IDENTIFICATION OF PLANT VIRAL SYNERGISM GENES

IDENTIFICATION OF PLANT VIRAL SYNERGISM GENES

Xing Ming Shi, Gail Pruss, Xin Ge, Verl Sriskanda and Vicki Bowman Vance

Department of Biological Sciences, University of South Carolina, Columbia, SC 29208, FAX: (803)777-3179, vance@biol.sc.edu

SUMMARY

Plants are commonly exposed to multiple viral infections and interactions between co-infecting pathogens may be antagonistic or synergistic. Antagonistic reactions may render a host plant infected with one virus resistant to subsequent infection with a related virus (cross protection). The mechanisms involved in such antagonistic interactions are currently the focus of intense research in a variety of virus/host systems. Using a transgenic plant approach, investigators have determined that the expression of individual viral genes can result in resistance to subsequent virus infection. This line of investigation has yielded valuable clues to the basic mechanisms of cross protection, as well as being of practical significance. However, since the mechanisms by which virally encoded resistance genes mediate protection are not completely understood, the question of the safety of this biotechnological breakthrough has been raised. One potential risk in the use of virus encoded genes to confer protection involves another class of interactions which occurs very commonly in plants, viral synergism. In contrast to cross protection, the importance of the synergistic-type viral interactions in understanding viral disease has not been extensively studied and is just beginning to come to light. Many plant diseases are caused by the interaction of two unrelated viruses in the same host plant. Our recent work indicates that at least one of these viral synergistic diseases, the interaction between potato virus X (PVX) and any of a variety of members of the potyvirus group of plant viruses, can be mimicked by expressing of a subset of the potyviral genome in transgenic plants, and then infecting the plant with PVX, the second virus of the synergistic pair. Thus, it is possible that viral genes expressed in plants for the purpose of cross protection may interact with an unrelated virus to produce a synergistic viral disease instead of the expected resistance. The research described here should provide some of the necessary background information for a realistic appraisal of the risk involved in using virally encoded resistance genes to engineer protection in plants.

INTRODUCTION

Mixed viral infections are common in plants and interactions may occur between viruses within the same host cell. Such viral interactions may be either antagonistic or synergistic. Often the multiplication of one virus interferes with the subsequent replication or movement of another virus in the same host and renders the plant resistant to the second of the two invading viruses. This antagonistic interaction is called cross protection and is restricted to closely related viruses. The mechanisms involved in cross protection and other such antagonstic viral interactions are currently the focus of intense research in a variety of virus/host systems (see Wilson, 1993 for a recent review).

In the second class of interaction, co-infection of a host plant with two unrelated viruses elicits disease symptoms that are more severe than the sum of those induced in either single infection. Many plant diseases are caused by the interaction of two unrelated viruses in the same host plant. Such synergistic diseases have been known since 1925 and more examples are reported each year (for example, Blood, 1928; Calvert and Ghabriel, 1983; Clark et al., 1980; Garces-Orejuela and Pound, 1956; Kuhn and Dawson, 1973; Poolpol and Inouye, 1986; Rochow and Ross, 1955; Vance, 1991). The importance of this synergistic-type of viral interaction in understanding plant viral disease is just beginning to come to light.

Virally encoded resistance genes can induce cross protection when expressed in transgenic plants. In 1986 it was demonstrated that the expression of a viral coat protein in a transgenic plant may mimic cross protection, offering some protection from subsequent infection with the same virus (Powell et al., 1986). This technology has since been applied to many virus infections, resulting in a large body of data concerning the mechanism for coat protein mediated protection as well as the obvious practical significance of engineered plant protection. More recently, this prolific area of research has expanded to the identification of nonstructural viral genes which may also serve as resistance genes to protect the plant from the same or related virus (Braun and Hemenway, 1992; Longstaff et al., 1993; MacFarlane and Davies, 1992). Interestingly, despite the concentration of research effort in this area, the mechanism for viral cross protection remains unresolved. In the absence of a comprehensive understanding of the phenomenon of viral protection mediated by virally encoded resistance genes, the question of the safety of engineered cross protection has been raised (DeZoeten, 1991; Farrinelli et al., 1992).

