|DING, FANG - Huazhong Agricultural University|
|YUAN, QING - Luzhou Medical College|
Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2016
Publication Date: 7/6/2016
Citation: Ding, F., Duan, Y., Yuan, Q., Shao, J.Y., Hartung, J.S. 2016. Serological detection of ‘Candidatus Liberibacter asiaticus’ in citrus, and the identification of a promising secreted chaperone protein responding to cellular pathogens. Scientific Reports. 6:29272. doi: 10.1038/srep29272.
Interpretive Summary: A bacterium causes HLB, a very serious disease of citrus but it is very difficult to detect. We produced antibodies in rabbits against a particular protein that is found on the surface of the bacterial cells. We used the antibodies to create an assay to detect the bacterium in infected orange trees. Samples from infected trees developed purple spots where the bacteria were located. The assay was specific for the bacterium that causes HLB and did not react with other pathogens commonly found in citrus. Our method is simple, can be used on large numbers of samples and may facilitate future research on the disease. It is also faster, cheaper and better for field diagnosis of HLB than current methods.
Technical Abstract: Antibodies against ‘Candidatus Liberibacter asiaticus (CaLas) would be useful in inexpensive tissue print assays to detect the pathogen in infected plants. Such assays would provide low cost detection and a level of spatial and anatomical detail not possible with other methods like qPCR. We used ScFv748 and ScFv932, single chain antibodies selected to bind to surface exposed fragments of the major outer membrane protein, OmpA, and the flagellar basal ring protein, FlgI, respectively. To detect the scFv bound to CaLas in tissue prints, we used secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary monoclonal antibody produced apparently positive results with CaLas diseased samples when the primary scFv were not used. The anti-FLAG monoclonal antibody (Mab) also identified plants infected with two genotypes of Citrus tristeza virus, Citrus chlorotic dwarf virus and Xylella fastidiosa. We also prepared a conventional anti-OmpA rabbit polyclonal antibody against the same fragment of OmpA. The rabbit anti-OmpA polyclonal antibody combined with a goat anti-rabbit polyclonal antibody (Pab) conjugated with alkaline phosphatase produced very strong purple color in individual phloem cells, preserving the anatomical distribution expected for this pathogen. These results were entirely specific for CaLas infected citrus. We also identified by GeLC-MS/MS a paralogous group of secreted chaperone proteins belonging to the HSP-90 family that contained an amino acid sequence DDDDK identical to the carboxy-terminal sequence of the FLAG epitope and that responded to cellular pathogens. The simplicity, cost and ability to scale the tissue print assay with the polyclonal antibody makes this an attractive assay to complement PCR-based assays currently in use. The FLAG- like epitope may itself be useful as a molecular marker for the rapid screening of citrus plants for the presence of vascular pathogens.