Genetic analysis of cowpea mosaic virus mutants by supplementation and reassortment tests

VIROLOGY

70, 151-163

(1976)

Genetic Analysis of Cowpea Mosaic Virus Mutants Supplementation and Reassortment Tests

by

C. P. DE JAGER Department

of Virology,

Agricultural

University,

Wageningen,

The Netherlands

Accepted October 13, 1975

Three nitrous acid induced mutants were derived from the yellow strain isolate Sb of cowpea mosaic virus. The mutants, designated as N123, N163, and N140, were differentiated by atypical local lesions on “Pinto” beans and “Early Red” cowpeas and defective symptom development on the systemic hosts “Noordhollandse bruine” bean and “Blackeye” cowpea. Mutants N123 and Nl40 attained only low titers in “Blackeye” cowpeas and could not be purified in workable quantities. Three tests were applied to locate the mutations in either the middle or bottom components of the mutants: (1) In vitro recombination tests, involving the screening of hybrid isolates obtained from heterologous mixtures of components of wild and mutant strains; (2) supplementation tests, in which wild-type components were separately added to mutant preparations; restoration of a wild-type character to one of the mixtures indicated in which component the mutation (or mutations) occurred, (3) reassortment of components tests, based on the assumption that a mixture of two defective mutants may only show a wild-type character if the mutants have mutations in different components With all mutants, the results of the different test methods agreed. In mutants N123 and N163, the mutations were located in the middle component. Nl40 appeared to be a bottom component mutant. The results suggest that changes in the reaction of all differential hosts to a specific mutant are due to the same mutational event. Since N123 and NI40 carry mutations for defective symptom development in different components. this pair of mutants may be used to locate mutations of other defective mutants by means of simple reassortment tests. INTRODUCTION

The multipartite nature of many plant viruses offers the possibility of assigning phenotypic properties to specific parts of the genome. As phenotypic properties are expressions of genomic functions, such a grouping of biological markers in defined linkage groups may be useful for studying the role of the plant virus genome in the infection, replication, and symptomatological processes. Determinants for a variety of biological properties have been located for several multicomponent viruses; tobacco rattle virus (Ghabrial and Lister, 1973; Robinson, 19731, alfalfa mosaic virus (Dingjan-Versteegh et al., 1972), tobacco streak virus (Fulton, 1972), cowpea chlorotic mottle and brome mosaic viruses (Bancroft and Lane, 151 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

19731, raspberry ringspot virus (Harrison et al., 1974), and viruses of the cowpea mosaic virus group (Bruening, 1969; De Jager and Van Kammen, 1970; Moore and Scott, 1971; Wood, 1972; Kassanis et al., 1973). In most cases naturally occurring strains of the viruses were used. Artificially induced mutants may have advantages over naturally occurring strains. Observed differences between natural strains may be polygenic, and as a result, it may be difficult to associate marker properties with functional genetic units or molecular events. Induced mutants, however, may often carry single mutations resulting in defective functions, which may be characterized biochemically. The present paper reports the location of defective mutations in three nitrous acid

152

C. P. DE JAGER

induced mutants of cowpea mosaic virus (CPMV), a virus with a bipartite genome. For this purpose, three kinds of tests were performed. The tests were based on: (i) construction of hybrid strains by mixing purified components of the different strains (in vitro recombination); (ii) restoration of wild-type characters to a mutant preparation by the addition of the wildtype component corresponding to the component of the mutant carrying the mutation (supplementation); and (iii) generation of wild-type virus from unmutated components present in a mixture of strains carrying mutations in different components (reassortment of components).

