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Restricted virus multiplication and movement of tomato mosaic virus in resistant tomato somaclones
Plant Science, 89 (1993) 113-122 Elsevier Scientific Publishers Ireland Ltd.
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Restricted virus multiplication and movement of tomato mosaic virus in resistant tomato somaclones Sandra Schiller Smith and Harry H. Murakishi Department of Botany and Plant Pathology, Michigan State UniversiO,, East Lansing, M1 48824-1312 (USA) (Received August 24th, 1992; revision received October 27th, 1992; accepted November 4th, 1992)
Somaclonally-derived resistance from fully tomato mosaic virus (ToMV)-susceptible tomato (Lycopersicon esculentum, Mill.) was compared to resistance conferred by other known ToMV resistance genes in tomato. Plants were challenged by the common tomato mosaic virus strain (ToMV-O) under different conditions, by different tobamovirus strains and by tobacco etch virus (TEV) and cucumber mosaic virus (CMV). Resistance was limited to ToMV-O and the closely related Flavum strain of TMV (TMV-FI) and was found to be stable at high temperatures (28-30°C). The systemic movement of the virus was restricted in resistant somaclone lines as compared to susceptible tomato plants. The movement of virus was also monitored in reciprocal grafts of resistant and susceptible plants. Transport of virus from inoculated rootstock was limited in both positions, with resistant material as scion or rootstock. The presence of virus was detected at the cellular level by releasing protoplasts from inoculated plants and using immunofluorescent staining to detect virus coat protein. In resistant somaclones, the rate of virus infection was very low (< 0.1% of protoplasts with virus). In some resistant plants the percentage of infected protoplasts was high (65%) but the infected protoplasts had only moderate levels of virus present.
Key words: tomato; mosaic virus resistance; somaclonal variation
Introduction Resistance to ToMV was obtained in somaclones by regenerating tomato plants through adventitious shoot formation from leaf discs of ToMV-susceptible (GCRI-26) tomato seedlings [1]. Resistance shown by the somaclones appeared to be a delay in symptom expression and virus multiplication. Further characterization of this resistance through study of virus movement, effect of high temperature and reaction to other viruses and another strain of ToMV is presented here. Comparison is made to previously described resistance in tomato.
Correspondence to: H.H. Murakishi (Professor Emeritus), Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824-1312, USA.
T o M V resistance genes in tomato
There are three well-characterized ToMV resistance genes in tomato, each of which has been incorporated into cultivated tomato from wild tomato species. The gene Tm-1 is co-dominant for limitation of virus replication and dominant for suppression of symptom formation [2]. At a different locus, the allelic genes Tm-2 [3] and Tm-2 2 [4] effectively prevent infection of ToMV by a hypersensitive response, usually evident as local lesions. At higher temperature or in heterozygous plants, the hypersensitive resistance response may cause systemic necrosis. These three genes have been bred into many commercial cultivars as Tm1/Tm-1, Tm-2/Tm-2 2 to give complete resistance to date [5]. Also, each of these alleles has been incorporated into isogenic lines of tomato and has been tested with different virus strains [6]. Such lines can serve as a model system for study of
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
114 resistance mechanisms. Because the resistant somaclones in this study were derived from GCRI26, one of Pelham's near-isogenic plant lines, direct comparison of resistance was made between the somaclones and the other resistance alleles.
Temperature sensitivity When resistant tomato plants are inoculated with virus and incubated at high temperature (28-30°C), there is a breakdown in the resistance. With Tm-1, the results have been ambivalent with reports of temperature sensitivity [6] and stability [2]. The genes Tm-2 and Tin-22 both lose resistance at higher temperature [6-8], allowing systemic virus multiplication and spread. When such plants are moved to normal temperatures, the plants become systemically necrotic. Grafting experiments Study of virus movement in reciprocally grafted tomato has been done for each resistance gene. Selman and Yahampath [9] made reciprocal grafts between a cultivar with the Tm-1 gene and a susceptible cultivar with a different genetic background. The scion was inoculated with ToMV and virus was assayed by inoculation on N. glutinosa. When the rootstock was susceptible, the resistant scion was less resistant, allowing the multiplication of virus. When the rootstock was resistant, the susceptible scion became infected with virus, indicating that there was no viral inhibitor being transported upward. In either case, the virus moved freely across the graft and replicated throughout the plant, indicating that the Tm-1 gene did not inhibit transport of virus or cause systemic resistance. Pilowsky [10] studied reciprocal grafts between plants with Tm-2 e and infected susceptible plants and found that in either position (rootstock or scion) the Tm-2: plants became systemically necrotic, even when the graft was severed after 7 days. Stobbs and MacNeill [7] studied virus movement when grafts were made between plants expressing Tm-2 and susceptible plants. Stem inserts of one genotype were made between rootstock and scion of the other genotype and ToMV was inoculated on the rootstocks after the graft unions were established. The resistant insert did not alter virus movement; when a leaf of the susceptible
rootstock was inoculated, the virus moved into the scion, but when a leaf of the resistant rootstock was inoculated, the virus remained in the inoculated leaf. When grafted as rootstock or scion, the resistant plants developed systemic necrosis when the grafted partner was TMV-infected. In the same study, virus movement was found to occur through the phloem when examined using electron microscopy. It was suggested that the virus by-passes the normal genetic resistance mechanisms by entering the resistant mesophyll from the phloem. The virus was localized if the only inoculum was on a resistant leaf. Methods
Response of somaclones to different tobamovirus strains Tomato seeds were planted and cultivated as described previously [1]. Groups of tomato plants representing the R 1 generation of all six somaclonal lines and the susceptible line, GCRI-26, were divided and inoculated with ToMV-O, TMVF1 or a strain that overcomes the resistance of the Tm-1 gene, TMV-1 (described by Pelham [6] and kindly provided by F. Motoyoshi, Tsukuba, Japan). Assay of the inoculated plants was done at 10 days post infection (p.i.) and continued for 20, 30, 50 and 70 days for ToMV-O and TMV-FI. Plants that became infected with TMV-1 were discarded immediately after assaying to prevent unintentional spread to other tomato plants. Presence of ToMV-O and TMV-F1 was detected using double antibody sandwich microplate enzyme-linked immunosorbent assay (DASELISA) as described [1]. For all DAS-ELISA, duplicate samples were assayed for each plant and compared to uninfected and heavily infected control samples. Presence of TMV-1 was detected by observation of symptoms and inclusion bodies in leaf hair cells. Comparisons of ToMV-O and TMV-F1 were repeated three times with 6-16 plants of each line. Comparison of TMV-I to ToMV-O was done once with twelve plants of each line. Response of somaclones to inoculation by different viruses Groups of tomato seedlings representing the R i
