The effect of polyacrylic acid treatment on the susceptibility of Nicotiana tabacum cv. xanthi-nc to tobacco mosaic virus

PhysiologicalPlant Pathology(1978) 13, 13-21

The effect of polyacrylic acid treatment on the susceptibility of Mcotiana tabacum cv. xanthi-nc to tobacco mosaic virus A. C. CASSELLS, A. BARNETT and M. BARLASS Departmentof Plant Sciences,Wye College(Uniuersityof London), Wye, Kent ZV2.5 5AH, U.K. (Accepdfw publicationJamuuy1978)

Lesion sizes in leaves of Ni~otiana tabacumcv. xanthi-nc treated with polyacrylic acid (PA), and showing 60% reduction in lesion numbers, did not differ significantly from those in untreated control plants. Protoplasts isolated from treated plants did not differ significantly in 0/0 infection or in the production of infectious virus at 24 or 48 h after inoculation in vitro, compared to the controls. Polyacrylic acid treatment was shown to have a differential effect on the susceptibility of xanthi-nc plants grown in different light intensities; highest resistance was induced in plants grown in low light. A study of the water relations of low light grown plants indicated increased osmotic potential and decreased pressure potential, compared with the controls. The transpiration resistance of treated plants was increased. Spraying of treated plants with an antitranspirant abolished PA-induced resistance. It is suggested that P A treatment may interact with the plant mechanism(s) affecting susceptibility to virus infection.

INTRODUCTION Polyacrylic acid (PA), an interferon inducer in animals [ 41, has induced resistance to tobacco mosaic virus (TMV) in .Nicotianu tabacum cv. xanthi-nc [5], to TMV in N. glutinosa, to tobacco ringspot virus in N. tubucum White Burley and to pelargonium leaf curl virus (PLCV) in Duturu strumonium (Cassells & Flynn, unpublished). Four “new” proteins have been reported to occur in PA-treated xanthi-nc plants [5J. Some workers consider that induced resistance to virus infection in plants is analogous to the induction of interferon in animal virus infections [S, 91 and that the primary effect is on the virus-localizing mechanism in the plant and not on the surface infectible site [II]. In preliminary studies, while observing that P A treatment reduced the number of TMV local lesions by up to 90% in N. glutinosu and .N. tubucum cv. xanthi-nc, and TRW ringspots by up to 60% in inoculated leaves of N. tubaurn White Burley, in few cases was a reduction observed in the sizes of the lesions which formed in the inoculated leaves. This suggested that the primary effect of PA treatment might be on the surface infectible site. The present study was made to elucidate further the phenomenon of polyacrylic acid induced resistance in view of the potential significance of an interferon-like mechanism in plants. 0048-4059/78/0701-0013

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A. C. Cassells, A. Barnett and M. Barlass

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MATERIALS AND METHODS Plant cultivation Ncotiana tabacum cv. xanthi-nc plants were grown in the glasshouse to the 20 to 25 leaf stage in 12 cm pots in an acid peat supplied with a complete nutrient solution (Bio P Base; Pan Britannica Industries, Herts., U.K.). The mean daily solar radiation was in the range 70 to 300 m W h/cm2, day length was 15 h, supplemented when appropriate by mercury vapour lamps, and the minimum temperature was 15 “C. Some experiments were made in a growth room which was maintained at 22 “C, 8.6 W m-s, 15 h photoperiod unless otherwise stated. Virus The TMV

isolate used was a type 0 isolate purified

as described previously

[I].