Virally encoded genes may also induce synergism when expressed in transgenic plants. One potential risk in the engineering of cross protection using viral resistance genes, is that such genes may interact to induce a synergistic viral disease instead of the expected cross protection. The actual risk, if any would depend on a number of factors: Are there, in fact, virally encoded "synergism genes", just as there are virally encoded resistance genes? If so, do any of the resistance genes also function as synergism genes? Do certain classes of viral genes serve as synergism genes in many cases, as for example the coat protein gene acts to induce resistance in many different virus infections? Or are there different classes of synergism which are induced by different classes of viral genes? In contrast to the situation with cross protection, research into the mechanism of synergistic viral interaction has been limited. Due to the absence of basic background information about viral synergism, the actual risk of accidentally inducing one disease in engineered plants while protecting them from another disease is unknown.

Potyvirus-associated synergisms. Interestingly, a large number of reported plant viral synergisms involve a member of the potyvirus group of plant viruses as one of the synergistic pair. Several of these potyvirus-associated synergisms have been examined in some detail: 1. corn lethal necrosis caused by co-infection with maize chlorotic mottle virus (MCMV) and maize dwarf mosaic virus (MDMV, potyvirus)(Goldberg and Brakke, 1987; Uyemoto et al., 1981), 2. the interaction of bean pod mottle virus (BPMV) with soybean mosaic virus (SMV, potyvirus) (Anjos et al., 1992; Calvert and Ghabriel, 1983; Lee and Ross, 1972) in soybean and 3. the classic interaction of potato virus X (PVX) and potato virus Y (PVY, potyvirus) in tobacco and other solaneous plants (Damirdagh and Ross, 1967; Ford and Ross, 1962a and b; Goodman and Ross, 1974 a and b; Rochow and Ross, 1955; Ross, 1968; Vance, 1991). More recently we have shown that PVX synergistic disease is caused by co-infection with not only PVY, but also a variety of other potyviruses including pepper mottle virus (PepMoV), tobacco vein mottling virus (TVMV) and tobacco etch virus (TEV) (Vance et al., 1995). In each of these potyvirus-associated synergisms, the level of the nonpotyvirus in the synergistic pair (MCMV, BPMV and PVX) increases 5-10 fold in co-infected plants, while the level of the potyvirus is unchanged from that seen in singly infected plants. The increase in the non-potyvirus accumulation is correlated with the increased symptom severity typical of the co-infection. However, it is not clear that the positive correlation between nonpotyviral accumulation and symptom severity reflects a cause and effect relationship, and, in fact, the basis of synergistic disease is not understood.

It is not clear how co-infection with a potyvirus influences PVX pathogenesis, but active replication and/or the concomitant gene expression from the potyviral genomic RNA is critical (Goodman and Ross, 1974 a and b). Analysis of the genomic RNA sequence of a number of potyviral genomic RNAs reveals a single long open reading frame encoding a large polyprotein with a common organization of the eight known potyviral proteins (for review see Riechmann et al., 1992). The mature viral proteins are subsequently released in a complex series of protein processing events involving three known viral proteases. One or a subset of these mature potyviral gene products may be sufficient to induce the synergistic response to PVX infection or the entire process of potyviral gene expression and replication may be necessary. Here we report the results of stable transformation experiments to determine if synergistic disease is mediated by expression of potyviral "synergism genes".

MATERIALS AND METHODS

Viruses and Plants. The virus strains used in these experiments were as follows: PVX (PV54, American Type Culture Collection), TEV and TVMV (the kind gift of Dr. Simon Scott, Clemson University). Identities of the potyviral isolates were confirmed by ELISA analyses using a panel of monoclonal antibodies specific for a range of different potyviruses (Jordan, and Hammond, 1991). Construction, selection and analyses of the TVMV transgenic lines 1-6 (Graybosch et al., 1989; Maiti et al., 1993), TVMV lines 10 and 11 (Berger et al., 1989; and TEV line U-6B (Carrington et al., 1990) used in this study have been described previously. The TVMV constructs were transformed into Nicotiana tabacum cv Burley 21 and the TEV construct is in N. tabacum cv Havana 425. TVMV lines 8 and 9 have been previously described (Vance et al., 1995). All transgenic plants were grown from seed germinated in the presence of kanamycin at a concentration of 600 ug/ml. Nontransformed Burley 21 tobacco plants were used as controls for experiments with the TVMV transgenic lines and vector-only transformed Havana 425 plants were used as controls for the experiments with the TEV transgenic line.

