Transient expression of heterologous RNAs using tomato golden mosaic virus

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Abstract
The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component contains the only virally-encoded function required for autonomous replication in infected plant cells. We used agroinoculation of petunia leaf discs with the A component to develop a transient expression system which permits direct examination of viral transcripts by S1 nuclease protection. The AR1 gene, which encodes the TGMV coat protein, was transcribed transiently in leaf discs after agroinoculation of TGMV A DNA. Synthesis of AR1 RNA was dependent on T-DNA transfer and TGMV DNA replication, demonstrating that it is a plant transcription product. The AL open reading frames of TGMV A were also expressed transiently in leaf discs. The ratio between AR1 RNA and the major leftward RNA was constant and was used to normalize AR1 transcription for viral DNA copy number. The bacterial genes encoding chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) were transiently expressed in leaf discs from the AR1 promoter in TGMV A. The levels of AR1 and GUS RNAs were similar in leaf discs after adjusting for viral DNA copy number, while CAT RNA was less abundant. The geminivirus transient expression system allows rapid analysis of RNAs transcribed from foreign genes and can serve as a preliminary screen in the construction of transgenic plants.