lhe Sainsbury Laboratory, John lnnes Centre, Colney, Norwich NR4 7UH, United Kingdom INTRODUCTION In 1985, Sanford and Johnston developed the simple and ele- gant concept of parasite- or pathogen-derived resistance (Sanford and Johnston, 1985). Subsequently, there have been numerous attempts to generate virus resistance in transgenic plants based on this concept through the expression of virus- derived genes or genome fragments (Beachy, 1993; Wilson, 1993; Baulcombe, 1994b; Lomonossoff, 1995). Many of these attempts have been successful, and some have led to the de- velopment of virus-resistant potato and squash cultivars for commercial application. In this article, the major emphasis is on the mechanisms of pathogen-derived resistance rather than on the practicali- ties of using this technology for crop improvement. These mechanisms have proven difficult to unravel largely because a resistance mechanism related to transgene silencing can override the direct phenotype of virus-derived transgenes. lhe examples discussed first are those in which it is clear whether the mechanism involves gene silencing. In later sections, some less well understood examples are reviewed. The final sec- tion anticipates likely future developments in pathogen-derived resistance.

Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants.

The common phenotype of different resistant plants are described and individual examples are reviewed in order to compare and contrast the characteristics of resistance to different viruses. The limited number of experiments that explore the fundamental basis of resistance are described. Ideas are presented for the types of experiments that are needed to more completely understand the mechanisms of resistance and to extend the resistance to other important plant viruses. Topics covered include: definition; strategy to create coat protein-mediated resistance; phenotype of coat protein-mediated resistance; examples of coat protein-mediated resistance; understanding the mechanisms of CP-mediated resistance; field tests of coat protein-mediated resistance; and conclusions and future directions

Coat Protein-Mediated Resistance Against Virus Infection

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16:PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1999.00508.x

Stable chloroplast transformation in potato : use of green fluorescent protein as a plastid marker

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

Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3 terminal nucleotides of the TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 micrograms/ml or 300 micrograms/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 micrograms/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to approximately 5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequence-specific RNA transcript of the expected size, but no protein product was detected.

Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus.

Potato virus X (PVX) and potato virus Y (PVY) infection in potato may result in the loss of certification of seed potatoes and affect quality and yield of potatoes in commercial production. We transformed a major commercial cultivar of potato, Russet Burbank, with the coat protein genes of PVX and PVY. Transgenic plants that expressed both CP genes were resistant to infection by PVX and PVY by mechanical inoculation. One line was also resistant when PVY was inoculated with viruliferous green peach aphids. These experiments demonstrate that CP protection is effective against mixed infection by two different viruses and against mechanical and aphid transmission of PVY.

Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank.

Transgenic Russet Burbank potato plants expressing the coat protein genes of potato virus X (PVX) and potato virus Y (PVY) were transplanted into the field after propagation in tissue culture. Virus resistance, plant growth and tuber yield were determined. Our results show that expression of PVX and PVY CP genes confer a very high level of resistance to PVX and PVY infection in clone 303, one of the four different transgenic potato clones tested. After inoculation with PVX and PVY, tuber yield of control Russet Burbank plants decreased, while the yield of clone 303 was unaffected. Four different uninoculated transgenic clones had tuber yields as high as control Russet Burbank. These results confirm that resistance to PVX and PVY is effective in the field and can prevent yield losses due to dual infection by these viruses.

Cliff Lawson

Papers

Engineering resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank.

Field resistance of transgenic russet burbank potato to effects of infection by potato virus X and potato virus Y