEA, for M. grisea ferulic acid esterase A) has high level of homology to ferulic acid esterase A (EC 3.1.1.73) from Penicillium funiculosum. The two proteins share a conserved bacterial poly(3-hydroxybutyrate)depolymerase domain. Tested by the Signal3.0,TMHMM2.0 and ProtComp6.1 programs, MgFAEA contains a secretory signal peptide, without transmembrane structure, and is located mainly in extracellular matrix. Expression of the recombinant MgFAEA-HIS₆ confirmed that the MgFAEA is a secreted protein. Assayed by FESM (feruloyl esterase screening medium) agar plate showed that ferulic acid esterase activity was enhanced in MgFAEA over-expressed transformants.Analysis of RT-PCR shows that MgFAEA constitutively expressed in asexual development of M.grisea. During infection stage, the expression level of MgFAEA was greatly increased at 168hpi. MgFAEA over-expression transformants and knockout muants were shown to be as virulent as the wild type strains in infecting their rice host. On the FESM agar plate, feruloyl esterase activity was indistinguishable between⊿mgfaeA mutant and wild type strain Guy11, which suggested that redundancy may exist in ferulic acid esterase from M. grisea.Furthermore, based on BLASTP search in M. grisea genome database, three hypothetical proteins with high homology to MgFAEA were retrieved. RT-PCR was used to analyze expression profile of these genes in the wild type strain and⊿mgfaeA mutant. The result indicated that MGG₁0040.5 transcripts were greatly increased in⊿mgfaeA-infected rice leaves, which implied that MGG₁0040.5 would have complementary function for MgFAEA. The finding in our study will provide the basis for further study of the biochemical properties of the ferulic acid esterase and its relative function in fungal pathogenesis on plants.