Isolation of an Arabidopsis thaliana Mutant in Which the Multiplication of both Cucumber Mosaic Virus and Turnip Crinkle Virus Is Affected

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1 JOURNAL OF VIROLOGY, Nov. 1998, p Vol. 72, No X/98/$ Copyright 1998, American Society for Microbiology. All Rights Reserved. Isolation of an Arabidopsis thaliana Mutant in Which the Multiplication of both Cucumber Mosaic Virus and Turnip Crinkle Virus Is Affected MOTOYASU YOSHII, NORIMICHI YOSHIOKA, MASAYUKI ISHIKAWA,* AND SATOSHI NAITO Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo , Japan Received 13 April 1998/Accepted 18 July 1998 During the systemic infection of plants by viruses, host factors play an important role in supporting virus multiplication. To identify and characterize the host factors involved in this process, we isolated an Arabidopsis thaliana mutant named RB663, in which accumulation of the coat protein (CP) of cucumber mosaic virus (CMV) in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in RB663 plants was controlled by a monogenic, recessive mutation designated cum2-1, which is located on chromosome III and is distinct from the previously characterized cum1 mutation. Multiplication of CMV was delayed in inoculated leaves of RB663 plants, whereas the multiplication in RB663 protoplasts was similar to that in wild-type protoplasts. This suggests that the cum2-1 mutation affects the cell-to-cell movement of CMV rather than CMV replication within a single cell. In RB663 plants, the multiplication of turnip crinkle virus (TCV) was also delayed but that of tobacco mosaic virus was not affected. As observed with CMV, the multiplication of TCV was normal in protoplasts and delayed in inoculated leaves of RB663 plants compared to that in wild-type plants. Furthermore, the phenotype of delayed TCV multiplication cosegregated with the cum2-1 mutation as far as we examined. Therefore, the cum2-1 mutation is likely to affect the cell-to-cell movement of both CMV and TCV, implying a common aspect to the mechanisms of cell-to-cell movement in these two distinct viruses. Systemic infection of plants by viruses occurs through complex interactions between virus-encoded and host-encoded factors. Although much information on the roles of viral factors involved in the infection process is available, little is known about the host factors involved. To understand the molecular mechanism of virus multiplication in plants, it is necessary to identify and characterize such host factors. During the process of systemic infection, positive-strand RNA viruses uncoat to release genomic RNA in the cytoplasm of host cells and replicate by using replication proteins that are translated from the genomic RNA. Then the viruses move from an infected cell to adjacent cells via plasmodesmata, a process mediated by virusencoded movement proteins (MPs). In many virus-host combinations, cell-to-cell movement is the key step which determines susceptibility. Finally, the viruses enter the phloem and rapidly move to noninfected tissues at some distance from the inoculated leaf (long-distance movement). At present, several host factors necessary for efficient plant viral RNA replication have been identified (31, 33), but those necessary for the local or systemic spreading of plant viruses remain unknown. Cucumber mosaic virus (CMV) has three individual segments of capped messenger-sense RNA as a genome, RNAs 1, 2, and 3 (reviewed in reference 32). RNAs 1 and 2 encode proteins 1a and 2a, respectively. Both 1a and 2a are necessary for viral RNA replication in a single cell (15, 29), and they have amino acid sequence similarities to the replication proteins of other alpha-like viruses, including tobacco mosaic virus (TMV) (1, 12, 14). RNA 2 also encodes a protein designated 2b (9), which is suggested to be involved in host-specific long-distance * Corresponding author. Mailing address: Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo , Japan. Phone: Fax: ishikawa@abs.agr.hokudai.ac.jp movement of the virus (10). RNA 3 encodes two proteins, 3a and the coat protein (CP). The 3a protein is translated directly from RNA 3 and has an approximate molecular weight of 30,000, whereas CP is translated from subgenomic RNA 4, which is synthesized during replication (35). Both the 3a protein and CP are necessary for efficient cell-to-cell movement of CMV (4, 6, 8, 37). Arabidopsis thaliana is widely regarded as an ideal model plant for genetic and molecular biological studies (reviewed in references 7, 21, 28, and 36). Furthermore, the Y strain of CMV (CMV-Y), the Cg strain of TMV (TMV-Cg), and the B strain of turnip crinkle virus (TCV-B) systemically infect A. thaliana ecotype Col-0 plants without causing visible hypersensitive reactions (17, 25, 38, 43). Therefore, a complete set of host factors which support efficient multiplication of CMV-Y, TMV-Cg, and TCV-B are present in this ecotype. As a first step to the identification of host factors which support virus multiplication, we have previously isolated mutants of A. thaliana in which TMV-Cg or CMV-Y cannot multiply efficiently (17, 30, 43). The tom1 and tom2 (tobamovirus multiplication) mutations affect TMV multiplication within a single cell, whereas the cum1 (cucumovirus multiplication) mutation affects the local spreading of CMV within the inoculated leaf. Each mutation is single, recessive, nuclear, and virus specific; i.e., tom1 and tom2 mutations do not affect the multiplication of CMV or TCV, and the cum1 mutation does not affect that of TMV or TCV. These characteristics suggest that the corresponding wild-type gene product supports virus multiplication through specific interactions with virus-encoded factors. In this study, we have analyzed a second mutant of A. thaliana, RB663, in which the multiplication of CMV is delayed and show that the causal mutation, cum2, is likely to affect the local spreading of both CMV and TCV. The genome of TCV is thought to encode two separate nonstructural proteins with Downloaded from on July 9, 2018 by guest

2 8732 YOSHII ET AL. J. VIROL. molecular weights of ca. 8,000 and 9,000, which are both necessary for virus movement (13, 24). Despite the differences in molecular weight and number of movement proteins between CMV and TCV, our results suggest that the wild-type CUM2 gene product is involved in the cell-to-cell movement of these two distinct viruses. MATERIALS AND METHODS Viruses and antisera. CMV-Y (37), TMV-Cg (26, 42), and TCV-B (16) were propagated, and their virus particles and virion RNAs were purified as described by Ishikawa et al. (17, 18). Rabbit antiserum against TMV-Cg was obtained from E. Shikata, rabbit antiserum against TCV-M was provided by A. E. Simon and C. Zhang, and rabbit antiserum against CMV-D was obtained from American Type Culture Collection (Manassas, Va.). The rabbit antiserum against either CMV-D and TCV-M efficiently cross-reacts with the CP of CMV-Y and TCV-B, respectively. Plant materials and growth conditions. A. thaliana (L.) Heynh. Columbia (Col-0) and Landsberg erecta (Ler) were used as the wild-type strains. A. thaliana RB568 (the cum1-1 mutant) and RB663, in which the accumulation of CMV-Y CP is delayed, were isolated from an M 2 population derived from ethyl methanesulfonate-mutagenized A. thaliana Col-0 seeds as described by Yoshii et al. (43). Seeds were sown on rockwool soaked in distilled water, incubated in the dark for 2 days at 4 C, and grown at 22 to 23 C under continuous fluorescent illumination. The plants were watered with a nutrient medium as described by Fujiwara et al. (11). Plant inoculation. Mechanical inoculation of A. thaliana plants with CMV-Y, TMV-Cg, and TCV-B was carried out as described by Yoshii et al. (43). Growth conditions after virus inoculation for examination of viral CP accumulation in inoculated or uninoculated leaves were as described by