ch.e?nicals The polyacrylic acids (PA)used were a gift from Allied Colloids Ltd, Bradford, U.K. Polymer ranging in molecular weight from 13.2 x lo6 to 1445 were used in prehminary trials. The lowest mol. wt. polymers were found to be the most efficient inducers of resistance and consequently PA 1445 and 1750 were used in the experiments reported here. These chemicals were neutralized before use and applied to the plants either as foliar spray at 20 mg/ml, with O*05°,$Tween 20 asawetting agent, or watered on to the soil [q in the pots as 100 ml of O-02 M solutions. When applied as sprays, the controls were sprayed with the wetting agent alone, and both control and treated leaves were washed with distilled water prior to inoculation, to remove residual PA which reduces virus infectivity. The dosage of PA watered on was determined for plants grown under the conditions described above. Doubling of the dosage resulted in wilting and premature senescence of the leaves; higher concentrations also induced vein-clearing and rapid death of the plant. Treated plants were left for at least 3 days before being tested for induced resistance. Virus inonclation Plants were inoculated with a stock preparation of virus containing 6 pg/ml TMV in 50 rmf sodium phosphate buffer at pH 7-O to which 3 mg/ml Celite was added as abrasive. The stock preparation gave approximately 100 to 200 lesions per half leaf. Inoculations were carried out in the late morning or afternoon unless otherwise stated. Protoplast isolation and infection Protoplasts were isolated as follows: the epidermis was removed by peeling and the leaves floated on 0.66 M manmtol overnight at 4 “C in the dark. After 15 h, the mannitol was removed and replaced with a solution of O*5o/o w/v Macerozyme pectinase, 1 y. w/v Onozuka R-10 cellulase in 0.66 rd mannitol, pH 5.8. The tissue was incubated in the dark at 25 “C for 2 h. Cell and protoplast release and viability were determined as before [J1 and the protoplasts were washed and infected as follows. The protoplasts were allowed to settle under gravity, then resuspended in

The effect of polyacrylic acid

15

0.66 M manmtol (1 x lo6 protoplasts per 0.1 ml) and mixed with 50 ~JJof TMV suspension (6 mg/ml) plus O-4 ml of 8% w / v solution of polyethylene glycol (PEG M W 6000). The mixture was then diluted with 2 ml of O-1 M sodium phosphatebuffered mannitol (O-66 M) at pH 7.0. The infection mixture was incubated at 25 “C in the dark for 1 h. The protoplasts were allowed to settle and the supernatant removed and replaced with 10 ml of Takebe’s medium [I.?] without hormones. The protoplasts were again allowed to settle and the supernatant removed. The protoplasts were resuspended in 5 ml of Takebe’s medium (as above) containing 4 PM zeatin and incubated in a growth room (22 “C, 15 h photoperiod, 8.6 W mT2). Protoplast viability during and at the end of the incubation period was determined by staining with fluorescein diacetate 133, samples were taken at 24 and 48 h for infectious virus production and at 48 h for fluorescent antibody staining. The percentage of protoplasts containing TMV antigen was determined by staining with lissamine rhodamine B-conjugated antibody [I]. Infectious virus production was assayed as follows: the protoplasts were harvested by sedimentation, the pellet was resuspended in O-6 ml of 10 nm sodium phosphate buffer at pH 7-O and frozen; it was then allowed to thaw, 3 mg/ml Celite was added and the suspension was ground in a glass homogenizer. The ground protoplast extracts was assayed on half leaves on 8. glutinosa, opposite half-leaves being inoculated with standard virus preparation. Water relations of leaf tissue The water relations of the leaves were studied by psychrometry 1143 as follows. Discs (0.5 cm) were punched from the interveinal areas of control and PA-treated plants. Two discs were taken at each time, and one from each treatment placed in the chamber of a dew-point, thermocouple hygrometer (Wescor Inc., Logan, Utah, U.S.A.). The second disc from each sampled leaf was immediately placed on a small plastic bag and frozen at - 17 “C (see “frozen disc” below). The chambers containing the fresh discs were equilibrated for 30 min and readings made. With fresh discs, readings were made hourly and the data subjected to least squares analysis to obtain the line of best fit, from which the equilibrium value was obtained. In the case of frozen discs, care was taken at all stages in handling to avoid water loss to the atmosphere, consequently the frozen discs were transferred rapidly to the psychrometer chamber and allowed to equilibrate before readings were made (as above). The water potential of the tissue was obtained from the readings for fresh discs, the osmotic potential of the sap was determined from the readings for frozen discs and the pressure potential calculated by difference. The plants used in this investigation were taken at random from a batch in which induced resistance had been demonstrated by prior inoculation with TMV. Discs were taken at 9.30 a.m. and 2.30 p.m. on consecutive days. It has been confirmed that the PA-induced resistance persists for at least 30 days in treated plants (Cassells & Flynn, unpublished). Leaf transpiration resiktance Transpiration resistance was measured with an automatic diffusion porometer [14] (Crump Scientific, Rayleigh, Essex, U.K.). Repeated measurements were

