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Increase and spread of cowpea chlorotic mottle virus in resistant and fully susceptible cowpeas
I’hy.ciological
Plant Pathology
(1984)
24, 339-345
Increase and spread of cowpea chlorotic mottle virus in resistant and fully susceptible cowpeas S. D.
WYATT
I@artment
of Plant
and T. C. Pathology,
( ArceptedJolbr publication
February
WILKINSON
Washington
State University,
Pullman,
WA 99164-6430,
USA
1984)
The resistance of cowpea plant introduction (PI) 186465 to infection by cowpea chlorotic mottle virus, strain T, involves reduced virus replication and restricted virus movement. At the cellular level, it is difficult to distinguish between uniformly reduced virus replication and a high virus-replicating capacity in only a few cells. Therefore, protoplasts were isolated at intervals from inoculated leaves and stained with virus-specific fluorescent antibody. Protoplasts from PI 186465 leaves inoculated with strain T showed a steady increase in the fraction of stained cells to a value of approximately 25% at 25 days after inoculation. In contrast, the more virulent strain R caused the fraction stained to rapidly increase to approximately 40% at day 7 and remain close to that value through day 25. In both systems virus accumulation and fraction of stained protoplasts increased roughly in parallel, and the amounts of virions per stained protoplast approached similar maxima. Treatment of plants with 2-thiouracil increased virus yield per protoplast but not the fraction of protoplasts stained by fluorescent antibody. When isolated PI 186465 protoplasts were inoculated with strains T and R the viruses increased with equal efficiency. We conclude that leaf cells of this cowpea strain reduced both the accumulation and spread of strain T but lose the ability to reduce accumulation when isolated as protoplasts.
INTRODUCTION
Although cowpea cultivars are generally susceptible to CCMV, the reaction of cowpea PI 186465 is strain-dependent. Virus strain T replicates minimally in PI 186465, is restricted to the inoculated leaf, and produces no symptoms, local or systemic [IO]. Strains have been selected that can overcome PI 186465 resistance and cause a systemic infection [II]. One strain, R, has been very useful in genetic and biochemical studies. Both replication and spread of virus in PI 186465 cowpeas have been linked to the information carried by RNA 1 of the three-part viral genome [II]. Our results and those of others [S] suggest that viruses must specify some factor necessary for systemic spread. However, the biochemical function of the RNA 1 gene product is not known. Because both replication and spread are involved, it has been difficult to different iate between several possible resistance mechanisms : minimal replication per cell, Scientilic Paper No. 6395, College Pullman, WA, IJSA. Abbreviations used in text: CCMV, fluorescein isothiocyanate. (048-4059/84/030339
+ 07 SOS.OO/O
of Agriculture cowpea
chlorotic
Research mottle 0
Center, virus;
Washington
FA, fluorescent
1984 Academic
Press
Inc.
State
University,
antibodies; (London)
FITC, Limited
340
S. D. Wyatt and T. C. Wilkinson
localization of replication at initial infection sites or slow spread of virus within the inoculated leaf. In this study our approach has been to determine the extent of virus spread in cells of inoculated leaves by staining isolated protoplasts with fluorescent antibodies specific for the virus. The amount of spread of strain T (resistant reaction) and strain R (susceptible reaction) within the inoculated leaf were compared. Results were partially presented previously [ 121. MATERIALS AND METHODS Virus and plant manipulation
