RISK ASSESSMENT OF RECOMBINATION AND TRANSCAPSIDATION OF TOBACCO MOSAIC VIRUS WITH THE CUCUMBER MOSAIC VIRUS COAT PROTEIN

Peter Palukaitis and Lee Zhang

Dept. of Plant Pathology, Cornell University, Ithaca, NY 14853 USA. email: pfp1@cornell.edu

SUMMARY

To assess the risks associated with recombination involving viral sequences in transgenic plants, we have constructed two hybrids between tobacco mosaic tobamovirus (TMV) and cucumber mosaic cucumovirus (CMV), designated T/CMV-0 and -1. In T/CMV-0, a fragment containing most of the coat protein (CP) gene of CMV was inserted out of frame into the CP gene of TMV, so that neither CP could be expressed. In T/CMV-1, the entire CP gene of CMV precisely replaced that of TMV. These hybrid viruses have been used to assess risks associated with recombination and transcapsidation. In Nicotiana tabacum, both viruses could infect the inoculated leaf and move cell-to-cell, but neither virus could move systemically. In N. benthamiana, T/CMV-0 did not show systemic symptoms and moved systemically very poorly. In the same host, T/CMV-1 gave a systemic infection, in which the leaves showed mosaic symptoms, although the kinetics of infection suggested that the route of systemic invasion was not via the vasculature. Extracts of the systemically-infected leaves were infectious upon passage to either the same host or to N. tabacum cv. NN. CMV-like particles could be purified from the T/CMV-1-infected leaves, with a yield of ca. 10% that obtained from CMV-infected N. benthamiana. However, the virus particles were either poorly infectious or non-infectious. Analysis of the encapsidated RNAs showed the presence of numerous species ranging in size from CMV RNA 1 (3.35 kb) down to CMV RNA 4 (1.0 kb), with little-to-no detectable full-length T/CMV-1 (6.7 kb). These encapsidated viral sequences were fragments of T/CMV-1, and not plant ribosomal RNAs. Since only encapsidated RNAs can be transmitted by the aphid vectors of CMV, these data demonstrate that RNAs of heterologous viruses will only be encapsidated efficiently if the RNAs are similar in size to the CMV RNAs. Thus, these data indicate that transgenic plants expressing CMV CP, at levels orders of magnitude lower than observed here for T/CMV-1, do not pose a risk of transcapsidation and transmission of heterologous viral genomes. These data also demonstrate that recombinant viruses formed between TMV and the CMV CP gene are debilitated for long-distance movement via the vascular system. Thus, recombinant viruses formed between the CMV CP gene and most other viral genomes are unlikely to survive in nature.

Key words: Transgenic, recombination, transcapsidation, viral fitness

INTRODUCTION

The issue of whether transgenic plants expressing the coat protein (CP) gene(s) of a virus are likely to be sources of genetic material for recombination with unrelated viruses, generating unique, and by inference, more virulent viruses has been of concern to a number of critics and some researchers in the field (Hull, 1990; de Zoeten, 1991; Chasan, 1993; Rissler and Mellon, 1993; Schoelz and Wintermantel, 1993; Tepfer, 1993; Greene and Allison, 1994). This is one of several issues that has led to delays in the application of this aspect of agricultural biotechnology. With the generation of unique sources of resistance and the rapid ability to introduce such resistance genes into a variety of plant species, the concerns raised have led to a heightened awareness of a number of processes in which viruses are involved, including transcapsidation (Palukaitis, 1991) and recombination (Rissler and Mellon, 1993; Schoelz and Wintermantel, 1993; Greene and Allison, 1994). While transcapsidation occurs with some closely related plant viruses, it occurs rarely, if at all in other circumstances (Palukaitis, 1991). Recombination has been observed to occur among plant viruses (White and Morris, 1994), and also between a plant virus and similar viral sequences expressed from a transgene, under strong or moderate selection pressure (Schoelz and Wintermantel, 1993; Greene and Allison, 1994; Wintermantel and Schoelz, 1996). However, this is not the main cause of concern, which is whether an unrelated virus could obtain viral sequences from a transgene and alter its pathogenicity, host range, or method of transmission; i.e., virus-vector interactions (de Zoeten, 1991; Rissler and Mellon, 1993). Thus, transgenic plants are being examined for recombination between viral transgenes and various viruses to see if such recombinant viruses will form and whether the recombinant viruses display any selective advantage and/or altered phenotype.