Yoshii et al. (43). Detection and quantification of viral CP. Extraction of total protein from A. thaliana plants and protoplasts, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and quantification of viral CP were performed as described by Yoshii et al. (43). The dot enzymelinked immunosorbent assay (ELISA) method was also carried out as described by Yoshii et al. (43) and used to determine the CMV multiplication phenotype of the F 2 lines in Table 1. To determine the CMV multiplication phenotype of F 2 lines as shown in Table 2 or of F 2 mapping lines, we inoculated 10 to 12 F 3 plants per line with either CMV-Y or TCV-B, bulk harvested the aerial parts of the inoculated plants at 5 or 6 days postinoculation (p.i.), and determined the concentration of viral CPs in the protein extract by using SDS-PAGE and Coomassie brilliant blue R-250 (CBB) staining. By using this bulk-harvesting assay, lines homozygous for the cum2-1 mutation showed CMV-Y or TCV-B CP accumulation similar to that of infected RB663 plants (i.e., almost undetectable [see Fig. 4]) and those with either cum2-1/cum2 or CUM2/CUM2 genotypes showed a higher accumulation of viral CPs (see Fig. 4). This method was relatively quick and easy, and as mentioned in the footnote to Table 2, gave equivalent results to those obtained by the dot ELISA method shown in Table 1. Genetic mapping. To map the cum2-1 mutation, reciprocal crosses were performed between RB663 (derived from Col-0) and wild-type Ler plants. Individual F 2 mapping lines were established by harvesting F 3 seeds from each of the self-pollinated F 2 plants. From these lines, we selected 14 lines in which the accumulation of TCV-B was reduced at 5 or 6 days p.i., by using the bulkharvesting method mentioned above. In these 14 lines, CMV-Y accumulation was also reduced at 6 days p.i. We then extracted genomic DNA (2) from approximately 18 combined F 3 plants per selected line. The simple sequence length polymorphism (SSLP) or cleaved amplified polymorphic sequence (CAPS) markers nga63 and nga280 on chromosome I, nga168 on chromosome II, nga162, GAPA, GL1, nga112, and nga6 on chromosome III, nga8 on chromosome IV, and nag106 and nga76 on chromosome V were subsequently used to examine the genotype of each line. Information on the nucleotide sequences of PCR primers and map positions of DNA markers were obtained from the A. thaliana database ( in March 1998 or from the literature (3, 20). Primers for the DNA markers were purchased from Research Genetics (Huntsville, Ala.). The PCR conditions were as described by Bell and Ecker (3) when SSLP markers were used and as described by Konieczny and Ausubel (20) when CAPS markers were used. Preparation and inoculation of A. thaliana protoplasts. A. thaliana protoplasts were prepared from suspension-cultured calli and inoculated with virion RNAs by electroporation as described by Ishikawa et al. (18). Northern blot analysis. Total nucleic acids were extracted from protoplasts, purified, denatured with glyoxal, separated in 1% agarose gels, transferred onto GeneScreen membranes (DuPont-NEN, Boston, Mass.), and hybridized with 32 P-labelled probes, as described by Ishikawa et al. (18). Probes used for detection of virus-related RNAs were prepared as described by Yoshii et al. (43). A ubiquitin gene-specific probe was prepared as follows. UBQ5-specific DNA was amplified by PCR from genomic DNA of A. thaliana Col-0 with the primers dgtggtgctaagaagaggaaga and dtcaagcttcaactccttcttt (34) and cloned into pcr2.1 with an Original TA cloning kit (Invitrogen, San Diego, Calif.) to obtain pcr-ubq5. pgem-ubq5 was then constructed by inserting the 266-bp EcoRI fragment from pcr-ubq5 into the EcoRI site of pgem-7zf( ). The nucleotide sequence of the insert DNA was confirmed by dye terminator cycle sequencing with an automated DNA sequencer (PRISM 373; Applied Biosystems, Foster City, Calif.). 