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generation somaclonal lines and GCRI-26 were divided and inoculated with the pepper strain of CMV or with the wild tobacco strain of TEV, which were both provided by T. Zitter, Cornell University, New York. The inoculum was prepared from frozen, infected tobacco tissue which was ground at 1 g/20 ml of 0.01 M NaKphosphate buffer (pH 7.0). Detection of the virus was done using DAS-ELISA, with gammaglobulin specific for each virus. Each gammaglobulin was purified from rabbit antiserum (from American Type Culture Collection, Rockville, MD) as described previously [1]. Plants were assayed in duplicate at 10 and 20 days p.i. Twelve plants of each line were tested with each virus. Response of somaclones to inoculation and incubation at different temperatures Groups of tomato plants representing R l and R 2 generations of somaclones, GCRI-26 (+/+) and the resistant tomato line GCRI-237 (Tm1/Tm-1) were inoculated with ToMV-O and divided into sets which were incubated at 25°C or 30°C with otherwise similar conditions. Plants were assayed for the presence of virus at 10, 20 and 30 days p.i. (and one group was assayed at 50, 70 and 150 days p.i. as well) using DAS-ELISA in duplicate with positive and negative controls. The first experiment included six plants of each line for each temperature and was replicated using 6 - 9 plants of each line for each temperature. Virus movement & tomato Plants were transplanted into 4-inch pots at 12 days. The groups consisted of R2 plants from somaclone lines 12 and 247 and the susceptible line GCRI-26. One group was inoculated with ToMVO when the plants were 32-day-old and the second group was inoculated when 25-day-old. The outermost leaflet of the first true leaf was marked by punching a hole with a wide-bore needle and after dusting with carborundum, it was inoculated with virus. The inoculum was not rinsed off, but was allowed to dry in situ. Ten to 12 plants of GCRI26 and somaclone 247 were tested in each replication. At each time point (4, 7, 10 and 20 days p.i.), leaflets were removed from each leaf of the plant. Two or three plants of each line were sampled for each time point. Once plants had been sampled,
they were not used for further assay to eliminate the chance that plants might inadvertantly be inoculated during sampling. Leaf samples were assayed using DAS-ELISA and the absorbance values for leaflets from each corresponding leaf were averaged. Plants from the second repetition were used to isolate protoplasts for detection of infection at the cellular level. As controls, susceptible and resistant plants were mock-inoculated with buffer only and assayed. Virus movement in grafted tomato plants To study the movement of virus between reciprocal grafts of resistant and susceptible plant material, several types of grafts were established. Whip grafts were done using young seedlings by wetting a clean, new razor blade and removing the top of 18-day-old plants with a V-shaped cut. Tops were removed from 2 plants and exchanged, resulting in a rootstock with cotyledons only and a scion with 3 young leaves. The graft was held in place using 1/2-inch wide self-adhesive tape. Plants were sealed in polyethylene bags to increase the humidity and were allowed to bond for several weeks. A cotyledon of the rootstock of the grafted plant was then inoculated. Of the 10 whip grafts made, three grafted plants survived for the assays. The second set of plants was grafted using older plants (20-30 days). Again, V-shaped cuts were made near the tops of 2 plants and the tops were interchanged. After the whip graft was established (25-30 days), there were 2 true leaves remaining below the graft plus secondary shoots and 2-3 young leaves on the scion. The outermost leaflet of the first true leaf of the rootstock was inoculated. Of the 10 grafts made, again only 3 were successful and used for the assays. The third type of graft was established using an approach graft which incorporated a tongue (whip and tongue graft). This method was used successfully on older plants (33-39 days) and was less stressful to the plants. The graft was made on the stem between the third and fourth true leaves in most cases and was bound with self-adhesive tape. After 2 weeks, one stem was severed below the graft and the opposing top was removed. The outermost leaflet of the first true leaf was inoculated a week after that. Five whip and tongue grafts were used for assay.
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Assay of the grafted plants was done using DAS-ELISA at 0, 5, 7 and 10 days p.i. Some plants were assayed further at 21, 34, 39 and 44 days p.i. For the first group of grafted plants every leaf of both rootstock and scion were assayed in duplicate for each time point. Later groups were assayed from 1 or 2 leaves from both parts. Three pairs of reciprocal grafts were established between somaclone 247 (R2) and GCRI-26, and one pair between between somaclone 12 (R3) and GCRI26. Two control grafts were made between two plants of GCRI-26 to eliminate the possibility that delayed virus spread could be due to the graft itself.
Protoplast isolation Tomato plants were grown in the laboratory (22-25°C) under cool white fluorescent lights supplying 60 /xE m -2 s -t on a 16-h/day light cycle. The plants were treated with fertilizer daily when they were watered with one tablespoon of Peters 20-20-20 per gallon. At about 4 weeks, plants were used for isolation of protoplasts. Protoplast isolation procedures were adapted from Kassanis and White [11]. After leaves were surface sterilized and rinsed, leaves were stacked and sliced transversely in 1-mm thin sections. Leaf strips were placed in a petri dish containing 20 ml of an enzyme solution consisting of 1% (w/v) cellulysin, 0.5'7,, (w/v) macerase, (Calbiochem, Behring Diagnostics, San Diego, CA) and 0.4 M sorbitol in cell protoplast wash (CPW) salts [12] at pH 7.0. The strips were incubated on a slow shaker in the dark at room temperature for 12-18 h. The enzyme solution containing protoplasts and leaf debris was filtered through a 60-#m sieve into a petri plate. Protoplasts were transferred to a 30-ml roundbottomed centrifuge tube and centrifuged at 35 x g for 10 min. The supernatant fluid was removed by suction, the pellet was gently resuspended by rolling in a small amount of washing buffer and the protoplasts were moved to a 15-ml tube. The protoplasts were washed in 15 ml of sorbitol-CPW buffer, centrifuged again at 35 x g and the supernatant fluid was removed by suction. The protoplasts were rinsed a total of three times in this manner. The final step in purification of the protoplasts was to float the intact protoplasts on
a solution made of Lymphoprep (Accurate Chemical and Scientific Corp., Westbury, NY) and 0.5 M sucrose in CPW salts at 1:2 (v/v) for healthy protoplasts [13] or at 2:1 (v/v) for protoplasts which were isolated from virus-infected plants. Concentration of protoplasts was adjusted and viability was determined by microscopic examination after the addition of several drops of Evan's blue dye (0.05% (w/v) Evan's blue in 0.7 M mannitol, pH 7).
Preparation and staining of injected protoplasts Following the procedure by Otsuki and Takebe [14], the infected protoplasts were labelled with ToMV antibody which was conjugated to fluorescein isothiocyanate (FITC). The FITC-conjugate was prepared using the method of Spendlove [15] and cross-adsorbed with acetone powder of healthy tobacco leaves using the method of Otsuki and Takebe [16]. FITC-labelled cells were viewed with a Zeiss G F L epiluminescent microscope equipped with exciter filters KP-490 and LP-445, dichroic reflector 510 and barrier filter 520. Photographs of the fluorescing cells were taken with ASA 400 daylight film. Exposures were between 12 rain (for heavily infected cells) and 28 min (for uninfected cells). Results
Response of somaclonal lines to different viruses The six somaclonal lines displayed resistance to ToMV-O and TMV-FI for more than 70 days post-inoculation as detected by ELISA (Fig. 1). All six lines were susceptible to the strain TMV-1 and were infected by 10 days post-inoculation (not shown). There were plants among somaclone lines which showed a delay in infection after inoculation with CMV, but all plants were infected by 20 days p.i. (Fig. 1). When inoculated with TEV, all plants had high levels of virus at 10 days p.i. (Fig. 1).