16

A. C. Cassells, A. Barnett and M. Barlass

made until consecutive readings agreed to within 10%. Measurements were made on one area of the leaf and usually were completed in 10 set, where the first and last reading differed by more than 15%, the readings were disregarded. Application of anti-transpirant Plants were sprayed on the underside of the leaf with S-600 (85% modified polyvinyl resin; Synchemicals Ltd, London, U.K.) in water (1 : 9, v/v) containing 0.05% Tween 20 as a wetting agent. Plants were left for 4 h after treatment before inoculation on the upper leaf surface for measurement of transpiration resistance,

RESULTS

The e$ct of polyaqlic

acid treatment on local lesion size

Two aspects of PA-induced resistance to virus infection have been reported, viz. a reduction in lesion size in treated leaves and a reduction in lesion numbers. However, we observed no obvious differences in the size of those lesions which developed in treated leaves. These qualitative observations were confirmed by taking pairs of leaves at random from equivalent positions on control and PA-treated plants (pot watered with 100 ml of 0.02 M PA, mol. wt. 1750) and measuring lesion areas as described previously [Z]. In a typical experiment involving eight leaves on four pairs of plants, in which control leaves developed c. 200 lesions per half leaf and the treated c. 90 lesions, the average lesion size on the control was OS96mm2 and on the treated leaves 1.0 mm2 (not significantly different). Thus PA treatment significantly reduced local lesion numbers (P = 0.01) without affecting lesion size. Toxic concentrations of PA greatly reduced leaf area and under these conditions lesion size was reduced. Tobacco mosaic virus replication in protoplastsfrom N. tabacum cu. xanthi-nc plants showing PA-induced resistance To determine whether PA-induced resistance operated at the cell level, as does interferon in induced animals cells [II], mesophyll protoplasts were isolated from control plants and plants which had been sprayed with PA 1750. Following PA treatment the plants were left for 8 days in a shaded glasshouse, the leaves were washed with distilled water to remove residual PA, and plants from each batch, chosen at random, were inoculated with TMV to test for induced resistance. Following lesion development, leaves were removed from non-inoculated PA-treated plants from similar positions to those in which induced resistance had been confirmed, and also leaves from similar positions on non-treated, uninoculated control plants. The results (Table 1) did not indicate any differences between the number of infected protoplasts from control plants or PA-treated plants in which a 90% reduction in lesion numbers was found, nor was there a reduction in virus production in protoplasts from induced plants at 24 or 48 h after infection. This experiment was repeated in an exactly similar manner, but with the inclusion of 1 mg/ml PA 1750 in the protoplast incubation medium, and a similar result obtained.

The effect of polyacrylic

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acid TABLE 1

Tobaccomosaic virus replicationin ~oto&sts isolatedfrom the kaws of N. tabacum cu. xanthi-nc plants showing~ly&ic acid-inducedr&stance. Protoj&astswereinoculatedin vitro

Plant treatment=

Hours after inoculation

Release of mesophyll protoplasts (%Jb

Viability of protoplasts (%)”

Local lesions/ 10’ protoplastsO

80 75 75

593 583

192x 10” 1.2x 10s

65

z 60

812 812

2.0x 10’ 2.0x 10s

63

Control plants (non-induced)

0 24 48

90

PA-treated (induced)

0 24 48

66

TMV particles/ protopla&

Protoplasts fluorescing (%I’

a Protoplasts isolated from untreated plants and PA-treated plants showing 90% reduction in lesion numbers in inoculated leaves. * Cf. [3]. c Mean of duplicate experiments, three replicates of each assay (mean of three half leaves). d Based on EM particle counts of standard TMV preparation. * Protoplasts stained with lissamine rhodamine B-conjugated antiserum [I].