32 P-labelled ubiquitin gene-specific RNA probe was synthesized from XbaI-digested pgem-ubq5 with SP6 RNA polymerase (27). After hybridization and washing, Northern blots were analyzed with a Bio Imaging Analyzer (BAS1000; Fuji Photo Film, Tokyo, Japan). RESULTS Isolation of an A. thaliana mutant, RB663, in which the accumulation of CMV-Y CP is delayed. To identify host factors involved in CMV multiplication, we previously screened for A. thaliana mutants in which the accumulation of CMV-Y CP in upper uninoculated leaves was reduced to low levels and isolated two such mutants from 4,800 M 2 plants derived from ethyl methanesulfonate-mutagenized Col-0 seeds (43). In this paper, we report the characterization of one of these mutants, RB663. RB663 plants grew normally under our growth conditions. To examine the accumulation pattern of CMV-Y in RB663 plants, aerial tissues of CMV-infected plants were harvested at 1, 3, 5, 8, 10, and 14 days p.i. and divided into four regions as illustrated in Fig. 1A (the first to third leaves [R1], the fourth to sixth leaves [R2], the seventh to ninth leaves [R3], and a mixture of cauline leaves, stems, and unexpanded rosette leaves that lack petioles [Cau]), and the accumulation of the CP in each region was determined (Fig. 1). Inoculated leaves withered within a few days after inoculation and were excluded from this analysis. In wild-type Col-0 plants, CMV-Y CP was first detected in the combined region of Cau and R3 at 3 days p.i. The concentration of CMV-Y CP in the upper regions of infected Col-0 plants was higher at 5 days p.i. but lower at 14 days p.i. than in the lower regions (Fig. 1B). In contrast, the accumulation of CMV-Y CP in RB663 plants was almost negligible until 5 days p.i. The concentration of CMV-Y CP in the upper regions of infected RB663 plants was higher than in the lower regions at 8, 10, and 14 days p.i. The accumulation pattern of CMV-Y CP in RB663 plants at 10 and 14 days p.i. was similar to that in wild-type Col-0 plants at 5 days p.i. Thus, CMV multiplication in RB663 plants was delayed compared to that in wild-type plants. Under our growth conditions, no apparent local necrotic lesions that would indicate one of the hypersensitive responses were observed on inoculated leaves of RB663 plants (data not shown). Delayed accumulation of CMV CP in RB663 plants is controlled by a monogenic, recessive trait which is distinct from the cum1 mutation. To determine the genetic basis of the phenotype of delayed accumulation of CMV-Y CP in RB663 plants, crosses were carried out between RB663 and wild-type Col-0 plants. The level of CMV-Y CP accumulation in F 1 plants was similar to that in Col-0 plants at 6 days p.i. (Fig. 2), indicating that the mutant allele is not dominant. We then examined the segregation of the phenotype in the F 2 generation (F 2 plants were obtained by self-pollination of the F 1 plants). Since plants infected with CMV-Y have poor fertility, the phenotype of F 2 plants cannot be reconfirmed in the F 3 generation if F 2 plants are directly inoculated. Therefore, we harvested F 3 seeds from each F 2 individual separately (hereafter, we refer to each pool of F 3 seeds or plants derived from a single F 2 individual as an F 2 line) and determined the phenotype of these F 2 lines as follows: 10 to 12 F 3 plants per F 2 line were inoculated with CMV-Y, the R3 leaves (Fig. 1A) were harvested separately at 6 days p.i., and the accumulation of CMV-Y CP was detected separately by the dot ELISA method. When RB663 direct descendants were assayed for CMV accumulation by this method, all the plants showed sig-