Virus Purification Components

and

Separation

of

Wild-type and mutant CPMV were propagated in Blackeye cowpeas. Virus was purified from the primary leaves by the polyethyleneglycol-NaCl method, as described by Van Kammen (1967). Purified middle (M) and bottom (B) components of the virus were prepared by centrifugation in a linear 15-30% sucrose gradient in 0.01 M phosphate buffer, pH 7.0, using BXIV and BXV rotors of a MSE centrifuge (Measuring and Scientific Equipment, England). The BXIV rotor was loaded with a maximum of 40 mg of virus and run for 4 hr at 30,000 rpm. The BXV rotor was loaded with a maximum of MATERIALS AND METHODS 70 mg of virus and run at 23,000 rpm for 17 Viruses and Test Plants hr. A yellow strain isolate of CPMV, origiAfter the first separation the peak fracnally designated as Sb (Agrawal, 1964), tions of M and B components were pooled was used as wild-type. Mutant strains and recentrifuged one to four times in the N123, N163, and N140 were obtained from zonal rotor. The final M and B component this wild-type CPMV by treatment of the preparations had very little or no residual virus (N123 and N140) or the purified mid- infectivity. dle component (N163) with nitrous acid. The concentrations of the unfractionThe wild-type and mutant viruses were ated virus and the purified components differentiated on Phaseolus vulgaris L. were determined spectrophotometrically var. “Pinto” and var. “Noordhollandse using the following extinction coefficients bruine” (NHB) and on Vigna unguiculata (1 mg/ml; 1 cm light path): E,,, = 8.1 for (L.) Walp var. “Blackeye Early Barnshorn” unfractionated virus, E,,, = 6.2 for M, and and var. “Early Bed.” The Pinto bean vari- E,,, = 10.0 for B components. ety was a suitable local lesion host. The wild-type and mutant strains were Infectivity Tests Virus infectivity was measured by local maintained by serial local lesion transfer onto Pinto beans with simultaneous test- lesion assay on the primary leaves of Pinto ing on the other differential hosts as a beans. The preparations to be tested were check for the retention of the specific inoculated onto one-half of eight detached symptom characteristics and for the bio- leaves. The opposite halves were inoculated with a standard wild-type inoculum logical purity. Whenever a contaminant was suspected on the basis of symptoms (0.5-l Fg virus/ml). The leaves were kept produced, that isolate was discarded and on moist filter paper in petri dishes under replaced by another local lesion isolate constant illumination at 20”. The lesions were counted after 4 or 5 days and the from Pinto. Primary leaves were inoculated using infectivity was expressed as: (total number glass spatulas with a roughened flat end of lesions on eight test halves/total numand 500-mesh Carborundum as an abra- ber of lesions on the control halves) x 100. sive. Inocula from infected leaves were prepared by grinding leaf tissue in 0.01 M Isolation of Mutants Mutagenic treatment with nitrous acid phosphate buffer, pH 7.0, on roughened watch glasses using the spatulas. Special was done according to Mundry and Gierer care was taken to ensure that only virus (1958). Reaction mixtures consisted of 1 vol of 1 M acetate buffer pH 5.0, 1 vol of 4 M free glassware and buffers were used.

COWPEA

MOSAIC

NaNO,, and 2 vol of virus suspension in 0.01 M phosphate buffer, pH 7.0 (100-300 pglml) . Diluted samples of the reaction mixtures were inoculated onto primary Pinto leaves. N163 and N123 were isolated from atypical local lesions on such leaves at relatively high survival levels (SO-SO%>. N140 was isolated from one of the few lesions appearing at a survival level of less than 0.1%. The mutants were carried through at least 9 local lesion transfers before they were used in experiments.

VIRUS

MUTANTS

153

mogenate of a wild-type local lesion was inoculated for comparison. The appearance of local symptoms of wild and mutant types was recorded and local infection centers of either type were counted where possible. (3) Reassortment of components (ROC) test. This test was performed with mutants

showing defective symptom expression in the same differential host. One or more local lesions of any two mutants were homogenized in 0.01 M phosphate buffer, pH 7.0. The homogenates were inoculated singly onto the opposite halves of one attached primary Pinto leaf. The two halves Localization of Mutations of the opposite primary leaf were inoculated with a 1:l mixture of the mutant (1) In vitro recombination (ZVR) test. homogenates and a homogenate of a wildHomologous and heterologous mixtures of the M and B components of wild and mu- type lesion, respectively. When systemically reacting Blackeye tant strains were inoculated onto Pinto cowpeas were used as test plants, the four beans at a concentration which gave wellinocula were applied on different plants. separated lesions. The type of these lesions provided the first evidence for the location RESULTS of the mutation (or mutations) for lesion Characterization of the Mutants character. Ten or fifteen lesions produced by heterIn Table 1 the characteristic symptoms ologous mixtures were punched out and produced by the mutants N163, N123, and subcultured on the four differential hosts. N140 are described and compared with The symptom characteristics of these le- those of the wild-type. The three mutants sion isolates indicated in which component could be readily distinguished from each the aberrant phenotype was inherited. other and from the wild type by local le(2) Supplementation test. Local lesions sion type on Pinto beans and by symptoms of mutant isolates, retaining their specific on the inoculated leaves of the other differsymptom characteristics during serial lo- ential hosts. Generally, the mutants incal lesion transfer on Pinto leaves, were duced less well-developed local symptoms ground in a few drops of 0.01 M phosphate than the wild-type virus. buffer, pH 7.0. An equal volume of the The poor symptom development was reabove buffer was added to a part of the flected by a decreased virus multiplicahomogenate. Another part of the homoge- tion. Local lesions of all three mutants nate was supplemented with an equal from Pinto beans had to be homogenized in volume of either M or B component of wild about 10 times less buffer than wild-type type in the same buffer at a suitable con- lesions to obtain comparable lesion numcentration. bers upon reinoculation onto healthy Pinto For testing on Pinto beans, these prepa- plants. rations were inoculated onto opposite Primary Blackeye leaves inoculated with halves of attached primary leaves. The a relatively high concentration of N123 two halves of the opposite leaf were inocu- showed sometimes superficial etching. lated with, respectively, wild-type M or B Leaves inoculated with N140 always recomponent and a 1:l mixture of these com- mained symptomless. However, there was ponents in the same concentration. For some multiplication of these mutants in testing on the other differential hosts, the the inoculated leaves. The dilution end preparations were inoculated onto series of point of sap from these leaves was in the four separate plants. In some cases a ho- order of 10e4,for both mutants. Although