Strain T of CCMV was maintained in cowpea cultivar California Blackeye and strain R was maintained in cowpea introducti.on 186465. Cowpeas were grown in plastic pots (8 cm) in commercial soil mixture amended weekly with a complete fertilizer. The plants were maintained at 27 “C in the greenhouse with fluorescent light supplement when the photoperiod was less than 12 h. The susceptibility of PI 186465 cowpeas to initial infection by CCMV was increased by a 24-h dark treatment before inoculation. Primary leaves were inoculated before the formation of trifoliate leaves, usually 9-10 days after seeding, by rubbing leaves with virus in 0.05 M phosphate buffer, pH 7.0, containing O*5o/oCelite. In some experiments it was necessary to prevent the age related abscission of primary leaves by removing the trifoliate leaves as they expanded. Protoplast
isolation
Protoplasts were isolated from cowpeas enzymatically using a Carborundum-brush technique [I]. In preliminary protoplast experiments the protoplasts from PI 186465 cowpea primary leaves generally did not survive the isolation technique, while those from California Blackeye survived well. A 24-h dark treatment of PI 186465 plants before protoplast isolation as suggested by G. Bruening (pers. comm.) increased viability as indicated by a completely spherical shape. Figure 1 shows the number of viable protoplasts isolated per gram of strain T- and R-inoculated tissues. The protoplast yield declined somewhat with increasing age of the tissue during the experiments which lasted 25 days, but the decline was linear. It is assumed that protoplasts isolated are a representative sample of the population. In some experiments protoplasts were inoculated with CCMV (1 pg ml-l) pretreated with polyomithine (2 pg ml-l). The inoculum was incubated at 25 “C for 10 min, then added to freshly pelleted protoplasts at a final concentration of 5 x lo5 protoplasts ml-‘. During the inoculation procedure the protoplasts and polyomithinetreated virus were in 0.02 M potassium citrate, 0.45 M mannitol, pH 5.2. The protoplasts were incubated with inoculum for 10 min and washed by centrifugation at 1OOg. The protoplasts when appropriate were incubated 24-48 h at 25 “C in 0.45 M mannitol containing White’s salts solution [9], 2-4-D (0.25 pg ml-l, chlorotetracycline (4 mg 100 ml-‘), and griseofulvin (2 mg 100 ml-l). The pH of the medium was 5.8 and there were 5 x 105 protoplasts ml-‘. Immunojuorescence
Antiserum to CCMV strain. T was prepared in rabbits as previously described [Ill. The IgG was purified by precipitation with (NH,),SO, and reacted with fluorescein
Resistance
of cowpeas
to cowpea
a
chlorotic
22
4
I 6 Time
1. Number of inoculation with CCMV FIG.
mottle
virus
341
I I I I I I I I 8 IO 12 14 16 18 2022 after
inoculation
protoplasts isolated from strains T (a) and R (A).
PI
I 24:
(days)
186465
cowpea
primary
leaves
after
isothiocyanate (FITC) [6]. Th e conjugate was separated from unreacted FITC by ,:hromatography on a Bio-Gel P-10 column (1 x 15 cm) using saline phosphate Duffer, pH 7.4, for elution. The FITC-serum was cross-absorbed with acetone powder of cowpea tissue before use [6]. The protoplasts to be stained with fluorescent ,antibodies (FA) were affixed to Mayer’s albumin-coated glass slides and dried immediately with a hair drier. They were then fixed sequentially in acetone (30 min) for 2 h at 30 “C and and 95% ethanol (30 min), stained with FITC-antibodies ‘destained overnight in saline phosphate, pH 7.4. Analytical
methods
Virus was extracted from leaves or pelleted protoplasts (10 OOOg, 10 min) by homogenization in 0.2 M acetate buffer (pH 5.0) containing cysteine-HCl (0.01 M), sodium diethyldithiocarbamate (0.01 M) and MgCl, (0.01 M). Following clarification of the extract by chloroform-butanol emulsification and freezing, the virus was separated From remaining host material by the centrifugation of the extracts on sucrose density gradients ( lO-lOo/o w/v) in 0.2 M sodium acetate, pH 5.0, for 4 h at 27 000 rev min-’ in a Spinco SW 27 rotor. The virus content was calculated based on an extinction coefficient (E&f0 = 5.8). RESULTS Spread of virus
