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.
Highlights
ACP population with area-wide application of a pesticide program to limit disease spread, and the application of aggressive plant nutrition programs to support production from infected trees[12,13]
We have recently developed a library of antibodies against CaLas that can be screened to isolate antibodies against any defined and cloned CaLas antigen. This library of single chain antibodies was made using phage display technology, and includes unique mouse antibodies that recognize antigens present in psyllids infected by CaLas that were used to immunize mice as the first step in the creation of the library. scFv are available that recognize antigens expressed on the surface of CaLas cells, including the major outer membrane protein, OmpA and a protein component of the basal flagellar apparatus, FlgI21
The anatomical detail of the plant is preserved in tissue prints, and the technique has been used to study the distribution of plant viruses in infected plants[23] and in particular a direct tissue blot immunoassay (DTBIA) has been used for the detection of Citrus tristeza virus (CTV)[24]
Summary
ACP population with area-wide application of a pesticide program to limit disease spread, and the application of aggressive plant nutrition programs to support production from infected trees[12,13]. Tissue printing was first used to study the distribution of the cell wall protein extensin in soybean plants[22]. The anatomical detail of the plant is preserved in tissue prints, and the technique has been used to study the distribution of plant viruses in infected plants[23] and in particular a direct tissue blot immunoassay (DTBIA) has been used for the detection of Citrus tristeza virus (CTV)[24]. We used a polyclonal antibody (Pab) produced in a rabbit against the major outer membrane protein, OmpA, in tissue print assays of similar plant tissues. The Pab was in turn detected by a goat anti-rabbit IgG polyclonal antibody This allowed a direct comparison between the polyclonal and scFv recombinant antibodies for DTBIA
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