Cloning of a Homologous Gene of Magnaporthe grisea PMK1 Type MAPK from Ustilaginoidea virens and Functional Identification by Complement in Magnaporthe grisea Corresponding Mutant

Abstract:

Abstract: [Objective] Cloning of a Homologous Gene of PMK1 Type Mitogen-Activated Protein Kinase (MAPK) from the rice false smut fungus Ustilaginoidea virens. [Methods] According to the conserved amino acid sequence of several filamentous fungus MAPKs, which were homologous to Magnaporthe grisea PMK1, degenerate PCR primers were de-signed to amplify the MAPK internal DNA fragment from Ustilaginoidea grisea. The complete UVMK1 DNA and cDNA sequences were obtained using Thermal Asymmetric Interlaced–PCR (TAIL-PCR) and RT-PCR methods. Functional Iden-tification was done by using the M. grisea ΔPMK1 mutant stain nn78, including appressoria differentiation assay and bar-ley infection test. [Results] The total length of UVMK1 was 1435 bp. It contained 3 introns and encoded 355 amino acids. The induced amino acid sequence showed identical to Magnaporthe grisea PMK1, Fusarium oxysporum FMK1, Fusarium solani FsMAPK, Colletotrichum lagenarium CMK1, Botrytis cinerea BMK1, Claviceps purpurea CMPK1. After transfor-mation of the ΔPMK1 mutant of M. grisea using a complement vector with the complete cDNA of UVMK1 (under the M. grisea MPG1 promoter), five transformants were obtained. Furthermore, the selected two transformants fully restored their ability to form appressoria and infect a barley leaf. [Conclusion] In this study, we characterized the frst MAPK protein from U. virens, and that UVMK1 is a homologue of M. grisea PMK1.