The extent of spread was determined in primary leaves of PI 186465 cowpeas inoculated with strain R (susceptible reaction) and strain T (resistant reaction) at intervals of 1,4, 7, 11, 18 and 25 days after inoculation. This determination was made by staining protoplasts isolated from the leaves with fluorescent antibodies specific for CCMV. The amount of virus in strain T-inoculated tissue (resistant reaction) and the proportion of FA positive protoplasts changed with time quite differently compared with the susceptible reaction (strain R) . Protopkkts from PI 186465 leaves inoculated with strain T showed a slow but steady increase in the fraction of stained cells to a value of approximately 25% at 25 days after inoculation (Table 1). In
342
S. D. Wyatt and T. C. Wilkinson TABLE D~#&nces
in intercellular spread in piimary leaves of a cowpea cultivar between CCMV have a resistant or susceptible relationship with the cultiuars
Virus
Infection (days)
Susceptible reaction
1
ND” 31 66 130 166 123
4 7 11 18 25
1
concentration in tissue (pg g-l tissue) (R)
Resistant (T) reaction ND 1 1 12 21 34
Infected
protoplasts cowpeas
Susceptible reaction
(R)
2(9/481)b 17(132/776) 41(268/656) 40(204/508) 48(218/458) 38(212/556)
=No virus was detected by sucrose density gradient analysis. bAverage number protoplasts infected, number counted and total number
strains
that
from PI 186465 (T/o) Resistant (T) reaction 0(0/728) 2(15/741) 4(29/681) 10(42/424) 13(54/410) 24( 138/573)
counted
contrast, the more virulent strain R caused the fraction stained to rapidly increase to approximately 40% at day 7 and remain close to that value through day 25. In both systems virus accumulation and fraction of stained protoplasts increased roughly in parallel, and the amounts of virion per stained protoplast approached similar maxima. Based on infectivity, purification experiments [IO] and ELISA (unpublished results), strain T does not move systemically in PI 186465 cowpeas. No infected protoplasts were detected by FA when trifoliolate tissue from strain T-inoculated plants was used. In comparison, 39% of the protoplasts from the trifoliolates from R-inoculated plants were FA positive. The relationship of spread to virus accumulation The relationship of spread to virus accumulation was investigated by testing protoplasts from 2-thiouracil-treated cowpeas. This base analog enhances CCMV replication several-fold without extending the length of the replication period [3]. PI 186465 cowpeas were inoculated with strain T and 4 days after inoculation were treated with 2-thiouracil by adding 50 ml (0*005 M) to each pot on three successive days. The results of two experiments are given in Table 2. Generally, there was up to threefold more virus in 2-thiouracil-treated leaves as determined by the amount of virus isolated by sucrose density gradient centrifugation of leaf extracts. However, the number of FA positive protoplasts was unchanged. 2-Thiouracil appears to stimulate CCMV replication but has no effect on spread of the virus in infected leaves.
of FA stain Virus must accumulate before detection by FA is possible, therefore marginally or newly infected protoplasts might appear FA negative. It was hoped that virus replication during an in vitro 48 h pre-stain incubation of protoplasts from strain T and R inoculated PI 186465 leaves would increase the number of FA positives.
Sensitivity
Resistance
of cowpeas
to cowpea
chlorotic
mottle
TABLE Proportion
of epidermal
Infection
and mesophyll
length
1
Expt
2
11 11 11 11
2
infected with 2-thiourocil
2-Thiouracil treatment (days after inoculation)
(days) Expt
cells
CCMV
treatment
with
Infected protoplasts from PI 186465 cowpea
( %I) 13 13 7 8
treatment
The average numbers of virus particles
3
per protoplast
isolatedfrom
CCMV (w Infection
length
(days)
4
5 x 106 protoplasts) Susceptible 1.6 8.2 18.2 20.9 10.5
4 7 11 18 25
Resistant -a
’
7 11 18 25
virus
T after
151 59 82 39
TABLE
‘No
strain
CCMV @g/g tissue)
4 -* 4
“iKo 2-thiouracil
343
virus
was detected
Virus particles/ protoplast (strain
R) 4.18 2.14 4.76 5.47 2.74
(strain
density
10” lo5 lo5 105 lo5
leaves
Particles/FA positive protoplast
2.45 5.2 1.18 1.15 0.72
x x x x x
lo5 lo5 lo6 108 lo6
ri -~
3.9 2.7 11.1
by sucrose
x x x x x
inoculated
1.0 x 105 0.7 x Ioj 2.9 x 105
gradient
1.01 x 105 0.5 x 106 1.2 x 106
analysis.
