Symptoms of Cauliflower Mosaic Virus Infection in Arabidopsis thaliana and Turnip

Symptoms of Cauliflower Mosaic Virus Infection in Arabidopsis thaliana and TurnipAuthor(s): Ulrich MelcherReviewed work(s):Source: Botanical Gazette, Vol. 150, No. 2 (Jun., 1989), pp. 139-147Published by: The University of Chicago PressStable URL: http://www.jstor.org/stable/2995230 .Accessed: 02/10/2012 08:11

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BOT. GAZ. 150(2): 139-147. 1989. (C) 1989 by The University of Chicago. All rights reserved. 0006-8071 /89/5002-0013$02.00

SYMPTOMS OF CAULIFLOWER MOSAIC VIRUS INFECTION

IN ARABIDOPSIS THALIANA AND TURNIP

ULRICH MELCHER

Department of Biochemistry, Oklahoma State University, Stillwater, Oklahoma 74078-0454

The type of symptoms of cauliflower mosaic virus (CaMV) infection observed in Arabidopsis thaliana and turnip depend on host species and viral isolate. Inflorescences of infected A. thaliana plants were chlorotic, often in sectors that also involved axillary shoots, cauline leaves, and siliques. Infection also affected silique and seed morphology and, in some shoots, the ability of seeds to germinate and the sensitivity of seeds to bleach. Isolate NY8153 and isolates derived from CM4-184 caused stunting of rosette leaves, while isolate CM4-184 did not. CaMV nucleic acid was detected in seed coats but not in embryos of seeds from infected plants. The symptoms on turnip leaves (chlorotic spotting, chlorotic mottling, fine vein clearing, necrotic spotting, coarse vein clearing, stunting, rugosity, senescence, and pale greenness) depended on the age of the leaf at infection and on the viral isolate used. Symptoms occurred in left-right and tip-to-base sectors. The observations suggest that viral infection, to cause symptoms, acts primarily on differentiating cells.

Introduction

Arabidopsis thaliana is a small cruciferous plant with a haploid genome size of 70 pg and little re- petitive DNA (PRUITT and MEYEROWITZ 1986; MEYEROWITZ 1987). It has a short generation time and it is prolific. A genomic library (PRUITT and MEYEROWITZ 1986) and a leaf cDNA library (MES- NARD et al. 1985) are available. Several genes have been cloned and sequenced (MEYEROWITZ and PRUITT 1985). Mutagenesis of A. thaliana has been well investigated, and a large number of mutants are available (REDEI 1975; MEYEROWITZ and PRUITT 1985). These factors make A. thaliana an ideal plant for genetic and molecular studies of the interaction of viruses with host genomes.

Arabidopsis thaliana is a host for cauliflower mosaic virus (CaMV) (BALAZS and LEBEURIER 1981), a well characterized caulimovirus, that con- tains an 8-kbp double-stranded circular DNA as its genetic material (COVEY 1985; MAULE 1985). CaMV DNA exists in nuclei of infected cells as covalently closed minichromosomal DNA (MENISSIER et al. 1982; OLSZEWSKI et al. 1982). The replication of DNA occurs in the cytoplasm by reverse transcrip- tion of a 35S RNA transcript. Although CaMV is not seed-transmitted, virus can be found in the testa of some infected species (TOMLINSON and WALKER 1973). Symptoms caused by CaMV in A. thaliana have been briefly described (BALAZS and LEBEU- RIER 1981) and include vein clearing, chlorotic spotting, stunting, and a diminished seed yield. Symptoms of CaMV infection of turnip plants (BROADBENT 1957; AL ANI et al. 1979; HOWELL et al. 1980; DAUBERT et al. 1983; DAUBERT et al. 1984; MAULE 1985) can include chlorotic spotting, chlorotic mottling, vein clearing, vein banding, leaf stunting, necrotic flecking, senescence, and rugos-

Manuscript received June 1988; revised manuscript received January 1989.

