Applying the pthA effector protein of Xanthomonas citrisubsp. citrifor production of specific antibodies and its application for detection of infected plants

Bacterial citrus canker, caused by Xanthomonas citrisubsp. citri, is a major disease of citrus throughout the world. The pthA bacterial effector protein, as an essential element for pathogenesis, is present within the infected plants. Here, development of a specific antibody raised against pthA as an early diagnostic tool is reported. For this aim, partial sequence at the C-terminus of pthAgene (606 bp), was PCR-amplified and cloned. The coding region of truncated pthA, t-pthA,was inserted into the pET28a(+) bacterial expression vector. The recombinant t-pthA was expressed in Escherichia colistrain Rosetta (DE3) and purified via affinity chromatography. The purity and integrity of expressed protein was confirmed by SDS-PAGE followed by Western blot analysis using anti-His antibody. The purified recombinant t-pthA was used for immunization of rabbits. Immunoglobulin was purified using protein A Sepharose. The binding ability of prepared IgG was confirmed via a suppression assay performed by direct injection of purified IgG into tobacco leaves followed by infiltration of purified t-pthA protein into leaves. This assay revealed that the presence of anti-pthA IgG prevents hypersensivity response (HR) of t-pthA within the infiltrated leaves. Further in vitro assays such as PTA-ELISA, Dot Immunobinding Assay (DIBA), and Western blot analysis proved the ability of prepared antibody for efficient detection of infected citrus plants. To the best of our knowledge, this is the first report for applying bacterial effector protein, such as pthA, as an antigen for developing a diagnostic approach against a plant bacterial disease.