However, incubation of protoplasts before staining did not affect the fraction of FA positive protoplasts. To determine how much virus was in individual protoplasts isolated from infected PI 186465 tissue, virus was recovered by sucrose density gradient centrifugation from homogenized protoplasts and the average number of particles per protoplast was calculated (Table 3). In strain R-inoculated protoplasts at 4 days the average number of virus particles per protoplast and per FA-positive protoplast were 41 000 and 245 000, respectively and both reached a plateau by 11 days post-inoculation (approximately 500 000 and 1 200 000). In contrast, no strain T nucleoprotein was found by sucrose density gradient centrifugation until 11 days after inoculation. By 25 days the average number of strain T particles per protoplast and per FA-positive
344
S. D. Wyatt and T. C. Wilkinson
protoplast was 290 000 and 1 200 000, respectively. These data suggest that the rate of cell-to-cell virus spread and the replication rate per cell in the resistant reaction is less than that observed in susceptible reactions. Susceptibility of PI 186465protoplasts to infection In vitro inoculation of protoplasts from PI 186465 cowpeas with strains T and R was
tested as a way to study the resistance mechanism. Protoplasts were isolated from healthy PI 186465 cowpeas and inoculated with strains T and R. The inoculated protoplasts were incubated at 25 “C and assayed at 24 and 48 h with virus specific fluorescent antibody. There was very little difference in the proportion of strain R and strain T inoculated protoplasts which stained. The percent of protoplasts infected was 47 and 54 for R and T, respectively (48 h), suggesting there is little if any difference in susceptibility of PI 186465 protoplasts to initial infection. It has previously been shown that, from a mixture, strain R is selected over strain T in PI 186465 [II]. Therefore, the virus produced in inoculated protoplasts was characterized to determine if strain T was being replicated. Protoplasts (5 x lo6 protoplasts) were inoculated with either strain T or strain R and incubated for 48 h. Extracts were infectious on the local lesion host Bragg soybean. The lesions transferred to California Blackeye and PI 186465 (16 per strain) caused typical reactions [lZ]. Virus from strain T inoculated protoplasts caused bright, chlorotic mottle on California Blackeye and no symptoms on PI 186465. Virus from strain R inoculated protoplasts (16 lesions) caused a mild mottle on California Blackeye and a chlorotic mottle on PI 186465 cowpeas. These reactions are typical of the two strains. DISCUSSION
We previously demonstrated that resistance to CCMV strain T in PI 186465 cowpeas involves reduced virus-replicating capacity of the host which was associated with a quantitative alteration in the proportion of the four encapsidated RNAs [IO] and restricted virus movement. Paradoxically, strain T accumulation continues until senescence of the leaves, whereas strain R (susceptible reaction) replication in the inoculated leaves is exponential and ceases by 7-10 days post-inoculation at 27 “C [II]. Apparently, in the resistant tissues virus is not restricted to a few cells but spread and/or replication is slow. A lack of spread could result from either a lack of movement of virus from cell to cell or a failure to initiate replication once virus transfer has occurred. Though speculative, the latter would explain why both replication and spread of strain T are genetically linked to RNA 1 in PI 186465 cowpeas [II]. The mechanisms that regulate the amount of virus replication and spread are not known but inheritance studies indicate that they are controlled by several host genes [4] and the virus [II]. Genetic experiments with CCMV [II] and temperature-sensitive mutants of TMV [5] have shown that spread is dependent on some mechanism provided by the virus genome. In the case of CCMV, the addition of RNA 1 from strain R to the strain T genome is required for systemic infection of PI 186465 [If]. The provision by the virus. of some mechanism for spread is also suggested by experiments with 2-thiouracil. The treatment of strain T inoculated PI 186465 cowpeas with 2-thiouracil enhanced replication but did not produce greater cell to cell or systemic spread of replicational activity.
