title: Heterologous production, purification, and immunodetection of Magnaporthe oryzae avirulence protein AVR-Pia.
abstract: The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.
authors.affiliation: a Graduate School of Agriculture, Hokkaido University , Sapporo , Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Satoh
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Miki
authors.forename: Toyoyuki
authors.initials: T
authors.lastname: Ose
authors.forename: Azusa
authors.initials: A
authors.lastname: Oikawa
authors.forename: Katsumi
authors.initials: K
authors.lastname: Maenaka
authors.forename: Ryouhei
authors.initials: R
authors.lastname: Terauchi
authors.forename: Kozo
authors.initials: K
authors.lastname: Asano
authors.forename: Teruo
authors.initials: T
authors.lastname: Sone
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D005818:Genetic Engineering; D007118:Immunoassay; D020082:Magnaporthe / Q000235:genetics*; D058849:Protein Refolding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins
keywords: antibody; avirulence protein; effector; quantification; refolding