Although many plants are infected by several plant viruses at one time, there is no evidence for new viruses having been formed recently by recombination between two viruses that are taxonomically different (Falk and Bruening, 1994). Hence, obtaining and identifying potential recombinant plant viruses as replication and movement competent entities is likely to have a low probability. Therefore, while such data are being sought, we have adopted an alternative strategy and generated a recombinant plant virus by genetic engineering and asked what are the fitness and properties of this virus compared to its two parents? That is, we have bypassed the first step: the need to obtain a recombinant, hybrid virus, which would have to be selected for during replication and movement of the infecting virus for the recombinant virus to be detectable. This allows us to assess the biological properties and fitness of the hybrid virus. To facilitate this analysis, we have taken two well-characterized viruses, tobacco mosaic virus (TMV) and cucumber mosaic (CMV) and created a hybrid virus referred to as T/CMV, in which the CP gene of TMV has been replaced by the CP gene of CMV.

TMV is a rod-shaped plant virus with a single-stranded RNA genome of 6.4 kb, encoding five proteins involved in virus replication, movement and encapsidation . TMV is transmitted very efficiently mechanically, but not by any known virus vector (Palukaitis and Zaitlin, 1986). CMV is an isometric virus, containing three genomic RNAs of 3.35 kb, 3.0 kb and 2.2 kb, encoding five proteins. CMV is transmitted mechanically, and by a number of aphid species, as well as in some instances through the seed (Palukaitis et al., 1992). The host range of CMV is larger than that of TMV, but they also share a number of common hosts. In both cases, the CP has been shown to have a role in long-distance movement of the virus, and for CMV the CP is the sole determinant of transmission by aphids (Perry et al., 1994). Thus, the hybrid virus, T/CMV, might be expected to have shared properties common to both parents and some properties unique to one parent, or even unique to itself. We report here the construction and characterization of two variants of T/CMV, and compare their fitness with that of the parental viruses, TMV and CMV.

MATERIALS AND METHODS

Construction of T/CMV-0. All cloning manipulations were carried out using standard procedures described Sambrook et al. (1989). T/CMV-0 was constructed using a full-length cDNA clone of the TMV genome (pTMV304), that had been modified with an SP6 RNA polymerase promoter replacing the E. coli RNA polymerase promoter in the vector from plasmid pTMV204 (Dawson et al., 1986; W.O. Dawson, personal communication). The modified TMV cDNA clone was digested with the restriction endonucleases PacI and AccI, which cleave at residues 5785 and 6064, respectively, of the TMV genome, and the ends were made blunt by treatment with the Klenow fragment of E. coli DNA polymerase I. A blunt-end fragment containing all of the CMV CP gene sequences, except those encoding the N-terminal 7 amino acids, was obtained by digesting pLSCMV3, a full-length cDNA clone of RNA 3 from the LS-strain of CMV (Zhang et al., 1994), with the restriction enzymes AlwI and NruI, which cleave at nucleotides 1240 and 1922, respectively, of the LS-CMV RNA 3 sequence. The blunt-end CMV CP gene fragment was ligated into the larger fragment of the above digested pTMV304, containing most of the TMV genome, and the resulting cDNA clone, pT/CMV-0, was obtained after transformation of the ligated plasmid in E. coli. pT/CMV-0 encodes 22 amino acids of the TMV CP gene, followed sequentially by an insertion of the out-of-frame CMV CP gene fragment, part of the TMV CP sequence, and the TMV 3' non-translated region (Figure 1B).