Resistance
of cowpeas
to cowpea
chlorotic
mottle
virus
345
REFERENCES 1. BEER, H. & BRUENING, G. (1975). The use of an abrasive in the isolation of cowpea leaf protoplasts which support the multiplication of cowpea mosaic virus. Virology 64, 272-276. 2. DAWSON, W. 0. & KUHN, C. W. (1972). Enhancement of cowpea chlorotic mottle virus biosynthesis and in UZUDinfectivity by 2 thiouracil. Virology 47, 21-29. 3. KUHN, C. W. (1977). Differential effect of P-thiouraril on synthesis of two plant viruses in the same host. Interuirology 8, 37-13. 4. KUHN, C. W., WYATT, S. D. & BRANTLEY, B. B. (1981). Genetic control ofsymptoms, movement, and virus accumulation in cowpea plants infected with cowpea chlorotic mottle virus. Phytopathology 71,1310~1315. 5. NICHIGUCHI, M., M~TOYOSHI, F. & OSHIMA, N. (1978). Behavior of a temperature sensitive strain of TMV in tomato leaves and protoplasts. Journal of General Virology 39,53-61, 6. OTSUKI, Y. & TAKEBE, I. (1969). Fluorescent antibody staining of tobacco mosaic virus antigen in tobacco mesophyll protoplasts. Virology, 38,497--199. 7. OTSOKI, Y., SHIMOMURA, T. & TAKEBE, I. (1972). Tobacco mosaic virus multiplication and expression of the pi gene in necrotic responding tobacco varieties. Virology 50,45-50. 8. S~LZINSKI, M. A. & ZAITLIN, M. (1982). Tobacco mosaic virus replication in resistant and susceptible plants: in some resistant species virus is confined to a small number of initially infected cells. Virology 121, 12-l 9. 9. WHITE, P. R. (1954). The Cultimtion ofAnimal and Plant Cells. Ronald Press, New York. IO. \YYAI.T, S. D. & KUHN, C. W. (1979). Replication and properties of cowpea chlorotic mottle virus in resistant cowpeas. Phytopathology 69, 125-129. Il. WYAI.?., S. D. & KUHN, C. W. (1980). Derivation of a new strain of cowpea chlorotic mottle virus from resistant cowpeas. Journal ofGenera Virology 49, 289-296. 12. MIYATT, S. D. & WILKINSON, T. C. (1981). Movement of virus within cowpeas resistant to cowpea chlorotic mottle virus. (Abstr.) Phyfopathology 71, 266.
Plant Pathology
(1984)
24, 339-345
Increase and spread of cowpea chlorotic mottle virus in resistant and fully susceptible cowpeas S. D.
WYATT
I@artment
of Plant
and T. C. Pathology,
( ArceptedJolbr publication
February
WILKINSON
Washington
State University,
Pullman,
WA 99164-6430,
USA
1984)
The resistance of cowpea plant introduction (PI) 186465 to infection by cowpea chlorotic mottle virus, strain T, involves reduced virus replication and restricted virus movement. At the cellular level, it is difficult to distinguish between uniformly reduced virus replication and a high virus-replicating capacity in only a few cells. Therefore, protoplasts were isolated at intervals from inoculated leaves and stained with virus-specific fluorescent antibody. Protoplasts from PI 186465 leaves inoculated with strain T showed a steady increase in the fraction of stained cells to a value of approximately 25% at 25 days after inoculation. In contrast, the more virulent strain R caused the fraction stained to rapidly increase to approximately 40% at day 7 and remain close to that value through day 25. In both systems virus accumulation and fraction of stained protoplasts increased roughly in parallel, and the amounts of virions per stained protoplast approached similar maxima. Treatment of plants with 2-thiouracil increased virus yield per protoplast but not the fraction of protoplasts stained by fluorescent antibody. When isolated PI 186465 protoplasts were inoculated with strains T and R the viruses increased with equal efficiency. We conclude that leaf cells of this cowpea strain reduced both the accumulation and spread of strain T but lose the ability to reduce accumulation when isolated as protoplasts.
INTRODUCTION
Although cowpea cultivars are generally susceptible to CCMV, the reaction of cowpea PI 186465 is strain-dependent. Virus strain T replicates minimally in PI 186465, is restricted to the inoculated leaf, and produces no symptoms, local or systemic [IO]. Strains have been selected that can overcome PI 186465 resistance and cause a systemic infection [II]. One strain, R, has been very useful in genetic and biochemical studies. Both replication and spread of virus in PI 186465 cowpeas have been linked to the information carried by RNA 1 of the three-part viral genome [II]. Our results and those of others [S] suggest that viruses must specify some factor necessary for systemic spread. However, the biochemical function of the RNA 1 gene product is not known. Because both replication and spread are involved, it has been difficult to different iate between several possible resistance mechanisms : minimal replication per cell, Scientilic Paper No. 6395, College Pullman, WA, IJSA. Abbreviations used in text: CCMV, fluorescein isothiocyanate. (048-4059/84/030339
+ 07 SOS.OO/O
of Agriculture cowpea
chlorotic
Research mottle 0
Center, virus;
Washington
FA, fluorescent
1984 Academic
Press
Inc.
