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Different forms of interference between two tobamoviruses in two different hosts I. Aguilar³, F. SaÂnchez and F. Ponz*² INIA, Departamento de Mejora GeneÂtica y BiotecnologõÂa, Autopista A-6, km 7, 28040 Madrid, Spain
Oilseed rape mosaic (ORMV) and tobacco mild green mosaic (TMGMV) tobamoviruses interfered with each other when infecting the same host, and interference was host-dependent. In tobacco cross-protection was obtained in two ways: the protecting virus prevented the accumulation of the challenging virus, even in the inoculated leaf; in Arabidopsis, protection was obtained only when the protecting virus was TMGMV. The protecting mechanism in Arabidopsis appeared to differ from that operating in tobacco. Although ORMV could be detected in the inoculated leaf, TMGMV prevented systemic infection by ORMV. Thus the host appears to play an important role in this type of cross-protection. Keywords: Arabidopsis, cross-protection, ORMV, TMGMV, tobacco, tobamovirus
Introduction Different kinds of host±virus interaction have been described in mixed infections of plant viruses. A virus may stimulate the replication or the distribution rate of another virus (Atabekov & Dorokhov, 1984). A nonhost for one virus can even become infected with the help of another virus (Dodds & Hamilton, 1972). However, the presence of one virus can sometimes prevent the infection of a second. The phenomenon where a first virus protects from infection by a second virus which causes severe symptoms in the same host is known as cross-protection (Fraser, 1998). This was first described in 1929 (McKinney, 1929) and has been used in the control of some virus diseases (e.g. Rast, 1972; Costa & Muller, 1980; Lecoq & Lemaire, 1991; Wang et al., 1991; Fraser, 1998). Different mechanisms have been proposed to explain cross-protection (Sherwood & Fulton, 1982; Ponz & Bruening, 1986; Matthews, 1991). These can be placed in three main groups: (i) general: competition for replication sites or host factors such as host components of the viral replicase; (ii) due to the coat protein: adsorption from the first virus particles, encapsidation of the superinfecting RNA, inhibition of the early disassembly events, etc.; and (iii) nucleic acid
*To whom correspondence should be addressed. ²E-mail:
[email protected] ³Present address: Dpto BiotecnologõÂa, ETS Ingenieros AgroÂnomos, 28040 Madrid, Spain. Accepted 28 June 2000. Q 2000 BSPP
interactions: genetic recombination, negative strand capture or competition for the translation machinery. From results obtained in pathogen-derived resistant (PDR) transgenic plants (Lomonossoff, 1995), there is probably more than one mechanism or gene involved in cross-protection. The relative importance of the different mechanisms may be a function of the viruses and the particular host species. Rezende et al. (1992) suggested that the host may play a role in cross-protection between tobamoviruses. We report here cross-protection between two members of the tobamovirus genus: oilseed rape mosaic virus (ORMV; Aguilar et al., 1996) and tobacco mild green mosaic virus (TMGMV; SolõÂs & GarcõÂa-Arenal, 1990). Tobamoviruses are often used as model systems in plant virology as the viruses are easily handled. As the two viruses belong to the same genus but have a relatively distant genetic relationship (Aguilar et al., 1996), any type of interaction is theoretically possible: synergistic, interference or neutral (Dodds & Hamilton, 1972; Sherwood & Fulton, 1982; Atabekov & Dorokhov, 1984; Zinnen & Fulton, 1986). In order to study whether the host was active or passive in this process, the interaction was studied in two hosts, tobacco and Arabidopsis, from different botanical families. Tobacco is a classical host in studies of virus±plant interactions, and Arabidopsis is an excellent candidate for studies of the role of the host in plant±pathogen interactions (Dangl, 1993; Simon, 1994). After single inoculation with each virus, the two hosts behaved differently. Tobacco plants were good hosts for both viruses, which were able to replicate and move systemically in this species. Arabidopsis was a good host for ORMV, but 659
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Inoculation with the protecting and challenging viruses
Figure 1 Designs of the cross protection experiments. (a) Study of the behaviour of both viruses in each host. (b) Coinoculation. (c) Inoculation in different leaves and at different times (example for t7; the same was done for 14, 21 and 28 d.a.i.). Each experiment was repeated twice with a different number of plants. Abbreviations: il, inoculated leaf; ul, uninoculated leaf; T, TMGMV; O, ORMV; il T 1 O, coinoculated leaf; ul T 1 O, leaf not coinoculated; il 1, leaf inoculated with virus 1; il 2, leaf inoculated with virus 2. Italic: inoculating time (i.e. t0); bold: harvesting time (i.e. t7, 7 d.a.i.).
TMGMV accumulation level was lower and the virus moved much more slowly than in tobacco. These results made this system (two different hosts and two different viruses) suitable for an analysis of the interaction.