ity (distortion). Symptoms observed depend on the strain of CaMV (DAUBERT et al . 1984; MAULE 1985). Leaf pigment symptoms are sometimes sectored (BROADBENT 1957; MAULE 1985) . Several CaMV mutants, constructed in vitro, that affect the type of symptom are known (DAUBERT et al. 1983). However, no well-defined mutation has been identified that prevents the production of symptoms without interfering with replication and spread of the virus (MELCHER et al. 1986). I report further observations on the symptoms of CaMV infection in A. thaliana and turnips; these observations pro- vide background for studies of the effects of CaMV infection on host gene expression.

Material and methods

Turnip (Brassica rapa [L. ] 'Just Right' ) and Arabidopsis thaliana (L. ) Heynh., ecotypes Co- lumbia and La-O, plants were grown in a growth chamber with a 12-h light-21C and 12-h dark-19C cycle. Arabidopsis thaliana plants were irrigated daily with a nutrient solution (HEATH et al. 1986). Plasmid DNAs containing CaMV DNA sequences (table 1) were digested with SaNl (pLW414, CaBB1, CaNB2, Ca37, and pCMS31) or XhoI (pLW303X). Turnips were inoculated with the plasmid digests or with virions (D/H, CabbB-D) as previously described (SUN et al. 1988). Virion stocks were prepared from systemically diseased leaves by the method of HULL et al. (1976).

Arabidopsis thaliana leaves were inoculated with cotton-tipped applicators dipped in 1-3 mg/L (or 20 mg/L) suspensions of CaMV virions of isolates NY8153, CabbS, CabbB-D, D/H, CaNB2, CaBB1, CM4- 184, and W (table 1) in 1 % dibasic potassium phosphate containing Celite. The supported leaf surface was stroked once or twice with the cotton-tipped applicator. The adaxial surfaces of the third and fourth leaves of 2 1 /2-3-wk-old plants

139

TABLE 1

CAMV ISOLATES USED

Isolate Source Comments References

CM4-184 ................... pLW414, S. HOWELL ................ ORFII deletion; sequence HOWELL et al. 1980;

known HOWARTH et al . 1981; DIXON et al . 1986

CaBBl .......................... Plasmid CaBBl, T. HOHN Derived from CM1841 BRISSON et al. 1984 CaNB2 .......................... Plasmid CaNB2, T. HOHN Derived from CaBBl by BRISSON et al. 1984

insertion of dihydrofolate reductase coding sequence

Cabbage S (CabbS) .......... Plasmid Ca37, K. RICHARDS ....... Sequence known LEBEURIER et al. 1982;

FRANCK et al . 1980 D/H . . ..... .. . .. . . . . . . . . . . . ........ G . LEBEURIER Sequence known BALAZS et al . 1982 NY8153 .......................... pCMS31 Viral strain originally from ARMOUR et al. 1983

R. SHEPHERD Cabbage B-D (CabbB-D) .......................... R. SHEPHERD ... DAUBERT et al. 1982 W .......................... pLW303X, S. HOWELL Chimera of Cabbage B-JI WALDEN and HOWELL 1982;

and another isolate CHOE et al. 1985

140 BOTANICAL GAZETTE [JUNE

were chosen for inoculation, since the highest fre- quency of infection without premature death of the plants was obtained in this manner. Visible disease symptoms appeared on noninoculated leaves 9 d after inoculation. Three- to four-week-old turnip plants with their first two leaves removed were inoculated with CaMV virions of the isolates listed in table 1 by rubbing with a gloved finger 20 I1L of a 2 ,ug mL-1 suspension of virions in 1% dibasic potassium phosphate containing Celite on each third, fourth, and fifth leaves. Plants were misted with water after inoculation and were returned to the growth chamber where they were inspected pe- riodically for symptoms. The number of turnip plant leaf initials at various stages of growth was deter- mined by dissecting the growing point under a compound microscope.