State
University,
antibodies; (London)
FITC, Limited
340
S. D. Wyatt and T. C. Wilkinson
localization of replication at initial infection sites or slow spread of virus within the inoculated leaf. In this study our approach has been to determine the extent of virus spread in cells of inoculated leaves by staining isolated protoplasts with fluorescent antibodies specific for the virus. The amount of spread of strain T (resistant reaction) and strain R (susceptible reaction) within the inoculated leaf were compared. Results were partially presented previously [ 121. MATERIALS AND METHODS Virus and plant manipulation
Strain T of CCMV was maintained in cowpea cultivar California Blackeye and strain R was maintained in cowpea introducti.on 186465. Cowpeas were grown in plastic pots (8 cm) in commercial soil mixture amended weekly with a complete fertilizer. The plants were maintained at 27 “C in the greenhouse with fluorescent light supplement when the photoperiod was less than 12 h. The susceptibility of PI 186465 cowpeas to initial infection by CCMV was increased by a 24-h dark treatment before inoculation. Primary leaves were inoculated before the formation of trifoliate leaves, usually 9-10 days after seeding, by rubbing leaves with virus in 0.05 M phosphate buffer, pH 7.0, containing O*5o/oCelite. In some experiments it was necessary to prevent the age related abscission of primary leaves by removing the trifoliate leaves as they expanded. Protoplast
isolation
Protoplasts were isolated from cowpeas enzymatically using a Carborundum-brush technique [I]. In preliminary protoplast experiments the protoplasts from PI 186465 cowpea primary leaves generally did not survive the isolation technique, while those from California Blackeye survived well. A 24-h dark treatment of PI 186465 plants before protoplast isolation as suggested by G. Bruening (pers. comm.) increased viability as indicated by a completely spherical shape. Figure 1 shows the number of viable protoplasts isolated per gram of strain T- and R-inoculated tissues. The protoplast yield declined somewhat with increasing age of the tissue during the experiments which lasted 25 days, but the decline was linear. It is assumed that protoplasts isolated are a representative sample of the population. In some experiments protoplasts were inoculated with CCMV (1 pg ml-l) pretreated with polyomithine (2 pg ml-l). The inoculum was incubated at 25 “C for 10 min, then added to freshly pelleted protoplasts at a final concentration of 5 x lo5 protoplasts ml-‘. During the inoculation procedure the protoplasts and polyomithinetreated virus were in 0.02 M potassium citrate, 0.45 M mannitol, pH 5.2. The protoplasts were incubated with inoculum for 10 min and washed by centrifugation at 1OOg. The protoplasts when appropriate were incubated 24-48 h at 25 “C in 0.45 M mannitol containing White’s salts solution [9], 2-4-D (0.25 pg ml-l, chlorotetracycline (4 mg 100 ml-‘), and griseofulvin (2 mg 100 ml-l). The pH of the medium was 5.8 and there were 5 x 105 protoplasts ml-‘. Immunojuorescence
Antiserum to CCMV strain. T was prepared in rabbits as previously described [Ill. The IgG was purified by precipitation with (NH,),SO, and reacted with fluorescein
Resistance
of cowpeas
to cowpea
a
chlorotic
22
4
I 6 Time
1. Number of inoculation with CCMV FIG.
mottle
virus
341
I I I I I I I I 8 IO 12 14 16 18 2022 after
inoculation
protoplasts isolated from strains T (a) and R (A).