Response of somaclonal resistance to high temperature The somaclonal resistance to ToMV was not changed when inoculated plants were grown at high temperature. Ninety-six percent of the in-
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Fig. 1. Proportion of plants free of virus after challenge with ToMV-O, TMV-F1, CMV or TEV. Tomato line GCRl-26 ('26') is the susceptible control. Other lines (215, 219, 247, 322, 330) are somaclonal lines which were selected for resistance to TMV. Plants were sampled for ELISA at 10 and 20 days post inoculation.
oculated seedlings of the 6 somaclone lines were virus-free after 30 days at 25°C and at 28-30°C. However, the plants grown in the warmer conditions were slightly stunted. Susceptible GCRl-26 plants were infected at 10 days post-inoculation and severe stunting was seen in infected GCRI-26 plants at the higher temperature. Two plants of line 237 (Tm-l/Tm-1) at 28-30°C which had a medium virus titer of 0.35 absorbance units (AU) at 10 days died after necrotic collapse before they could be sampled again at 20 days, but the other seven plants were virus-free at 30 days.
Systemic movement of virus in susceptible and resistant plants The movement of ToMV-O was fairly rapid in susceptible plants. At 4 days, virus was detected in the inoculated leaflet and in neighboring leaflets. By the seventh day after inoculation, there was a high titer of virus in the upper leaves, which were
rapidly expanding. The virus spread through the rest of the plant and at 20 days the whole plant contained a high virus titer. When this experiment was repeated using somaclonal line 247, high levels of virus did not develop anywhere in the plant. The inoculated leaf had a detectable level of virus and over time, traces of virus were detected in the upper parts of the plants, but at the lower limits of detection by ELISA (values in the range of 0.02-0.09 AU). Intermediate levels of virus (ELISA values in the range of 0.05-0.32 AU) were detected in plants of line 12 at 15-20 days after inoculation. It was evident that the virus did multiply and move systemically in these plants, but only to a limited extent.
Movement of virus in grafted plants In the control grafts that were made with GCRI26 on GCRI-26, there was no delay in virus move-
118 ment (Fig. 2A). Virus appeared in the rootstock leaves and the scion leaves in equal amounts at 5 days (ELISA values from 0.50 to 0.89 AU) and at 10 days (values from 1.11 to 1.20 AU). Because the virus multiplied and spread to all parts of the grafted plant quickly, the possibility that virus movement was limited by the graft union was eliminated. When the susceptible line GCRI-26 was used as the inoculated rootstock and the resistant line 247 as the scion, the virus replicated and moved throughout the leaves of the susceptible rootstock to attain a high titer (ELISA values from 1.70 to 1.75 AU) by 7 - 1 0 days (Fig. 2B). In each case, accumulation of virus was delayed in both rootstock and scion compared to the control grafts. In one graft, the virus was delayed in multiplying to a high titer in the resistant scion, even with the presence of the large virus concentration in the rootstock. In two other grafts of this type, there was a lower titer of virus present in the rootstock and virus moved into the resistant scion and reached a high titer in 7-21 days. In each grafted plant, the delay seen in virus multiplication in the resistant scion was notable when compared to the control graft of susceptible to susceptible plants. In the reciprocal graft of GCRI-26 (scion) and somaclone line 247 (rootstock), the inoculated
resistant rootstock did not show the presence of virus (except for the virus in the inoculated leaf, with ELISA values from 0.00 to 0.14 AU) (Fig. 2C). The virus did not appear in the susceptible scion for more than 21 days p.i. and when it did appear (with ELISA values of over 1.00 AU), the rootstock became infected as well. In one grafted plant, the virus did not multiply in the susceptible scion for more than 40 days. The reciprocal grafts between GCRI-26 and somaclonal line 12 were similar to the above grafts in their response, but the resistance did not seem as strong in general, which was consistent with the results from the study of virus movement in plants of line 12. When GCRI-26 was the rootstock, there were high levels of virus present in the lower leaves, but only intermediate levels of virus accumulated in the leaves of the resistant scion (ELISA values from 0.24 to 0.45 AU) for longer than 20 days. When somaclonal line 12 served as the rootstock, a fairly high level of virus was detected in the inoculated leaf at 5 days (0.68 AU), but did not multiply in the susceptible scion for more than 10 days.
Protoplasts Uninfected cells exhibited slight light green fluorescence and no bright spots. Protoplasts
Fig. 2. Results of experimentsin which grafted plants were inoculated in the outermost lower leaflet of the rootstock (open triangle). Movement of the virus was followed using ELISA to measure virus titer in leaves of the rootstock and scion. Grafts were made between two susceptible GCRI-26 plants as a control (A) and of resistant (line 247) onto susceptible (GCRI-26) rootstock (B) and susceptible (GCRI-26)onto resistant (line 247) rootstock (C). Illustrations represent grafts 10 days after inoculation; shading indicates high virus titer.
119 isolated from infected GCRI-26 (ELISA value of 1.85 AU) were smaller than protoplasts from healthy plants and were associated with more cellular debris. After staining with FITC-anti-coat protein antibody conjugate, 51.4% of the protoplasts prepared from infected GCRI-26 were highly fluorescent, where the entire protoplast fluoresced with bright yellow-green, indicating the presence of virus (Fig. 3). Uninfected protoplasts appeared darker (not shown). Protoplasts which were isolated from inoculated resistant somaclones differed in two ways. Plants from lines 12, 215 and 247 which had moderately low ELISA values (0.2-0.8 AU), yielded only a few infected protoplasts out of thousands of healthy protoplasts (Fig. 4). In a second response, protoplasts isolated from plants from lines 12 and 247 with very low ELISA values (0.05 AU) appeared to contain a small amount of virus in many of the protoplasts (Fig. 5). Protoplasts isolated from one plant from line 247 which was symptomless had an ELISA value of 0.05 AU at 15 days p.i. Of these protoplasts, 65.6% were labelled with the FITCantibody. The fluorescent labelling was not seen completely throughout each protoplast, unlike protoplasts from infected GCRI-26 plants. In-
stead, the virus seemed to be localized within compartments in the cytoplasm (Figs. 4 and 5). This type of labelling was seen in about one half of the plants tested in several experiments. Discussion
The resistance of the six somaclonal lines seemed in general to be similar. The ToMV-resistant tomato somaclonal lines in this study were all susceptible to a more invasive tomato strain, TMV-1 and to TEV and CMV. Their resistance was limited to the common strain of tomato mosaic virus (ToMV-O) and to TMV-FI. The somaclonal lines were similar in response to high temperature incubation after inoculation, remaining resistant at 28-30°C for more than 30 days. The somaclonal resistance was similar to the resistance given by the Tin-1 gene; each line limited viral multiplication to varying degrees and suppressed symptoms, but was overcome by the more invasive tomato mosaic virus strain, ToMV-1 and was not affected by high temperature incubation. Of the six somaclonal lines, the degree of resistance found in somaclonal line 247 was the highest and most consistent. For comparison,
Fig. 3. Fluorescenceimage of ToMV-infectedprotoplasts isolated from susceptible tomato line GCRI-26 and treated with FITCantibody to ToMV coat protein. Most protoplasts were brightly labelled throughout. (600x ).