Infection and sampling. In stable transformation experiments, transgenic lines and control plants of the same variety or vector-only transformed derivatives of the same variety were mechanically inoculated on a single lower leaf with PVX at a concentration of 0.5 mg/ml. All plants used in the experiments reported here were germinated and grown in an insect-vector free growth chamber, and plants for use in a given experiment were chosen to be the same size and in the same stage of development. Nucleic acid and protein samples were taken from the third leaf above the inoculated leaf at ten days post-inoculation. Equivalent portions of that leaf, not including the midvein, were used to isolate total nucleic acid. Portions of the opposite half leaf were used to isolate protein. Plants from transgenic lines TVMV 10 and 11 and TEV U-6B were assayed for the presence of coat protein of PepMoV, TEV, TVMV and PVY, to insure that the synergistic response of these plants to PVX infection was not due to inadvertent co-infection with any of these viruses.

RESULTS AND DISCUSSION

The 5'-proximal regions of both the TVMV and the TEV genomes function in PVX/potyviral synergism. In order to determine if expression of a subset of a potyviral genome is able to induce synergism with PVX, stably transformed tobacco plants expressing various regions of the TVMV genome were infected with PVX and examined for symptoms of PVX/potyviral synergism. A number of transgenic tobacco lines were tested, comprising six different constructs each expressing a single TVMV gene or a subset of the TVMV genome encoding more than one protein. The TVMV genes introduced into each of the transgenic lines, as well as their positions along the TVMV genomic RNA, are shown in Figure 1. Transgenic lines expressing an internal region of the TVMV encoded polyprotein were engineered with appropriate translation start and stop codons. In the cases where specific antibodies were available, the size and amount of protein from the introduced gene(s) was determined by western blot analyses of total protein extracts from plant leaves. For lines 1-6 the transgene protein levels varied from approximately 0.1 to 0.3% of total soluble protein (Maiti et al., 1993), while those of lines 8 and 9 were approximately 10-fold lower (Vance et al., 1995). This percentage of viral-encoded soluble protein is estimated to range from 0.5 to 5% of the level in infected plants. The level of HC-Pro in lines 10 and 11 was approximately 1% of that measured in TVMV infected plants (Berger et al., 1989).

Upon infection with PVX, two of the transgenic lines (10 and 11) consistently displayed symptoms more severe than those in nontransgenic plants infected with PVX, but similar to those characteristic of PVX/potyvirus synergism. The other eight TVMV transgenic lines displayed the very mild mottling symptoms typical of PVX infection in nontransgenic plants. Although the symptoms in lines 10 and 11 were fairly typical of PVX/potyviral disease, the timing of the response was different than in doubly infected plants: Vein clearing began at around day 7 post-inoculation rather than at day 5 as in doubly infected plants, and symptoms developed more slowly. Figure 2 shows the symptoms in a line 10 transgenic plant infected with PVX (center plant) compared to those in control plants infected singly with PVX (rightmost plant) or doubly with PVX and TVMV (leftmost plant). The symptoms in the doubly infected plant are typical of PVX/potyviral synergism and similar to those seen in the transgenic plant infected with PVX.

The two transgenic lines (10 and 11) which respond synergistically to single infection with PVX are derived from independent transformations with the same TVMV construct containing the 5'-proximal 3544 nucleotides of the TVMV genome (Berger et al., 1989). This portion of the TVMV genome contains the 5' untranslated region (5'UTR) and encodes the first three mature proteins of the TVMV polyprotein: protease-1 (P1), helper component-protease (HC-Pro) and protein-3 (P3). HC-Pro is properly cleaved from this abbreviated polyprotein and is functional in aphid transmission of TVMV (Berger et al., 1989). Our results indicate that the N-terminal 3544 nucleotides of the potyviral RNA, or one or more of the encoded proteins plays a role in mediating the synergistic response. The results also suggest that the remainder of the genome is not required for synergism, since transgenic lines expressing other portions of the potyviral genome do not support any aspect of the synergism. However, a role for these other potyviral sequences in synergism cannot yet be excluded since their level of expression in the transgenic plants is much lower than that seen in infected plants.