Arabidopsis and tobacco plants were mechanically inoculated with 200 ng purified virus per leaf diluted in 50 mm phosphate buffer pH 7´0, to a final concentration of 40 ng m L21. Arabidopsis plants were inoculated 15 days after transplanting, and tobacco plants were inoculated at the four-leaf stage. To study virus behaviour in both hosts, each virus was inoculated separately on different plant species (Fig. 1a). In tobacco and for each set of experiments, four different plants were used and the experiments repeated twice. In Arabidopsis, five independent plants were analysed in two independent experiments. The interaction between the two viruses was studied in two types of experiments. In the first set of experiments, the two viruses were coinoculated (Fig. 1b). In the second set the two viruses were inoculated separately (Fig. 1c). To coinoculate the plants, equal volumes of the same virus concentration were mixed and inoculated on one leaf (Fig. 1b). In the second set of experiments (Fig. 1c), the two viruses were inoculated on different leaves, at different times and in two ways. TMGMV was used as the protecting virus (virus 1) and ORMV as the challenging virus (virus 2), and vice versa. The inoculations were made at different times to study whether the establishment of the protecting virus had any effect on infection by the challenging virus. The challenging virus was inoculated 7, 14, 21 or 28 days after inoculation (d.a.i.) with the protecting virus (Fig. 1c, example for t7). For each treatment, four tobacco plants and six Arabidopsis plants were used. All the experiments were repeated twice.
Conditions of sample harvesting
Materials and methods Virus material ORMV was propagated and purified as described previously (Aguilar et al., 1996). Purified TMGMV was kindly provided by Dr F. GarcõÂa-Arenal (Departamento de BiotecnologõÂa, ETSI AgroÂnomos, Madrid, Spain). Virus concentrations were determined spectrophotometrically.
Plant material Seeds of Arabidopsis thaliana ecotype RLD were sterilized, sown in GM medium Petri dishes, and kept for 24 h at 48C. After 15 days in a growth chamber (16 h photoperiod, light intensity 100 m E m22 s21) they were planted in soil and kept in a greenhouse at 258C. Seeds of Nicotiana tabacum L. cv. Samsun seeds were germinated in soil, and the seedlings transplanted 3 weeks later.
For dot-blot analysis the inoculated leaf (il) was harvested 7 d.a.i. and an uninoculated leaf (ul) at 28 d.a.i. (Fig. 1a). In the coinoculated plants, the coinoculated leaf (il T 1 O) was harvested at 7 d.a.i. and an uninoculated one (ul T 1 O) at 28 d.a.i. (Fig. 1b). These time points were chosen because the interval 7±28 d.a.i was sufficient for both viruses to infect plants systemically. When the viruses were inoculated on different leaves and at different times, the samples were taken as shown in Fig. 1(c). In tobacco plants, the leaves inoculated with the two viruses separately (il 1, il 2) were analysed at 7 d.a.i. for the corresponding virus (t7 for virus 1; t14 for virus 2). An uninoculated leaf (ul) was analysed 7 days after inoculating for the challenging virus (t14). The same analysis was made for the different challenging virus inoculation times. In Arabidopsis, the leaves inoculated with ORMV were analysed at different times to assess virus presence. The uninoculated leaves were analysed 21 days after ORMV inoculation. In this host, leaves inoculated with TMGMV only were not Q 2000 BSPP
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Figure 2 TMGMV and ORMV infection in tobacco. (a) Separately: leaves were analysed in four different plants for each kind of inoculum (1±4 for virus T; 1 0 -4 0 for virus O). Time of analysis, il 7 d.a.i.; ul 28 d.a.i. (b) Co-inoculation: leaves were analysed in four different plants (1±4). The filter was duplicated, one for each probe. Time of analysis, il 7 d.a.i.; ul 28 d.a.i. (c) At different times and in different leaves (example for t7). T/O, TMGMV protecting virus (virus 1), ORMV challenging virus (virus 2) (plants 1±2); O/T, ORMV protecting virus (virus 1), TMGMV challenging virus (virus 2) (plants 3±4). Time of analysis, 7 d.a.i. after inoculation with the challenging virus. The filter was duplicated, one for each probe. Abbreviations: il, inoculated leaf; ul, uninoculated leaf; T, TMGMV; O, ORMV; T 1 O, coinoculation.
harvested for tests of virus presence at 7 d.a.i., because preliminary experiments had shown that the virus is not detectable at that stage (data not shown).