154

C. P. DE JAGER TABLE 1 SYMPTOMS PRODUCED BY WILD-TYPE CPMV AND THE DERIVED MUTANT STRAINS ON FOUR DIFFERENTIAL HOSTS” Host

Virus

Phaseolus vulgaris var. Pinto

L.

Wild-type

N163

N123

N140

P. vulgaris var. Noordhollandse bruine

Wild-type

N163

N123

Vigna unguiculata CL.1 Walp var. Blackeye Early Ramshorn

N140 Wild-type

N163

N123 N140 V.

unguiculata Early Red

var.

Wild-type

N163

N123 N140 a Lot., local symptoms;

this end very fied

is not point small from

Sys., systemic

Symptoms Lot.: large round areas with netlike necrosis of the smallest veins, later becoming necrotic with dark brown rings; diameter: l-3 mm Sys.: no systemic infection Lot.: small white spots with diffuse borders and a few veinal necroses, the center later becoming light brown necrotic; diameter: 0.5-l mm Sys.: no systemic infection Lot.: small brown necrotic spots with sharp borders; diameter: 0.5-l mm Sys.: no systemic infection Lot.: like the wild-type but becoming chlorotic rather than necrotic; diameter: 1-3 mm Sys.: no systemic infection Lot.: large round chlorotic spots (diameter: 2-5 mm) with chlorosis extending in the surrounding veins, later dispersed necrosis. Sys.: yellow starlike spots and mosaic, necrotic specks in the veins, deformation of lamina Lot.: small chlorotic spots (diameter: 1-3 mm) and some veinal chlorosis, no necrosis Sys.: yellow mosaic, no necrosis. Little distortion of lamina Lot.: small distinct chlorotic spots (diameter: l-2 mm), rapidly becoming necrotic, some necrosis of adjoining veins Sys.: no symptoms Not tested Lot.: Large yellow chlorotic spots with diffuse borders; diameter: l-3 mm Sys.: fine yellow or light green mosaic Lot.: light green chlorotic spots, smaller than wild-type, diameter: 1-2 mm Sys.: as wild-type but mosaic developing less rapidly Lot.: either no symptoms or light green superficial etching Sys.: no symptoms Lot.: no symptoms Sys.: no symptoms Lot.: small dark brown local lesions, sometimes white; diameter: 0.5-l mm Sys.: no symptoms Lot.: very small white and light brown local lesions; diameter: SO.5 mm Sys.: no symptoms Lot.: no symptoms Sys.: no symptoms Lot.: small white local lesions; diameter: 0.5-l mm Sys.: no symptoms symptoms

much lower than the dilution for wild-type virus (1O--5), only amounts of virus could be purileaves inoculated with either

in trifoliate

leaves.

N123 or NI40. In contrast, N163 gave good yields upon purification. Although the chlorotic lesions produced by N163 on inoculated Blackeye leaves were less pro-

COWPEA

MOSAIC

VIRUS

155

MUTANTS

The differences in lesion type between N163 and the wild strain did not show up clearly on detached leaves, but the lesion types could be readily identified when homologous and heterologous mixtures of components were inoculated onto intact Pinto bean plants. The types of lesions produced on Pinto beans are indicated in Table 2. The lesion IVR Tests type appeared to be associated with the M N163 was the only mutant of which the component of the parent strain. Fifteen lesions of each of the combinanucleoprotein components could be obtained in a purified form. Consequently, tions MwildBlcs and M>asBwildwere subculIVR tests could only be performed with tured onto Pinto and NHB bean plants. From Table 2 it appears that in all but two this mutant. In the first IVR test, attention was given cases the isolates produced in both bean to the altered symptoms of N163 in the two varieties gave the symptoms of the parent bean varieties. The residual infectivity of strain donating the M component. Mutations in N163 for altered symptoms the separated components of the wild and mutant strains and their ability to yield on Blackeye and Early Red cowpeas were infections in heterologous mixtures were located in a similar IVR test. Again the determined in infectivity tests (Table 2). heterologous mixtures induced the lesion Single components produced lesions only type of the parent strain from which the M sporadically or not at all. In contrast, the component originated. Upon subculturing, four mixtures of components gave 80 to 170 single lesion isolates of the heterologous times as many lesions as the respective mixtures on Blackeye and Early Red cowcomponents alone when compared to the peas, all isolates derived from the combicontrol numbers. Thus, the purity of the nation MwildBlaa produced wild-type local components was satisfactory and the het- symptoms on both cowpea varieties. The erologous components were compatible. mutant type lesions of the combination nounced than those of wild-type virus, there was no significant difference in virus yield between the two strains. Wild-type virus and mutant N163 both had M and B component ratios of approximately 1:l. Thus, separation of nucleoprotein components of N163 was possible as for wild-type virus.

