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.     

Expression of the potato leafroll luteovirus coat protein gene in transgenic potato plants inhibits viral infection

Transgenic potato plants, cultivar Désirée, were produced that contained the coat protein gene of potato leafroll luteovirus (PLRV). The transformed potato plants expressed the PLRV coat protein (CP) RNA sequences but accumulation of coat protein in transgenic tissues could not be detected. Upon inoculation with PLRV, the PLRV CP RNA expressing potato plants showed a reduced rate of virus multiplication.     

High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs

The application of RNA-mediated resistance against Cucumber mosaic virus (CMV) by using single transgene constructs generally results in only a small portion of resistant individuals. Inverted repeat constructs encoding self-complementary double-stranded RNA have been demonstrated a potential way to obtain RNA-mediated resistance at high efficiency. To test this observation as a possible method for high frequency induction of CMV resistance, Nicotiana benthamiana plants were transformed with transgenes designed to produce double strand RNA molecules of CMV RNA 2 or coat protein (CP) gene sequences. Seventy-five percent of the tested R₀ plants transformed with an RNA 2-derived inverted repeat construct (1534 nt CMV sequence) showed extreme resistance to CMV, while a lower percentage of resistance (30%) was observed in R₀ lines transformed with a similar construct of a shorter viral RNA 2 sequence (490 nt). The resistance level conferred by CP sequences was also efficient by using a dsRNA construct, reaching a level of 50%. Self-pollinated (S₁) progenies obtained from most resistant R₀ plants all showed resistance levels of 100%, perfectly correlating with the expression of transgenic siRNAs. The results indicate that the use of inverted repeat viral transgenes is a highly efficient approach to obtain CMV resistant transgenic plants. Consequently, only a handful of transgenic plants will have to be generated using such constructs for successful resistance, which enables the implementation of this protocol for crops that are difficult to transform, such as ornamental plants in which CMV is an important pathogen.     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene

Summary Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and -glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.     

Genetically engineered resistance to potato virus X in four commercial potato cultivars

The genes for the capsid protein (CP) and the 8K movement protein of PVX were introduced into potato (Solanum tuberosum L.) and expressed under the control of CaMV 35S promoter using a binary vector andAgrobacterium tumefaciens. Four commercial potato cultivars (Russet Burbank, Shepody, Desirée and Bintje) have been efficiently transformed. Eleven independent transgenic clones, with CP expression levels higher than 0.05% of the soluble leaf proteins, were analyzed for resistance to inoculation with PVX (5 and 50µg/ml). The resistance of the transgenic plants to PVX was observed with the lower titer of virus inoculation (5 µg/ml) but not with higher titer (50 µg/ml). A significant reduction in the accumulation of virus in the inoculated transgenic potato plants has been observed under greenhouse and field conditions. Furthermore, the CP gene is very stable and is transferred to new plants originated from stem cuttings or from tubers. The transgenic plants appeared to be phenotypically identical to the nontransformed controls.Abbreviations BAP benzyl-aminopurine - BCIP 5-bromo-4-chloro-3-indolylphosphate p-Toluidine salt - CaMV cauliflower mosaic virus - CP capsid protein - GA₃ gibberellic acid - Kbp kilobase pair - NAA naphthalene acetic acid - NBT nitroblue tetrazolium chloride - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PMSF phenyl methyl sulfonyl fluoride - PVX potato virus X - PVY potato virus Y     

High resistance to cucumber mosaic virus conferred by satellite RNA and coat protein in transgenic commercial tobacco cultivar G-140.

A chimeric vector was constructed to express cucumber mosaic virus (CMV) satellite (Sat) RNA and coat protein (CP). Transgenic lines of tobacco cultivar G-140 expressing CP and Sat-RNA were obtained; these lines had high resistance to CMV. Fifty to 70% of the transgenic plants were symptomless 90 days after inoculation with 25-50 micrograms/ml of CMV. Resistance was about twice that conferred by the Sat-RNA or the CP gene alone in transformed plants.     