Leaf-blot hybridization in which blots of tissue on filter paper are hybridized with radioactive DNA probes was performed as described by MELCHER et al. (1986). Hybridization of radioactive probe DNAs to leaf skeletons and nick translation of DNA was performed as previously described (MELCHER et al. 1981). CaMV DNA prepared according to GARD- NER and SHEPHERD ( 1980) and psAt2 105, a plasmid clone of the 12S storage protein gene of the Columbia ecotype of A. thaliana (PRUITT et al. un- published), were used as probes. CaMV antigen was assayed in extracts of leaves (MELCHER et al. 1980) using rabbit anti-CaMV (MELCHER et al. 1980) in an indirect ELISA (EDWARDS and COOPER 1985). Arabidopsis thaliana seeds were surface- sterilized by soaking for 30 sec in 95% ethanol, followed by 6 min in 2.6% sodium hypochlorite. Bleach hypersensitivity was scored by observing seeds under the compound microscope for changes in pigmentation during hypochlorite treatment.

Surface-sterilized seeds were placed on 0.8% agar containing minimal salt nutrients (MURASHIGE and SKOOG 1962) for germination. In some germination experiments seeds were spread on filter paper moistened with sterile water in petri dishes.

Results

ARABIDOPSIS THALIANA LEAVES AND STEMS

Virions of all but three of the CaMV isolates tested induced symptoms in Arabidopsis thaliana, Co- lumbia ecotype, with high efficiency (table 2). The CM4-184 and D/H isolates were not always ef- fective in producing infection, while at 2,ug mL-' the CabbS isolate, which reproducibly infected turnips 'Just Right,' did not infect Columbia or La- O. Columbia and La-O plants were infected after inoculation with 20 ,ug mL-9 CabbS virions. Sys- temically CaMV-infected rosette leaves of A. thaliana exhibited vein clearing and occasional chlorotic spots. In some plants, the chlorosis proceeded to necrosis and eventual death of the plants. Some plants died without flowering. Symptoms on cauline leaves included a few chlorotic spots, vein clearing, mottled chlorotic mosaic, and totally chlorotic leaves. Stems of infected plants were either entirely green, a mosaic of green and chlorotic stripes parallel to the stem axis, or completely chlorotic. When stems were striped, cauline leaves

. . r

arlslng trom green stripes were green, sometimes exhibiting vein clearing. Cauline leaves that arose from chlorotic sectors of the stem were chlorotic. Immature siliques, green when arising from green stem tissue, were light yellow to brown when the siliques arose from chlorotic sectors. In shoots bearing a mixture of normal and distorted siliques the distorted siliques originated from chlorotic stem

TABLE 2

ISOLATE-SPECIFIC VARIATION IN INFECTIVITY AND SYMPTOM APPEARANCE IN CAMV INFECTION OF

ARABIDOPSIS THALIANA ECOTYPE COLUMBIA

Polarity

CaMV isolate Infectiona Stunting Chlorosis extent of chlorosis

CabbS .... . . . . . . . . . . .......... . . . . . . . . ......... 0/ gb * - - ... ...

CabbB-D ...................................... 5/5 0/5 3/5 5/5

W ...................................... 6/6 0/6 5/5 l /5

CM4-184 ...................................... 3/5 0/3 2/3 0/3

CaBB1 ...................................... 5/5 4/5 0/5 0/5

CaNB2 ...................................... 6/6 6/6 0/5 0/5

NY8153 ...................................... 6/6 6/6 3/6 2/3

D/H ...................................... 3/5 0/3 3/3 0/2

aNo. of plants with the trait/no. of plants evaluated inoculated with 2 1lg mL i of virions of the indicated isolates. Evaluation

was performed 3-5 wk postinoculation.

bInfection was obtained, however, when plants were inoculated with 20 1lg mL-} virions.

MELCHER CAMV IN TURNIP AND ARABIDOPSIS 141

1989]

tissue. Axillary shoots were also green or chlorotic depending on the sector of origin. The green axillary shoots nevertheless had vein-clearing symptoms.

