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.     

Viral protein synthesis in plants infected with broadbean mottle virus.

The development of virus protein in Vicia faba plants following inoculation with broadbean mottle virus (BBMV) was studied by means of immunoradioautography. Antigen was found in the nucleus and the cytoplasm on the first day of infection. It then accumulated rapidly in the cytoplasm as infection progressed, and high concentrations of antigen were present in the cytoplasm by the fourth day after infection. Chloroplasts were not labeled. Viral antigen was always associated with viral inclusion bodies. The results with BBMV antigen are compared with those in earlier reports on the appearance of tobacco mosaic virus and clover yellow mosaic virus antigen.     

Partial sequence of the N-terminal portion of the protein of cowpea chlorotic mottle virus

The action of trypsin on cowpea chlorotic mottle virus (CCMV) at pH 7.4 caused the release of 25 amino acid residues from the N-terminal of the protein. Separation of peptides released during this digestion on cation exchange resins and determination of their N-termini gave 6 major peptides: the N-terminal peptide CH₃C(0) NH(Lys, Thr₂, Ser, Gly₂, Val₂); AlaAlaAlaArg; AsnThrArg; AlaGlnArg; LeuThrArg; and LysAsnLysArg. Some of these peptides were cleaved by nontryptic enzymes in the digestion. Peptides cleaved by trypsin from chymotrypsintreated virus were Lys, Arg, and AsnThrArg.     

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.     

Identification of an amino terminal domain required for the transforming activity of the Rous sarcoma virus src protein

Transformation of chicken cells by Rous sarcoma virus (RSV) requires the functional expression of the viral src protein, a tyrosine protein kinase, pp60src. Variants of RSV containing deletions within the amino terminal one-third of the src protein have been identified that exhibit either temperature-sensitive or transformation-defective phenotype when used to infect chicken embryo cells. To define the regions within the amino terminal portion of pp60src that influence morphological transformation, a series of overlapping deletion mutations in the src gene of Prague A RSV (Pr A RSV) were constructed and their biological and biochemical properties were analyzed. Deletions within the src gene which remove amino acid residues 38 to 142 had minimal effects on the ability of the mutant viruses to induce cellular transformation. However, deletions, which impinged upon the region of the src gene encoding residues 142 to 169, inhibited cellular transformation. A variant containing a deletion of amino acid residues 169 to 225, was temperature sensitive for transformation. Structurally altered src proteins recovered from cells infected with transformation-defective variants exhibited a somewhat reduced tyrosine protein kinase activities when assayed in the immune complex kinase assay. Analysis of the in vivo phosphorylation of a pp60src substrate, the 36-kDa protein, revealed virtually wild-type levels of phosphorylation in cells infected with the transformation-defective mutants. These studies suggest that the region of the Pr A RSV src protein delineated by amino acid residues 142 to 169 is essential for initiation and maintenance of morphological transformation of chicken cells in culture.     

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.     

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."     

Identification and subcellular localization of a putative cell-to-cell transport protein from red clover mottle virus

To investigate the mode of gene expression of red clover mottle virus (RCMV) middle component (M) RNA, we have synthesized an oligopeptide corresponding to the predicted carboxy-terminus of the RCMV counterparts of the cowpea mosaic virus (CPMV) 48K and 58K proteins. Using an antiserum raised against this synthetic oligopeptide, we have detected a 43-kDa protein in the 30,000 g pellet from extracts of RCMV-infected cowpea protoplasts. Immunogold cytochemistry further localized this protein to the plasmodesmata of RCMV-infected pea tissue. This subcellular location, taken together with other evidence, suggests that this 43-kDa protein has a role in the cell-to-cell spread of RCMV.     

