Decreased levels of TMV coat protein in transgenic tobacco plants at elevated temperatures reduce resistance to TMV infection

Transgenic tobacco plants that accumulate tobacco mosaic virus (TMV) coat protein (CP) are resistant to TMV infection under standard growth conditions. The amount of CP accumulated and the degree of resistance to TMV were found to be temperature dependent. Exposure to continuous high temperatures (30-35 degrees) results in a sharp decrease in the amount of CP within 6 hr with no further change for at least 6 days. Under these conditions the transgenic plants developed typical systemic disease symptoms when inoculated with TMV although disease development was delayed. Transgenic plants which were moved from 35 to 22 degrees accumulated the normal level of CP within several hours. Transgenic tobacco plants inoculated and held at 35/25 degrees day/night cycles retained resistance to TMV infection. The level of CP mRNA was constant at each temperature and was associated with polyribosomes. On the basis of these results we suggest that the low level of CP under elevated temperature is due to instability of the TMV CP. In contrast, TMV CP levels in transgenic tomato plants also dropped under elevated temperatures yet retained high resistance to TMV.     

Tissue-specific expression of the TMV coat protein in transgenic tobacco plants affects the level of coat protein-mediated virus protection

Transgenic tobacco plants were produced that express a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) under the control of the promoter from a ribulose bisphosphate carboxylase small subunit (rbcS) gene. Plant lines expressing comparable levels of CP from the rbcS and cauliflower mosaic virus 35S promoters were compared for resistance to TMV. In whole plant assays the 35S:CP constructs gave higher resistance than the rbcS:CP constructs. On the other hand, leaf mesophyll protoplasts isolated from both plant lines were equally resistant to infection by TMV. This indicated that the difference in resistance between the lines in the whole plant assay reflects differences at the level of short- and/or long-distance spread of TMV. Therefore, we propose that the difference in tissue-specific expression between the 35S and rbcS promoters accounts for greater resistance in the plant lines that express the 35S:CP chimeric genes.     

Lesions and virus accumulation in inoculated transgenic tobacco plants expressing the coat protein gene of tobacco mosaic virus

The objective of this work was to identify steps in virus infection which were inhibited in transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) that express the coat protein (CP) gene of the U, strain of tobacco mosaic virus (TMV). These plants were shown to be protected against disease development after inoculation with U,-TMV (P. Powell Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, 1986, Science 232, 738-743). Experiments were also conducted to allow comparison between the protection observed for the transgenic plants and cross-protection. In addition to protection against U1-TMV, the CP-expressing transgenic plants were protected against symptom development of infection after inoculation with a severe TMV strain, PV230, a strain which is immunologically related to the U1 strain. The numbers of chlorotic lesions produced on inoculated leaves of CID-expressing transgenic Xanthi plants infected with PV230 were 30%-or-less of those on leaves of control plants. Likewise necrotic lesion numbers produced on inoculated leaves of CP-expressing transgenic Xanthi 'nc' plants infected with U1 were 5%-or-less of those on leaves of control plants. Virus accumulation in the inoculated leaves of the CP-expressing Xanthi plants was substantially lower than that in leaves of control plants and thus correlated well with the lesion numbers. These results indicate that the delay in disease development includes prevention of virus accumulation in the inoculated leaves. Furthermore, there was a substantial reduction in accumulation of virus in the first leaf above the inoculated leaves in transgenic plants compared with control plants. Inoculation with viral RNA rather than virus largely overcame the protection, leading to the conclusion that the presence of the CID on virus particles in the challenge inoculum was necessary for maximum protection. As shown by these studies, expression of the TMV-CP coding sequence in transgenic plants mimics several of the characteristics of classical cross-protection previously reported by other researchers. We therefore refer to the protection observed for the transgenic plants as "genetically engineered cross-protection."     

Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein

Tobacco mosaic virus (TMV)-like pseudovirus particles containing mRNA for Escherichia coli beta-glucuronidase (GUS) were electroporated into mesophyll protoplasts from control or TMV coat protein (CP)-transgenic tobacco (Nicotiana tabacum cv. Xanthi). GUS-particles were expressed 100-fold less efficiently in CP-transformed than in control protoplasts whereas unencapsidated GUS mRNA was expressed only 2.8-fold less efficiently. Lower transient expression of packaged GUS mRNA is probably due to inhibited disassembly of nucleocapsids in CP-transgenic protoplasts. Control and U1 CP-transformed protoplasts are equally susceptible to infection by cowpea strain TMV (Cc), as well as unencapsidated Cc or U1 RNA. In contrast, native or in vitro reconstituted U1 TMV particles result in 5- to 6-fold fewer infected CP-transgenic than control protoplasts. When Cc RNA was transcapsidated in U1 CP in vitro, the hybrid virions were equally infectious in both classes of protoplasts. We conclude that although compatible U1 protein-protein interactions significantly inhibit (GUS) nucleocapsid disassembly in CP-transgenic protoplasts, the endogenous CP must also interfere with a later stage of infection involving the homologous viral RNA.     