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Fig. 4. Fluorescence image of protoplasts isolated from a resistant somaclonal line after inoculation with ToMV. One ToMVinfected protoplast was found among thousands of healthy protoplasts after treatment with FITC-antibody to ToMV coat protein. (600 x ).
Fig. 5. Fluorescence image of protoplasts isolated from a resistant somaclonal line alter inoculation with ToMV. Alter treatment with FITC-antibody to ToMV coat protein, many protoplasts show localized areas of FITC-labelled ToMV even though the plant was symptomless and had a very low virus titer when sampled using ELISA. (600x).
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somaclonal lines 12 and 247 were chosen for studies of virus movement. In these studies, after inoculation with ToMV, very low virus titers (0.02-0.09 AU) were present in inoculated leaves of somaclonal line 247, but the virus did not move into the plant systemically. This restriction of virus movement could have been due to deficient levels of virus or to a deficiency in the transport function. However, somaclone 12 allowed a medium virus titer (0.05-0.32 AU) to accumulate throughout the plant but did not allow a high virus titer. In contrast, susceptible control tomato seedlings allowed the virus to spread throughout the plant in high titer ( > 1.50 AU) in 7 days. This result was similar to the classical work done on virus movement in tomato by Samuel [17] and the more recent report of movement of potato spindle tuber viroid in potato [18]. The long-distance, intraplant transport was consistent with the pattern of movement of photosynthetic products moving in the phloem from fully expanded leaves to developing upper leaves. Differences in the resistance gene from each line may explain the consistently greater virus titer in somaclone line 12 compared to line 247. It would be of interest to test for complementation between these two lines. Perhaps both somaclonal lines limit replication, but line 247 may limit systemic transport of the virus as well. At this point, however, it is difficult to separate the two effects. In grafts of resistant on susceptible tomato seedlings, inoculated susceptible rootstock allowed multiplication of virus, but the upper, resistant scion delayed multiplication of virus. Somaclonal line 247 completely delayed multiplication for more than 10 days, even when the rootstock had a high virus titer. The resistant scion was infected by 20 days. Somaclonal line 12 allowed medium virus titer to accumulate, which was maintained for more than 20 days. The inhibition of virus multiplication in somaclone material was significant in both cases. The accumulation of virus in the somaclonal material could have been due to the selection of a more invasive virus substrain which was able to overcome the somaclonal resistance [191. In the reciprocal grafts of susceptible on resistant tomato seedlings, inoculated resistant root-
stock restricted the virus to the inoculated leaf lor more than 21 days. In susceptible control grafts, virus levels were high throughout the scion and rootstock at 5 days. Because of the long delay in accumulation of virus in the scion, it was concluded that the resistant rootstock was restricting the movement of the small amount of virus that was present in the rootstock. If any infective virus had moved into the susceptible scion, there would have been a high virus titer in 5 days. When susceptible GCRI-26 plants infected with ToMV were used as a source of protoplasts, it was found that the protoplasts were reduced in size and a high percentage were fluorescent. In the resistant plants from somaclonal lines, there was a lower percentage of infected protoplasts. Also, the virus (coat protein) appeared to be limited to discrete areas in the cytoplasm. The presence of virus coat protein has been detected in inclusion bodies in the cytoplasm [20] and has also been shown to accumulate in thylakoid membranes [21]. Thus, it would be interesting to determine more precisely the location of the viral coat protein in protoplasts of the tomato somaclones. The resistant plants seemed to limit the multiplication of ToMV-O. Protoplasts released from inoculated, resistant plants had lower levels of virus present in each cell, suggesting that there could be an inhibition of replication at the cellular level. There was also a restriction of virus movement, especially evident in the grafting experiments. In crosses with isogenic plants expressing known Tm resistance genes, it was determined that the somaclonal resistance was additive with Tm-1 but not with Tin-2 [22]. It was proposed that the resistance trait in each somaclonal line may be inherited as a single, incompletely dominant nuclear gene, with a maternally transmitted factor as well. The role of each of these components can only be ascertained by more complete characterization of the somaclonal lines to examine mechanisms of resistance.
Acknowledgements Research was supported by the Michigan Agricultural Experiment Station and The Upjohn Company, Kalamazoo, MI.
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3 4
5 6 7
8
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10 11
K.A. Barden, S. Schiller Smith and H.H. Murakishi, Regeneration and screening of tomato somaclones for resistance to tobacco mosaic virus. Plant Sci., 5 (1986) 209-213. R.S.S. Fraser and S.A.R. Loughlin, Resistance to TMV in tomato: effects of the Tm-1 gene on virus multiplication. J. Gen. Virol., 48 (1980) 87-96. T.J. Hall, Resistance at the Tm-2 locus in the tomato to ToMV. Euphytica, 29 (1980) 189-197. L.J. Alexander, Transfer of a dominant type of resistance to the 4 known Ohio pathogenic strains of tobacco mosaic virus (TMV) from Lycopersicon peruvianum to L. esculentum. Phytopathology, 53 (1963) 869. G.E. Russell, Plant Breeding for Pest and Disease Resistance. Buttersworth, London, 1978, p. 485. J. Pelham, Strain-type interaction of TMV in tomato. Ann. Appl. Biol., 71 (1972) 219-228. L.W. Stobbs and B.H. MacNeill, Increase of tobacco mosaic virus in graft inoculated TMV-resistant tomatoes. Can. J. Plant Pathol., 2 (1980) 217-221. M. Cirulli and L.J. Alexander, Influence of temperature and strain of tobacco mosaic virus on resistant tomato breeding line derived from Lycopersicon peruvianum. Phytopathology, 59 (1969) 1287-1297. I.W. Selman and A.C.I. Yahampath, Some physiological characteristics of two tomato cultivars, one tolerant and one susceptible to tobacco mosaic virus. Ann. Bot., 37 (1973) 853-865. M. Pilowsky, Grafting studies with Tm-2 a stock. Tomato Genet. Coop. Rep., 21 (1971) 36. B. Kassanis and R.F. White, A simplified method of obtaining tobacco protoplasts for infection with TMV. J. Gen. Virol., 24 (1974) 447-452.
