RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation.

Transgenic Nicotiana tabacum cv. Burley 49 plants were generated that express the 5' untranslated region of the tobacco etch potyvirus (TEV) genome ligated to a mutated version of the TEV coat protein gene sequence that rendered it untranslatable. Eight different transgenic plant lines were analyzed for transgene expression and for resistance to TEV. Three different responses were noted when the transgenic plant lines were inoculated with TEV: 1) some were highly resistant, and no virus replication occurred; 2) some were susceptible but able to recover from systemic TEV infection; and 3) some were susceptible to TEV infection. Plant tissue displaying the recovery phenotype was analyzed for virus replication and transgene expression. Recovered tissue could not be infected with TEV and had steady-state transgene RNA levels which were five- to eightfold lower than those of unchallenged transgenic plant tissue. Nuclear runoff assays suggested a post-transcriptional reduction in specific RNA levels. The highly resistant and recovery phenotypes associated with TEV challenge inoculation and the reduction of steady-state RNA levels in recovered transgenic leaf tissue may be manifestations of a common mechanism.

Analysis of transgenic tobacco plants expressing a truncated form of a potyvirus coat protein nucleotide sequence.

Transgenic Nicotiana tabacum cv. Burley 49 plants were generated which expressed a tobacco etch virus (TEV) coat protein (CP) gene construct containing a stop codon positioned at codon 147. This gene construct was expected to produce a TEV CP lacking the carboxy-terminal 118 amino acids of the full-length 264 amino acid CP. TEV CP gene transcripts of the expected size could be detected in transgenic plants but the expected truncated CP could not be detected. Ten independent transgenic lines expressing this form of the TEV CP gene were examined in detail. Two transgenic plant lines were resistant to aphid- or mechanically transmitted TEV and one line was highly resistant. Protoplasts derived from the highly resistant plant line did not support virus replication. The data suggested that the expression of this mutated form of the TEV CP gene could interfere with TEV replication and displayed features associated with RNA-mediated virus resistance.

Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance.

Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typical systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenic plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced in the recovered tissue. The resistance was virus specific. Recovered plant tissue could not be infected with TEV, but was susceptible to the closely related virus, potato virus Y. The resistance phenotype was functional at the single-cell level because protoplasts from recovered transgenic tissue did not support TEV replication. Surprisingly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studies suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation.

Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts.

Transgenic tobacco plants which express untranslatable sense or antisense forms of the tobacco etch virus potyvirus (TEV) coat protein (CP) gene sequence have been generated. One of seven transgenic plant lines expressing a CP gene antisense transcript showed an attenuation of symptoms when inoculated with TEV. Three of ten transgenic plant lines expressing untranslatable sense transcripts did not develop symptoms when inoculated with TEV. These lines were resistant to either aphid or mechanically transmitted TEV. In contrast to CP-mediated resistance reported for other viruses, resistance was (1) mediated by an RNA molecule; (2) TEV-specific (i.e., "broad-spectrum resistance" was not observed); (3) independent of inoculum levels; (4) not dependent on plant size and; (5) due to decreased levels of virus replication. Protoplast experiments were used to demonstrate that resistant plant lines did not support the production of virus protein and progeny virus at wild-type levels.

Pathogen-derived resistance to a potyvirus: immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence.

Transgenic Nicotiana tabacum 'Burley 49' plants containing one of six different forms of the tobacco etch virus (TEV) coat protein (CP) nucleotide sequence have been generated. In whole plant studies, R1 and R2 progeny were inoculated mechanically with TEV, and the appearance and severity of symptoms were recorded. Symptom phenotype was altered, ranging from near wild type susceptibility to apparent immunity. Protoplasts derived from wild type and transgenic Burley 49 plant lines were transfected with TEV RNA. Protoplasts from transgenic plants expressing full-length or truncated forms of TEV CP supported virus replication. Protoplasts from certain transgenic plants, producing plus- or minus-sense CP transcripts but no CP, did not support virus replication at wild type levels. A model is proposed to account for these observations.