RESUMEN
The mechanism of translocation of RxLR effectors from plant pathogenic oomycetes into the cytoplasm of their host is currently the object of intense research activity and debate. Here, we report the biochemical and thermodynamic characterization of the Phytophthora infestans effector AVR3a in vitro. We show that the amino acids surrounding the RxLR leader mediate homodimerization of the protein. Dimerization was considerably attenuated by a localized mutation within the RxLR motif that was previously described to prevent translocation of the protein into host. Importantly, we confirm that the reported phospholipid-binding properties of AVR3a are mediated by its C-terminal effector domain, not its RxLR leader. However, we show that the observed phospholipid interaction is attributable to a weak association with denatured protein molecules and is therefore most likely physiologically irrelevant.
Asunto(s)
Fosfolípidos/metabolismo , Phytophthora infestans/metabolismo , Multimerización de Proteína , Factores de Virulencia/química , Factores de Virulencia/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Mutación , Fosfolípidos/química , Phytophthora infestans/genética , Enfermedades de las Plantas/microbiología , Unión Proteica , Señales de Clasificación de Proteína/genética , Solanum tuberosum/microbiología , Factores de Virulencia/genéticaRESUMEN
A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 microg liter(-1)) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 micro g of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.