Construction of T/CMV-1. T/CMV-1 was constructed using a cDNA clone of the TMV genome, from which the CP gene had been deleted and replaced with sequences of the restriction enzyme XhoI (pTMV S3-28; Lehto et al., 1990), and which had been further modified with an SP6 RNA polymerase promoter replacing the E. coli polymerase promoter in the vector, by exchange of the 3' half of the TMV insert sequences with pTMV304. The CP gene of LS-CMV was amplified by PCR from the cDNA clone pLSCMV3, using primers corresponding to the 5' and 3' termini of the CP gene, flanked by sequences containing cleavage sites for the restriction enzyme XhoI. The PCR fragment was digested with XhoI and was ligated into the modified, XhoI-digested pTMV S3-28. After transformation into E. coli, the resulting cDNA clone, pT/CMV-1, was isolated. T/CMV-1 (Figure 1C) contains the complete LS-CMV CP gene inserted into the location previously occupied by the TMV CP gene in the TMV genome (Figure 1A).

Transcription of T/CMV-0 and T/CMV-1 and infectivity assays. Infectious RNAs were transcribed from the cDNA clones pT/CMV-0 and pT/CMV-1 using SP6 RNA polymerase, after linearization of the plasmids with the restriction enzyme KpnI. The RNA transcripts were inoculated to plants (N. tabacum cv. Samsun nn and cv. Samsun NN, N. benthamiana, and N. clevelandii) dusted with Carborundum. The plants were maintained in environmentally controlled growth chambers, and were examined for symptom production over several weeks. Inoculated leaves from the above plants as well as leaves above the inoculated leaves (i.e., systemic leaves) were ground in a mortar and pestle in 50 mM potassium phosphate, pH 7, and the extracts were assessed for infectivity, by inoculation either to N. benthamiana or to N. tabacum cv. Samsun NN. The latter plant is a hypersensitive host for TMV, and produces necrotic local lesions when infected by TMV. These lesions provide a quantitative estimate of the concentration of TMV-like RNAs present in the extracts (see Table 2). Total RNAs were also extracted from T/CMV-1-infected N. benthamiana leaves using an RNA extraction kit obtained from QIAGEN, according to the manufacturers instructions.

Purification and analysis of T/CMV-1. T/CMV-1 particles were purified from systemically-infected N. benthamiana leaves using the standard CMV purification procedure (Palukaitis et al., 1992). The particles were analyzed under the electron microscope, after staining with uranyl acetate (Palukaitis et al., 1992). RNA extracted from either the virus particles or infected leaves was analyzed by agarose gel electrophoresis and northern blot hybridization using TMV RNA-, CMV RNA- or (tomato) ribosomal RNA-specific probes (Sambrook et al., 1989).

RESULTS

Construction of T/CMV-0 and T/CMV-1. To determine whether a hybrid virus potentially formed in a transgenic plant expressing the CMV CP gene would be viable, we generated two variants of TMV (Figure 1A) containing the CMV CP gene. In the first instance, we incorporated most of the CMV CP gene into the TMV CP gene; albeit out of frame (Figure 1B). This is the type of recombinant, hybrid virus most likely to form in nature, in which neither CP gene is intact. This construct was designated T/CMV-0. The second variant precisely replaced the TMV CP gene with that of CMV (Figure 1C), so that there were no extra amino acids associated with the CMV CP, although the latter is now expressed from the TMV rather than CMV genome.