Expression of alfalfa mosaic virus and tobacco rattle virus coat protein genes in transgenic tobacco plants

Using the Agrobacterium tumefaciens binary vector system, a chimeric gene consisting of the cauliflower mosaic virus 35 S promoter, alfalfa mosaic virus (AIMV) coat protein (CP) cistron, and the nopaline synthase polyadenylation signal was integrated into the genome of Nicotiana tabacum cv. Samsun NN. In 70% of the transgenic tobacco plants the chimeric mRNA and its translation product could be detected. CP accumulated to levels up to 0.05% of the soluble leaf protein. The accumulation was independent of leaf age. The same approach was undertaken for the CP of tobacco rattle virus (TRV). The chimeric gene was integrated in the genome of Nicotiana tabacum cv. Xanthi nc. The results with respect to the accumulation of the chimeric mRNA and TRV CP in leaves of transgenic tobacco plants were comparable to those with AIMV transformed plants. Plants accumulating AIMV CP were highly resistant to infection with AIMV nucleoproteins but could be infected with a mixture of AIMV RNAs 1-4. Moreover, a mixture of AIMV RNAs 1, 2, and 3 was infectious to these plants but not to nontransformed control plants.     

High resistance and control of biological risks in transgenic plants expressing modified plum pox virus coat protein

Transgenic plums transformed with the plum pox virus coat protein (PPV CP) gene displayed a resistance to the sharka disease (Ravelonandro et al., 1997). However, the expression of PPV CP in transgenic plants may lead to complementation of deficient characteristic of an incoming potyvirus. Indeed, an aphid-intransmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) could be transmitted when encapsidated by the engineered PPV CP (Lecoq et al., 1993). To control such a risk, new PPV CP constructs were designed and introduced into Nicotiana benthamiana genome. In the first construct, the DAG amino acid triplet involved in the potyvirus aphid-transmission was deleted. The second construct encoded a truncated PPV CP lacking its first 140 amino acids. In the last construct, the nucleotides encoding the charged amino-acids R220, Q221 and D264 localized in the core of the PPV CP were removed. A bacterial expression system was developed to show that these deletions prevent the assembly of the PPV CP subunits. For each construct, several transgenic lines were produced and first challenged with several strains of PPV. Two phenotypes of resistance were observed: recovery and immunity. Their biochemical characterization showed that the resistance was RNA-mediated and therefore can be classified as homology-dependent (Jacquet et al., 1998a). Resistant lines producing high level of wild type or modified PPV CP were then inoculated with ZYMV-NAT to perform an aphid-transmission assay. Results of these experiments demonstrated that the use of modified forms of PPV CP genes in transgenic plants provide a good way to control the biological risks associated with heteroencapsidation (Jacquet et al., 1998b).     

Inhibition of uncoating of tobacco mosaic virus particles in protoplasts from transgenic tobacco plants that express the viral coat protein gene

The uncoating of tobacco mosaic virus (TMV) particles in protoplasts isolated from leaves of transgenic tobacco plants that express the TMV coat protein gene was investigated. Extracts of these protoplasts collected up to 1 hr after inoculation with TMV contained fewer of the complexes ("striposomes") thought to be involved in cotranslational disassembly of virus particles than did extracts of protoplasts that do not express the viral coat protein gene. These results are consistent with the hypothesis that TMV coat protein-mediated resistance is at least in part the result of inhibition of the uncoating of the virus particles in the inoculum.     

Correlation between amino acid exchanges in coat protein of TMV mutants and the nature of the mutagens

This paper represents a comparative study of the action of various reagents of different mutagenicity on the messenger activity of TMV RNA, as monitored by amino acid analyses of the coat protein of 225 mutants. No such comparative data appear to be available from other laboratories.The stable mutants with single amino acid exchanges obtained with nitrous acid, hydroxylamine, and methoxyamine showed the expected predominance of those changes which would be predicted on the basis of their reaction specificity (C → U and A → G; C → U).The amino acid exchanges caused by bromination and alkylation indicated a greater variety of mutagenic events. Recent studies of the many reactions caused by alkylating agents, indicating that all bases may be modified at more than one site, supply an explanation for this finding.The results of photochemical reactions which destroy bases resemble those of spontaneous mutations in giving a completely random exchange pattern.     

