title: Serological detection of 'Candidatus Liberibacter asiaticus' in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens.
abstract: We describe experiments with antibodies against 'Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with other vascular pathogens. We then identified a paralogous group of secreted chaperone proteins in the HSP-90 family that contained the amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope. A rabbit polyclonal antibody against one of the same epitopes combined with a goat anti-rabbit secondary antibody produced very strong purple color in individual phloem cells, as expected for this pathogen. These results were entirely specific for CaLas-infected citrus. The simplicity, cost and ability to scale the tissue print assay makes this an attractive assay to complement PCR-based assays currently in use. The partial FLAG epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens.
authors.affiliation: Hubei Key Laboratory of Plant Pathology, Huazhong Agricultural University, Wuhan 430070 P. R. China.|USDA ARS MPPL 10300 Baltimore Ave., Beltsville, MD 20705 USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Ding
authors.affiliation: USDA ARS USHRL 2001 South Rock Road, Fort Pierce, FL 34945 USA.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Duan
authors.affiliation: Sichuan Medical University, Luzhou, Sichuan, 646000 P. R. China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: USDA ARS MPPL 10300 Baltimore Ave., Beltsville, MD 20705 USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Shao
authors.affiliation: USDA ARS MPPL 10300 Baltimore Ave., Beltsville, MD 20705 USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Hartung
mesh_terms: D000907:Antibodies, Bacterial / Q000378:metabolism; D000942:Antigens, Bacterial / Q000032:analysis*; D002853:Chromatography, Liquid; D002957:Citrus / Q000382:microbiology*; D054884:Host-Pathogen Interactions; D007118:Immunoassay / Q000379:methods; D018832:Molecular Chaperones / Q000032:analysis*; D010935:Plant Diseases / Q000382:microbiology*; D010940:Plant Proteins / Q000032:analysis*; D012230:Rhizobiaceae / Q000276:immunology / Q000302:isolation & purification*; D057127:Single-Chain Antibodies / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D018832:Molecular Chaperones; D010940:Plant Proteins; D057127:Single-Chain Antibodies