Infectivity of T/CMV-0 and T/CMV-1 in tobacco. RNAs transcribed from both hybrid virus cDNA clones were inoculated to tobacco plants (Nicotiana tabacum cv. Samsun nn), a systemic host for both TMV and CMV. In both cases, no symptoms were observed in either the inoculated leaves or the systemic leaves. Back inoculation from both types of leaves to the hypersensitive host NN-tobacco (N. tabacum cv. Samsun NN) showed that there was virus present in the inoculated tobacco leaves, but not the upper, non-inoculated leaves, for both T/CMV-0 and T/CMV-1 (Table 1). The infectivity observed (less than five lesions per leaf) was very low compared to the number of lesions obtained from tobacco infected by TMV (several hundred lesions), consistent with either a low titer of infectious material, or the presence of naked RNA, which is very labile in plant sap. The latter situation has been described for mutants of TMV that either do not produce CP or do not assembly into virus particles (Siegel et al., 1962; Dawson et al., 1988). Our inability to recover infectivity from necrotic lesions of NN-tobacco inoculated with T/CMV-0 or T/CMV-1 is consistent with the above observations.

Infectivity of T/CMV-0 and T/CMV-1 in other Nicotiana species. Inoculation of N. benthamiana and N. clevelandii plants with T/CMV-0 and T/CMV-1 present in infected-tobacco sap or using RNA transcripts resulted in infection in the inoculated leaves of both hosts (Table 1), but the systemic leaves showed different results, depending on the inoculum and the host (Tables 1and 2). In N. benthamiana, T/CMV-1 induced systemic dark-green/light-green mosaic symptoms about two weeks post-inoculation, and T/CMV-0 did not induce any systemic symptoms, whereas TMV would have induced systemic symptoms 6 days p.i., and killed the plants by two weeks p.i. (data not shown). While no infectivity was observed using sap extracts from systemically infected N. benthamiana leaves inoculated with T/CMV-0 (Table 1), when total RNAs were extracted and analyzed for infectivity, there was a very low level of infectivity present (Table 2), much less so than in N. benthamiana leaves systemically infected with T/CMV-1 (Table 2). These data show that T/CMV-0 and T/CMV-1 were moving systemically very slowly, and probably not via the vascular system, but rather cell-to-cell up the stem. By contrast, in N. clevelandii, where the internodal distances are very small, both T/CMV-0 and T/CMV-1 more rapidly infected the plants systemically (Table 1), although here again, neither hybrid virus was able either to infect the plants as rapidly as TMV or CMV, or to cause the lethal symptoms associated with infection by TMV.

Characterization of T/CMV-1 in N. benthamiana. Using the CMV purification procedure, virus particles could be isolated from T/CMV-1 infected N. benthamiana plants. The yield of virus was about 10% of that obtained from N. benthamiana infected with LS-CMV or TMV. The T/CMV-1 particles resembled CMV under the electron microscope (data not presented), although they were either non-infectious or very poorly infectious, producing 0 or 2-3 lesions per leaf inoculated with virus at ca. 2 mg / ml. The infectivity did not change after one or two cycles of differential centrifugation, suggesting that the infectivity was not due to T/CMV-1 RNA bound to the outside of the virus particles, where it would be expected to be more labile.

Northern blot hybridization of T/CMV-1 RNA extracted from virus particles showed only traces of full-length (6.7 kb) T/CMV-1 RNA, if any at all. Most or all of the encapsidated RNAs from T/CMV-1 particles were in the size range of the CMV RNAs (3.35 - 1.0 kb), and hybridized to cloned probes of both the CMV CP gene and the TMV genome, but not to plant ribosomal RNAs (data not presented). Thus, the data show that either predominantly full-length T/CMV-1 RNA is encapsidated by the CMV CP in vivo, but the RNA is very labile and is cleaved to yield discrete fragments, or little-to-no full-length T/CMV-1 RNA is encapsidated by the CMV CP (due to size constraints for encapsidation), but rather specific fragments of T/CMV-1 RNA are encapsidated, equal in size to the CMV RNAs naturally packaged by this CP.

