The specific involvement of coat protein in tobacco mosaic virus cross protection

VIROLOGY119, I%158 (1982)

The Specific Involvement

of Coat Protein in Tobacco Cross Protection

Mosaic Virus

JOHN I.,. SHERWOOD’ AND ROBERT W. FULTON Department of Plant Pathology, University of W~consin, Madison, Wiswn.6 55706 Received 0ctobe-r 5, 1981;accepted Januar,y 27, 1982 Nicotiana s&z&-is infected by strains of tobacco mosaic virus (TMV) causing mosaic can be superinfected in the dark green leaf tissue, but not light green tissue, by necrotizing strains of TMV. The dark green tissue, however, is much less susceptible than healthy tissue, to some extent, even to unrelated viruses. The RNA of necrotizing strains of TMV was relatively more infectious than intact virus on mosaic than on healthy leaves and caused lesions in both light and dark green tissues. The same relationship was found in Nicotianu lon&Iora and, when the protecting strain in N. sglvestris could be used as a challenge, in Cap&urn kc&urn. The efficiency of superinfection by RNA was not found with viruses unrelated to TMV. When bentonite at 1 mg/ml, which is known to strip protein from TMV, was included in the inoculum of intact TMV it superinfected in the same manner as RNA. RNA of a necrotizing strain of TMV, encapsidated in brome mosaic virus protein and used as a challenge, superinfected in the same manner as RNA. When encapsidated in common TMV protein, however, it behaved as native virus. Cross protection apparently results from the prevention of uncoating of related challenge virus in light green tissue of N. sylve&is. Locally inoculated N. syZve..strisleaves were insusceptible to challenge RNA or intact virus when the protecting virus was increasing. After increase ceased, RNA was more infectious than intact virus. INTRODUCTION

Cross protection, the protection of a virus-infected plant against infection by other strains of the same virus, has been the subject of theoretical as well as practical interest (Hamilton, 1980; Fulton, 1982). Various theories have been proposed to explain it, but most involve assumptions unsupported by experimental evidence. Kiihler and Hauschild (1947) suggested that the protecting virus monopolized a metabolite essential for a challenge virus. This necessitates assuming a different, specific metabolite for each virus. Gibbs (1969) and Ross (1974) suggested that replicase recognition might be the basis of specificity. Kavanau (1949) suggested that challenge virions were adsorbed by aggregates of protecting virions, thus immobilizing them. DeZoeten and Fulton (1975) hypothesized that free coat 1Present address: Department of Plant Pathology, Oklahoma State University, Stillwater, Okla. 74078.

protein of the protecting virus might coat the challenge RNA and prevent its replication. Zaitlin (1976), however, showed that this was probably not the case. The Nicotiana sylvestris-tobacco mosaic virus (TMV) cross-protection system as first used by Kunkel(1934) is particularly suitable for experimental manipulation and was used in the present work. Isolates of TMV that systemically infect and cause mosaic, or that produce necrotic lesions, are readily available and can be purified in high concentrations. Kunkel (1934) found that mosaic leaves, or locally inoculated leaves, were protected against subsequent infection by necrotizing strains of TMV. They were not protected against cucumber mosaic or tobacco ringspot virus. The specificity of this and similar reactions of other host-virus systems has been used widely as an indication of relatedness of viral isolates. A feature of the N. sylvestris-TMV system, as well as some others, is that su-

150

COAT PROTEIN TABLE

IN VIRUS

1

TOBACCOMOSAICVIRUS STRAINSAND THE REACTIONSOF SELECFEDHOSTS Reaction of hosts” Strain or isolate

NiZWtiiWMZ &bacwmcv. N. @es- N longi- capsicllm. tris Xanthi-nc &n-a bclcmtum

151

CROSS PROTECTION

sisting of RNA provided evidence indicating that cross protection in the N. ~&XXt&s-TMV system results from an inability of challenge virus to uncoat when inoculated on light green areas of mosaic leaves. MATERIALS