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E.M. Frearson, J.B. Power and E.C. Cocking, The isolation, culture and regeneration of Petunia leaf protoplasts. Dev. Biol., 33 (1973) 130-137. 13 A. Guri, M. Volokita and K.C. Sink, Plant regeneration from leaf protoplasts of Solanum torvurn. Plant Cell Rep., 6 (1987) 302-304. 14 Y. Otsuki and I. Takebe, Fluorescent antibody staining of tobacco mosaic virus antigen in tobacco mesophyll protoplasts. Virology, 38 (1969) 497-499. 15 R.S. Spendlove, Microscopic techniques, in: K. Maramorosch and H. Hoprowski (Eds.), Methods in Virology, Vol. 3, Academic Press, 1967, pp. 475-520. 16 Y. Otsuki and I. Takebe, Production of mixedly coated particles in tobacco mesophyll protoplasts doubly infected by strains of tobacco mosaic virus. Virology, 84 (1978) 162-171. 17 G. Samuel, The movement of tobacco mosaic virus within the plant. Ann. Appl. Biol., 21 (1934)90-111. 18 P. Palukaitis, Potato spindle tuber viroid: investigation of the long-distance, intra-plant transport route. Virology, 158 (1987) 239-241. 19 J.R.O. Dawson, Contrasting effects of resistant and susceptible tomato plants on tomato mosaic virus multiplication. Ann. Appl. Biol., 56 (1965) 485-491. 20 G.J. Hills, K.A. Plaskitt, N.D. Young, D.D. Dunigan, J.W. Watts, T.M.A. Wilson and M. Zaitlin, lmmunogold localization of the intracellular sites of structural and nonstructural tobacco mosaic virus proteins. Virology, 161 (1987) 488-496. 21 A. Reinero and R.N. Beachy, Association of TMV coat protein with chloroplast membranes in virus-infected leaves. Plant Mol. Biol., 6 (1986) 291-301. 22 S.S. Smith and H.H. Murakishi, Genetic characterization of resistance to tomato mosaic virus (ToMV) in tomato somaclones. Phytopathology, 80 (1990) 966 (Abstract).
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Restricted virus multiplication and movement of tomato mosaic virus in resistant tomato somaclones Sandra Schiller Smith and Harry H. Murakishi Department of Botany and Plant Pathology, Michigan State UniversiO,, East Lansing, M1 48824-1312 (USA) (Received August 24th, 1992; revision received October 27th, 1992; accepted November 4th, 1992)
Somaclonally-derived resistance from fully tomato mosaic virus (ToMV)-susceptible tomato (Lycopersicon esculentum, Mill.) was compared to resistance conferred by other known ToMV resistance genes in tomato. Plants were challenged by the common tomato mosaic virus strain (ToMV-O) under different conditions, by different tobamovirus strains and by tobacco etch virus (TEV) and cucumber mosaic virus (CMV). Resistance was limited to ToMV-O and the closely related Flavum strain of TMV (TMV-FI) and was found to be stable at high temperatures (28-30°C). The systemic movement of the virus was restricted in resistant somaclone lines as compared to susceptible tomato plants. The movement of virus was also monitored in reciprocal grafts of resistant and susceptible plants. Transport of virus from inoculated rootstock was limited in both positions, with resistant material as scion or rootstock. The presence of virus was detected at the cellular level by releasing protoplasts from inoculated plants and using immunofluorescent staining to detect virus coat protein. In resistant somaclones, the rate of virus infection was very low (< 0.1% of protoplasts with virus). In some resistant plants the percentage of infected protoplasts was high (65%) but the infected protoplasts had only moderate levels of virus present.
Key words: tomato; mosaic virus resistance; somaclonal variation
Introduction Resistance to ToMV was obtained in somaclones by regenerating tomato plants through adventitious shoot formation from leaf discs of ToMV-susceptible (GCRI-26) tomato seedlings [1]. Resistance shown by the somaclones appeared to be a delay in symptom expression and virus multiplication. Further characterization of this resistance through study of virus movement, effect of high temperature and reaction to other viruses and another strain of ToMV is presented here. Comparison is made to previously described resistance in tomato.
Correspondence to: H.H. Murakishi (Professor Emeritus), Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824-1312, USA.
T o M V resistance genes in tomato
There are three well-characterized ToMV resistance genes in tomato, each of which has been incorporated into cultivated tomato from wild tomato species. The gene Tm-1 is co-dominant for limitation of virus replication and dominant for suppression of symptom formation [2]. At a different locus, the allelic genes Tm-2 [3] and Tm-2 2 [4] effectively prevent infection of ToMV by a hypersensitive response, usually evident as local lesions. At higher temperature or in heterozygous plants, the hypersensitive resistance response may cause systemic necrosis. These three genes have been bred into many commercial cultivars as Tm1/Tm-1, Tm-2/Tm-2 2 to give complete resistance to date [5]. Also, each of these alleles has been incorporated into isogenic lines of tomato and has been tested with different virus strains [6]. Such lines can serve as a model system for study of
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114 resistance mechanisms. Because the resistant somaclones in this study were derived from GCRI26, one of Pelham's near-isogenic plant lines, direct comparison of resistance was made between the somaclones and the other resistance alleles.
