Transgenic tobacco plants carrying a genetic cassette including an antisense DNA sequence of the virally encoded ALI gene of the geminivirus tomato golden mosaic virus (TGMV) were constructed; ALI encodes a protein absolutely required for TGMV DNA replication. These genetic cassettes also contained, on the same transcription unit, a gene encoding hygromycin resistance, which allowed selection for concomitant expression of the antisense gene. In transgenic lines, RNA transcripts of the predicted size and strand specificity were detected in antisense plants and sense controls. After infection of plants with TGMV, by agroinoculation, the frequency of symptom development was very significantly reduced in a number of antisense lines and correlated, broadly, with the abundance of antisense RNA transcript and with a reduction in viral DNA harvested from infected leaf tissue. We used an in vitro assay to study viral DNA replication in the absence of cell-to-cell spread; no replication was seen in five of the six antisense lines studied, in contrast to controls.Antisense RNA, complementary to a target RNA (e.g., mRNA), has recently been exploited to artificially suppress gene expression with high specificity (1, 2). Both previously introduced and endogenous genes have successfully been targeted by this technique. The variety of models accounting for the mechanism include antisense RNA-DNA interaction interfering with transcription, rapid degradation of the sense RNA-antisense RNA duplex, a block in the transport of duplex RNA into the cytoplasm, and inhibition oftranslation. Because antisense RNA acts at the transcript level, it is effective against multiple gene copies and, in addition, acts in trans. It would thus seem to be ideal for the suppression of viral infection; viruses invade the cell from outside and multiply to a high copy number.Antisense RNA has been successfully used to suppress viral infection in bacteria and in animal tissue (1). In plants, cross-protection between RNA viral strains has historically been used as a strategy for achieving virus resistance. This has led to engineering resistance by construction of transgenic plants that overexpress the viral coat protein and plants that contain replicating copies of a "satellite RNA" (3). The reported applications of antisense technology to the suppression of viral infection in plants have, so far, met with only limited success (4, 5). This is probably due to the mode of action of antisense RNA and the viruses chosen as targets (i.e., RNA viruses that replicate in the cytoplasm).The single-stranded DNA viruses known as geminiviruses are serious plant pathogens, yet little has been done to engineer plants with resistance. Recently, however, Stanley et al. (6) have shown that transgenic plants containing a defective geminiviral DNA show some decrease in symptom severity following infection. Because geminiviruses replicate in the nucleus, they would seem to be ideal targets to apply antisense technology to engineer resistance. To our knowledge, this p...