Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/1995
Publication Date: N/A
Interpretive Summary: Rapid diagnosis of virus infection is important to the control of virus diseases of citrus, including those caused by citrus tristeza virus. Serological tests, which are based on the use of a specific antibody obtained from animals immunized with purified virus, can be used to detect viruses in extracts from infected plants, and provide a rapid and convenient means for virus diagnosis. One of the factors which limits greater application of sero-diagnostic procedures for many plant viruses, including citrus tristeza virus, has been the difficulty in purifying sufficient virus to produce large amounts of high quality antisera. This paper describes an approach which eliminated the need to purify tristeza virus from infected plants. Using molecular technology, the tristeza virus gene which controls production of the virus coat protein was expressed in bacteria which could be easily grown in the laboratory, and caused these bacteria to produce large quantities of virus protein. The protein was easily purified and used for production of antisera. The antisera successfully detected tristeza virus infection in citrus trees. This technology facilitates large scale production of antisera and can also allow future development of antisera custom engineered to recognize specific strains.
Technical Abstract: Using specific primers based on the sequence of the Florida isolate T36 of citrus tristeza virus (CTV), the coat protein (CP) gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from the severe California isolate SY568 of CTV. The RT-PCR product was cloned, sequenced, and sub-cloned into an expression vector pMAL-c2. The CTV CP was expressed as a fusion product containing a fragment of the E. coli maltose-binding protein (MBP). This MBP-CP fusion protein reacted with CTV-specific antisera in immunoblotting and enzyme linked immunosorbent assay (ELISA). After cell disruption, the MBP CP fusion protein was purified to near homogeneity by amylose resin affinity column chromatography giving a yield of 1 mg fusion protein per 10 ml of E. coli culture. Antisera obtained from rabbits after injection with MBP CP protein were specific to CTV, with a titer of about 10**5 in an indirect ELISA, and were suitable in ELISA for trapping. These polyclonal antisera reacted with a wide range of CTV isolates from different geographic sources and of different biological properties.