Sample analysis by dot-blot hybridization Leaf samples were homogenized in 1 : 5 (w/v) extraction buffer (formamide 50% v/v, formaldehyde 6% v/v, 20 mm MOPS (3-N morpholino propanesulphonic acid) pH 7´0, 5 mm sodium acetate, 1 mm EDTA; Frenkel et al., 1992). The virus RNA was denatured for 10 min at 658C, 1 vol. 20 £ SSPE (3´6 m NaCl, 0´2 m Q 2000 BSPP
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NaH2PO4, 0´02 m EDTA pH 7´7) was added, and 5 mL of the sample blotted onto nitrocellulose membranes (Hybond C extra, Amersham Pharmacia Biotech International, Little Chalfont, UK). The filters were baked for 2 h at 808C. The filters to analyse the plants inoculated with both viruses (Figs 2b,c and 4b,c) were duplicated for differential probe hybridization. A 2´2 kb cDNA probe corresponding to its replicase gene was used to detect ORMV (Aguilar et al., 1996). To detect TMGMV, a 0´8 kb cDNA from the 3 0 terminal region of its replicase gene and the 5 0 -terminal region of its movement protein gene was used (SolõÂs &
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exposures were carried out at 2808C for different times with intensifying screens on Hyperfilm (Amersham).
Results TMGMV prevents ORMV accumulation in tobacco and vice versa ORMV (O) and TMGMV (T) systemically infected tobacco. Both viruses were detected in inoculated (il T, il O) and uninoculated (ul T, ul O) leaves (Fig. 2a). In the coinoculation experiments (Fig. 2b), ORMV was detected in the inoculated leaves (il T 1 O) of all plants. There was only a faint hybridization signal of TMGMV in one of the plants (il T 1 O 3). ORMV interfered with TMGMV accumulation in the coinoculated leaves. Interference was also observed in the uninoculated leaves (ul T 1 O). When ORMV was present TMGMV was not, and vice versa (Fig. 2b). It appeared that the virus to establish first (ORMV in il T 1 O) or that which progressed more quickly (TMGMV in ul T 1 O 3, ORMV in the rest) prevented the accumulation of the other virus. In order to study whether the establishment of the protecting virus always inhibited the accumulation of the challenging virus, each was inoculated on a different leaf at different times (Fig. 2c). The results indicated that the protecting virus (virus 1) interfered very strongly with the multiplication of the challenging virus (virus 2). Thus the T-probe filter in Fig. 2(c) did not show a signal for plants 3 and 4, which were first inoculated with ORMV. The converse is true for the Oprobed filter, except for a very faint signal in the inoculated leaves, probably due to residual inoculum. The results show, regardless of the time that elapsed between the inoculation of both viruses (7, 14, 21, 28, 35 and 42 d.a.i., data not shown), that the protecting virus always inhibited the accumulation of the challenging one. Observation of symptoms (Fig. 3) showed that O/T plants behaved like those inoculated with ORMV alone. T/O plants were protected from the severe symptoms caused by ORMV, but showed more severe symptoms than those inoculated with TMGMV alone.
Figure 3 Symptoms of tobacco plants inoculated with both viruses spatially and temporally separated. (a) ORMV infection; (b) O/T plants; (c) T/O plants; (d) TMGMV infection. The analysis was done at 42 d.a.i.
GarcõÂa-Arenal, 1990). The probes were labelled with dCTP a32P (3000 m Ci mmol21, Nuclear IbeÂrica, Madrid, Spain) with the Megaprime Labelling System (Amersham) and purified with Microspin S-200 columns (Amersham). Hybridization was carried out following the HybondC extra (Amersham) protocol. Autoradiographic
TMGMV prevents ORMV accumulation in Arabidopsis When Arabidopsis plants were infected with TMGMV (Fig. 4a), virus was detected in the inoculated leaves only at 14 d.a.i., and only in an uninoculated leaf out of the five plants analysed at 28 d.a.i. (Fig. 4a). This virus infected and accumulated slowly, and infection was symptomless. ORMV was detected in inoculated leaves at 7, 14, 21 and 28 d.a.i., and in the uninoculated leaves from 21 d.a.i. onwards (Fig. 4a). The symptoms were yellowing and stunting (MartõÂn MartõÂn et al., 1997). In coinoculation experiments (Fig. 4b), only ORMV infected Arabidopsis; TMGMV was not detected. Q 2000 BSPP
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Figure 4 TMGMV and ORMV infection in Arabidopsis. (a) Separately: analysis at 7, 14, 21 and 28 d.a.i. (b) Co-inoculation: il T 1 O analysed at 7 d.a.i.; ul T 1 O analysed at 28 d.a.i. Leaves were analysed in three different plants (1±3). The filter was duplicated, one for each probe. (c) At different times and in different leaves. T/O, TMGMV protecting virus (virus 1), ORMV challenging virus (virus 2) (plants 1±2); O/T, ORMV protecting virus (virus 1), TMGMV challenging virus (virus 2) (plants 3±4). 7, 14, 21 and 28 d.a.i. inoculation time of the challenging virus, with respect to the inoculation of the protecting virus. Time of analysis, 21 d.a.i. of the challenging virus. The filter was duplicated, one for each probe. Abbreviations: il, inoculated leaf; ul, uninoculated leaf; T, TMGMV; O, ORMV.
