Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

应用RNAi技术培育抗TMV病毒转基因烟草

利用烟草花叶病毒(TMV)外壳蛋白基因构建RNAi干涉载体, 通过叶盘法转化至烟草K326 和龙江911两个栽培品种。对转基因株系的荧光定量PCR分析表明, 不同转基因株系的病毒RNA靶序列都得到一定程度的降解, 抗病性鉴定结果证实, 转基因K326和龙江911两个栽培品种的转基因材料分别有83％和90％转基因株系对TMV呈现免疫级抗性。     

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.     

Phosphodiesterase activities in transgenic tobacco plants associated with the movement protein of tobacco mosaic virus

Summary Hydrolytic activities of leaf extracts from normal and transgenic plants, with (+ MP) and without (-MP) the movement protein of tobacco mosaic virus, were examined. In the + MP transgenic plants, as compared with non-transgenic and — MP plants, higher hydrolytic activities were found on the following substrates: bis-(nitrophenyl)-phosphate (BPNPP, phosphodiesterase), p-nitrophenyl-(phenyl)-phosphate (PNPPP, nucleotidephosphodiesterase) and thymidine-3-monophosphate p-nitrophenyl ester (T₃MPP; 3nucleotide phosphodiesterase.) The + MP plant lines, as compared with other transgenic plants, exhibited higher nucleotide-phosphodiesterase activity in the soluble as well as in the membrane fraction. Substrate concentration kinetic studies revealed the presence of a nucleotide-phospho-diesterase with a high substrate affinity in the +MP extracts in addition to the enzyme with a relatively low substrate affinity present also in the — MP transgenic plants. This high affinity enzyme could be removed from the soluble fraction by precipitation with anti-MP serum, indicating its possible association with the movement protein.     

Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants.

Expression of the tobacco mosaic virus 30-kD movement protein (TMV MP) gene in tobacco plants increases the plasmodesmatal size exclusion limit (SEL) 10-fold between mesophyll cells in mature leaves. In the present study, we examined the structure of plasmodesmata as a function of leaf development. In young leaves of 30-kD TMV MP transgenic (line 274) and vector control (line 306) plants, almost all plasmodesmata were primary in nature. In both plant lines, secondary plasmodesmata were formed, in a basipetal pattern, as the leaves underwent expansion growth. Ultrastructural and immunolabeling studies demonstrated that in line 274 the TMV MP accumulated predominantly in secondary plasmodesmata of nonvascular tissues and was associated with a filamentous material. A developmental progression was detected in terms of the presence of TMV MP; all secondary plasmodesmata in the tip of the fourth leaf contained TMV MP in association with the filamentous material. Dye-coupling experiments demonstrated that the TMV MP-induced increase in plasmodesmatal SEL could be routinely detected in the tip of the fourth leaf, but was restricted to mesophyll and bundle sheath cells. These findings are discussed with respect to the structure and function of plasmodesmata, particularly those aspects related to virus movement.     

Effect of yeast CTA1 gene expression on response of tobacco plants to tobacco mosaic virus infection

The response of tobacco (Nicotiana tabacum L. cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV). These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase gene CTA1 under the control of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions. Cellular localization of the CTA1 protein was established using immunocytochemical analysis. Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space. The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight. No significant differences were recorded in comparison with non-transgenic tobacco plants. The 3,3'-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H(2)O(2)) in the TMV infected tissue. Presence of H(2)O(2) could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants. In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants. Changes in the distribution of H(2)O(2) and in lesion formation were not reflected by changes in salicylic acid production. In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar. This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.     

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.     

Hairpin RNA derived from viral NIa gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants

Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An RNAi construct was designed to target the nuclear inclusion protein ‘a’ (NIa) gene of WSMV. Wheat was stably cotransformed with two plasmids: pStargate‐NIa expressing hairpin RNA (hpRNA) including WSMV sequence and pCMneoSTLS2 with the nptII selectable marker. When T₁ progeny were assayed against WSMV, ten of sixteen families showed complete resistance in transgenic segregants. The resistance was classified as immunity by four criteria: no disease symptoms were produced; ELISA readings were as in uninoculated plants; viral sequences could not be detected by RT‐PCR from leaf extracts; and leaf extracts failed to give infections in susceptible plants when used in test‐inoculation experiments. Southern blot hybridization analysis indicated hpRNA transgene integrated into the wheat genome. Moreover, accumulation of small RNAs derived from the hpRNA transgene sequence positively correlated with immunity. We also showed that the selectable marker gene nptII segregated independently of the hpRNA transgene in some transgenics, and therefore demonstrated that it is possible using these techniques, to produce marker‐free WSMV immune transgenic plants. This is the first report of immunity in wheat to WSMV using a spliceable intron hpRNA strategy.     

A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco

Ribozymes of the hammerhead class can be designed to cleave a target RNA in a sequence-specific manner and can potentially be used to specifically modulate gene activity. We have targeted the tobacco mosaic virus (TMV) genome with a ribozyme containing three catalytic hammerhead domains embedded within a 1 kb antisense RNA. The ribozyme was able to cleave TMV RNA at all three target sites in vitro at 25°C. Transgenic tobacco plants were generated which expressed the ribozyme or the corresponding antisense constructs directed at the TMV genome. Six of 38 independent transgenic plant lines expressing the ribozyme and 6 of 39 plant lines expressing the antisense gene showed some level of protection against TMV infection. Homozygous progeny of some lines were highly resistant to TMV; at least 50% of the plants remained asymptomatic even when challenged with high levels of TMV. These plants also displayed resistance to infection with TMV RNA or the related tomato mosaic virus (ToMV). In contrast, hemizygous plants of the same lines displayed only very weak resistance when inoculated with low amounts of TMV and no resistance against high inoculation levels. Resistance in homozygous plants was not overcome by a TMV strain which was altered at the three target sites to abolish ribozyme-mediated cleavage, suggesting that the ribozyme conferred resistance primarily by an antisense mechanism.     

Protection of transgenic tobacco plants expressing bovine pancreatic ribonuclease against tobacco mosaic virus

Transgenic tobacco plants (Nicotiana tabacum cv. SR1) expressing extracellular pancreatic ribonuclease from Bos taurus and characterized by an increased level of ribonuclease activity in leaf extracts were challenged with tobacco mosaic virus. The transgenic plants exhibited a significantly higher level of protection against the virus infection than the control non-transformed plants. The protection was evidenced by the absence (or significant delay) of the appearance of typical mosaic symptoms and the retarded accumulation of infectious virus and viral antigen. These results demonstrate that modulation of extracellular nuclease expression can be efficiently used in promoting protection against viral diseases.     

Production of virus resistant and insect tolerant transgenic tobacco plants

The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis -endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with Agrobacterium tumefaciens A12 carrying recombinant pE14. Transgenic r₀ and R₁ tobacco plants expressing CMV-CP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering.Abbreviations CMV cucumber mosaic virus - Bt toxin Bacillus thuringiensis -endotoxin - CaMV cauliflower mosaic virus - NOS nopaline synthase - Kan Kanamycin - Spe spectinomycin - Carb Carbenicillin     

Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.