PI
I 24:
(days)
186465
cowpea
primary
leaves
after
isothiocyanate (FITC) [6]. Th e conjugate was separated from unreacted FITC by ,:hromatography on a Bio-Gel P-10 column (1 x 15 cm) using saline phosphate Duffer, pH 7.4, for elution. The FITC-serum was cross-absorbed with acetone powder of cowpea tissue before use [6]. The protoplasts to be stained with fluorescent ,antibodies (FA) were affixed to Mayer’s albumin-coated glass slides and dried immediately with a hair drier. They were then fixed sequentially in acetone (30 min) for 2 h at 30 “C and and 95% ethanol (30 min), stained with FITC-antibodies ‘destained overnight in saline phosphate, pH 7.4. Analytical
methods
Virus was extracted from leaves or pelleted protoplasts (10 OOOg, 10 min) by homogenization in 0.2 M acetate buffer (pH 5.0) containing cysteine-HCl (0.01 M), sodium diethyldithiocarbamate (0.01 M) and MgCl, (0.01 M). Following clarification of the extract by chloroform-butanol emulsification and freezing, the virus was separated From remaining host material by the centrifugation of the extracts on sucrose density gradients ( lO-lOo/o w/v) in 0.2 M sodium acetate, pH 5.0, for 4 h at 27 000 rev min-’ in a Spinco SW 27 rotor. The virus content was calculated based on an extinction coefficient (E&f0 = 5.8). RESULTS Spread of virus
The extent of spread was determined in primary leaves of PI 186465 cowpeas inoculated with strain R (susceptible reaction) and strain T (resistant reaction) at intervals of 1,4, 7, 11, 18 and 25 days after inoculation. This determination was made by staining protoplasts isolated from the leaves with fluorescent antibodies specific for CCMV. The amount of virus in strain T-inoculated tissue (resistant reaction) and the proportion of FA positive protoplasts changed with time quite differently compared with the susceptible reaction (strain R) . Protopkkts from PI 186465 leaves inoculated with strain T showed a slow but steady increase in the fraction of stained cells to a value of approximately 25% at 25 days after inoculation (Table 1). In
342
S. D. Wyatt and T. C. Wilkinson TABLE D~#&nces
in intercellular spread in piimary leaves of a cowpea cultivar between CCMV have a resistant or susceptible relationship with the cultiuars
Virus
Infection (days)
Susceptible reaction
1
ND” 31 66 130 166 123
4 7 11 18 25
1
concentration in tissue (pg g-l tissue) (R)
Resistant (T) reaction ND 1 1 12 21 34
Infected
protoplasts cowpeas
Susceptible reaction
(R)
2(9/481)b 17(132/776) 41(268/656) 40(204/508) 48(218/458) 38(212/556)
=No virus was detected by sucrose density gradient analysis. bAverage number protoplasts infected, number counted and total number
strains
that
from PI 186465 (T/o) Resistant (T) reaction 0(0/728) 2(15/741) 4(29/681) 10(42/424) 13(54/410) 24( 138/573)
counted
contrast, the more virulent strain R caused the fraction stained to rapidly increase to approximately 40% at day 7 and remain close to that value through day 25. In both systems virus accumulation and fraction of stained protoplasts increased roughly in parallel, and the amounts of virion per stained protoplast approached similar maxima. Based on infectivity, purification experiments [IO] and ELISA (unpublished results), strain T does not move systemically in PI 186465 cowpeas. No infected protoplasts were detected by FA when trifoliolate tissue from strain T-inoculated plants was used. In comparison, 39% of the protoplasts from the trifoliolates from R-inoculated plants were FA positive. The relationship of spread to virus accumulation The relationship of spread to virus accumulation was investigated by testing protoplasts from 2-thiouracil-treated cowpeas. This base analog enhances CCMV replication several-fold without extending the length of the replication period [3]. PI 186465 cowpeas were inoculated with strain T and 4 days after inoculation were treated with 2-thiouracil by adding 50 ml (0*005 M) to each pot on three successive days. The results of two experiments are given in Table 2. Generally, there was up to threefold more virus in 2-thiouracil-treated leaves as determined by the amount of virus isolated by sucrose density gradient centrifugation of leaf extracts. However, the number of FA positive protoplasts was unchanged. 2-Thiouracil appears to stimulate CCMV replication but has no effect on spread of the virus in infected leaves.
of FA stain Virus must accumulate before detection by FA is possible, therefore marginally or newly infected protoplasts might appear FA negative. It was hoped that virus replication during an in vitro 48 h pre-stain incubation of protoplasts from strain T and R inoculated PI 186465 leaves would increase the number of FA positives.
Sensitivity
Resistance
of cowpeas
to cowpea
chlorotic
mottle
TABLE Proportion
of epidermal
Infection
and mesophyll
length
1
Expt
2
11 11 11 11
2
infected with 2-thiourocil
2-Thiouracil treatment (days after inoculation)
(days) Expt
cells
CCMV
treatment
with
Infected protoplasts from PI 186465 cowpea
( %I) 13 13 7 8
treatment
The average numbers of virus particles
3
per protoplast
isolatedfrom
CCMV (w Infection
length
(days)
4
5 x 106 protoplasts) Susceptible 1.6 8.2 18.2 20.9 10.5
4 7 11 18 25
Resistant -a
’
7 11 18 25
virus
T after
151 59 82 39
TABLE
‘No
strain
CCMV @g/g tissue)
4 -* 4
“iKo 2-thiouracil
343
virus
was detected
Virus particles/ protoplast (strain
R) 4.18 2.14 4.76 5.47 2.74
(strain
density
10” lo5 lo5 105 lo5
leaves
Particles/FA positive protoplast
2.45 5.2 1.18 1.15 0.72
x x x x x
lo5 lo5 lo6 108 lo6
ri -~
3.9 2.7 11.1
by sucrose
x x x x x
inoculated
1.0 x 105 0.7 x Ioj 2.9 x 105
gradient
1.01 x 105 0.5 x 106 1.2 x 106
analysis.