Isolate-specific differences in symptoms were noted (table 2). Only isolates CaNB2, CaBB 1, and NY8153 produced stunting and increased serration of rosette leaves. Plants infected with W and D/H isolates invariably had cauline leaves that were more than half chlorotic, while extensive chlorosis was not seen on CaBB 1- and CaNB2-infected plants. Chlorosis was more predominant in the basal half of cauline leaves of plants infected with CabbB-D and NY8153 isolates than in the leaf tips. Little or no polarity was found in the chlorosis of cauline leaves of plants infected with the other isolates tested. When rosette leaves, cauline leaves, stems, inflorescences, and immature siliques were tested for the presence of CaMV nucleic acid by tissue- blot hybridization, all healthy-appearing tissues failed to bind above background levels of CaMV DNA probe. All positive-hybridization spots were from visibly diseased tissue. Blots of some symptomatic tissues did not bind enough probe for detection. Plasmid psAt2 105, which bears a fragment of A. thaliana-unique sequence DNA, failed to hybridize detectably to blots, indicating that only high concentrations of DNA are detected by this method.

ARABIDOPSIS THALIANA FLOWERS AND SILIQUES

No consistent abnormalities were noted in the flowers of CaMV-infected A. thaliana. Both normal and abnormal siliques were found on infected plants. Some siliques were shorter than those of healthy plants but were otherwise normal in appearance. The surfaces of other siliques conformed to the pattern of seeds inside, producing an abnormal appearance. Such siliques were often contorted rather than straight and were not dehiscent.

In terminal flower clusters very small contorted siliques were often found. One silique had an an- ther-like structure emanating from the middle of the silique. A petal-like structure was found at the analogous position in another silique.

ARABIDOPSIS THALIANA SEED SYMPTOMS

Infected plants produced normal seeds and two kinds of shrunken seeds: small seeds with angular, wrinkled seed coats, and flat seeds. Occasional seeds failed to develop. The incidence of shrunken seed varied in seed collected from individual shoots from 1.7Wo to 70.9%. Healthy plants produced less than 1% shrunken seeds. The occurrence of shrunken seeds was correlated with position of the seed on the plant and in the silique. Older siliques tended to have normal seeds while younger ones contained predominantly shrunken seeds. In siliques that contained mixtures of normal and shrunken seeds, the normal seeds were found usually at the base of the silique and the shrunken seeds at the tip. Shrunken seeds contained embryos arrested in their development. In one typical batch of 42 such seeds, there were one heart-stage embryo, 13 torpedo-stage embryos, eight curled-cotyledon-stage embryos, and 20 mature embryos that were less than full size.

Some infected plants produced seeds of normal size with a coat pigment that was more sensitive to bleaching than that in seeds from healthy plants. Within 3-6 min of immersion of these seeds in bleach, extensive patches of the seed coat became transparent. Some seed coats became completely transparent. Seeds of healthy plants required more than 15 min in bleach to become transparent. The hypersensitivity to bleach was found in seeds from three of seven plants examined that were infected with different CaMV isolates. While seeds from one shoot of a plant infected with the CM4-184 isolate were hypersensitive to bleach, seeds of a

142 BOTANICAL GAZETTE

[JUNE

neighboring diseased shoot of the same plant were not.

Germination of seeds from infected stems varied from 0% to 100%. Seeds from healthy plants tested in parallel always gave 100% germination. Ger- mination patterns of seeds of infected plants were reproducible in separate trials both when seeds were germinated on agar containing mineral salt medium and when germination was on moist filter paper. The variation in germination percentage ex- tended to batches of seed from plants infected with the same isolate of CaMV. Some shoots produced poorer germination frequencies than other shoots of the same plant. Although shrunken seeds had a lower ability to germinate, seed size was not per- fectly correlated with poor germination. Some shrunken seeds did germinate, and some normal seeds did not germinate. All seeds with bleach-hypersensitive coats failed to germinate, perhaps because of injury to the embryo. Two days after germination the seedlings from shrunken seeds were shorter than those from normal seeds. The seedlings nevertheless developed into fertile plants.