TABLE 2 IVR TEST WITH Nl63: INFECTIVITY OF INDIVIDUAL COMPONENTS AND MIXTURES OF COMPONENTS OF SYMPTOMS PRODUCED IN PINTO AND NHB BEANS Inocnlum

M,mBwi,c, N163 Wild-type

Concentration @g/ ml)

N,“f;e’ sions” on test halves

Number of lesions* on control halves”

1 1 1 1 1+1 1+1 1+1

0 1 1 3 81 676 312

565 408 409 778 460 636 481


1+1 0.5

295 636

860 705

34 90

Percentage of control

Pinto lesion type direct inoculation

AND THE TYPE

__ .---_ Symptom. ty i?duc$ by subcultured sing e lesion wolates on: Pinto beans

NHB beans’

0

Wild Mutant Wild Mutant Mutant Wild

15/15” wild 15/15 mutant

13/15 wild Z/15 mutant 15/15 mutant

” Lesion numbers are totals of eight half leaves. ’ Control halves were inoculated with wild-type virus at a concentration of 0.5 pg/ml. ’ On NHB beans, local as well as systemic symptoms were recorded. ’ Numerator, number of isolates showing symptom type indicated; denominator, total number of isolates tested.

156

C. P. DE JAGER

M1BzBWildpredominantly yielded isolates giving mutant symptoms on cowpea plants. From the above results it was concluded that the alterations in symptom expression of N163 on the four differential hosts resulted from one or more mutations in the M component RNA. Supplementation

Tests

With N163, only the differential reactions of the two bean varieties were used as markers in supplementation tests. On Pinto beans, nine single lesion homogenates of N163 were separately tested with added wild-type M component and nine other lesion homogenates with added wild-type B component. The results are summarized in Table 3 in which the numbers of wild- and mutant-type lesions are given. From Table 3, it is clear that only the addition of M component to the mutant virus resulted in the appearance of wildtype lesions in addition to mutant lesions. The number of wild-type lesions given for the inoculum N163 + M is probably an underestimate since only lesions of doubtless wild phenotype were scored. No wildtype lesions were observed after addition of bottom component. This was not due to TABLE

3

SUPPLEMENTATION OF N163: EFFECT ON THE LESION TYPE ON PINTO BEANS Inoculum

Lesion type N163

Wild

N163 N163 + M” (2)’ M (2) Wild-type

620” 550 0 0

0 71 0 290

N163 N163 + Bd (32) B (32) Wild-type

330 350 0 0

0 0 I 134

M (2) + B (32) Wild-type

0 0

160 156

‘I Lesion numbers are totals of nine half leaves. h M, wild-type M component. c Numbers in parentheses indicate final concentrations in micrograms per milliliter. d B, wild-type B component.

lack of activity of the B component since a mixture of M and B components produced as many lesions as the control wild-type inoculum used in this experiment. On NHB beans, five separate homogenates, each of two mutant lesions, were supplemented with each of the two wildtype components. Due to the coalescense of chlorotic areas it was impossible to count the local symptoms, especially those of wild type occurring together with mutant symptoms. Nevertheless, the results were unequivocal. Typical symptom expression is pictured in Fig. 1. Wild-type infection centers appeared after addition of the M component but not after addition of the B component. The added components, in comparison, showed no or very little residual infectivity when inoculated separately. However, they were both fully active as a mixture, M and B components together producing many lesions of the wild type. It is evident that only addition of wildtype M component restored the ability to produce wild-type symptoms on the two bean varieties. This indicates that one or more mutations in the middle component of N163 prevented normal symptom induction in these hosts. This conclusion agrees with the results of the IVR test with N163 in which the inheritance of bean symptom expression appeared to be through the M component. No supplementation tests were done with N163 on the cowpea varieties since the mutant- and wild-type lesions on these hosts were difficult to differentiate when intermingled. The reactions of N123 on all the differential hosts were sufEciently distinct from the wild-type to be used as markers in supplementation tests. On Pinto beans, the local lesions induced by the mutant were much smaller than those of the wild strain. This was true also of the chlorotic spots of N123 on inoculated leaves of NHB beans. N123 produced no visible local or systemic reaction on the two cowpea varieties. On Pinto and NHB beans, the test procedure for N123 was the same as for N163 except that, because of the low virus content of N123 lesions, only one bulk inocu-