To determine if the 5'-proximal region of the potyviral genome also mediates other PVX/potyviral synergisms, we used a similar approach to analyze the PVX/TEV interaction. A previously constructed transgenic tobacco line expressing the 5'-proximal region of the TEV genome (line U-6B, Carrington et al., 1990) was infected with PVX and the symptoms were compared to those of a vector-only transformed line infected with PVX. The TEV transgene contains 2670 nucleotides of TEV sequence, including the 5'UTR, the entire P1 and HC-Pro genes, and approximately one quarter of the P3 gene. Western blot analysis indicates that HC-Pro expressed in these plants co-migrates with that produced in TEV infected plants, suggesting that both P1 and HC-Pro protease are properly processed from this abbreviated polyprotein (Carrington et al., 1990; Verchot et al., 1991). Furthermore, in contrast to the TVMV transgenic lines, the TEV transgenic line expresses HC-Pro at a high level, approaching that seen in wild type TEV infections of tobacco. Presumably P1 protein is present in similar high amounts in the transgenic line since it is expressed as part of the same polyprotein as HC-Pro. Infection of the TEV transgenic line with PVX results in a rapid and dramatic increase in symptoms over those seen in the vector-only transformed plant infected with PVX. The first systemically infected leaves show initial vein clearing at day 5 post-inoculation, rapidly become necrotic, and typically die by day 10 post-inoculation (data not shown). This result indicates that expression of the 5'-proximal region of the TEV genome is sufficient to induce a synergistic-like response to PVX and suggests that analogous regions of the two potyviral genomes mediate this response in both PVX/TVMV and PVX/TEV synergisms. Since the TEV transgene contains a smaller region of the genome than the TVMV transgene, this experiment also suggests that the potyviral sequences required for the synergistic response extend only through the first quarter of the P3 gene.

Synergistic viral infections in plants have been known for a long time and the mechanisms by which co-infecting viruses interact to alter host response have been a matter of speculation for almost as long (Ross, 1974). One possibility is that one virus makes use of the other's replication system, using it to replicate a heterologous genome. The data reported here eliminate that possibility as the mechanism by which the PVX/potyviral synergisms operate, since the 5' proximal region of the potyviral genome which mediates synergism does not include the potyviral replicase. An alternate theory, that the interaction is mediated by expression of a subset of one viral genome, is supported by the data presented here. Thus, this potyviral sequence is behaving as a viral "synergism sequence", inducing PVX/potyviral synergistic disease in a plant infected singly with PVX. This is the first such synergism sequence to be reported. It remains to be seen if the potyviral synergism sequence identified here is common to all the potyviral associated synergistic diseases.

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Figure 1. Diagrammatic representation of the TVMV polyprotein showing the localization of the individual genes and a listing of the TVMV transgenic lines indicating the genes included in the construct, the segregation characteristics ofthe transgene as measured by selection on kanamycin, and the protein level ofthe encoded gene product(s) in plant leaves. The following abbreviations not used in the text are: Km, kanamycin; Cl, cytoplasmic inclusion body protein; Nla, nuclear inclusion body protein a; gInH, E. coli glutamine binding protein; and Nlb, nuclear inclusion body protein b.

* indicates that these seed are homozygous and do not segregate on kanamycin plates. # indicates that no appropriate antibodies were available for this measurement. Expression levels for lines 1-9 refer to the percentage of transgene-derived protein/total soluble protein. ^a indicates that expression levels for lines 10 and 11 refer to percentage of transgene expression as compared with a wild type viral infection. In all cases, plants used to determine the level of gene expression were grown from the same generation of seed used to produce plants for the synergism studies.

Figure2. Symptoms displayed by nontransgenic Nicotiania tabacum (cv Burley 2l ) lnfected with PVX (rightmost plant) or with both PVX and TVMV (leftmost plant) or transgenic line 10 expressing three genes from the 5'-proximal end of the TVMV genome and infected singly with PVX (middle plant).