The presence of CaMV nucleic acid in seeds and seed parts was tested by hybridization. Blots of seeds, of seed coats, and of embryos from healthy plants did not bind detectable levels of CaMV DNA. CaMV DNA hybridization was readily detected in blots of seeds and seed coats from infected plants. Not all seeds bound probe detectably. No hybridization, however, was detected in embryos of seeds from infected plants even when blots of the seed coats of those embryos hybridized. Several seedlings of seeds from CaMV-infected plants were grown into mature plants. Death of the first few leaves occurred on four of 15 plants after the plants had acquired four to 10 leaves. No CaMV DNA was detected in leaves of these plants by squish hybridization. Mature plants appeared healthy, flowered, and set normal seed.

TURNIP LEAF SYMPTOMS

The following symptoms of CaMV infection of turnips were observed after infection of turnips with eight strains of five isolates of CaMV: chlorotic spotting, chlorotic mottling, vein clearing, vein banding, leaf stunting, necrotic flecking, senescence, and rugosity (distortion). Vein clearing affected the minor veins (fine vein clearing) or the major veins (coarse vein clearing). Some leaves were uniformly pale green except for a region of vari- able size in the center of the leaf which was normal green. Dark green spots or patches were occasion- ally found in younger leaves. In two of 30 plants, a dark green patch was found that bordered sharply on a yellow patch of comparable size and shape. Symptoms on a given leaf did not change with in- creasing age of the leaf.

The symptom types observed on a plant were dependent on the strain used to inoculate the plant (table 3) under the conditions of plant growth used. Fine and coarse vein clearing were characteristic of CM4-184 and related strains. Necrosis of midribs was limited to CabbB-D. Rugosity, notable with CabbB-D, CabbS, D/H, and NY8153 isolates, was barely evident with W, CM4- 184, and related strains. Stunting was either severe (CabbB-D), moderate (D /H, NY8 153, CabbS), or slight (CM4- 184, CaBB1), depending on the CaMV strain used.

There was considerable variation from plant to plant in the leaf number on which a particular symptom first appeared. Nonetheless, pigment pattern symptoms on oldest to youngest leaves of each plant followed this order: healthy, chlorotic spotting, chlorotic mottling, fine vein clearing, coarse vein clearing, pale greenness (fig. 1). When a strain did not cause vein clearing, the number of leaves exhibiting a chlorotic mottle was correspondingly greater. Necrotic flecks, when they occurred, occurred on one to four leaves, generally between leaves 9 and 14 (table 3, fig. 1). Midrib necrosis, where found, began at leaf 15-19 and continued to occur on all subsequent leaves. Since the de- pendence of symptom type on leaf age could be a reflection of the stage of leaf development at the time the leaf was first infected, the number of leaves or leaf initials was counted under a dissecting microscope. At the time of inoculation, plants had 17.9 + 1.8 leaves or leaf initials. Seven days after inoculation there were 23.7 + 1.0 leaves and initials. Inoculated leaves had to be left on the plant for 4 or more d for the plant to develop systemic symptoms. Thus, at the time the growing point became infected, leaves younger than ca. leaf 20 had not developed sufficiently for them to be distin- guished with the compound microscope.

Leaf pigment pattern symptoms were sometimes sectored. Sectoring was both left-right and radial. When sectoring was radial, symptoms progressed invariably from tip to base in the same order as from older to younger leaves. For example, one leaf had chlorotic spots at the tip, fine vein clearing in the center, and coarse vein clearing at the base. The pigment patterns on halves of left-right sectored leaves were the same as those of their phyl- lotactic neighbors (fig. 2). When only one leaf (the fifth) was inoculated, symptoms appeared on leaves emerging within about 60° of the point of attach- ment of the inoculated leaf. Leaves at the edges of this region had symptoms only on the half closest to the inoculated leaf.

