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.

Field testing for virus resistance and agronomic performance in transgenic plants.

Resistance to specific virus diseases may be added as heritable characteristics to susceptible drop 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.

Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin.

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars.

Reduced Field Spread of Potato Leafroll Virus in Potatoes Transformed with the Potato Leafroll Virus Coat Protein Gene.

Russet Burbank potato was transformed with plant expression vectors containing the potato leafroll luteovirus (PLRV) coat protein (CP) gene. Transgenic potato lines contained a gene expression cassette with two copies of a PLRV CP gene in which the nucleotide sequence was modified to improve expression of the gene. In addition, the two copies of the PLRV CP gene were each driven by a different promoter. Field test screening for PLRV resistance identified 15 lines which showed moderate resistance to PLRV infection and virus titer build-up and a longer incubation period for systemic infection. By conducting field resistance assays during a period when the vector of PLRV was not present, it was possible to test whether the observed resistance was sufficient to restrict aphid transmission of PLRV in a field test. Two years of field testing demonstrated that PLRV-spread from an infected plant to adjacent healthy plants of the same line was severely restricted in nearly all the transgenic lines in the field. These lines have useful resistance to PLRV and could aid in managing PLRV disease in Russet Burbank potato.

Effects of acute exposure to hyperbaric oxygen on the rheology and morphology of the red blood cells in the rat.

The effects of acute exposure to hyperbaric oxygen (HBO) on the rheology and morphology of red blood cells (RBC) were studied in three groups of Sprague-Dawley rats: a control (CON) group comprised rats not exposed to HBO, a second group was exposed to HBO at 2.8 atm for 6 hr and studied immediately after the exposure (HBO), and a third group was examined after being allowed to recover in room air for 24 hr after exposure to HBO (REC). RBC deformability was assessed by two different techniques, the ektacytometer and the micropore filters. While the ektacytometer did not detect a significant difference among the three groups, the filtration method showed that acute exposure to HBO causes a significant increase in the filtration index (FI), denoting a significant reduction in RBC deformability. However, this reduction returned to the control level after 24 hr of recovery in air (FI, 7.1 +/- 1.2 in CON and 9.5 +/- 2.6 in REC compared to 36.2 +/- 3.7 in HBO, P < 0.0001). The morphology of RBC was studied by scanning electron microscopy. Immediately after exposure to HBO there was a marked increase in the echinocytes (41.6 +/- 5.9%) compared to a control level of 16.7 +/- 4.8%, P < 0.05. The increase in the echinocytes became less pronounced after 24 hr of recovery (23.5 +/- 9.3%). In conclusion, acute exposure to HBO causes significant alterations in RBC rheology and morphology. Due to the sensitivity of the micropore filters, we hypothesize that the decrease in RBC deformability might affect their flow through small-caliber blood vessels. These alterations, however, are reversible after a relatively short period of recovery in air.

Cellular deformability of normoxic and hypoxic mammalian red blood cells.

The deformability of red blood cells is important in the microcirculation where capillary diameters are often smaller than those of the red blood cells. In the present study, ektacytometry was used to examine the effect of hypoxia on the deformability of red blood cells from five mammalian species: Human, cat, rat, rabbit, and dog. Deformability was characterized in both normoxic (PO2 = 129 +/- 6 mm Hg) and hypoxic (PO2 = 47 +/- 6 mm Hg) conditions in two different ways. First, we used the Elongation Index (EI) which quantitates the extent of elongation of red blood cells in response to increasing fluid shear stress; second, we used the Elongation Constant (EC), which quantitates the exponential dependence of the fraction of maximal elongation on the varying shear stress. The EI was measured at high shear stresses (150-250 dyn/cm2), as well as at lower shear stresses (15, 32 and 64 dyn/cm2) that occur in the microcirculation. In response to hypoxia at high shear stresses, the EI of the rat red blood cells decreased by 9.3% (P < 0.05), but was not altered in the other four species studied. Moreover, in all five species, the EC and EI at the lower shear stresses were unaltered in response to hypoxia. These ektacytometry experiments indicate that (1) the elongation constant is a new and useful parameter for characterizing the deformability of red blood cells and (2), the deformability of human, cat, dog, and rabbit red blood cells is unaltered by hypoxia. The results constrain the possible mechanisms that could account for the observation that hypoxia decreases the filterability of certain species of red blood cells, which was reported previously.

Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein.

Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection. A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens. Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses. Resistance was effective against both mechanical and aphid transmission. Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection. Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses. To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic line. Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection.

Transgenic plants expressing the coat protein (CP) of alfalfa mosaic virus (AIMV) are resistant to infection by AIMV. A mutation was introduced into the second amino acid of the cDNA for the CP of AIMV. Three different transgenic tobacco lines expressing the mutant CP and two different transgenic tobacco lines expressing the wild-type CP at similar levels were challenged with AIMV virions and viral RNA. Whereas the lines expressing the wild-type CP were highly resistant to infection by AIMV virions and viral RNA, the lines expressing the mutant CP were susceptible to infection by both. The binding affinity of the mutant and the wild-type CPs for the 3' terminal protein binding site on AIMV RNAs was similar, as determined by electrophoretic mobility shift assay. A mixture of AIMV genomic RNAs 1-3 was infectious on the plants expressing the mutant CP but not on vector control plants or plants expressing the wild-type CP, indicating that the mutant CP can activate the AIMV genomic RNAs for infection. These results demonstrate that the second amino acid of the AIMV CP is critical for protection from AIMV but not for the initial interaction between the AIMV RNA and CP, suggesting that this initial interaction does not play a major role in CP-mediated protection.

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.

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

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.

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.