COWPEA MOSAIC VIRUS MUTANTS

N140

M

N140 + M

M+B

N14O+B N140 B M+B FIG. 1. Supplementation test with mutant N163 on NHB beans. Upper row: A mixture of noninfectious M-component CM) and N163 gives wild-type symptoms in addition to mutant symptoms. Bottom t’ow: A mixture of the slightly infectious wild-type B-component (B) and N163 induces only mutant symptoms. Activity of the two wild-type components is evident from the production of wild-type infection centers by the M + B mixture. FIG. 2. Supplementation test with mutant Nl40 on Blackeye cowpeas. Inability of the mutant to produce symptoms in the inoculated leaves is compensated for by added wild-type B-component (B) but not by added wild-type M-component (Ml. Activity of the two wild-type components is evident from the production of spots by the M + B mixture.

158

C. P. DE JAGER

lum of N123 lesions was tested on NHB beans instead of separate lesion homogenates. Since on Pinto and NHB beans there was a clear distinction between mutantand wild-type symptoms, the results of the inoculations could be compared on the basis of the numbers of lesions of wild and mutant type. The data are given in Table 4. Similar results were obtained using either bean variety. The effects observed were essentially the same as those in the preceding experiments with N163, indicating that only the M component of the wildtype was able to restore the wild-type symptom production. Supplementation tests with N123 on both cowpea varieties were performed with five separate single-lesion homogenates for each of the components to be tested. In all cases, the same treatments gave similar results. No visible local reaction occurred on Blackeye cow-peas following inoculation with N123 or wild-type M component. Wild-type B component, whether inoculated singly or mixed with N123, produced visible infection centers only sporadically. In contrast, a mixture of N123 and wildtype M component induced many chlorotic spots which were indistinguishable from those evoked by a mixture of the two wildtype components. On Early Bed cowpeas, the results of the supplementation test with N123 were obscured by the occurrence on the primary leaves of brown spots not induced by virus infection. These “pseudolesions” were also observed on noninoculated control plants. Unfortunately, these spots could not be distinguished from wild-type local lesions. Consequently, their numbers had to be included in the lesion counts. Nevertheless,

the results of this test were unequivocal. Data are presented in Table 5. Judging from the number of spots on leaves inoculated with the noninfectious M component and the weakly infectious B component, the total number of pseudolesions on five plants could not have exceeded 100. AddiTABLE

Beans

OF N123:

IIlOCUlUIIl

Lesions”

of‘ infection tersb of:

N123-type

Pinto

NHB

cen-

Wild-type

N123 N123 + M’ (4)” M (4) Wild-type

1513 375 0 0

0 648 0 858

N123 N123 + B’ (321 B (32) Wild-type

615 690

0 2

0

10

0

470

M (41 + B (321 Wild-type

0 0

300 600

N123 N123 + M (4) N123 + B (32) M (4) B (32) M (4) + B (32) Wild-type

1402 807 1355 0 0 0 0

0 50 1 0 5 99 x*

” Number of lesions on Pinto beans are totals of nine half leaves. h Numbers of infection centers on NHB beans are totals of 14 primary leaves on 7 plants. c M, wild-type M component; B, wild-type B component. d Numbers in parentheses indicate final concentrations in micrograms per milliliter. e 5, too many infection centers to count.

TABLE SUPPLEMENTATION

4

SUPPLEMENTATION OF N123: EFFECT ON LESION TYPE ON PINXI BEANS AND LOCAL SYMPTOMS ON NHB BEANS

5

EFFECT ON LESION

NUMBERS

IN EARLY

RED COWPEAS

Inocula N123

N123 + compo-

Component”

nent”

Wild-type Wild-type

M added B added

45” 82

690 119

53 82

Mixture of componenta” 463 487

U Final concentrations of wild-type components were 4 pg/ml for M and 32 pg/ml for B in all inocula. h Lesion numbers are totals of 10 leaves of 5 plants and include numbers of pseudolesions.

