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Science 14 March 1986:
Vol. 231. no. 4743, pp. 1294 - 1297
DOI: 10.1126/science.231.4743.1294
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Articles

Bacterial Gene Inserted in an Engineered RNA Virus: Efficient Expression in Monocotyledonous Plant Cells

ROY FRENCH 1, MICHAEL JANDA 1, and PAUL AHLQUIST 1

1 Biophysics Laboratory and Department of Plant Pathology, University of Wisconsin, Madison 53706

Brome mosaic virus (BMV) is a plant virus whose genome consists of three RNA components. A previously described viral complementary DNA expression system has been used to express both wild-type and altered genomic RNA's in barley protoplasts. Variants of BMV RNA3 were constructed in which the coat gene had been removed or replaced with sequences encoding chloramphenicol acetyltransferase (CAT). CAT sequences were also inserted near the 5' end of the intact coat gene. When inoculated on protoplasts together with transcripts of BMV RNA's 1 and 2, all of these RNA3 derivatives were replicated and produced subgenomic RNA's analogous to the normal subgenomic coat protein messenger RNA. RNA3 derivatives in which the CAT coding sequences were oriented with the same polarity as viral genes produced significant CAT activity in protoplasts. CAT expression was improved by inserting the CAT gene in frame with the upstream coat protein initiation codon, and exceeded expression in plant cells transformed with Ti plasmid-based vectors.

Submitted on May 28, 1985
Accepted on November 15, 1985


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