The e&t of polyacrylic acid treatment on the susceptibility of N. tabacum plants grown under dz@rent light intensities

cu. xanthi-nc

Exposure of test plants to a period of darkness or low light intensity before inoculation usually increases susceptibility to virus infection [IO]. To test the effects of PA treatment on this phenomenon, 32 uniform xanthi-nc plants were selected and placed in a growth room at 22 “C. Four light regimes were used : two light intensities, 8.6 W m-2 (“high”) and O-8 W rnw2 (“low”) combined with continuous light or a 15 h photoperiod. There were four control and four PA-treated plants (PA sprayed, conditions as above) in each batch. After 3 days both sprayed and control plants were washed and six comparable half-leaves were inoculated on each plant with TMV. After a further 4 days, the lesion numbers were counted, leaves detached and their areas measured. The data are given in Table 2. Polyacrylic acid treatment reduced lesion numbers in continuous high light intensities by 35% compared to TABLE

The e&t of @yaqlic

Photoperiod Continuousa 15h”

Light intensity (W m-a)

2

acid on lesion productionin N. tabacum cu. xanthi-nc grown underfour light regimesin a growth room Mean lesion numbers/leaf Control Treated o~ change

Control

Leaf area (cm*) c Treated o/0 change

8.6 0.8

99 199

66** 27*

-35 -86

54 63

64 57

+18

8.6 0.8

103 359

68, 44,

-34 -88

58 66

56 62

-4 -6

Twenty-four replicate half-leaves per experiment, %nean of three experiments, *mean of two experiments. Significant at P = O*Ol*, 0.02**. * Mean of 24 leaves per experiment, leaf area was measured 6 days after PA treatment. 2

-10

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A. C. Cassells, A. Barnett and M. Barlass

the control (mean of three experiments, P= 0.02). PA induced a significant reduction (P=O*Ol) in lesion numbers on leaves receiving continuous low light, lesions decreasing by 86%. At these concentrations PA had no effect on leaf area. A similar response pattern to PA was noted in plants grown with a 15 h photoperiod. The untreated 15 h photoperiod control plants were more susceptible than those grown under continuous light and in both batches there were no significant differences in leaf area. PA significantly reduced lesion numbers by 88% at low light intensities and by 34% at high light intensities. These results were confirmed using plants grown in the glasshouse in spring and autumn or in the shade in the glasshouse in summer. Th efict of polyacrylic acid treatment on the susceptibility and water relations of N. tabacum cu. xanthi-nc plants The differential effects of PA treatment on plants grown in high and low light intensities, and the known changes in water relations and susceptibility which can be associated with light intensities [IO], suggested that PA treatment might be affecting susceptibility through changes in the water relations of treated plants. To test this hypothesis, batches of 20 plants (10 PA-treated and 10 controls) (as above) were grown under low light intensities in the growth room. Five comparable leaves per plant were inoculated with virus to confirm the induction of resistance and discs were punched from similar leaves of the plants for the determination of water potential and osmotic potential. The results (Table 3) are given for samples taken at approximately 3 and 7 h after the end of the dark period to gain an indication of the possible effect of diurnal changes on the phenomenon and their magnitude. The data (mean of duplicate experiments) show no significant variation between the morning and afternoon samples within the treatments. However, at both times, the water potential and osmotic potential of the PA-treated induced plants were higher than the controls and the pressure potential was lower. These results were observed in subsequent experiments. TABLE 3 The effect of pdyamylk acid treatment an the water relations of the kaf Control W-4

PA-treated

Water potential Osmotic potential Wall potential

a.m.O a.m. a.m.

o-o - 10.2 + 10.2

-4.1 - 13.4 +9.3

Water potential Osmotic potential Wall potential

p.m. p.m. p.m.

