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In vivo expression of the 29000 M_r protein from RNA-2 of pea early browning tobravirus

¹ Danish Research Service for Plant and Soil Science, Lottenborgvej 2, DK-2800 Lyngby, Denmark
and² Genetic Engineering Group, Building 227, The Technical University of Denmark, DK-2800 Lyngby, Denmark

The purpose of this work has been to investigate in vivo transcription and translation products from an open reading frame (ORF) located downstream of the coat protein (CP) cistron on RNA-2 of pea early browning virus (PEBV). This work was initiated as a step towards elucidation of the significance of this putative gene. Sequence data on RNA-2 suggest that a 29600 M_r (29·6K) protein is translated from the ORF in question. Hybridization with ORF-specific probes on Northern blots with RNA from infected plants showed that PEBV synthesizes a subgenomic RNA encoding CP (RNA-2a) with a size of 3000 nucleotides (nt) and that a putative subgenomic RNA (RNA-2b) encoding the 29·6K protein appears to have a size of 1600 nt. This is 300 nt less than the size predicted from the sequencing data. For antibody production, a cDNA fragment harbouring 85% of the 29·6K ORF was cloned into the pUEX3 expression vector. The resulting plasmid expresses the 29·6K protein as a fusion protein with -galactosidase and this protein was used for raising antiserum containing specific anti-29·6K protein antibodies. By using these antibodies on immunoblots it was demonstrated that the 29·6K protein is expressed in infected plants.

Received 12 October 1990; accepted 12 February 1991.

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