3 VOL. 72, 1998 A. THALIANA MUTANTS WITH DELAYED CMV AND TCV SPREADING 8733 FIG. 1. Time course of accumulation of CMV-Y CP in wild-type Col-0 and RB663 plants. (A) The aerial tissues of four inoculated individuals were harvested separately at various times p.i. and dissected into four regions: R1 (the first to third leaves), R2 (the fourth and sixth leaves), R3 (the seventh to ninth leaves), and Cau (a mixture of cauline leaves, stems, and unexpanded rosette leaves that lack petioles). Prior to harvesting, CMV-Y was inoculated on the fifth leaf, indicated by I. (B and C) The accumulation of CMV-Y CP in Col-0 (B) and in direct descendants (M 6 generation) of RB663 (C) was quantitated for each region (Cau, R1, R2, and R3) as described by Yoshii et al. (43). Briefly, totalprotein samples were prepared and separated by SDS-PAGE, CMV CP was detected by CBB staining or the immunoblotting method with anti-cmv CP antibodies, and the concentration of the CP was estimated by comparing the band intensity with that of a known amount of purified CMV CP standards. Means and standard deviations of CMV-Y CP concentrations calculated from the data obtained with four individuals are shown. Similar results were obtained in triplicate experiments. The asterisks in panels B and C indicate that the regions Cau and R3 could not be harvested separately and hence were combined for analysis. Inoculated leaves withered at 2 to 3 days p.i. under the conditions used for this experiment, and thus the accumulation of CMV-Y CP in inoculated leaves was not examined. FIG. 2. Accumulation of CMV-Y CP in F 1 plants. Thirteen individuals each of RB663 (M 6 ), RB568 (M 6 ), wild-type Col-0, and F 1 plants derived from the reciprocal crosses between Col-0 and RB663 (M 5 ) plants and 20 individuals of F 1 plants derived from the reciprocal crosses between RB568 (M 5 ) and RB663 (M 5 ) plants were inoculated with CMV-Y. At 6 days p.i., one of the R3 leaves (Fig. 1A) was harvested separately from each plant, and the level of CMV-Y CP accumulation was determined, as in Fig. 1. Means and standard deviations of CP concentrations are shown. The M 6 plants of RB568 and RB663 were direct descendants of the M 5 plants of RB568 and RB663 used for the crosses. which carries a monogenic, recessive mutation designated cum1-1 (43). The accumulation of CMV-Y CP in the F1 plants was similar to that in wild-type Col-0 plants (Fig. 2), suggesting that the causal mutation in RB663 plants is distinct from cum1. We named the causal mutation in RB663 plants cum2-1. The map position of the cum2 locus was determined by using another set of F 2 lines (these F 2 lines are referred to below as the mapping lines) that were derived from F 1 plants obtained by the crosses between the RB663 plants (derived from Col-0) and Ler, a wild-type ecotype distinct from Col-0. The multiplication of CMV-Y and TCV-B in Ler plants was similar to that in Col-0 plants. We selected mapping lines in which the accumulation of CMV-Y CP was reduced to low levels at 6 days p.i. (for details, see Materials and Methods) and prepared genomic DNA from each of these lines. The genotype at PCRbased polymorphic DNA markers covering the A. thaliana genome (see Materials and Methods) was then determined for the DNA preparations. The numbers of chromatids recombined between a DNA marker and cum2-1 mutation with respect to the total numbers of chromatids examined were 13 of 28, 13 of 28, 12 of 28, 8 of 28, 8 of 28, 2 of 28, 1 of 28, 1 of 28, nals similar to that derived from noninfected plants, or only 1 or 2 of the 10 plants showed positive signals weaker than that of CMV-infected Col-0 plants at 6 days p.i. In approximately one-fourth of the F 2 lines, all F 3 plants accumulated similar levels of CMV-Y CP to that in RB663 plants (these F 2 lines are classified as low level), whereas in the other F 2 lines, more than half of the F 3 plants accumulated similar levels of CMV-Y CP to that in infected Col-0 plants (these F 2 lines are classified as high level) (Table 1). These results are consistent with the hypothesis that the phenotype of delayed accumulation of CMV-Y CP in RB663 plants is controlled by a monogenic, recessive trait. Next, crosses were carried out between RB663 and RB568, another mutant plant with delayed multiplication of CMV-Y TABLE 1. Segregation of the phenotype of low-level CMV-Y CP accumulation in F 2 generations Cross (female male) No. of F 2 lines in which CMV- Y CP accumulated to b : High levels Low levels Col-0 RB663 a d RB663 Col d a RB663 (M 5 ) plants were used for crosses. b The phenotype of F 2 lines was determined by the dot ELISA method as described in the text. c The chi-square values were calculated on the basis of the expected ratio of high-level to low-level accumulation 3:1. d Not significantly different from a 3:1 segregation (P 0.05). 2c