0.0 -11.9 +11*9

-4.1 -15.2 +11*1

Data are for 10 plants per experiment, mean of two experiments. o Samples were taken 3 h after end of the dark period (a.m.) or 7 h after the dark period (p.m.). Water and osmotic potential were determined by psychrometry (details in Materials and Methods) ; wall pressure was calculated as follows : water potential = osmotic potential + wall potential.

The effect of polyacrylic acid

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77~ efects of polyactylic acid treatment on the transpiration resistanceof leavesof N. tabacum cu. xanthi-nc Uniform batches of six plants were selected, one batch was watered with PA 1445 (application as for PA 1750 a b ove), a second served as control. These were placed in the growth room under low light intensities for induced resistance to develop; this was confirmed by inoculation with TMV (as above). Uniform leaves were chosen on each plant and the transpiration resistance measured using a diffusion porometer. The transpiration resistance of the leaves from PA treated plants was 9.4 s cm-l and for the control 7.4 s cm-l (mean of duplicate experiments), The effect of an anti-tran.@ rant on the suscefitibi&y of control and PA treated N. tabacum cu. xanthi-nc plants. The results reported above suggested that PA treatment interacted with endogenous mechanism affecting water relations and influencing susceptibility to virus inoculation To test this hypothesis, an experiment was carried out in an attempt to alter the water relations of control and treated plants. Twenty-four uniform xanthi-nc plants were selected and divided into two groups of 12. One group was treated with PA 1750 (watered on), the others were controls. After resistance had developed, six plants from each group were selected at random and the transpiration resistance measured (as above). They were then inoculated with TMV using the standard procedure. The remaining plants, PA-treated and controls, were sprayed on the undersurface of the leaves with S-600 and subsequently inoculated on the upper leaf surface with TMV. The plants were left in the growth room for 4 days. Lesion numbers were then counted. The transpiration resistance of unsprayed, control and PA-treated plants were 3.1 and 4.5 s cm-l respectively (mean of three plants) ; lesion counts were 486 and 248 respectively (mean of six leaves, spread over three plants). The transpiration resistances of sprayed, control and PA-treated plants were 39.9 and 42.7 s cm-1 respectively and lesion counts 331 and 368 respectively (replication as for unsprayed batch). DISCUSSION

Interferons in animal cells can result in the induction of an antiviral state which results in the slowing of virus replication, possibly by affecting the rate of synthesis of virus-coded proteins [II]. No such effect was recorded in the present work for TMV replication in protoplasts from PA-treated, induced plants, despite the fact that comparable leaves showed approximately 90% reduction in lesion numbers. In addition, there was no correlation between reduction in lesion numbers, and reduction in the sizes of those lesions which formed in the leaves of induced plants. Localization of virus in plants is known to depend on cell interactions [9], which presumably cannot occur in isolated protoplasts. Thus it is conceivable that PA treatment facilitates the localization mechanism by affecting cell-to-cell movement of virus. However, the “all or nothing” nature of the response (i.e. no gradation in lesion size is apparent) is difficult to interpret in this way uriless it is postulated that there are two different types of infectible sites, one of which is affected by the