However, incubation of protoplasts before staining did not affect the fraction of FA positive protoplasts. To determine how much virus was in individual protoplasts isolated from infected PI 186465 tissue, virus was recovered by sucrose density gradient centrifugation from homogenized protoplasts and the average number of particles per protoplast was calculated (Table 3). In strain R-inoculated protoplasts at 4 days the average number of virus particles per protoplast and per FA-positive protoplast were 41 000 and 245 000, respectively and both reached a plateau by 11 days post-inoculation (approximately 500 000 and 1 200 000). In contrast, no strain T nucleoprotein was found by sucrose density gradient centrifugation until 11 days after inoculation. By 25 days the average number of strain T particles per protoplast and per FA-positive
344
S. D. Wyatt and T. C. Wilkinson
protoplast was 290 000 and 1 200 000, respectively. These data suggest that the rate of cell-to-cell virus spread and the replication rate per cell in the resistant reaction is less than that observed in susceptible reactions. Susceptibility of PI 186465protoplasts to infection In vitro inoculation of protoplasts from PI 186465 cowpeas with strains T and R was
tested as a way to study the resistance mechanism. Protoplasts were isolated from healthy PI 186465 cowpeas and inoculated with strains T and R. The inoculated protoplasts were incubated at 25 “C and assayed at 24 and 48 h with virus specific fluorescent antibody. There was very little difference in the proportion of strain R and strain T inoculated protoplasts which stained. The percent of protoplasts infected was 47 and 54 for R and T, respectively (48 h), suggesting there is little if any difference in susceptibility of PI 186465 protoplasts to initial infection. It has previously been shown that, from a mixture, strain R is selected over strain T in PI 186465 [II]. Therefore, the virus produced in inoculated protoplasts was characterized to determine if strain T was being replicated. Protoplasts (5 x lo6 protoplasts) were inoculated with either strain T or strain R and incubated for 48 h. Extracts were infectious on the local lesion host Bragg soybean. The lesions transferred to California Blackeye and PI 186465 (16 per strain) caused typical reactions [lZ]. Virus from strain T inoculated protoplasts caused bright, chlorotic mottle on California Blackeye and no symptoms on PI 186465. Virus from strain R inoculated protoplasts (16 lesions) caused a mild mottle on California Blackeye and a chlorotic mottle on PI 186465 cowpeas. These reactions are typical of the two strains. DISCUSSION
We previously demonstrated that resistance to CCMV strain T in PI 186465 cowpeas involves reduced virus-replicating capacity of the host which was associated with a quantitative alteration in the proportion of the four encapsidated RNAs [IO] and restricted virus movement. Paradoxically, strain T accumulation continues until senescence of the leaves, whereas strain R (susceptible reaction) replication in the inoculated leaves is exponential and ceases by 7-10 days post-inoculation at 27 “C [II]. Apparently, in the resistant tissues virus is not restricted to a few cells but spread and/or replication is slow. A lack of spread could result from either a lack of movement of virus from cell to cell or a failure to initiate replication once virus transfer has occurred. Though speculative, the latter would explain why both replication and spread of strain T are genetically linked to RNA 1 in PI 186465 cowpeas [II]. The mechanisms that regulate the amount of virus replication and spread are not known but inheritance studies indicate that they are controlled by several host genes [4] and the virus [II]. Genetic experiments with CCMV [II] and temperature-sensitive mutants of TMV [5] have shown that spread is dependent on some mechanism provided by the virus genome. In the case of CCMV, the addition of RNA 1 from strain R to the strain T genome is required for systemic infection of PI 186465 [If]. The provision by the virus. of some mechanism for spread is also suggested by experiments with 2-thiouracil. The treatment of strain T inoculated PI 186465 cowpeas with 2-thiouracil enhanced replication but did not produce greater cell to cell or systemic spread of replicational activity.
Resistance
of cowpeas
to cowpea
chlorotic
mottle
virus
345
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