CaMV nucleic acid in inoculated leaves, detected by leaf skeleton hybridization, was localized in symptomatic areas. Radioautographic images of systemically infected, chlorotically spotted leaves showed the chlorotic spots as rings slightly darker than the background. Leaf-skeleton hybridization

TABLE 3

ISOLATE-DEPENDENT VARIATION IN SYMPTOMS OF CAMV INFECTION IN TURNIP PLANTS

SYMPTOMS ISOLATE (no. of plants observed) CSP MOT FVC CVC PGN NEC MRN RUG STN

CM4-184 (5) ................. 5 (2-6) 5 (2-7) 3 (3-6) 6 (2-9) 0 (0-5) 1 (0-5) 0 (0-0) 1 (0-2) 9.7 + 2.3 CaBBl (5) ................. 5 (4-6) 3 (2-5) 4 (4-6) 7 (4-10) 0 (0-5) 2 (0-2) 0 (0-0) 1 (0-2) 9.0 + 3.6 CaNB2 (5) ................. 5 (4-8) 5 (0-6) 3 (1-6) 6 (5-7) 0 (0-0) 5 (0-6) 0 (0-0) 1 (0-6) 7.9 + 2.1 W (5) ................. 4 (1-6) 8 (6-9) 0 (0-0) 0 (0-0) 5 (3-7) 0 (0-1) 0 (0-0) 0 (0-3) 6.8 + 2.4 CabbB-D (5) ................. 4 (2-4) 11 (10-14) 0 (0-0) 0 (0-0) 4 (2-7) 4 (3-7) 7 (3-9) 7 (5-14) 2.3 + 1.2 CabbS (3) ................. 4 (2-6) 11 (6-12) 0 (0-0) 0 (0-0) 7 (0-9) 4 (2-5) 0 (0-0) 9 (8-10) 6.6 + 1.2 D/H (5) ................. 3 (2-5) 11 (5-13) 0 (0-0) 0 (0-0) 6 (3-9) 2 (1-4) 0 (0-1) 8 (5-12) 5.2 + 1.6 NY8153 (4) ................. 4.5 (3-6) 9.5 (3-12) 0 (0-0) 0 (0-0) 5 (0-6) 1.5 (0-5) 0 (0-0) 10 (10-10) 5.4 + 1.8

aDesignation of symptoms: CSP, chlorotic spotting; MC)T, mottling; FVC, fine vein clearing; CVC, coarse vein clearing; PGN, pale green; NEC, necrotic flecks on leaf blade; MRN, necrosis of the midrib; RUG, rugosity; STN, stunting. For all symptoms except STN the median number of leaves with the indicated symptom per infected plant is reported with the range of values in parentheses. For STN the mean blade length of leaves 15 through 20 is given in cm + standard deviation from the mean. For healthy plants this value was (9.5 + 3.0) cm. Plants had 25 to 30 leaves at the time of evaluation.

MELCHER CAMV IN TURNIP AND ARABIDOPSIS 143 1989]

Differences in the kinds of symptoms detected under the conditions of plant growth used depended on the species of the host, the age of the tissue when it was first infected, and the isolate of the virus used to infect the plants. Arabidopsis thaliana and turnip responded differently to inoculation with the same isolates (tables 2 and 3). The group of isolates that caused stunting of A. thaliana rosette leaves (CaNB2, CaBB1, NY8153) was different from the group that caused stunting of turnip rosette leaves (CabbB-D, NY8153, D/H, and to a lesser extent, CabbS and W). The CabbS isolate, while readily able to infect turnip plants when present in the inoculum at 2 1lg mL-t, only infected A. thaliana at higher inoculum concentrations. These differences indicate that there are sub- tle differences in the way that a given isolate interacts with the two hosts.