20

A. C. Cassells, A. Barnett and M. Barlass

treatment. The differential effect of PA treatment on plants grown in high and low light intensities supports an alternative hypothesis, namely that PA treatment interacts with the mechanism(s) affectmg the susceptibility of the plant to virus infection. Further support for this view comes from the finding that PA treatment prevents the diurnal increase in susceptibility which starts at the beginning of the photoperoid [IO] (Cassells & Roebuck, unpublished). Effects were reported both on the osmotic potential and the water potential of the tissues of treated plants, and by difference, on the pressure potential of these plants. These factors have previously been implicated in altered susceptibility to virus infection (e.g. see review [IO]). In preliminary trials, PA was found to induce wilting at higher concentrations but in the present work no evidence was found that PA exerted an effect on the water relations of the treated leaves by decreasing transpiration resistance, on the contrary transpiration resistance was increased. In summary, the abolition of PA-induced resistance following treatment with the anti-transpirant S-600 further supports the view that PA-induced changes in susceptibility are related to its effect on cell water relations. Failure to detect significant effects on leaf growth (Table 2) or increased wall or cuticle thickness (Cassells, unpublished) following PA treatment offer further support for this view. Previous workers [8] have failed to find a correlation between ectodesmata and increased susceptibility to virus on transfer of plants from high to low light conditions. The transient nature of those infectible sites, which are suppressed by PA treatment, suggests that they may not be associated with structural changes in the leaf but rather of the plasmareflect changes in cell turgor. It is speculated that “woundability” lemma (important in relation to the entry of viruses into isolated protoplasts [7j) may be increased in turgid cells. Electron microscope and other studies are in progress in an attempt to elucidate further the surface infectible site; polyacrylic acid may be a useful tool in this study. We are grateful to Professor I. W. Selman for the interest he has shown in this work, and to Dr S. Burrage for advice concerning the studies on water relations. A.C.C. acknowledges an equipment grant from the Central Research Fund of the University of London. REFERENCES 1. C&SELLS, A. C. & GATENEY, A. A. (1975). The use of lissamine rhodaminel conjugated antibody for the detection of tobacco mosaic virus antigen in tomato mesophyll protoplasts. ,@tschn$ fur Jvafurfwschung MC, 696-697. 2. CASSELU, A. C. & HERRICK, C. C. (1977). The identification of mild and severe strains of tobacco mosaic virus in doubly inoculated tomato plants. Annals of A#Ged Biology 86,37-46. 3. CASSELLS, A. C. & BAUAS, M. (1976). Environmentally induced changes in the cell walls of tomato leaves in relation to cell and protoplast release. Physiologin Pluntar~m 37,239-246. 4. DE CLERCQ, E., ECKSTEIN, F. & MERRIOAN, T. C. (1970). Structural requirements for synthetic polyanions to act as interferon inducers. Annals of the New Tork Au&my of Science173,-l. 5. GIANINAZZI, S. & KASANIS, B. (1974). Virus resistance induced in plants by polyacrylic acid. 3ournalOfGeaeral Virology 23, l-10. 6. KAS~, B. & Wnrra, R. F. (1975). Polyacrylic acid-induced resistance to tobacco mosaic virus in tobacco cv. xanthi. Annals of Applied Biology 79,2 15-220. 7. RASSANU, B., Wnrra, R. F., TURNER, R. H. & WOODS, R. D. (1977). The mechanism of virus entry during infection of tobacco protoplasts with TMV. Phytopathologi.rche.$&schrijl88, 215228.

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8. Lrrz, R. E. & KIMMINS, W. C. (1971). Interpretation of ectodesmata in relation to susceptibiity to plant viruses. Camrdian Joumul of Botany 49,201 l-2014. 9. LOEBENSTEW,G. (1972). Localization and induced resistance in virus-infected plants. Annual R&w of Phytojathologv 10, 177-206. 10. M~rrsnsws, R. E. P. (1970). Plant Virology, pp. 349-378. Academic Press, New York. 11. METZ, D. H. (1975). Discrimination between viral and cellular macro-molecular synthesis. In Control Processesin Virus Multiplication, Ed. by D. C. Burke & W. C. Russell. &i&y for Gmral Microbiology Sym@sium 25,323-355. Cambridge University Press. 12. NAGATA, T. & TAKEBE, I. (1970). Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Plan& 92, 301-308. 13. Ross, A, F. (1974). Interaction of viruses in the host. In Proceedings of the third Intemationul SVmpsium on Virus Direa.scs of Omam&ul Plants, Ed. by R. H. Lawson & M. K. Corbett, pp. 247-260. International Society for Horticultural Science, The Hague. 14. SLA~~K, B. (1974). Methodr of Sfuajing Plant Water Rel&ms. Chapman and Hall, London.