4 8734 YOSHII ET AL. J. VIROL. FIG. 3. Map location of cum2-1 mutation on chromosome III. The map positions (in centimorgans) of the markers are based on the data of Camilleri et al. (5) and are shown on the left. The markers on chromosome III that we tested for linkage analysis are indicated on the right. SSLP and CAPS markers are shown by open boxes and open circles, respectively. The number of the chromatids recombined between a marker and the cum2-1 locus with respect to the total number of chromatids examined is shown in parentheses. 13 of 28, 10 of 28, and 9 of 28 for DNA markers nga63, nga280, nga168, nga162, GAPA, GL1, nga112, nga6, nga8, nag106, and nga76, respectively. As shown in Fig. 3, some of the markers on chromosome III showed linkage with the cum2-1 mutation. The other markers showed no significant linkage with the mutation. From these data, the cum2-1 mutation was suggested to be located within a region between markers GL1 (map position 44.7 centimorgans [cm]) and nga112 (map position 77.0 cm) on chromosome III (Fig. 3). Susceptibility of RB663 plants to viruses other than CMV-Y. The accumulation of TMV-Cg and TCV-B CPs in RB663 mutant plants was examined to determine whether the mutation also affects the multiplication of viruses other than CMV. TCV and TMV belong to different taxonomic groups from CMV and have been shown to multiply systemically in wildtype Col-0 plants (17, 25). The time course of TMV-Cg CP accumulation in RB663 plants was similar to that in wild-type Col-0 plants (Fig. 4; TMV-Cg CP accumulation was also similar at 13 days p.i. [data not shown]), whereas the accumulation of TCV-B CP was lower than that in Col-0 plants at 6 or 8 days p.i. (Fig. 4). At 10 or 13 days p.i., TCV-B CP accumulation was still lower in RB663 plants than in Col-0 plants, but the difference was smaller than that in the earlier stages (data not shown), suggesting that TCV-B CP accumulation was delayed in RB663 plants. To determine the genetic basis of the phenotype of delayed accumulation of TCV-B CP, we examined the phenotype of plants derived from reciprocal crosses between RB663 and wild-type Col-0 plants. In all of the F 1 plants, the accumulation of TCV-B CP at 6 days p.i., as determined by SDS-PAGE and CBB staining of total protein, was similar to that in Col-0 plants (data not shown). Of the lines randomly selected from the F 2 lines established by performing crosses between RB663 FIG. 4. Accumulation of CP of CMV-Y, TMV-Cg, and TCV-B in wild-type Col-0 and RB663 plants. Ten individuals each of Col-0 (A) and RB663 (B) plants were inoculated with CMV-Y, TMV-Cg, or TCV-B virions. The RB663 plant line used was obtained through two cycles of backcrossing, i.e., selecting a cum2-1 line from F 2 lines resulting from the self-pollination of F 1 plants generated by the crossing of cum2-1 pollen to Col-0 flowers. Total protein extract was separately prepared from aerial tissues of inoculated plants that were harvested at 3, 6, and 8 days p.i., as indicated above the panels, equal volumes of the extracts for each inoculation period were mixed, and the samples derived from 0.6 mg (fresh weight) of tissue were analyzed by SDS-PAGE with 11% (for TMV-Cg and CMV-Y) or 9% (for TCV-B) polyacrylamide gels and CBB staining. Total proteins from mock-inoculated plants were concurrently electrophoresed (lanes M). The positions of viral CPs are indicated by arrows. Similar results were obtained