Transformation of a muskmelon ‘Galia’ hybrid parental line (Cucumis melo L. var. reticulatus Ser.) with an antisense ACC oxidase gene

‘Galia’ muskmelon (Cucumis melo L. var. reticulatus Ser.) has been recalcitrant to transformation by Agrobacterium tumefaciens. Transformation of the ‘Galia’ male parental line, ‘Krymka’, with an ACC oxidase (CMACO-1) gene in antisense orientation is described herein. Explants were transformed using A. tumefaciens strain ABI, which contained a vector pCmACO1-AS plasmid, bearing an antisense gene of CMACO-1 and the CP4 syn gene (glyphosate-tolerance). Both CMACO-1 and CP4 syn genes were assessed by a polymerase chain reaction method. Flow cytometry analysis was performed to determine plant ploidy level of primary transformants. Two completely diploid independent transgenic plants were obtained. Southern blot and segregation analysis in the T₁ generation determined that each independent transgenic line had one single insertion of the transgene. These transgenic muskmelon male parental lines have potential for use in the production of ‘Galia’ F₁ hybrids with improved shelf life.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 g ml^–1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.     

Quantitative studies on the uptake and retention of potato leafroll virus by aphids in laboratory and field conditions

Enzyme-linked immunosorbent assay (ELISA) was adapted for the efficient detection and assay of potato leafroll virus (PLRV) in aphids. Best results were obtained when aphids were extracted in 0.05 M phosphate buffer, pH 7.0, and the extracts incubated at 37 °C for 1 h before starting the assay. Using batches of 20 green peach aphids (Myzus persicae), about 0.01 ng PLRV/aphid could be detected. The virus could also be detected in single aphids allowed a 1-day acquisition access period on infected potato leaves. The PLRV content of aphids depended on the age of potato source-plants and the position of source leaves on them. It increased with increase in acquisition access period up to 7 days but differed considerably between individual aphids. A maximum of 7 ng PLRV/aphid was recorded but aphids more usually accumulated about 0.2 ng PLRV per day. When aphids were allowed acquisition access periods of 1–3 days, and then caged singly on Physalis floridana seedlings for 3 days, the PLRV content of each aphid, measured subsequently, was not strongly correlated with the infection of P. floridana. The concentration of PLRV in leaf extracts differed only slightly when potato plants were kept at 15, 20, 25 or 30 °C for 1 or 2 wk, but the virus content of aphids kept on leaves at the different temperatures decreased with increase of temperature. PLRV was transmitted readily to P. floridana at all temperatures, but by a slightly smaller proportion of aphids, and after a longer latent period, at 15 °C than at 30 °C. The PLRV content of M. persicae fed on infected potato leaves decreased with increasing time after transfer to turnip (immune to PLRV). The decrease occurred in two phases, the first rapid and the second very slow. In the first phase the decrease was faster, briefer and greater at 25 and 30 °C than at 15 and 20 °C. No evidence was obtained that PLRV multiplies in M. persicae. These results are compatible with a model in which much of the PLRV in aphids during the second phase is in the haemocoele, and transmission is mainly limited by the rate of passage of virus particles from haemolymph to saliva. The potato aphid, Macrosiphum euphorbiae, transmitted PLRV much less efficiently than M. persicae. Its inefficiency as a vector could not be ascribed to failure to acquire or retain PLRV, or to the degradation of virus particles in the aphid. Probably only few PLRV particles pass from the haemolymph to saliva in this species. The virus content of M. euphorbiae collected from PLRV-infected potato plants in the field increased from early June to early July, and then decreased. PLRV was detected both in spring migrants collected from the plants and in summer migrants caught in yellow water-traps. PLRV was also detected in M. persicae collected from infected plants in July and August, and in trapped summer migrants, but their PLRV content was less than that of M. euphorbiae, and in some instances was too small for unequivocal detection.