1.12.14
PRODUCTION OF VIRUS-LIKE PARTICLES IN TRANSGENIC CELLS EXPRESSING THE COAT PROTEIN GENE OF INDIAN PEANUT CLUMP VIRUS

C BRAGARD2, MA MAYO1 and GH DUNCAN1

1Scottish Crop Research Institute, Dundee, UK; 2Present address, Phytopathology Unit, UCL, Louvain, Belgium

Background and objectives
In work aimed at testing the possibility of using transformation to induce resistance to Indian peanut clump virus (IPCV), lines of Nicotiana benthamiana plants were obtained that had been transformed with the coat protein gene of IPCV. Lines that contained detectable coat protein gene RNA and detectable coat protein had a measure of multiplication resistance [1]. These lines were examined to assess the aggregation state of the expressed coat protein.

Materials and methods
Plants had been transformed [1] using cDNA corresponding to the sequence between nucleotides 13 and 1133 of RNA-2 of an isolate of the H serotype of IPCV [2]. Plant extracts were examined by immunosorbent electron microscopy using antiserum to an H serotype isolate of IPCV. Extracts were also assayed using immunocapture-reverse transcription-PCR (IC-RT/PCR), also using anti-IPCV-H. The primers used were specific for sequences immediately adjacent to the initiation and termination codons of the coat protein gene of IPCV-H [2]. Extracts were also made from E. coli cells [strain BL21(DE3)pLysS] that had been transformed with IPCV coat protein gene cDNA cloned into pET-15b (kindly supplied by Dr RA Naidu, ICRISAT). The expression of the coat protein gene was induced by supplying IPTG 4Naiduh after subculturing the cells.

Results and conclusions
Sap extracts of transgenic plants contained particles that closely resembled the rod-shaped particles of IPCV in the electron microscope. The particles were found on grids coated with antibodies to IPCV-H, but not on grids coated with heterologous antiserum. IC-RT/PCR showed that extracts from transgenic plants contained RNA corresponding in sequence to the coat protein gene of IPCV.

Buffer extracts of E. coli cells transformed with the IPCV-CP-PET-156 vector also contained virus-like particles. IC-RT/PCR showed that these particles contained the coat protein gene of IPCV.

The results show that, if there is a particular sequence in the RNA of IPCV that is responsible for the RNA being encapsidated, then this sequence is within the coat protein gene. The results also raise the possibility that if plants transformed with this construct were to become infected by the fungus vector of IPCV, then the encapsidated RNA might be transmissible by the fungus along with IPCV.

References
1. Cheung MK, Kumar AK, Mayo MA, Twell DE, 1995. Abstracts of ODA Plant Sciences Programme Conference on the Semi-Arid System, Manchester.
2. Wesley SV, Mayo MA, Jolly CA et al., 1994. Archives of Virology 134, 271-278.