AND METHODS

Cultures of most viruses were those used in previous work (Fulton, 1951). Several isolates of TMV were from a collection provided earlier by H. H. McKinney. Turnip mosaic virus (TuMV) was provided by T. P. Pirone and T. C. Hall supplied brome S mosaic virus (BMV). Host reactions of the ’ S, mosaic;L, local necrotic lesions; -, indistinct symp- TMV strains are given in Table 1. toms or not tested. TuMV inoculum was prepared in 0.02 M borate buffer, pH 7.5. BMV was suspended perinfection by related virus strains can in 0.05 Alacetate buffer, pH 5.0, containing be demonstrated and quantitatively as- 0.001 M magnesium acetate. Inocula of sessed (Matthews, 1970, p. 399). Superinother viruses were prepared in 0.03 M fection may occur also when “preventive phosphate buffer, pH 8.0. inoculation” is used for practical disease When measured amounts of virus were control (Fletcher and Rowe, 1975). Fulto be inoculated, purified preparations ton (1951) found that when mosaic leaves with known absorbance at 260 nm were of N. sylvestris were challenged with appropriately diluted. Inoculations were fairly concentrated extracts of necrotizing made with small four-ply gauze pads satstrains, superinfection occurred, but only urated with inoculum and rubbed lightly in the dark green areas of mosaic leaves. on leaves previously dusted with 225pm These areas contained much less virus corundum. Inoculated leaves were immethan the light green areas in which no su- diately covered for 6-12 hr with damp paperinfection occurred. The application to per. Plants were grown in 4-in. pots in a mosaic leaves of concentrated preparagreenhouse maintained at about 25”. tions of mosaic-type strains did not preCross-protection tests were done with vent subsequent infection by necrotizing N. sylvestris Speg & Comes and Nicotiana strains, as it did in healthy leaves. This hmgitbra Cav. that had developed mosaic suggested that superinfection was not the leaves 5-6 weeks after the first 6-cm leaves consequence of the presence of virus-free were inoculated with TMV-Common. When cells in the dark green tissue. More recent infectivities were to be compared, leaves results of Shalla and Peterson (1978), how- were used that had approximately equal ever, demonstrate that some cells in the amounts of light and dark green tissue on dark green tissue are free of virus. Pre- each side of the midrib. Cap&cum baccasumably mosaic-type strains are pre- turn L. was used 5-6 weeks after inoculavented by some mechanism from multition with TMV-PV227. Healthy plants of plying in these cells, although necrotizing similar age were inoculated as controls. strains can multiply, at least enough to TMV isolates that caused necrotic leinduce necrotic lesions. sions on N. sylvestris were purified from In the present work, known amounts of Nicotianu taboxum cv. Havana 307 or Hapurified virus or RNA were used as chal- vana 38. TMV-Common was purified from lenges in order to provide a quantitative systemically infected N. sylvestris. basis for a more complete examination of TMV was purified by grinding frozen factors involved in cross protection. The leaves in an equal amount (w/v) of 0.003 effects on superinfection of inoculum con- M EDTA, pH 7.0, containing 0.02 M 2-merCommon C KC P 49 PV227

L L L L L L

S s L L L L

S L L -

L -

152

SHERWOOD

AND FULTON

captoethanol and A1203 equal to 10% of the tissue weight. The mixture was heated to 55” for 10 min, then centrifuged at low speed (10,000 rpm) in a Spinco No. 30 rotor before pelleting the virus at high speed (30,000 rpm, or 40,000 rpm in a No. 40 rotor) for 2 or 1 hr. This cycle of low- and high-speed centrifugation was repeated. Virus was resuspended in 0.003 M EDTA, pH 7.0. Tobacco necrosis virus (TNV) was purified from N. tabacum cv. Xanthi-nc or Datura strum&urn L. by the method of Lesnaw and Reichmann (1969). TuMV was purified from Brassica rapa L. using the initial extraction buffer of Choi et al. (1977) and the method of Shepherd and Pound (1960). BMV was purified from Hordeum vulgare L. cv. Dickson by the method of Lane (1977). Cucumber mosaic virus (CMV) was purified from N. tabacum cv. Xanthi-nc by the method of Lot et al. (1972). Tomato ringspot virus (TmRSV) was purified from Cwxmis sat&us L. or N. s$vestris by the method of State-Smith (1966). Statistical analysis was carried out according to Ryan et al (1976). TABLE