Temperature sensitivity When resistant tomato plants are inoculated with virus and incubated at high temperature (28-30°C), there is a breakdown in the resistance. With Tm-1, the results have been ambivalent with reports of temperature sensitivity [6] and stability [2]. The genes Tm-2 and Tin-22 both lose resistance at higher temperature [6-8], allowing systemic virus multiplication and spread. When such plants are moved to normal temperatures, the plants become systemically necrotic. Grafting experiments Study of virus movement in reciprocally grafted tomato has been done for each resistance gene. Selman and Yahampath [9] made reciprocal grafts between a cultivar with the Tm-1 gene and a susceptible cultivar with a different genetic background. The scion was inoculated with ToMV and virus was assayed by inoculation on N. glutinosa. When the rootstock was susceptible, the resistant scion was less resistant, allowing the multiplication of virus. When the rootstock was resistant, the susceptible scion became infected with virus, indicating that there was no viral inhibitor being transported upward. In either case, the virus moved freely across the graft and replicated throughout the plant, indicating that the Tm-1 gene did not inhibit transport of virus or cause systemic resistance. Pilowsky [10] studied reciprocal grafts between plants with Tm-2 e and infected susceptible plants and found that in either position (rootstock or scion) the Tm-2: plants became systemically necrotic, even when the graft was severed after 7 days. Stobbs and MacNeill [7] studied virus movement when grafts were made between plants expressing Tm-2 and susceptible plants. Stem inserts of one genotype were made between rootstock and scion of the other genotype and ToMV was inoculated on the rootstocks after the graft unions were established. The resistant insert did not alter virus movement; when a leaf of the susceptible
rootstock was inoculated, the virus moved into the scion, but when a leaf of the resistant rootstock was inoculated, the virus remained in the inoculated leaf. When grafted as rootstock or scion, the resistant plants developed systemic necrosis when the grafted partner was TMV-infected. In the same study, virus movement was found to occur through the phloem when examined using electron microscopy. It was suggested that the virus by-passes the normal genetic resistance mechanisms by entering the resistant mesophyll from the phloem. The virus was localized if the only inoculum was on a resistant leaf. Methods
Response of somaclones to different tobamovirus strains Tomato seeds were planted and cultivated as described previously [1]. Groups of tomato plants representing the R 1 generation of all six somaclonal lines and the susceptible line, GCRI-26, were divided and inoculated with ToMV-O, TMVF1 or a strain that overcomes the resistance of the Tm-1 gene, TMV-1 (described by Pelham [6] and kindly provided by F. Motoyoshi, Tsukuba, Japan). Assay of the inoculated plants was done at 10 days post infection (p.i.) and continued for 20, 30, 50 and 70 days for ToMV-O and TMV-FI. Plants that became infected with TMV-1 were discarded immediately after assaying to prevent unintentional spread to other tomato plants. Presence of ToMV-O and TMV-F1 was detected using double antibody sandwich microplate enzyme-linked immunosorbent assay (DASELISA) as described [1]. For all DAS-ELISA, duplicate samples were assayed for each plant and compared to uninfected and heavily infected control samples. Presence of TMV-1 was detected by observation of symptoms and inclusion bodies in leaf hair cells. Comparisons of ToMV-O and TMV-F1 were repeated three times with 6-16 plants of each line. Comparison of TMV-I to ToMV-O was done once with twelve plants of each line. Response of somaclones to inoculation by different viruses Groups of tomato seedlings representing the R i
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generation somaclonal lines and GCRI-26 were divided and inoculated with the pepper strain of CMV or with the wild tobacco strain of TEV, which were both provided by T. Zitter, Cornell University, New York. The inoculum was prepared from frozen, infected tobacco tissue which was ground at 1 g/20 ml of 0.01 M NaKphosphate buffer (pH 7.0). Detection of the virus was done using DAS-ELISA, with gammaglobulin specific for each virus. Each gammaglobulin was purified from rabbit antiserum (from American Type Culture Collection, Rockville, MD) as described previously [1]. Plants were assayed in duplicate at 10 and 20 days p.i. Twelve plants of each line were tested with each virus. Response of somaclones to inoculation and incubation at different temperatures Groups of tomato plants representing R l and R 2 generations of somaclones, GCRI-26 (+/+) and the resistant tomato line GCRI-237 (Tm1/Tm-1) were inoculated with ToMV-O and divided into sets which were incubated at 25°C or 30°C with otherwise similar conditions. Plants were assayed for the presence of virus at 10, 20 and 30 days p.i. (and one group was assayed at 50, 70 and 150 days p.i. as well) using DAS-ELISA in duplicate with positive and negative controls. The first experiment included six plants of each line for each temperature and was replicated using 6 - 9 plants of each line for each temperature. Virus movement & tomato Plants were transplanted into 4-inch pots at 12 days. The groups consisted of R2 plants from somaclone lines 12 and 247 and the susceptible line GCRI-26. One group was inoculated with ToMVO when the plants were 32-day-old and the second group was inoculated when 25-day-old. The outermost leaflet of the first true leaf was marked by punching a hole with a wide-bore needle and after dusting with carborundum, it was inoculated with virus. The inoculum was not rinsed off, but was allowed to dry in situ. Ten to 12 plants of GCRI26 and somaclone 247 were tested in each replication. At each time point (4, 7, 10 and 20 days p.i.), leaflets were removed from each leaf of the plant. Two or three plants of each line were sampled for each time point. Once plants had been sampled,
they were not used for further assay to eliminate the chance that plants might inadvertantly be inoculated during sampling. Leaf samples were assayed using DAS-ELISA and the absorbance values for leaflets from each corresponding leaf were averaged. Plants from the second repetition were used to isolate protoplasts for detection of infection at the cellular level. As controls, susceptible and resistant plants were mock-inoculated with buffer only and assayed. Virus movement in grafted tomato plants To study the movement of virus between reciprocal grafts of resistant and susceptible plant material, several types of grafts were established. Whip grafts were done using young seedlings by wetting a clean, new razor blade and removing the top of 18-day-old plants with a V-shaped cut. Tops were removed from 2 plants and exchanged, resulting in a rootstock with cotyledons only and a scion with 3 young leaves. The graft was held in place using 1/2-inch wide self-adhesive tape. Plants were sealed in polyethylene bags to increase the humidity and were allowed to bond for several weeks. A cotyledon of the rootstock of the grafted plant was then inoculated. Of the 10 whip grafts made, three grafted plants survived for the assays. The second set of plants was grafted using older plants (20-30 days). Again, V-shaped cuts were made near the tops of 2 plants and the tops were interchanged. After the whip graft was established (25-30 days), there were 2 true leaves remaining below the graft plus secondary shoots and 2-3 young leaves on the scion. The outermost leaflet of the first true leaf of the rootstock was inoculated. Of the 10 grafts made, again only 3 were successful and used for the assays. The third type of graft was established using an approach graft which incorporated a tongue (whip and tongue graft). This method was used successfully on older plants (33-39 days) and was less stressful to the plants. The graft was made on the stem between the third and fourth true leaves in most cases and was bound with self-adhesive tape. After 2 weeks, one stem was severed below the graft and the opposing top was removed. The outermost leaflet of the first true leaf was inoculated a week after that. Five whip and tongue grafts were used for assay.
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Assay of the grafted plants was done using DAS-ELISA at 0, 5, 7 and 10 days p.i. Some plants were assayed further at 21, 34, 39 and 44 days p.i. For the first group of grafted plants every leaf of both rootstock and scion were assayed in duplicate for each time point. Later groups were assayed from 1 or 2 leaves from both parts. Three pairs of reciprocal grafts were established between somaclone 247 (R2) and GCRI-26, and one pair between between somaclone 12 (R3) and GCRI26. Two control grafts were made between two plants of GCRI-26 to eliminate the possibility that delayed virus spread could be due to the graft itself.
