title: Overexpression and purification of PWL2D, a mutant of the effector protein PWL2 from Magnaporthe grisea.
abstract: The rice blast disease caused by the ascomycete Magnaporthe grisea continues to cause a tremendous impact in rice (Oryza sativa) cultures around the world. Elucidating the molecular basis of the fungus interactions with its host might help increase the general understanding of the pathogen-host relationship. At the moment of invasion, the fungus secretes effectors that modify host defenses and cellular processes as they successively invade living rice cells. PWL2, an effector protein, is a known AVR (avirulence) gene product. The PWL2 gene prevents the fungus from infecting weeping lovegrass (Eragrostis curvula). In this study, we identified a PWL2 allele gene (which we termed PWL2D) in a strain of M. grisea. The sequence of PWL2D has only two bases different from that of PWL2, producing alterations in residue 90 and residue 142. However, the alteration of residue 90 (from D(90) to N(90)) is critical to gene function. Here, we cloned the gene PWL2D in a pET System vector, expressed the gene product in Escherichia coli and evaluated by spectroscopic techniques some aspects of the PWL2D structure. While TRX-tagged PWL2D is prone to aggregation, the solubility of PWL2D is improved when it is overexpressed without its original signal peptide. Expression and purification procedures for these constructs are described. Finally, we found out that the protein seems to be an intrinsically disordered protein. Results from these studies will facilitate structural analysis of PWL2D and might contribute to understanding the gene's function and of fungal/plant interactions.
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética (CBMEG), Universidade Estadual de Campinas, CP 6010, CEP 13083-970 Campinas, SP, Brazil.
authors.forename: D R S
authors.initials: DR
authors.lastname: Schneider
authors.forename: A M
authors.initials: AM
authors.lastname: Saraiva
authors.forename: A R
authors.initials: AR
authors.lastname: Azzoni
authors.forename: H R C A N
authors.initials: HR
authors.lastname: Miranda
authors.forename: M A S
authors.initials: MA
authors.lastname: de Toledo
authors.forename: A C
authors.initials: AC
authors.lastname: Pelloso
authors.forename: A P
authors.initials: AP
authors.lastname: Souza
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics; D020082:Magnaporthe / Q000235:genetics*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D016415:Sequence Alignment; D013879:Thioredoxins / Q000737:chemistry; D015854:Up-Regulation
chemical_list: D005656:Fungal Proteins; D050505:Mutant Proteins; C095732:PWL2 protein, Magnaporthe grisea; D013879:Thioredoxins