RNAs of TMV, TNV, and TuMV were prepared by the method of Ralph and Berquist (1967) except that three extractions with chloroform rather than ether were done to remove residual phenol. Only preparations with a 260/280-nm absorbance ratio of 2 or more were retained. Bentonite was prepared by the method of Fraenkel-Conrat et al, (1961). When inoculating with RNA, frozen samples were thawed over ice and dilutions made in chilled KHzPOl (0.073 M, pH 4.2). In comparing infectivities of intact virus and RNA, inocula of RNA were prepared with the same amount of RNA as in the intact virus preparation. These are referred to as equivalent concentrations; intact TMV at 20 pg/ml was considered equivalent to its RNA at 1 pg/ml. Inoculation with RNA was done as for virus except that hands were washed after inoculating each leaf and a new gauze pad was used for each leaf. RESULTS

Relative susceptibility to wcrotizing TMV strains and to unrelated viruses. The num2

RELATIVE INFE~TIVITIES OF TMV RNAs AND EQUIVALENT AMOUNTS OF INTACT VIRUS ON HEALTHY AND MOSAIC LEAVES OF SEVERAL HOSTS

Protecting strain

Common Common Common Common

Challenge strain, virion conen (&ml)

P PV227 49 KC

(41.7) (108.3) (166.7) (63.7)

Average lesions on mosaic leaves

Average lesions on healthy leaves Virus

153 rt 175 + 190 k 137 +

RNA

4 13 15 12

14 21 12 13

f * f *

3 7 3 3

Lesion ratio for virus (mosaic/healthy)

Lesion ratio (RNA/virus)

Virus

RNA

Lesion ratio (RNA/virus)

0.09 0.12 0.06 0.07

14 zt 2 2Of4 17 i 4 12 -t 2

4+1 lo+1 4+1 5+1

0.28 0.50 0.24 0.42

0.03 0.11 0.09 0.06

13 f 2

6fl

0.46

0.27

11 % 1 7*1

4+1 5*1

0.36 0.71

0.11 0.11

Capsicumbacmtum PV227

Common (60)

49-c 3

9.5 + 3

0.19

Ntimtimalagiffora Common Common

P 49

(54.3) (162.9)

96+ 6 69 * 12

7 +1 6 f2

0.07 0.09

Note. Lesion counts are averages of 12 half-leaves. Intact TMV at 26 ag/ml was considered equivalent to RNA at 1 pg/ ml.

153

COAT PROTEIN IN VIRUS CROSS PROTECTION TABLE 3

RELATIVE INFECWITIES OF TOBACCO NECROSIS VIRUS (TNV) (6.0 pg/ml), TURNIP MOSAIC VIRUS (TuMV) (714 kg/ml), TOMATO RINGSPOT VIRUS (100 pg/ml), AND EQWALENT AMOS OF RNAs OF TNV” AND TuMV* ON HEALTHY LEAVES AND MOSAIC LEAVES OF Nimtiana sylvestris INFECTED WITH TMV-COMMON

Average lesions on healthy leaves by

Average lesions on mosaic leaves by

Virus

Virus

RNA

Lesion ratio (RNA/virus)

TNV TuMV TmRSV

662 6 116 2 15 35+4

621 4+1 -

0.09 0.03 -

Virus

RNA

Lesion ratio (RNA/virus)