Protoplast isolation Tomato plants were grown in the laboratory (22-25°C) under cool white fluorescent lights supplying 60 /xE m -2 s -t on a 16-h/day light cycle. The plants were treated with fertilizer daily when they were watered with one tablespoon of Peters 20-20-20 per gallon. At about 4 weeks, plants were used for isolation of protoplasts. Protoplast isolation procedures were adapted from Kassanis and White [11]. After leaves were surface sterilized and rinsed, leaves were stacked and sliced transversely in 1-mm thin sections. Leaf strips were placed in a petri dish containing 20 ml of an enzyme solution consisting of 1% (w/v) cellulysin, 0.5'7,, (w/v) macerase, (Calbiochem, Behring Diagnostics, San Diego, CA) and 0.4 M sorbitol in cell protoplast wash (CPW) salts [12] at pH 7.0. The strips were incubated on a slow shaker in the dark at room temperature for 12-18 h. The enzyme solution containing protoplasts and leaf debris was filtered through a 60-#m sieve into a petri plate. Protoplasts were transferred to a 30-ml roundbottomed centrifuge tube and centrifuged at 35 x g for 10 min. The supernatant fluid was removed by suction, the pellet was gently resuspended by rolling in a small amount of washing buffer and the protoplasts were moved to a 15-ml tube. The protoplasts were washed in 15 ml of sorbitol-CPW buffer, centrifuged again at 35 x g and the supernatant fluid was removed by suction. The protoplasts were rinsed a total of three times in this manner. The final step in purification of the protoplasts was to float the intact protoplasts on
a solution made of Lymphoprep (Accurate Chemical and Scientific Corp., Westbury, NY) and 0.5 M sucrose in CPW salts at 1:2 (v/v) for healthy protoplasts [13] or at 2:1 (v/v) for protoplasts which were isolated from virus-infected plants. Concentration of protoplasts was adjusted and viability was determined by microscopic examination after the addition of several drops of Evan's blue dye (0.05% (w/v) Evan's blue in 0.7 M mannitol, pH 7).
Preparation and staining of injected protoplasts Following the procedure by Otsuki and Takebe [14], the infected protoplasts were labelled with ToMV antibody which was conjugated to fluorescein isothiocyanate (FITC). The FITC-conjugate was prepared using the method of Spendlove [15] and cross-adsorbed with acetone powder of healthy tobacco leaves using the method of Otsuki and Takebe [16]. FITC-labelled cells were viewed with a Zeiss G F L epiluminescent microscope equipped with exciter filters KP-490 and LP-445, dichroic reflector 510 and barrier filter 520. Photographs of the fluorescing cells were taken with ASA 400 daylight film. Exposures were between 12 rain (for heavily infected cells) and 28 min (for uninfected cells). Results
Response of somaclonal lines to different viruses The six somaclonal lines displayed resistance to ToMV-O and TMV-FI for more than 70 days post-inoculation as detected by ELISA (Fig. 1). All six lines were susceptible to the strain TMV-1 and were infected by 10 days post-inoculation (not shown). There were plants among somaclone lines which showed a delay in infection after inoculation with CMV, but all plants were infected by 20 days p.i. (Fig. 1). When inoculated with TEV, all plants had high levels of virus at 10 days p.i. (Fig. 1).
Response of somaclonal resistance to high temperature The somaclonal resistance to ToMV was not changed when inoculated plants were grown at high temperature. Ninety-six percent of the in-
117
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Fig. 1. Proportion of plants free of virus after challenge with ToMV-O, TMV-F1, CMV or TEV. Tomato line GCRl-26 ('26') is the susceptible control. Other lines (215, 219, 247, 322, 330) are somaclonal lines which were selected for resistance to TMV. Plants were sampled for ELISA at 10 and 20 days post inoculation.
oculated seedlings of the 6 somaclone lines were virus-free after 30 days at 25°C and at 28-30°C. However, the plants grown in the warmer conditions were slightly stunted. Susceptible GCRl-26 plants were infected at 10 days post-inoculation and severe stunting was seen in infected GCRI-26 plants at the higher temperature. Two plants of line 237 (Tm-l/Tm-1) at 28-30°C which had a medium virus titer of 0.35 absorbance units (AU) at 10 days died after necrotic collapse before they could be sampled again at 20 days, but the other seven plants were virus-free at 30 days.
Systemic movement of virus in susceptible and resistant plants The movement of ToMV-O was fairly rapid in susceptible plants. At 4 days, virus was detected in the inoculated leaflet and in neighboring leaflets. By the seventh day after inoculation, there was a high titer of virus in the upper leaves, which were
rapidly expanding. The virus spread through the rest of the plant and at 20 days the whole plant contained a high virus titer. When this experiment was repeated using somaclonal line 247, high levels of virus did not develop anywhere in the plant. The inoculated leaf had a detectable level of virus and over time, traces of virus were detected in the upper parts of the plants, but at the lower limits of detection by ELISA (values in the range of 0.02-0.09 AU). Intermediate levels of virus (ELISA values in the range of 0.05-0.32 AU) were detected in plants of line 12 at 15-20 days after inoculation. It was evident that the virus did multiply and move systemically in these plants, but only to a limited extent.
Movement of virus in grafted plants In the control grafts that were made with GCRI26 on GCRI-26, there was no delay in virus move-
118 ment (Fig. 2A). Virus appeared in the rootstock leaves and the scion leaves in equal amounts at 5 days (ELISA values from 0.50 to 0.89 AU) and at 10 days (values from 1.11 to 1.20 AU). Because the virus multiplied and spread to all parts of the grafted plant quickly, the possibility that virus movement was limited by the graft union was eliminated. When the susceptible line GCRI-26 was used as the inoculated rootstock and the resistant line 247 as the scion, the virus replicated and moved throughout the leaves of the susceptible rootstock to attain a high titer (ELISA values from 1.70 to 1.75 AU) by 7 - 1 0 days (Fig. 2B). In each case, accumulation of virus was delayed in both rootstock and scion compared to the control grafts. In one graft, the virus was delayed in multiplying to a high titer in the resistant scion, even with the presence of the large virus concentration in the rootstock. In two other grafts of this type, there was a lower titer of virus present in the rootstock and virus moved into the resistant scion and reached a high titer in 7-21 days. In each grafted plant, the delay seen in virus multiplication in the resistant scion was notable when compared to the control graft of susceptible to susceptible plants. In the reciprocal graft of GCRI-26 (scion) and somaclone line 247 (rootstock), the inoculated
resistant rootstock did not show the presence of virus (except for the virus in the inoculated leaf, with ELISA values from 0.00 to 0.14 AU) (Fig. 2C). The virus did not appear in the susceptible scion for more than 21 days p.i. and when it did appear (with ELISA values of over 1.00 AU), the rootstock became infected as well. In one grafted plant, the virus did not multiply in the susceptible scion for more than 40 days. The reciprocal grafts between GCRI-26 and somaclonal line 12 were similar to the above grafts in their response, but the resistance did not seem as strong in general, which was consistent with the results from the study of virus movement in plants of line 12. When GCRI-26 was the rootstock, there were high levels of virus present in the lower leaves, but only intermediate levels of virus accumulated in the leaves of the resistant scion (ELISA values from 0.24 to 0.45 AU) for longer than 20 days. When somaclonal line 12 served as the rootstock, a fairly high level of virus was detected in the inoculated leaf at 5 days (0.68 AU), but did not multiply in the susceptible scion for more than 10 days.
