Wojciech K. Kaniewski

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.

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. 'Russet Burbank'.

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. ‘Russet Burbank’. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.

Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene.

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the ‘recovery’ type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.

Extreme resistance to Potato leafroll virus in potato cv. Russet Burbank mediated by the viral replicase gene

High levels of field resistance to Potato leafroll virus (PLRV; Genus: Polerovirus; Family: Luteoviridae) were achieved by expression of the unmodified, full-length PLRV replicase gene in potato plants cv. Russet Burbank. A high degree of resistance was also achieved, but less frequently, by expression of a truncated construct of the replicase gene. In limited testing, neither miss-frame nor antisense constructs of the replicase gene conferred resistance. The degree of resistance expressed among different transformant lines ranged from near immunity to full susceptibility. Resistance to the Colorado potato beetle (Leptinotarsa decemlineata Say) was combined with resistance to PLRV by expression of the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis in combination with the unmodified, full-length, viral replicase gene. Resistance was expressed as a reduced incidence of infection detectable by foliage symptoms or serological tests. Reduced incidence of infection was not associated with a decrease in virus antigen concentration in the few plants of resistant lines that became infected. Virus was not detected in the foliage of symptomless plants but was detected in progeny plants produced from the tubers of inoculated but symptomless test plants of some resistant lines. The resistance was effective under natural exposure and against plant-to-plant spread of PLRV by the aphid vector, Myzus persicae Sulzer. Three of the resistant lines selected in these studies were released and are now in commercial production.

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties.

Field testing for virus resistance and agronomic performance in transgenic plants

Resistance to specific virus diseases may be added as heritable characteristics to susceptible crop cultivars by transformation of the cultivars with specific virus-derived genes. In practice, however, resistance to the virus varies among transformed plants and transformation often changes cultivar yield and quality characteristics that are agronomically important. Therefore, rigorous selection among hundreds of different transformed plant lines is required to identify lines that are both virus resistant and also conform to or exceed standard characteristics of the original, susceptible cultivar. This paper describes methods we have developed for rapid selection of virus resistance, yield, and quality characteristics among transformed populations of plants.

A search for evidence of virus/transgene interactions in potatoes transformed with the potato leafroll virus replicase and coat protein genes

A search was conducted to detect evidence for interactions between potato leafroll virus (PLRV)-derived transgenes expressed in Russet Burbank potato and viruses to which the transgenic plants were exposed and by which they were infected. More than 25000 plants in 442 lines transformed with 16 different coat protein gene (CP) constructs and nearly 40000 plants in 512 lines transformed with seven different replicase gene (Rep) constructs of PLRV were exposed to field infection over a 6-year period. These plants were individually inspected for type and severity of virus symptoms. Heterologous viruses found infecting the plants were identified and examined for alterations in transmission characteristics, serological affinity, host range, and symptoms. Selected isolates of PLRV from field-infected plants were examined for unusual symptoms produced in diagnostic hosts and for alteration in sedimentation properties in density gradient tubes. Viruses that were propagated in selected transgenic lines in a greenhouse were examined for similar alterations. Transmission characteristics and serological properties were not altered when they replicated in potatoes containing CP constructs in the field or greenhouse. Potato plants expressing CP or Rep constructs of PLRV were not infected in the field or in the greenhouse with viruses that do not normally infect potato. New viruses or viruses with altered sedimentation characteristics, symptoms, or host range were not detected in field-exposed or greenhouse-inoculated potato plants expressing CP or Rep gene constructs of PLRV.

Resistance of transgenic tomato to cucumber mosaic cucumovirus under field conditions

Since the summer of 1993, transgenic tomato plants expressing the coat protein (CP) genes of cucumber mosaic cucumovirus have been tested under field conditions to assess the level of resistance and agronomic performance. Trials were performed in different areas in Italy and the target virus in the majority of tests was spread naturally by the indigenous aphid populations. Twenty-three homozygous lines of variety UC82B, transformed to contain four different CP genes of CMV, were evaluated. The lines were preselected for CP expression, single gene copy, and virus resistance in growth chamber experiments. In general, CMV resistance was confirmed under field conditions though resistance in the field was less effective than what was observed in growth chamber experiments. The resistance observed in multi-year and multi-location experiments is of commercial value for several of the most resistant lines. Engineered resistance upon transfer to Italian varieties by breeding or direct transformation will be used in tomato production in Italy or elsewhere.

NewLeaf Plus® Russet Burbank potatoes: replicase-mediated resistance to potato leafroll virus

Potato leafroll poleovirus and the Colorado potato beetle (Leptinotarsa decemlineata (Say)) are major pests of potato in the USA. The US Department of Agriculture estimates that over 50% of annual insecticide use on potato is applied to control the Colorado potato beetle and aphids that transmit potato leafroll virus (PLRV). To address this issue, Russet Burbank potatoes have been genetically modified for a high level of resistance to infection and the resulting disease symptoms caused by PLRV and to feeding damage caused by the Colorado potato beetle. This resistance was achieved by the expression of the unmodified full-length replicase gene of PLRV and the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis. Plant expression constructs containing various modifications of the PLRV replicase gene were produced during the development of this product. The genes in these constructs were a full-length unmodified replicase (open reading frame 2a/2b), an antisense orientation of the full-length cDNA, an open reading frame 1 translation of the full-length gene, and a gene truncation containing the 3′ sense coding portion of the replicase gene. Growth chamber experiments demonstrated that transformation of plants with the full-length and 3′ sense coding constructs substantially protected these potato plants from infection and disease symptoms caused by PLRV. The Russet Burbank potato expressing the full-length PLV replicase gene and the cry3A gene is a new potato product from NatureMark called NewLeaf Plus®.