DISCUSSION

The data presented here show that if recombination took place between the CP gene of CMV and the genome of TMV, then the most likely outcome would be a hybrid virus expressing the CP of neither parental virus (i.e., T/CMV-0). In general, such a virus would be debilitated for long-distance movement, and could not be transmitted easily to other hosts. The level of CP produced in transgenic plants expressing the CMV CP gene probably would be insufficient to encapsidate the defective virus into virus particles needed for transmission to other plants.

In the highly unlikely situation that perfect recombination occurred between the donor (CMV CP transgene) and the acceptor (TMV) virus, the hybrid virus (T/CMV-1) would also be debilitated for long-distance movement and further transmission, since little-to-no full-length virus is present in virus particles that might be transmitted by the aphid vectors of CMV. If transmission did occur, almost all of the transmitted virus particles would contain degraded RNA, and any infectious RNA that was transmitted would be debilitated for long-distance movement. As only encapsidated RNAs can be transmitted by the vectors of CMV, and since most plant viruses contain single or two component genomes where the genomic RNAs are much larger than CMV RNA 1 (3.35 kb), the risks associated with recombination and transcapsidation of unrelated viral genomes by transgenic plants expressing viral CPs are no greater from those associated with normal infections of plants by multiple, unrelated viruses, where no recombinant viruses have so far been detected. In fact, the risks probably are even less for recombinant viruses forming in transgenic plants, since only one of the recombining species is present as an intact virus. In conclusion, rather than generating novel viruses that would show enhanced pathology, host range, or virulence, our data indicate that hybrid viruses, generated in transgenic plants expressing viral CP sequences, are more likely to be debilitated and have a narrower host range than either parent virus. This may account for the inability so far to identify such a recombinant, hybrid virus in nature. The above conclusion also applies to other examples of hybrid viruses (constructed in vitro using unrelated viruses) that have been described in the literature, where the movement protein rather than CP has been exchanged (DeJong and Ahlquist, 1992; Giesman-Cookmeyer et al., 1995; Solovyev et al., 1996). The generation of a truly novel virus requires considerable adaptation, making such an occurrence extremely rare and unlikely.

ACKNOWLEDGMENTS

This work was supported in part by Grant No. 95-33120-1876 from the U.S. Department of Agriculture BRARGP.

REFERENCES

Chasan R. 1993. Harvesting virus recombinants. Plant Cell 5: 1489-1491.

Dawson WO, Beck DL, Knorr DA, Grantham GL 1986. cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. Proc. Natl. Acad. Sci. USA 83: 1832-1836.

Dawson WO, Bubrick P, Grantham GL. 1988. Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement, and symptomatology. Phytopathology 78: 7830789.

DeJong W, Ahlquist P. 1992. A hybrid plant virus made by transferring the noncapsid movement protein from a rod-shaped to an icosahedral virus is competent for systemic infection. Proc. Natl. Acad. Sci. USA 89: 6808-6812.

de Zoeten GA. 1991. Risk assessment: Do we let history repeat itself? Phytopathology 81: 585-586.

Falk BW, Bruening G. 1994. Will transgenic crops generate new viruses and new diseases? Science 263: 1395-1396.

Giesman-Cookmeyer D, Silver S, Vaewhongs AA, Lommel SA, Deom CM. 1995. Tobamovirus and dianthovirus movement proteins are functionally homologous. Virology 213: 38-45.

Greene AE, Allison RF. 1994. Recombination between viral RNA and transgenic plant transcripts. Science 262: 1423-1425.

Hull R. 1990. The use and misuse of viruses in cloning and expression in plants. In: Fraser RS (ed) Recognition and responses in plant-virus interactions. NATO ASI, Vol.H41. Springer-Verlag, Berlin, pp 443-457.

Lehto K, Grantham GL, Dawson WO. 1990. Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement. Virology 174: 145-157.

Palukaitis P. 1991. Virus-mediated genetic transfer in plants. In: Levin M, Strauss S (ed) Risk assessment in genetic engineering. McGraw-Hill, New York, pp 140-162.