Protoplasts Uninfected cells exhibited slight light green fluorescence and no bright spots. Protoplasts
Fig. 2. Results of experimentsin which grafted plants were inoculated in the outermost lower leaflet of the rootstock (open triangle). Movement of the virus was followed using ELISA to measure virus titer in leaves of the rootstock and scion. Grafts were made between two susceptible GCRI-26 plants as a control (A) and of resistant (line 247) onto susceptible (GCRI-26) rootstock (B) and susceptible (GCRI-26)onto resistant (line 247) rootstock (C). Illustrations represent grafts 10 days after inoculation; shading indicates high virus titer.
119 isolated from infected GCRI-26 (ELISA value of 1.85 AU) were smaller than protoplasts from healthy plants and were associated with more cellular debris. After staining with FITC-anti-coat protein antibody conjugate, 51.4% of the protoplasts prepared from infected GCRI-26 were highly fluorescent, where the entire protoplast fluoresced with bright yellow-green, indicating the presence of virus (Fig. 3). Uninfected protoplasts appeared darker (not shown). Protoplasts which were isolated from inoculated resistant somaclones differed in two ways. Plants from lines 12, 215 and 247 which had moderately low ELISA values (0.2-0.8 AU), yielded only a few infected protoplasts out of thousands of healthy protoplasts (Fig. 4). In a second response, protoplasts isolated from plants from lines 12 and 247 with very low ELISA values (0.05 AU) appeared to contain a small amount of virus in many of the protoplasts (Fig. 5). Protoplasts isolated from one plant from line 247 which was symptomless had an ELISA value of 0.05 AU at 15 days p.i. Of these protoplasts, 65.6% were labelled with the FITCantibody. The fluorescent labelling was not seen completely throughout each protoplast, unlike protoplasts from infected GCRI-26 plants. In-
stead, the virus seemed to be localized within compartments in the cytoplasm (Figs. 4 and 5). This type of labelling was seen in about one half of the plants tested in several experiments. Discussion
The resistance of the six somaclonal lines seemed in general to be similar. The ToMV-resistant tomato somaclonal lines in this study were all susceptible to a more invasive tomato strain, TMV-1 and to TEV and CMV. Their resistance was limited to the common strain of tomato mosaic virus (ToMV-O) and to TMV-FI. The somaclonal lines were similar in response to high temperature incubation after inoculation, remaining resistant at 28-30°C for more than 30 days. The somaclonal resistance was similar to the resistance given by the Tin-1 gene; each line limited viral multiplication to varying degrees and suppressed symptoms, but was overcome by the more invasive tomato mosaic virus strain, ToMV-1 and was not affected by high temperature incubation. Of the six somaclonal lines, the degree of resistance found in somaclonal line 247 was the highest and most consistent. For comparison,
Fig. 3. Fluorescenceimage of ToMV-infectedprotoplasts isolated from susceptible tomato line GCRI-26 and treated with FITCantibody to ToMV coat protein. Most protoplasts were brightly labelled throughout. (600x ).
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Fig. 4. Fluorescence image of protoplasts isolated from a resistant somaclonal line after inoculation with ToMV. One ToMVinfected protoplast was found among thousands of healthy protoplasts after treatment with FITC-antibody to ToMV coat protein. (600 x ).
Fig. 5. Fluorescence image of protoplasts isolated from a resistant somaclonal line alter inoculation with ToMV. Alter treatment with FITC-antibody to ToMV coat protein, many protoplasts show localized areas of FITC-labelled ToMV even though the plant was symptomless and had a very low virus titer when sampled using ELISA. (600x).
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somaclonal lines 12 and 247 were chosen for studies of virus movement. In these studies, after inoculation with ToMV, very low virus titers (0.02-0.09 AU) were present in inoculated leaves of somaclonal line 247, but the virus did not move into the plant systemically. This restriction of virus movement could have been due to deficient levels of virus or to a deficiency in the transport function. However, somaclone 12 allowed a medium virus titer (0.05-0.32 AU) to accumulate throughout the plant but did not allow a high virus titer. In contrast, susceptible control tomato seedlings allowed the virus to spread throughout the plant in high titer ( > 1.50 AU) in 7 days. This result was similar to the classical work done on virus movement in tomato by Samuel [17] and the more recent report of movement of potato spindle tuber viroid in potato [18]. The long-distance, intraplant transport was consistent with the pattern of movement of photosynthetic products moving in the phloem from fully expanded leaves to developing upper leaves. Differences in the resistance gene from each line may explain the consistently greater virus titer in somaclone line 12 compared to line 247. It would be of interest to test for complementation between these two lines. Perhaps both somaclonal lines limit replication, but line 247 may limit systemic transport of the virus as well. At this point, however, it is difficult to separate the two effects. In grafts of resistant on susceptible tomato seedlings, inoculated susceptible rootstock allowed multiplication of virus, but the upper, resistant scion delayed multiplication of virus. Somaclonal line 247 completely delayed multiplication for more than 10 days, even when the rootstock had a high virus titer. The resistant scion was infected by 20 days. Somaclonal line 12 allowed medium virus titer to accumulate, which was maintained for more than 20 days. The inhibition of virus multiplication in somaclone material was significant in both cases. The accumulation of virus in the somaclonal material could have been due to the selection of a more invasive virus substrain which was able to overcome the somaclonal resistance [191. In the reciprocal grafts of susceptible on resistant tomato seedlings, inoculated resistant root-
stock restricted the virus to the inoculated leaf lor more than 21 days. In susceptible control grafts, virus levels were high throughout the scion and rootstock at 5 days. Because of the long delay in accumulation of virus in the scion, it was concluded that the resistant rootstock was restricting the movement of the small amount of virus that was present in the rootstock. If any infective virus had moved into the susceptible scion, there would have been a high virus titer in 5 days. When susceptible GCRI-26 plants infected with ToMV were used as a source of protoplasts, it was found that the protoplasts were reduced in size and a high percentage were fluorescent. In the resistant plants from somaclonal lines, there was a lower percentage of infected protoplasts. Also, the virus (coat protein) appeared to be limited to discrete areas in the cytoplasm. The presence of virus coat protein has been detected in inclusion bodies in the cytoplasm [20] and has also been shown to accumulate in thylakoid membranes [21]. Thus, it would be interesting to determine more precisely the location of the viral coat protein in protoplasts of the tomato somaclones. The resistant plants seemed to limit the multiplication of ToMV-O. Protoplasts released from inoculated, resistant plants had lower levels of virus present in each cell, suggesting that there could be an inhibition of replication at the cellular level. There was also a restriction of virus movement, especially evident in the grafting experiments. In crosses with isogenic plants expressing known Tm resistance genes, it was determined that the somaclonal resistance was additive with Tm-1 but not with Tin-2 [22]. It was proposed that the resistance trait in each somaclonal line may be inherited as a single, incompletely dominant nuclear gene, with a maternally transmitted factor as well. The role of each of these components can only be ascertained by more complete characterization of the somaclonal lines to examine mechanisms of resistance.
Acknowledgements Research was supported by the Michigan Agricultural Experiment Station and The Upjohn Company, Kalamazoo, MI.
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5 6 7
8
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10 11
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