Functional analysis of the naturally recombinant DNA-A of the bipartite begomovirus Tomato chlorotic mottle virus

All geminiviruses found in Brazil belong to the Begomovirus genus with a bipartite genome that is split between two genomic components, DNA-A and DNA-B. The DNA-A of the bipartite begomovirus ToCMoV-[MG-Bt] (Tomato chlorotic mottle virus), however, possesses as a peculiar characteristic the capacity to systemically infect Nicotiana benthamiana. Here we further characterize this variant DNA-A and show that it also infects Solanum lycopersicum and other host plants, in the absence of DNA-B. The ToCMoV-[MG-Bt]-DNA-A encodes an additional ORF, designated AC5, but otherwise its genome organization is similar to other DNA-A from Western Hemisphere begomoviruses. We showed that this AC5 putative ORF is not essential for infection, as disruption of its coding capacity caused no effect on ToCMoV-[MG-Bt]-DNA-A-mediated infection process. Likewise, the ToCMoV-[MG-Bt]-DNA-A ac4 mutant was indistinguishable from its wild type counterpart in all hosts tested. In contrast, an av1 (coat protein) mutant was unable to infect systemically N. benthamiana and Chenopodium quinoa in the absence of DNA-B. However, inclusion of DNA-B in the infection assay fully rescued the movement defect of the ToCMoV-[MG-Bt]-DNA-A av1 mutant. These results suggest that at suboptimal conditions for infection the coat protein is required for ToCMoV-[MG-Bt] systemic movement.     

Analysis of the nucleotide sequence of bean pod mottle virus middle component RNA

The complete nucleotide sequence of the middle component RNA (M RNA) of the comovirus bean pod mottle virus (BPMV) has been determined. The sequence consists of 3662 nucleotides and contains a single long open reading frame sufficient to code for a protein of 113,353 Da. The proteolytic processing sites within this protein have been identified by comparison with the known three-dimensional structure of the virion and cleavage at these sites would lead to a range of products consistent with those observed during processing of the M RNA-encoded polyproteins in vitro. We have performed computer-aided searches for reiterated sequences within BPMV M RNA which might explain why ordered RNA is visible in the electron density map of BPMV middle component particles (Chen, Z., Stauffacher, C. V., Li, Y., Schmidt, T., Bomu, W., Kamer, G., Shanks, M., Lomonossoff, G., and Johnson, J. E., 1989, Science 245, 154-159). These searches revealed both the presence of overrepresented pentameric sequences and a consensus sequence which was repeated 15 times within the RNA sequence.     

Dispensability of 3' tRNA-like sequence for packaging cowpea chlorotic mottle virus genomic RNAs

The 3' ends of three genomic RNAs (gRNAs) of cowpea chlorotic mottle virus (CCMV) terminate in a highly conserved tRNA-like structure (3'TLS). To examine the intrinsic role played the 3'TLS in packaging, the competence of each gRNA lacking the 3' TLS (DeltaTLS-gRNA) to interact with dissociated coat protein (CP) subunits and form virions was assayed in vitro. In contrast to the well established requirement for the participation of either viral 3'TLS or host-tRNAs in the assembly of RNA-containing virions in brome mosaic virus (BMV; Choi, Y, G., Dreher, T. W., Rao, A. L. N. 2002. tRNA elements mediate the assembly of an icosahedral RNA virus. Proc. Natl. Acad. Sci. 99, 655-660), CCMV CP does not require the presence of viral TLS in cis or in trans. Similar in vitro assembly assays showed that CCMV CP subunits also packaged BMV RNAs lacking 3' TLS as well as two other non-bromoviral RNAs although with lesser efficiency. To characterize sequences of CCMV RNA3 (C3) required for packaging, a series deletions was engineered into C3 and their effect on virus assembly was examined. It was observed that, unlike BMV RNA3 whose packaging requires a bipartite signal (Choi, Y. G., Rao, A. L. N. 2003. Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal. J. Virol. 77, 9750-9757), packaging of C3 is independent of either movement protein (MP) ORF or CP ORF or 3' non-coding regions. Based on the differential prerequisites identified in this study for the assembly of BMV and CCMV, we hypothesize that the adaptive condition for movement in monocotyledonous host has made packaging a necessary co-requirement for BMV.