Cynthia Lou Hemenway

Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA

Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP‐antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP‐mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism.

Expression of Amino-Terminal Portions or Full-Length Viral Replicase Genes in Transgenic Plants Confers Resistance to Potato Virus X Infection.

The first open reading frame (ORF 1) of potato virus X (PVX) encodes a putative replicase gene. Transgenic tobacco lines expressing ORF 1 are resistant to PVX infection when inoculated with either PVX or PVX RNA. Analyses of lines containing various portions of the ORF 1 gene demonstrated that resistance is conferred to plants by expressing approximately the first half of the ORF 1 gene. One line expressing the untranslated leader and first 674 codons of ORF 1 is highly resistant to PVX infection. Conversely, lines expressing either approximately the third or fourth quarter of the ORF 1 gene, which contain the conserved nucleotide triphosphate (NTP) binding motif and Gly-Asp-Asp (GDD) motif, respectively, are not protected from PVX infection. In the resistant full-length and amino-terminal lines, lower numbers of local lesions were observed, and the virus accumulation in the inoculated and upper leaves was reduced when compared with the nontransformed control. When the performance of the most resistant ORF 1 line was compared with the most resistant coat protein (CP) line in a resistance test, the best ORF 1 line was more resistant to PVX infection than the best transgenic line expressing the PVX CP gene. These findings define a promising new approach for controlling plant viral infection.

Characterization of infectious transcripts from a potato virus X cDNA clone.

A full-length cDNA clone of potato virus X (PVX) has been constructed and fused to the bacteriophage T7 promoter in an in vitro transcription vector. Transcripts derived from this template (pMON 8660) were infectious when inoculated onto the local lesion host, Chenopodium amaranticolor. The infectivity of these transcripts was approximately 0.2% that of authentic PVX RNA. Lesions sampled from plants inoculated with these transcripts contained virus particles and virus aggregates typically observed in lesions from plants inoculated with authentic PVX RNA, as evidenced by electron microscopy. In addition, progeny virus isolated from these lesions was as infectious as progeny virus from an authentic PVX RNA infection when inoculated onto new local lesions plants. Infectious transcripts derived from PVX cDNA clones will facilitate analysis of the molecular aspects of PVX infection.

Interactions between plants, pathogens and insects: Possibilities for engineering resistance

Abstract Genetically engineered agricultural crops that resist viral and insect pathogens have been produced and tested under both environmentally controlled and field test conditions. Recent advances in creating plants that resist viruses, and plants that contain a bacterial protein, which confers selected insect resistance are reviewed. Recent studies on plant defense mechanisms are also discussed. In the future, plant defense mechanisms will be genetically modified to produce plants that resist insect and fungal pathogens.