When the two viruses were inoculated on different leaves at different times, the results differed depending on which virus was used for protection (Fig. 4c). When TMGMV was the protecting virus, it protected the plants from infection by ORMV (T/O plants). When ORMV was used as the protecting virus, TMGMV was not detected in either the inoculated or uninoculated leaves. However, and because of the infection features of TMGMV in Arabidopsis (see above), it is difficult to Q 2000 BSPP
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propose cross-protection in this direction (Fraser, 1998). A relevant difference with the cross-protection observed in tobacco was that ORMV could be detected in the leaves inoculated with this virus in the T/O plants (il O in T/O plants) (Fig. 4c). The plants protected by TMGMV were symptomless, whereas plants `protected' by ORMV showed symptoms typical of this virus. Dot-blot analyses were related to the symptomatology of the plants (Fig. 5).
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Figure 5 Symptoms in Arabidopsis plants inoculated with both viruses spatially and temporally separated. T/O, TMGMV protecting virus (virus 1), ORMV challenging virus (virus 2); O/T, ORMV protecting virus (virus 1), TMGMV challenging virus (virus 2). 7, 14, 21 and 28 d.a.i. inoculation time of the challenging virus, with respect to the inoculation of the protecting virus. Time of analysis, 21 d.a.i. of the challenging virus.
Discussion In the first set of experiments, the two viruses were coinoculated in the same leaf. The result of coinoculation was the same for the two hosts: there was strong interference, probably due to a direct competition between the two viruses or for host components. This phenomenon was the same independent of host, suggesting competition between the two viruses. The most obvious interpretation is that the virus with the greatest fitness `wins'. In this system, ORMV was almost always the more successful in both hosts. When the two viruses were inoculated on different leaves of the same host at different times, the results were more complex. Here, the virus first inoculated (protecting virus) influenced the type of infection that the second virus (challenging virus) was able to establish. However, the interference model cannot be easily interpreted, suggesting an important role for the host. In tobacco, a good host for both viruses, the influence of the protecting virus was evident, even in the leaf inoculated with the challenging virus. The dot-blot hybridization results show that at 7 d.a.i. the protecting virus inhibited accumulation of the challenging virus in inoculated leaves, indicative of protection. This was related to the symptoms observed in O/T plants. T/O plants showed more severe symptoms than plants inoculated with TMGMV alone; however, these symptoms were milder than those observed in plants inoculated with ORMV alone. This suggests that in these T/O plants cross-protection operated by delaying ORMV infection. These results indicate a clear direct competition between the two viruses in tobacco. In
Arabidopsis it was difficult to determine whether protection occurred when the protecting virus was ORMV, given the infection features of the two viruses in this host. Protection was observed when the protecting virus was TMGMV; in contrast to tobacco, this occurred with very low levels of TMGMV. Also in contrast to tobacco, the challenging virus was detected in the inoculated leaf of protected plants. This suggests that the cross-protection mechanism is different from that in tobacco. In Arabidopsis, TMGMV does not appear to prevent ORMV replication and accumulation, as the latter can replicate and accumulate in inoculated leaves. This phenomenon resembles the interference described in bean plants doubly infected with two different but closely related potyviruses (Khan et al., 1994). In a similar way, TMGMV possibly does not allow ORMV to move from the inoculated leaf to the phloem, delaying (as in bean) or impairing the transport of the challenging virus from inoculated to uninoculated leaves. Rezende et al. (1992) showed that the host plays a role in cross-protection. Our results support this view. In our experimental conditions and with these two tobamoviruses, the protection mechanism is different depending on the host. It is possible that in tobacco, as well as in Arabidopsis, the two viruses compete for host factors involved in the infection process. In virus±host interactions there must be host-specific factors that interact with the viral genes, the interaction of which is responsible for the success or failure of the infection process (Dawson & Hilf, 1992). ORMV and TMGMV would compete for the same factors, and crossprotection would be different depending on the Q 2000 BSPP
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behaviour of each virus in each host. The results suggest that the host is not passive in cross-protection. It is also possible that the different mechanisms proposed can explain cross-protection depending on the host. In this context, the fact that cross-protection has been found in Arabidopsis should permit analysis of the role of the host, taking advantage of its features as a model system.
Acknowledgements We thank Dr Aurora Fraile and Dr Fernando GarcõÂaArenal (ETSIA, U. PoliteÂcnica, Madrid, Spain) for the generous gift of oilseed rape mosaic virus, tobacco mild green mosaic virus, the ORMV purification protocol, and the probe to detect TMGMV. I.A. was supported by a fellowship from Comunidad de Madrid. This work was funded by grant BIO95-0766-C02-01Temp from the Spanish grant agency CICYT.
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