in triplicate experiments. and Col-0 plants and used to produce the data shown in Table 1, approximately one-fourth showed reduced accumulation of TCV-B CP (Table 2; for [29:11] segregation, 2 [3:1] 0.133, P 0.05; for [42:8] segregation, 2 [3:1] 2.16, P 0.05), suggesting that the causal mutation was a monogenic, recessive trait. Furthermore, all of the 19 F 2 lines which showed lowlevel accumulation of TCV-B CP showed low-level accumulation of CMV-Y CP, and the other 71 F 2 lines which showed high-level accumulation of TCV-B CP also showed high-level accumulation of CMV-Y CP (Table 2). None of the F 2 lines with high-level accumulation of TCV-B CP showed low-level accumulation of CMV-Y CP, and none of the F 2 lines with low-level accumulation of TCV-B CP showed high-level accumulation of CMV-Y CP. These results suggest that the cum2-1 mutation also affects TCV-B multiplication or that the casual mutation for the phenotype of reduced accumulation of TCV-B CP in RB663 plants is distinct from, but closely linked to the cum2-1 mutation (within 1.2 cm). Multiplication of CMV-Y, TCV-B, and TMV-Cg in inoculated leaves of RB663 plants and in RB663 protoplasts. To investigate the mechanisms of inhibition of CMV and TCV multiplication in RB663 plants, we first examined the CP accumulation within inoculated leaves. The accumulation of CMV-Y and TCV-B CPs in inoculated leaves of RB663 plants was decreased compared to that in Col-0 plants, whereas the

5 VOL. 72, 1998 A. THALIANA MUTANTS WITH DELAYED CMV AND TCV SPREADING 8735 TABLE 2. Segregation of the phenotypes of CMV-Y or TCV-B CP accumulation in F 2 generations Cross (female male) CMV-Y CP accumulation a No. of F 2 lines No. of F 2 lines in which TCV-B CP accumulated to a : High levels Low levels Col-0 RB663 High level Low level RB663 Col-0 High level Low level a A total 90 plant lines randomly selected from the F 2 lines used in the experiment in Table 1, which were derived from self-pollinated F 1 plants made by crosses between RB663 and Col-0 plants, were analyzed. The accumulation levels of CMV-Y or TCV-B CPs in the F 2 lines were determined by the bulkharvesting/sds-page method at 6 days p.i. as described in Materials and Methods. If the accumulation of CMV-Y or TCV-B CPs was similar to that in RB663 plants, the phenotypes of the corresponding F 2 line was counted as low level. If the accumulation of CMV-Y or TCV-B CPs were higher than that in RB663 plants, the phenotype of the corresponding F 2 plant was counted as high level. By using this assay method, all the F 2 lines displayed the same phenotype as determined by the dot ELISA method, confirming the accuracy of the two assay methods used for the experiments in Tables 1 and 2. time course and the level of CP accumulation of TMV-Cg was similar to that in wild-type Col-0 plants (Fig. 5), irrespective of whether the plants were inoculated with virion or virion RNA. The accumulation of CMV-Y and TCV-B CPs at 48 h p.i. in inoculated leaves of RB663 plants was approximately 1/5 and 1/10, respectively, of that in wild-type Col-0 plants (average of eight independent experiments [data not shown]). Thus, in RB663 plants, CMV and TCV multiplication is at least affected prior to the long-distance movement and after the uncoating of virus particles in an initially infected cell. Next, to determine whether the multiplication of CMV-Y and TCV-B within a single cell was affected in RB663, we examined the accumulation of virus-related RNAs in RB663 and wild-type Col-0 protoplasts inoculated with virion RNAs of CMV-Y, TCV-B, or TMV-Cg by electroporation. The time course and the level of accumulation of these virus-related RNAs and CPs were similar between RB663 and wild-type protoplasts. The accumulation of viral RNAs at early stages after inoculation is shown in Fig. 6 (consistent results were obtained in three independent experiments). The accumulation of each viral RNA at 24 h p.i. and the accumulation