Coat-protein-mediated resistance to tobacco mosaic virus: discovery mechanisms and exploitation

In 1986 we reported that transgenic plants which accumulate the coat protein of tobacco mosaic virus (TMV) are protected from infection by TMV, and by closely related tobamoviruses. The phenomenon is referred to as coat-protein-mediated resistance (CP-MR), and bears certain similarities to cross protection, a phenomenon described by plant pathologists early in this century. Our studies of CP-MR against TMV have demonstrated that transgenically expressed CP interferes with disassembly of TMV particles in the inoculated transgenic cell. However, there is little resistance to local, cell-to-cell spread of infection. CP-MR involves interaction between the transgenic CP and the CP of the challenge virus, and resistance to TMV is greater than to tobamo viruses that have CP genes more distantly related to the transgene. Using the known coordinates of the three-dimensional structure of TMV we developed mutant forms of CP that have stronger inter-subunit interactions, and confer increased levels of CP-MR compared with wild-type CP. Similarly, it is predicted that understanding the cellular and structural basis of CP-MR will lead to the development of variant CP transgenes that each can confer high levels of resistance against a range of tobamoviruses.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

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.     

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.     

Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.     

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。     

A new multigene family inducible by tobacco mosaic virus or salicylic acid in tobacco.

A previously undescribed cDNA family was isolated from tobacco challenged with tobacco mosaic virus (TMV). A cDNA library was constructed with mRNA from upper leaves of Xanthi nc tobacco plants that had been inoculated with TMV on the lower leaves 11 days previously. The library was screened differentially with radiolabeled cDNA synthesized with mRNA from upper, uninoculated leaves of either TMV-inoculated or mock-inoculated tobacco plants. The new cDNA family, designated SAR8.2, had at least five expressed members, one or more of which were inducible by TMV inoculation and by salicylic acid treatment. The cDNAs encoded small, highly basic proteins containing N-terminal hydrophobic signal peptides and highly conserved cysteine-rich C-terminal domains. One of the SAR8.2 family members contained a direct repeat of the C-terminal domain in tandem. Hybridization of SAR8.2 cDNA to tobacco genomic DNAs indicated a gene family of 10-12 members.     

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.     

The mechanism(s) of coat protein-mediated resistance against tobacco mosaic virus

The resistance of transgenic plants express genes encoding viral coat proteins to infection by the viruses from which the genes are derived was termed coat protein-mediated resistance (CP-MR) and has been demonstrated for a variety of virus/host combinations. The mechanism of CP-MR is perhaps best understood in the tobacco/TMV system. CP-MR against TMV requires accumulation of CP and does not seem to involve the induction of plant defense mechanisms. The resistance appears to be mainly based on the inhibition of virion disassembly in transgenic cells although there is evidence that a later step of infection is also affected. CP-MR of tobacco to TMV shares some features with classical cross-protection and with CP-MR in some, but not all other host/virus combinations.     

RNA silencing-mediated resistance is related to biotic / abiotic stresses and cellular RdRp expression in transgenic tobacco plants

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.