159

COWPEA MOSAIC VIRUS MUTANTS

tion of M component to N123 lesion homogenates increased the number of spots far beyond this level whereas addition of B component had little effect. A mixture of M and B components produced high lesion numbers. With all host plants, addition of wildtype M component to a N123 preparation resulted in the appearance of wild-type symptoms. Addition of B component had no effect whatsoever. This indicates that the M component of N123 contains one or more mutations which are responsible for the failure of N123 to grow normally and to produce symptoms resembling those of wild-type virus. In a supplementation test with N140, the inability of this mutant to produce visible infection centers on Blackeye cowpeas was used as a marker. Five separate single lesion homogenates were tested with each of the two wild-type components, The results are illustrated in Fig. 2, showing leaves from two series of four test plants. N140 inocula and the M component failed to produce chlorotic lesions when inoculated either singly or combined. The B component induced some spots when inoculated alone; however, addition of this component to N140 inocula greatly increased the number of spots. The resulting infection centers were of the same type as those produced by a mixture of the two wild-type components. Since addition of B component to a N140 preparation apparently restored a wildtype genome to the mixture, it was concluded that failure of N140 to multiply extensively and to produce symptoms in Blackeye cowpeas was due to one or more mutatiqns in the B component. ROC Tests

All three mutants produced poorly developed local lesions on Pinto in comparison with wild-type virus. In Blackeye cowpeas, the mutants induced either no symptoms or less pronounced local symptoms. Therefore, these hosts could be used to detect complete wild-type genomes in mixtures of two mutants. All possible combinations of mutants were tested, the results are summarized in Table 6.

TABLE 6 ROC TESTYWITH MUTANTS N163, N123, AND N140: EFFECT ON PINTO BEANS AND BUCKEYE COWPEA~

Mutants N163 + N123 N163 + N140 N123 + N140

Pinto beans

Blackeye cowpeas

- (14)”

- (2)

f (2) + (8)

+ (2) + (16)

” -, no wild-type symptoms observed, +, wildtype symptoms observed; numbers in parentheses indicate numbers of separately tested mixtures of lesion homogenates.

A mixture of N123 and N163 on Pinto beans gave hundreds of lesions of both mutant types; however, no wild-type lesions were observed. On Blackeye cowpeas the chlorotic spots induced by the mixture were indistinguishable from those produced by mutant N163 alone. The combination N163 + N140 produced, on Pinto beans, many wild-type lesions in addition to some lesions of each of the mutant types. On Blackeye, this combination evoked large yellow spots indistinguishable from those of wild-type CPMV. Inoculation of a mixture of lesion homogenates of N123 and N140 onto Pinto leaves resulted in the appearance of lesions of wild type and of both mutant types. On Blackeye cowpeas, the performance of this combination of mutants was particularly interesting since both mutants failed to give symptoms on this host. When mixed, the mutants produced large chlorotic spots on inoculated leaves (Fig. 3) which, however, developed more slowly than wild-type spots and were not as bright. The systemic symptoms were indistinguishable from those of wild-type viI-US.

A plausible explanation for the appearance of symptoms in plants infected with both N123 and N140 was the occurrence of wild-type virus, resulting from unmutated M and B components contributed by the two mutants. Two alternative explanations had to be considered, both assuming the occurrence of mutations in corresponding components of the strains involved. A first possibility was the occurrence of molecular recombination between mutated RNAs of either the two M or the two

160

C. P. DE JAGER

experiment, 13 out of 14 lesion isolates were of the wild-type and one appeared to be of the N123 type. It is improbable that at very high dilutions about 90% of the lesions start from the three types of particles necessary for complementation within the cell, and therefore, such a mechanism of genetic interaction cannot explain the observed phenomenon of abundant production of wildtype symptoms. NT23

N140

N123+N140 Sb FIG. 3. Reassortment of components test with mutants N123 and N140 on Blackeye cowpeas. Mutant preparations give no symptoms when inoculated separately but produce many infection centers when mixed. Wild-type control inoculum is indicated by Sb.

B components. This explanation was unlikely in view of the high frequency of the yellow spot induction and the lack of precedent for this type of genetic mechanism with plant viruses. A second alternative explanation might be the occurrence of complementation between mutated RNAs present within the same cell. To investigate this second alternative experiments were performed to see whether the wild strain or only the two mutant strains multiplied in primary Blackeye leaves infected with a mixed inoculum. A dilution series of sap from these leaves was inoculated onto Pinto beans. The 16 lesions which developed at limit dilution were subcultured onto Pinto and Blackeye plants. Of these lesions, 15 appeared to contain virus which behaved as normal wild-type. Only one isolate resembled N140 in the local lesion type and lack of symptoms on Blackeye. In a duplicate