2523 78 ?I 7 21k2

2 f 0.5 2 f 0.2 -

0.08 0.03 -

Lesion ratio for virus (mosaic/healthy) 0.38 0.67 0.63

Note. Lesion counts are averages of six or more half-leaves. ’ Intact TNV at 6.0 p&ml was considered equivalent to its RNA at 1.1 pg/ml. *Intact TuMV at ‘714pg/ml was considered equivalent to its RNA at 38.6 pg/ml.

bers of lesions produced on mosaic and healthy leaves by known amounts of purified necrotizing TMV of several strains, their RNAs, and several unrelated viruses were compared. As previously observed (Fulton, 1951), some necrotizing TMV strains (P and KC) were more efficient than others in superinfecting mosaic leaves (Table 2). Unrelated viruses also produced more lesions on healthy than on mosaic leaves, although the differences were much less than with TMV strains (Table 3). Lesions were produced by unrelated viruses in light green tissue, but these were fewer than lesions produced in dark green tissue. TMV inoculum produced lesions only in dark green tissue. There is apparently a minor, nonspecific reduction in susceptibility of TMV-infected N. sylvestris to unrelated viruses. Superi’rlfection of mosaic leaves with TMV RNA. Infection of leaves by RNA of necrotizing TMV was shown by Siegel et aL (1957) not to involve a lag period as intact virus did. This was interpreted as representing the time involved in removal of the protein coat of the virus before replication could proceed. The superinfecting capacity of TMV RNA was tested to see if the absence of a lag period, or the absence of coat protein, might affect the amount of superinfection. When healthy and mosaic leaves of N. sylvestris were inoculated with RNA prepared from necrotizing strains of TMV and opposite half-leaves inoculated with

an equivalent amount of intact TMV, each type of inoculum caused necrotic lesions. RNA inoculum, however, caused lesions in both light and dark green areas of mosaic leaves, whereas intact virus produced lesions only in dark green areas (Fig. 1). RNA was 6-12% as infectious as intact virus on healthy plants, but 2840% as infectious as intact virus on mosaic leaves (Table 2). This may have been due partly to the greater leaf area susceptible to RNA than to intact virus, but lesion numbers resulting from RNA inoculum also seemed to be greater in dark green areas. The same ratio of infectivities was also found when RNA and virus were inoculated to mosaic leaves at higher concentrations (RNA at 29.6 pg/ml), which would have saturated the infection sites on healthy plants. The relative infectivity of TMV RNA on healthy and mosaic leaves is similar to that of intact viruses unrelated to TMV (cf. Tables 2 and 3). Further purification of the RNA on sucrose density gradients or by hydroxyapatite chromatography did not alter its ability to cause lesions in light green tissue. Evidently necrotizing virus that cannot infect light green areas of mosaic leaves is able to do so after its coat protein is removed. Inoculation of other plant species, healthy and mosaic virus irlfected, with necrotizing TMV and its RNA. Previous results were obtained by challenging N. sylvestris infected with TMV-Common with necrotizing strains. To determine whether the re-

154

SHERWOOD AND FULTON

sults reflected peculiar properties of the strains or the host used, a system was sought in which the roles of the strains could be reversed, and TMV-Common used as the challenge inoculum. This was found with C. baccatum, which became systemically infected by PV227, but reacted with local necrotic lesions when inoculated with TMV-Common. When the infectivity of RNA of.TMVCommon at 3 pg/ml was compared with that of intact TMV-Common on healthy and systemically infected C. baccatum, the ratio of lesions produced (RNA/virus) was much higher in infected C! baccatum than in healthy C. buccatum (Table 2). Light and dark green areas of systemically infected C baccatum leaves were not sharply delineated. To test the response of another host, N. long$ora systemically infected with TMVCommon was challenged with RNA and intact virus of several necrotizing strains. This host reacted in the same way as N. sylvestris (Table 2), and lesions were produced in the light green areas by RNA. Thus the capability of superinfecting light green tissue of mosaic leaves was a typical property of the RNA of TMV in each system tested. Effect of inoculating with RNA of unrelated viruses. To determine whether the efficiency of superinfection by TMV RNA was specific, inoculations with other RNAs were made on healthy and mosaic leaves of N. sylvestris. Neither TNV RNA or TuMV RNA was any more efficient in infecting mosaic leaves than healthy leaves (Table 3). To determine whether the efficiency of TMV RNA in infecting mosaic leaves was expressed on plants infected with an unrelated virus, TMV-Common RNA at 3.1 pg/ml and an equivalent amount of intact virus were inoculated to opposite halves of N. tabacum cv. Xanthi-nc, healthy or systemically infected with CMV. Intact virus produced an average of 94 lesions on healthy leaves and 29 lesions on mosaic leaves. RNA produced five lesions per leaf on healthy leaves and two on mosaic leaves. Thus TMV RNA was 30-40% as infectious as intact virus on both healthy