CaMV infection does not cause visible disease symptoms by acting primarily on the physiology and biochemistry of already differentiated tissues. If it did, then all turnip leaves should have had identical symptoms regardless of leaf age, and sectoring of symptoms in turnip and A. thaliana should not have been observed. Rather, CaMV infection to cause visible symptoms must act mainly on differentiating cells. Leaf tissue that is mature at the time that infection begins does not exhibit symptoms of disease or contain significant amounts of virus (fig. 1, table 3, and MAULE 1985). The next oldest tissue exhibited chlorotic lesions. This symptom pattern mirrors the response of leaves to mechanical inoculation. Fully expanded leaves are refractory to inoculation with virions, while ex- panding leaves develop chlorotic spots (SUN et al. 1988). Turnip leaves developing at the time that CaMV infection began developed age-dependent symptom variations (fig. 1, table 3). The hypothesis of a primary effect of infection on differen-

of systemically infected leaves with coarse vein banding symptoms revealed a uniformly light bind- ing of the probe. The distribution of CaMV DNA in noninoculated leaves of CaMV-infected plants was also assayed by leaf-blot hybridization. Blots of leaf tissue of healthy plants and of tissue from asymptomatic leaves of infected plants uniformly failed to bind a CaMV DNA probe ( 18 and 19 blots tested, respectively). Blots from tissue of asymptomatic sectors of infected leaves only rarely bound detectable levels of probe (two of 20 blots). Green areas of leaves with coarse vein clearing bound the probe as well as the vein-cleared areas did. Pale green and dark green areas of leaves with the pale- green symptom bound the probe equally well. Ho- mogenates of asymptomatic leaves or asymptomatic regions of infected leaves did not react with antiserum to CaMV in indirect ELISA tests. The sensitivity of the test was sufficient to detect 4 1lg / gm tissue.

Discussion

TOMLINSON and WALKER (1973) found that CaMV was not transmitted through seeds of Capsella bursa- pastoris or Raphanus raphanistrum even though infectious virus were found in the testae of such seeds. My finding of CaMV nucleic acid in the seed coats but not the embryos of Arabidopsis thaliana is consistent with that report. As with most virus infections that are not seed-transmitted, most prog- eny of CaMV-infected A. thaliana plants showed no symptoms of infection. The absence of seed transmission and other parental effects makes it feasible to explore the use of CaMV DNAs bearing A. thaliana nucleotide sequences as agents muta- genic to A. thaliana via homologous recombination between virally carried and chromosomal sequences. Nonhomologous recombination of CaMV DNA with host DNA is not known to occur.

nec - NY8153

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nec _ D/H

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CabbS nec

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rug _

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Cabb B-D mrn _

nec _

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CaNB2 nec

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30 30

1 0 20 1 0 20

Leaf Number Leaf Number

FIG. 1. Distribution relative to leaf age of selected symptom types on rosette leaves of turnip plants infected with CaMV isolates. For each isolate the results of evaluation of one plant representative of at least four plants are given. Leaves were numbered from oldest to youngest. Leaves 3, 4, and 5 were inoculated. See table 3 for identification of symptom types.

tiating cells predicts that turnip leaves with clearing around minor veins did not also have clearing around major veins because the latter had com- pleted differentiation at the time infection reached those cells. The order of vein differentiation pre- dicted is consistent with the known order of dif-

ferentiation of major and minor veins in maize (ESAU 1943; SHARMAN 1942). The hypothesis also predicts that leaves in which both major and minor veins existed at the time of infection would not develop vein clearing, that leaves whose major veins were differentiating at infection time would de-

1989] MELCHER-CAMV IN TURNIP AND ARABIDOPSIS 145

SOLBERG and BALD 1962; REID and MArrHEws 1966; NILSSON-;REN et al. 1969; ATKINSON and MArrHEws 1970) are also consistent with the view that viral infection acts mainly on differentiating cells to cause symptoms. It is possible that these changes in normal patterns of development result from virally induced changes in host gene expression (ZArrLIN 1979).