DISCUSSION

All tests in which N163 and N123 were involved showed that the mutation responsible for the defective behavior of these mutants was in the M component. For mutant N163, this was not surprising since this mutant was isolated following nitrous acid treatment of separated wild-type M component. N123 and N140, obtained after nitrous acid treatment of complete virus, appeared to have mutations in different components. Thus, mutagenic treatment of unfractionated virus could yield both M and B component mutants. From the data presented it is clear that local lesion type on Pinto beans is affected by mutations in both M and B components. This is in agreement with the results of Wood (1972) who found that the type of lesions produced by two mutants of a severe strain of CPMV was controlled by the interaction of the gene products from both component RNAs. The mutants N123 and N140 grow poorly on Blackeye cowpeas. The multiplication and/or cell to cell movement of CPMV in this systemic host are apparently influenced by mutations in both M and B components. This implies that M as well as B component perform essential functions in the process leading to the accumulation of virus in cowpea plants. The biochemical characterization of these functions is the subject of further investigations. The mutants described here show changes in symptoms and multiplication in more than one host plant. However, in none of the mutants were mutations found in both components. The same holds true

COWPEA

MOSAIC

for eight out of nine mutants of the yellow strain that have been genetically analyzed (De Jager and Van Kammen, 1970; De Jager, 1973; unpublished results). This strongly suggests that the changes in the reaction of all differential hosts to a specific mutant result from the same mutational event. This would imply that there exist plant virus genes which govern several phenotypic properties. This is very likely in view of the limited size of the plant virus genome. A multiple effect of virus genes has been suggested by Harrison et al. (1974) for naturally occurring strains of raspberry ringspot virus. On the other hand, Sehgal and Krause (1968), working with tobacco mosaic virus mutants, observed that alterations in symptom expression in three tobacco varieties could occur both singly and in all the possible combinations. This suggested that the mutations occurred in different genetic units. Final proof for a pleiotropic effect of mutations in CPMV awaits the genetic or biochemical analysis of the separate genome segments. The naming of the different test methods is a matter of some concern. Bancroft and Lane (1973) used the name “hydribization test” for the construction of hybrid strains by the exchange of components in the test tube. However, this term is already used to denote experiments in the related field of research on the homologies of viral nucleic acid strands. In this study the test is named “in vitro recombination test” because of the analogy with spontaneous recombination in bacterial and animal DNA viruses. In both cases reciprocal hybrids arise, each carrying portions of the genomes of the parent strains. In the literature on multicomponent viruses, there is a tendency to use the word “complementation” in different contexts (Wood, 1972; Bancroft and Lane, 1973). Superficially, the ROC test bears some resemblance to complementation tests as used to analyze bacterial and animal viruses. There is, however, a basic difference. In ROC tests, wild-type virus is generated by reassortment of components. in the inoculum. In complementation tests, the mutants replicate as such by sharing

VIRUS

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161

unaffected genomic functions. To avoid confusion the term “complementation” shoud be reserved for the latter mechanism. This is the more important as it seems possible that in the future this type of interaction also may be used in the genetic analysis of plant viruses. In vitro recombination is a well-adopted method of locating biological markers on the genome segments of plant viruses with a multipartite genome (Jaspars, 1974). The method is laborious due to the necessity of isolating genome pieces of all strains involved. Application of the test is impossible for strains that cannot be obtained in a purified form. Supplementation and ROC tests, as described here, may be suitable alternatives. Supplementation tests have been reported earlier by Bancroft and Lane (1973) for mutants of cowpea chlorotic mottle and brome mosaic viruses. These authors diluted the mutant preparations just beyond the dilution end points. Addition of separate RNAs of the wild strain raised infectivity levels sufficiently to yield lesions. The type of these lesions provided evidence for the location of the mutations. In our experiments the mutant preparations were not diluted to the same degree. Consequently a positive effect of the addition of one of the wild-type components was to be recognized by the occurrence of wild-type infection centers among those of the mutant. This was only possible if there was a clear difference between the local symptoms of wild and mutant types. It was always essential to test both the M and B components of wild-type virus for their ability to restore a normal complete genome to a mixture. The results were considered to be unequivocal if only one of the added components had a positive effect. This proved to be the case in all tests. The B component preparations appeared always to contain slight residual contaminating homologous M component. In mixtures of the B component with mutants N163 and N123, this contamination always produced less wild-type symptoms than single B component preparations. Therefore, it may be assumed that in mixtures with mutant N140 on Blackeye cowpeas