Expression of Japanese encephalitis virus envelope protein in transgenic tobacco plants

The virus envelope (E) protein of Japanese encephalitis virus induces virus-neutralizing antibodies and is therefore a potential vaccine antigen. In a mammalian system, co-expression of another viral structural protein prM is necessary for proper expression and folding of E protein. Transgenic tobacco plants were produced carrying JEV cDNA encoding prM and E proteins under the control of the CaMV 35S promoter. E protein, however, was not detectable in these plants. In vitro translation studies showed that the presence of the prM sequence inhibited transgene expression in the plant system. Accordingly, JEV E protein could be expressed in transgenic tobacco plants only without the prM protein.     

Expression of rice yellow mottle virus coat protein enhances virus infection in transgenic plants

Summary. The disease caused by rice yellow mottle virus (RYMV) is a major, economically important constraint to rice production in Africa. RYMV is mechanically transmitted by a variety of agents, including insect vectors. The production of resistant rice varieties would be an important advance in the control of the disease and increase rice production in Africa. We produced transgenic plants of the Oryza sativa japonica variety, TP309, to express a RYMV coat protein gene (CP) and mutants of the CP under the control of a ubiquitin promoter. Transgenic plants expressing genes that encode wild-type CP (wt.CP), deleted CP (ΔNLS.CP), mRNA of the CP, or antisense CP sequences of the CP gene were characterised. Eighty per cent (80%) of independent transgenic lines analysed contained CP gene sequences. Transgenic plants were challenged with RYMV and produced two types of reactions. Most of the plants expressing antisense sequences of the CP and untranslatable CP mRNA exhibited a delay in virus accumulation of up to a week, and the level of virus accumulation was reduced compared with non-transgenic TP309 plants. Transgenic plants expressing RYMV wild-type CP (wt.CP) and deleted CP (ΔNLS.CP) accumulated the highest levels of virus particles. These results suggest that antisense CP and untranslatable CP mRNA induced moderate resistance, whereas transgenic CP enhanced virus infection.     

Altered local and systemic spread of movement deficient virus in transgenic tobacco plants expressing the cucumber mosaic virus 3a protein

Summary.  The 3a protein encoded by RNA 3 of cucumber mosaic virus has been identified as the viral cell-to-cell movement protein. The constitutive expression in transgenic tobacco plants of 3a protein from a subgroup I strain was able to complement in trans the short distance movement of a 3a defective CMV mutant belonging to a different taxonomic subgroup. This ability was dependent upon the accumulation levels of the 3a protein in transgenic tobacco plants. However, an initial delay in viral accumulation and spread of the defective virus as compared to the wild type virus was determined in complementation tests. Furthermore, a reduction in disease symptoms as well as a different pattern of systemic viral distribution from those of the wild type virus was detected. These results show that the early events in viral infection affect the long distance spread of the virus. Finally, the wild type virus moved faster in the 3a protein-expressing plants than in control plants, thus indicating that the constitutive expression of the 3a protein favours long-distance viral spread. Received June 29, 2000/Accepted July 18, 2000     

Influence of exogenous viral coat protein on the cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro

Free coat protein subunits (at 0.1-1.0 mg/ml) from tobacco mosaic virus (TMV) preferentially inhibit the cotranslational disassembly of pH 8-washed virions in vitro. The inhibitory effect is greater than in incubations programmed with naked TMV RNA. Other proteins tested, e.g., bovine serum albumin, had little or no effect on the expression of either template in vitro. Recently it has been proposed that cross-protection between viruses in plants is mediated by the ability of coat protein subunits to inhibit the disassembly of closely related virus strains. If cotranslational disassembly occurs in vivo, then our observations might lend some support to this hypothesis.     

TMV coat protein synthesis in vivo: analysis of the N-terminal acetylation

Nascent peptides from TMV infected leaves yield a product that behaves like the N-terminal peptide from TMV coat protein: a tryptic peptide precipitates at pH 4.7; a chymotryptic peptide, obtained from that tryptic peptide, passes through a Dowex 50 H⁺ column and has the same Rf as N-acetyl-seryl-tyrosine on paper chromatography. It is concluded that nascent TMV coat protein chains are N-terminally acetylated, and thus that acetylation occurs prior to completion of the peptide chain. Analyses of several possible transient intermediates suggest that the acetylation reaction occurs early in the synthesis.