FIG. 1. Lesions produced on mosaic Niootiana SW& V&T% inoculated with RNA (right half-leaf) and intact virions (left half-leaf) of a necrotizing strain of TMV. This leaf was not typical, but was selected for testing because most of each half-leaf was light green tissue.

and CMV-infected leaves. When the same test was done on half-leaves of Xanthi-nc 3 days after heavy inoculation with CMV, infection by both intact virus and RNA was 14% of that on healthy leaves. Inoculatti of mosaic leaves with intact viru.s and bentonite. Brakke and Van Pelt (1969) and Brakke (1971) showed that under certain conditions TMV degrades in the presence of small amounts of bentonite. This behavior provided an opportunity to confirm the infectious capabilities of TMV RNA which had not been subjected to deproteinization with phenol.

COAT PROTEIN

IN VIRUS

155

CROSS PROTECTION

TABLE

4

NUMBERSOFNECROTICLESIONSPRODUCEDBY NECROTIZINGSTRAINSOFTMV AND OTHERVIRUSESMIXED WITH BENTONITE(1 mg/ml) AND INOCULATEDTO HEALTHY AND MOSAICLEAVES OF Nicotiana sylvestrk Healthy leaves Virus inoculated

Mosaic leaves

With bentonite

Without bentonite

With hentonite

Without hentonite

TMV-P

104*14

108 + 2

29+3

10 f 2

TMV PV227 TNV

101 + 92+

102 + 4 91 + 3

41 r 5 31 -t 2

35 f 4

21 * 2

TmRSV

35+

7

2 3

12 f 1

32 f 2 22 + 2

Note. Lesion counts are averages of six half-leaves

tissue suggested that the inability of whole virus to infect was due to an inhibition of uncoating. If this is the case, the basis for specificity must lie in some sort of recognition of specific coat protein, since unrelated viruses do infect light green mosaic tissue. An attempt was made to test this by reconstituting RNA of a necrotizing strain (P) of TMV in protein subunits of other viruses or strains. Assembly of TMV RNA and BMV protein subunits and inoculation of the reconstituted virus were done by the method of Verduin and Bancroft (1969). Equivalent amounts of RNA and reconstituted virus were based on an c of 5.5 and 20% RNA for reconstituted virus and an E of 25 for RNA. Assembly of TMV-P RNA in TMV-Common coat protein was done according to Breck and Gordon (1970). Reconstituted virus was inoculated in the same way as native virus. The reconstituted virus consisting of TMV-P in BMV protein produced as many

Inoculum of intact virus of necrotizing strains of TMV (3-11 rg/ml) was prepared in O.O3M, pH 8.0, phosphate buffer containing 1 mg/ml bentonite. The inclusion of bentonite did not affect lesion numbers in healthy N. sylvestris but increased the numbers of lesions in mosaic leaves of N. sylvestris 2.9- to 3.4-fold (Table 4). Lesions appeared in both light and dark green areas of mosaic leaves inoculated with bentonite. Bentonite did not increase the infectivity of TNV or TmRSV in mosaic leaves of N. syhestris (Table 4). The effect of bentonite was greatest at 1 mg/ml, the amount used by Brakke and Van Pelt (1969). There was less effect at 0.1 or 10 mg/ml and no effect at 0.001 mg/ ml. The effect of bentonite was not reproduced by aluminum oxide, Celite, magnesium oxide, or magnesium trisilicate. Infectivity of virus reccmstituted from heterolog~ cumpcments. The infectivity of RNA inoculated to light green mosaic TABLE