Symptom types appear epistatic to types characteristic of infection of older leaves. Coarse and fine vein clearing were not observed together in the same blade sector. The youngest turnip leaves, pale green in appearance, exhibited none of the symptoms of older leaves. In turnips infected with isolates that do not induce vein clearing, leaves that should have had vein clearing were chlorotically mottled, a symptom characteristic of the next oldest leaves. Thus, infection-induced changes in meristematic tissues may preclude induction of later symptom types in cells that differentiate from that tissue. Alternatively, the initial phase of CaMV infection may cause symptom-inducing changes in differentiating cells that are not produced when chronically infected cells differentiate.

As has been noted (DAUBERT et al. 1984; MAULE 1985), the complex of symptom types induced in turnip plants by different isolates of CaMV is isolate-specific (table 3). It was thus not unexpected that isolate-specific differences in symptom patterns were also noted on A. thaliana (table 2). It was, however, surprising to find that CaNB2 and CaBBl, isolates derived from isolate CM4-184 by alteration of the DNA of open reading frame II (BRISSON et al. 1984), induced stunting of A. thaliana rosette leaves, while the parental CM4-184 did not. None of these isolates can produce an open reading frame II product functional in aphid transmission. The NY8153 isolate produces a functional polypeptide from frame II (ARMOUR et al. 1983) and caused the same stunting of rosette leaves. The absence of a correlation between the ability to produce a functional polypeptide and the ability to stunt rosette leaves suggests that nucleic acid sequences may play a role, other than via their protein coding function, in the induction of at least some symptoms. The CaMV structural gene for the inclusion body matrix protein has been identified as responsible for the induction of some symptoms (DAUBERT et al. 1984; SCHOELZ et al. 1986; BAUGHMAN et al. 1988), suggesting that other symptom induction may be mediated by virally encoded proteins.

Acknowledgments

I thank DAVID MEINKE for advice on care and handling of Arabidopsis thaliana, for encourage- ment, and for constructive criticism. I thank the

FIG. 2. Arrangement of leaves with pale green (striped areas) or coarse vein clearing (grey areas) symptoms on a tunlip plant infected with the CaBBl isolate of CaMV. Leaves are numbered from oldest to youngest.

velop coarse vein clearing, and that all leaves derived from tissues that had not differentiated at the time of infection would have the same symptoms pattern. The predictions are consonant with the observed leaf-age-dependent order of symptom types. The distribution of symptoms on turnip plants inoculated on a single leaf suggests that radial spread of infection was limited and that the infection path- way did not readily cross leaf midribs. Left-right sectoring of symptoms on turnip leaves (fig. 2) can thus be explained by infection reaching one-half of the leaf blade before infecting the other half. Sim- ilarly, tip-to-base sectoring of symptoms probably arose when tip tissue was more mature than base tissue when they were first infected, either because of earlier development of the tip tissue or because viral spread from base to tip was slow relative to maturation of the tissue.

Chimerism was also observed for several symptoms of infection of A. thaliana by CaMV. Some nonuniformly distibuted symptoms of infection (shrunken seeds, reduced internodes) are likely to be secondary effects of reduced photosynthesis re- sulting from chlorosis. Failure to germinate and bleach hypersensitivity were probably the result of physiological impairments since both full and shrunken seeds had these properties. The chimerism of chlorosis was seen both between different shoots of the same plant and within individual shoots. The chimerism within shoots could result from a difference in the times at which the infection began within the cluster of cells making up the shoot initial. Observations of the symptoms induced by RNA-containing viruses (ZECH 1952;


individuals listed in table 1 for providing CaMV isolates and variants, ELIOT MEYEROWITZ for a sample of psAt2105, MARY SCHATZ and ANN WIL LIAMS for technical assistance, and ROD PENNING

TON for helpful discussions. The study was supported by the Herrnan Frasch Foundation and the Oklahoma Agncultural Experiment Station, of which this is article J-5426.

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Symptoms of Cauliflower Mosaic Virus Infection in Arabidopsis thaliana and Turnip