Palukaitis P, Zaitlin M. 1986. Tobacco mosaic virus infectivity and replication. In: Van Regenmortel HM, Fraenkel-Conrat H (ed) The plant viruses. 2. The rod-shaped plant viruses. Plenum Press, New York, pp 105-131.

Palukaitis P, Roossinck MJ, Dietzgen RG, Francki RIB. 1992. Cucumber mosaic virus. Adv. Virus Res. 41: 281-348.

Perry KL, Zhang L, Shintaku MH, Palukaitis P. 1994. Mapping determinants in cucumber mosaic virus for transmission by Aphis gossypii. Virology 205: 591-595.

Rissler J, Mellon M. 1993. Perils amid the promise: Ecological risks of transgenic crops in a global market. Union of Concerned Scientists, Washington, DC.

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning. A laboratory manual. 2nd edn. Cold Spring Harbor Laboratory, New York.

Schoelz JE, Wintermantel WM. 1993. Expansion of viral host range through complementation and recombination in transgenic plants. Plant Cell 5: 1669-1679.

Siegel A, Zaitlin M, Seghal OP. 1962. The isolation of defective tobacco mosaic virus strains. Proc. Natl. Acad. Sci. USA 48: 1845-1851.

Solovyev AG, Zelenina DA, Savenkov EI, Grdzelishvili VZ, Morozov SY, Lesemann DE, Maiss E, Casper R, Atabekov JG. 1996. Movement of barley stripe mosaic virus chimera with a tobacco mosaic virus movement protein. Virology 217: 435-441.

Tepfer, M. 1993. Viral genes and transgenic plants. What are the potential environmental risks. Bio/Technology 11: 1125-1132.

White, KA, Morris TJ. 1994. Recombination between defective tombusvirus RNAs generates functional hybrid genomes. Proc. Natl. Acad. Sci. USA 91: 3642-3646.

Wintermantel WM, Schoelz JE. 1996. Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure. Virology 223: 156-164.

Zhang L, Hanada K, Palukaitis P. 1994. Mapping local and systemic symptom determinants of cucumber mosaic cucumovirus in tobacco. J. Gen. Virol. 75: 3185-3191.

Table 1. Infectivity of TMV and T/CMV hybrids on Nicotiana tabacum, N. benthamiana, and N. clevelandii.

Inoculum Species Leaf Infectivity
TMV N. tabacum Inoculateda

Systemic

+b

+

N. benthamiana Inoculated

Systemic

+

+

N. clevelandii Inoculated

Systemic

+

+

T/CMV-0 N. tabacum Inoculated

Systemic

+

-

N. benthamiana Inoculated

Systemic

+

-

N. clevelandii Inoculated

Systemic

+

+

T/CMV-1 N. tabacum Inoculated

Systemic

+

-

N. benthamiana Inoculated

Systemic

+

+

N. clevelandii Inoculated

Systemic

+

+

aBuffered sap extracts of the inolulated leaf or an upper (systemic)leaf were assayed for infectivity.

bInfectivity was measured as the presence or absence of necrotic local lesions on the hypersensitive host NN-tobacco.

Table 2. Infectivity of T/CMV-0 and T/CMV-1 in nucleic acids extracted from systemically infected N. benthamiana.
Total nucleic acid extracts Infectivity
T/CMV-0 Leaf no. 1a 2b
Leaf no. 2 2
Leaf no. 3 1
Leaf no. 4 1
T/CMV-1 Leaf no. 1 35
Leaf no. 2 38
Leaf no. 3 34
Leaf no. 4 11

aLeaf number one above the inoculated leaf.

bNumber of local lesions on NN-tobacco.

Figure 1. Diagram of the TMV genome and of the hybrid viruses T/CMV-0 and T/CMV-1. All of the CMV CP except the sequences encoding N-terminal 7 amino acids of the CMV CP were inserted out of frame into the TMV CP gene in T/CMV-0. The entire TMV CP gene was replaced by the entire CMV CP gene in T/CMV-1.