5

NUMBERSOFLESIONSPRODUCEDBY RNA OFTMV-P (NECROTIZING),ALONE ORENCAPSIDATEDIN DIFFERENT HETEROLOGOUS COATPROTEINSAND INOCULATEDTO HEALTHY ORMOSAICNimtiana q&x&-is LEAVES Average lesions/ half-leaves on Coat protein of capsid

Concn in inoculum (itahl)

Brome mosaic virus None (control) TMV-Common None (control)

36 1.8 14 0.7

Healthy leaves 41 34 184 37

Mosaic leaves 15 15 30 17

Lesion ratio (mosaic/healthy) 0.37 0.44 0.16 0.46

156

SHERWOOD

lesions on mosaic leaves as did nucleic acid alone (Table 5). The infectivity of TMV-P reconstituted in TMC-Common protein, however, was less infectious on mosaic plants and resembled intact virus in this respect. Necrotizing TMV RNA encapsidated in BMV protein caused lesions in both light and dark green tissues of mosaic leaves. The same RNA encapsidated in TMV-Common protein produced lesions only in dark green tissue. It thus appeared that virus reconstituted in TMV protein was inhibited from uncoating in light green tissue, but that virus reconstituted with an unrelated virus protein was not and proceeded to multiply. Superiqfection of local& protected halfleaves. Previous results were obtained by inoculating leaves that had developed mosaic symptoms and healthy controls. We wished to test the effect of inoculating RNA to N. sylvestris leaves heavily inoculated a few days previously with a mosaic-inducing strain of TMV. Kunkel(1934) showed that such tissue was almost completely protected against the effects of inoculation with necrotizing strains. In this way such tissue resembles the light green tissue of mosaic leaves. Leaves of healthy N. sylvestris plants that had been heavily inoculated on a succession of previous days with TMVTABLE

6

SUPERINFECPIONBY EQUIVALENT AMOIJNTS OF RNA AND VIRUS OF TMV-P IN Niwtimu @~estris LEAVES INFECXED FOR VARIOUS LENGTHS OF TIME BY TMVCOMMON (1 mg/ml) Lesions/half-leaf following inoculation with Days after first inoculation 0

1 5 10 15 20 25

Virus concn in tissue (n&g fresh wt) -0

0.09 1.69 3.52 4.10 4.24 4.15

Virus (200 pg/mI)

RNA (10 pg/ml)

128 35 1 0 1.5 4 5

96 65 0.5 0.5 4 14 16

No@. Virus concentration in previously inoculated leaves was determined in tissue samples removed just prior to challenge inoculation. a Used as a blank.

AND FULTON TABLE

7

CONCENTRATION OF TMV IN HEALTHY AND MOSAIC sylve&-ia FOLLOWING INOCULATION WITH TMV-COMMON RNA (1 mg/ml)

Niwtima

Concentration of virus (mg/g fresh wt) in Days after RNA inoculation

Healthy

Mosaic

0

-0

3 7 10 15

0.43 2.12 2.51 3.76

1.30 1.01 1.25 1.06 1.40

Note. Values for mosaic plants are averages from three plants/treatment. a Used as a blank.