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the wild-type infection centers were mainly due to multiplication of the unmutated M component particles of N140 and added B component particles. Apart from the intrinsic unambiguousness of the supplementation test, the reliability of this test was further demonstrated by the agreement of results gained using it with the results of the other test methods. The applicability of the ROC test demands that the mutants to be tested have defective symptom production or, even better, induce no symptoms at all. Under these conditions, the development of normal symptoms can be easily recognized providing the concentration of the mutants in the inoculum is sufficiently high (Fig. 3). Normal symptoms were expected for mixtures of mutants carrying mutations on different components and having, between them, a complete wild-type genome. Indeed production of normal symptoms appeared to occur in those cases in which the defects were assigned to different components by other tests. However, mutants having mutations on the same component, i.e., N123 and N163, did not give rise to wild-type symptoms. Thus, there was no evidence for intracellular complementation by the two M components. This type of genetic interaction was also excluded as the cause of symptom induction by the mixture of N123 and N140 on Blackeye. The virus produced in leaves inoculated with such a mixture proved to be mainly normal wild-type virus since the wild-type characteristics could not be eliminated by infection at high dilution. The slow development of local symptoms in Blackeye primary leaves infected with mixed mutants N123 and N140 may be attributed to the presence of the two mutated components which, by their low level of multiplication, interfered with the propagation of wild-type virus. The defectiveness of mutants N123 and N140 is most obvious on the local lesion host Pinto and the systemic host Blackeye. These mutants can be maintained as stable strains by local lesion transfer. These circumstances, together with the fact that the respective mutations occur on different

components, make N123 and N140 suitable as a set of standard mutants against which other mutants can be tested for the location of their mutations. Using these mutants, the location of defective mutations in the components of CPMV mutants may be simplified considerably. ACKNOWLEDGMENTS I wish to express my sincere appreciation to Professors J. P. H. van der Want and A. van Kammen for helpful and stimulating discussions and to the latter for his help in the preparation of the manuscript. Thanks are also due to Dr. A. Ziemiecki for correcting the English text and to Marianne van Woerden-Cramer for technical assistance. REFERENCES AGRAWAL, H. 0. (1964). Identification of cowpea mosaic virus isolates. Mededel. Landbouwhogeschool Wugeningen 64, NO.(~), l-53. BANCROFT, J. B., and LANE, L. C. (1973). Genetic analysis of cowpea chlorotic mottle and brome mosaic viruses. J. Gen. Viral. 19, 381-389. BRUENING, G. (1969). The inheritance of top component formation in cowpea mosaic virus. Virology 37, 577-584. DE JAGER, C. P. (1973). Genetical investigations on a multicomponent virus. In “Plant Virology, Proceedings of the 7th Conference of the Czechoslovak Plant Virologista, High Tatras 1971” (V. Bojfianskq, ed.), Publishing House of the Slovak Academy of Sciences, Bratislava. DE JAGER, C. P., and VAN KAMMEN, A. (1970). The relationship between the components of cowpea mosaic virus. III. Location of genetic information for two biological functions in the middle component of CPMV. Virology 41, 281-287. DINGJAN-VERSTEEGH, A., VAN VLOTEN-DOTING, L., and JASPARS, E. M. J. (1972). Alfalfa mosaic virus hybrids constructed by exchanging nucleoprotein components. Virology 49, 716-722. FULTON, R. W. (1972). Inheritance and recombination of strain-specific characters in tobacco streak virus. Virology 50, 810-820. GHABRIAL, S. A., and LISTER, R. M. (1973). Coat protein and symptom specification in tobacco rattle virus. Virology 52, 1-12. HARRISON, B. D., MURANT, A. F., MAYO, M. A., and ROBERTS, I. M. (1974). Distribution of determinants for symptom production, host range and nematode transmissibility between the two RNA components of raspberry ringspot virus. J. Gen. Viral. 22, 233-247. JASPARS, E. M. J. (1974). Plant viruses with a multipartite genome. Aduan. Virus. Res. 19, 37-149. KASSANIS, B., WHITE, R. F., and WOODS, R. D.

COWPEA MOSAIC VIRUS MUTANTS (1973). Genetic complementation between middle and bottom components of two strains of radish mosaic virus. J. Gen. Viral. 20, 277-285. MOORE,B. J., and SCOTT,H. A. (1971). Properties of a strain of bean pod mottle virus. Phytopathology 61, 831-833. MUNDRY, K. W., and GIERER, A. (1958). Die Erzeugung von Mutationen des Tabakmosaikvirus durch chemische Behandlung seiner Nucleinsaure in vitro. 2. Vererbungslehre 89, 614630. ROBINSON,D. J. (1973). Properties of two tempera-

ture-sensitive

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mutants of tobacco rattle virus. J.

Gen. Viral. 21, 499-506.

SEHGAL,0. P., and KRAUSE, G. F. (1968). Efficiency of nitrous acid as an inactivating and mutagenic agent of intact tobacco mosaic virus and its isolated nucleic acid. J. Viral. 2, 966-971. VAN KAMMEN, A. (1967). Purification and properties of the components of cowpea mosaic virus. Virology 31, 633-642. WOOD, H. A. (1972). Genetic complement&ion between the two nucleoprotein components of cowpea mosaic virus. Virology 49, 592-598.