Common were challenged on opposite halfleaves with equivalent amounts of virus or RNA of a necrotizing strain. Just prior to challenge inoculation, twelve 1%mm disks from the leaves of three plants for each treatment were removed to determine the concentration of the protecting strain by Schneider’s (1953) method. Locally inoculated half-leaves were not superinfected by either RNA or intact virus during the period that the protecting strain was increasing in concentration (Table 6). When the concentration of the mosaic strain remained constant, suggesting that multiplication had ceased, superinfection occurred, more with RNA than with an equivalent amount of intact virus. In an experiment designed to detect the presence of virus-free cells in the dark green areas of mosaic leaves, Fulton (1951) applied to the mosaic leaves an inoculum of the same (or other) strain causing mosaic. The concentration applied was about 250 times higher than that of a necrotizing strain which would have produced significant numbers of lesions. It was postulated that if there were virus-free cells in the dark green tissue, application of a mosaicinducing strain would infect them and protect them from the necrotic effects of subsequent application of necrotizing strains. This did not occur. This experiment was repeated by applying RNA of TMV-Common (1 mg/ml) to

COAT PROTEIN

IN VIRUS

mosaic or healthy leaves prior to challenge by a necrotizing strain at 92.5 pg/ml. This induced protection in healthy plants but not in mosaic plants. When the concentration of virus was measured in leaves following application of TMV-Common RNA, no increase could be detected in mosaic leaves (Table 7). In previously healthy leaves, however, virus concentration increased in 15 days to a level more than 2.5 times that in mosaic leaves. These results support other evidence indicating that complete cross protection occurs only when the protecting virus is actively multiplying. DISCUSSION

The induction of lesions in light green tissue of mosaic leaves by the RNA of necrotizing strains of TMV leads to several deductions. First, it is possible for necrotizing strains of TMV to replicate in light green tissue in the presence of large amounts of related virus. Second, the failure of intact virus to infect light green tissue must be due to the presence of coat protein around the RNA. Third, the protection of light green tissue against superinfection by intact virus must be due to prevention of initiation of infection rather than to inhibition of virus replication in the tissue. The initial interaction of a virus particle with a cell leading to uncoating is largely unknown. Dissociation of protein subunits of virus may be due to interaction with lipid-containing structures associated with the cell wall or cell membrane (Caspar, 1963). Niblet (1975) and Kurtz-Fritsch and Hirth (1972) concluded that membrane attachment reduced particle stability and that there was no need to postulate the involvement of an uncoating enzyme. Thus it may be that the site of protection is a lipid-containing structure at the boundary of the cell. Susceptibility of a plant to many different viruses probably involves the same mechanism for uncoating each one; yet uncoating of a related virus seems to be blocked in light green tissue. The basis of this specificity may involve the coat protein. Specificity might be regulated by the

CROSS PROTECTION

157

kind and amount of viral coat protein already present in the membrane. Cells of the light green tissue contain much more virus than those of dark green tissue; the cell membranes may contain correspondingly greater amounts of free coat protein. The effect of bentonite in promoting superinfection of light green tissue of mosaic leaves is consistent with its effect on uncoating of TMV. Brakke (1971) and Brakke and Van Pelt (1969) showed that bentonite did not adsorb TMV coat protein, and that uncoating of the virus was often not complete. They suggested that bentonite might result in stripping of protein subunits from the ends of otherwise intact TMV rods. Thus infection of light green tissue might be initiated by a small region of exposed RNA at one end of a virus rod. The demonstration that locally inoculated leaves are nearly completely immune from superinfection by either intact virus or RNA until their virus content reaches a constant level suggests that factors in addition to viral uncoating are involved in cross protection. Infection of locally inoculated tissue by unrelated viruses, however, is greatly reduced, so at least part of the insusceptibility may be nonspecific. Furthermore, it may be that more protein subunits are associated with cell membranes during periods of rapid virus replication than later, when virus may have ceased to replicate. Murakishi and Carlson (1976) and Shalla and Peterson (1978) have presented evidence of the presence of virus-free cells in dark green tissue of mosaic leaves infected by TMV. Because these cells remain virusfree although surrounded by virus-containing cells, Atkinson and Matthews (1970) postulated that a diffusible “dark green agent” kept virus from replicating in them. Our research found no evidence of replication of TMV-Common in dark green tissue when virus was inoculated to leaf surfaces. While a “dark green agent” may be responsible for maintaining some cells free of virus, it is insufficient to prevent superinfection by necrotizing virus. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wis-

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