of each viral CP at 24 and 48 h p.i. were also similar between RB663 and Col-0 protoplasts (data not shown). These results suggest that the cum2-1 mutation does not affect the multiplication of either CMV or TCV within a single cell but, rather, affects the local spreading of these viruses within an inoculated leaf. DISCUSSION We have characterized an A. thaliana mutant, RB663, in which the accumulation of CMV-Y CP in uninoculated leaves of infected plants is delayed compared to that in wild-type Col-0 plants. The causal mutation, cum2-1, was monogenic, recessive, and distinct from a previously identified cum1 mutation (43). The simplest interpretation of the recessiveness of the cum2-1 mutation is that the wild-type CUM2 gene product is necessary for the efficient multiplication of CMV and that the cum2-1 allele has lost the function to support CMV multiplication. However, at present, other possibilities, e.g., that the CUM2 gene product represses the activity (such as host defense functions induced by virus infection) that represses CMV multiplication, cannot be excluded. In RB663 plants, TMV multiplication was similar to that in wild-type plants. Thus, delayed multiplication of CMV-Y was not derived from a nonspecific effect on viral multiplication, such as the inhibition of initial invasion of virion or virion RNAs through changes of leaf surface structure or low metabolic activity of RB663 plants. The accumulation of CMV-Y CP within inoculated leaves of RB663 plants infected with either CMV virion or CMV virion RNA took place slowly compared to that in wild-type Col-0 plants (Fig. 5). In contrast, the accumulation of CMV-Y-related RNAs in RB663 protoplasts was similar to that in Col-0 protoplasts (Fig. 6). Such characteristics of the cum2-1 mutation are similar to those of the cum1-1 mutation. These results suggest that the cum2-1 mutation does not affect the uncoating of CMV virion in an initially infected cell or the amplification FIG. 5. Time course of the accumulation of CMV-Y, TCV-B, and TMV-Cg CPs in inoculated leaves of wild-type Col-0 and RB663 plants. Col-0 or RB663 plants were inoculated with virion (A) or virion RNA (B) of CMV-Y, TCV-B, or TMV-Cg. The RB663 plants used were from the same lines as those in the experiment in Fig. 4. Five inoculated leaves were harvested at time zero (immediately after the inoculum solution was washed off with distilled water) and at 24, 48, and 72 h p.i. (hpi) as indicated above the lanes. Total proteins were prepared from each sample and separated by SDS-PAGE, and the CPs were detected by the immunoblotting method (CBB staining was not used here because the amount of CP was relatively small). In the panels for CMV-Y or TCV-B virion-inoculated plants, total protein from 0.06 mg of leaf tissue was applied to each lane. In the panels for TCV-B RNA-inoculated plants, total protein from 0.12 mg of leaf tissue was applied to each lane. In the other panels, total protein from 0.6 mg of leaf tissue was applied to each lane. In lanes M, total protein from 0.6 mg of mock-inoculated plants was used. Similar results were obtained in eight independent experiments.

6 8736 YOSHII ET AL. J. VIROL. FIG. 6. Time course of the accumulation of CMV-Y, TCV-B, and TMV-Cg-related RNAs in wild-type Col-0 and RB663 protoplasts. (A) Protoplasts were prepared from liquid-cultured calli derived from seedlings of Col-0 or RB663 plants. The RB663 plants used were from the same line as that in the experiment in Fig. 4 (the yield and quality of protoplasts were low if they were prepared from direct descendants of RB663). Approximately five million Col-0 or RB663 protoplasts were mock inoculated (lanes M) or inoculated with either 7 g of CMV-Y RNA, 20 g of TMV-Cg RNA, or 5 g of TCV-B RNA by electroporation. Inoculated protoplasts were cultured for 2, 4, 6, and 8 h as indicated above the panels and harvested for analysis of RNA. Duplicate Northern blots of total RNA extracted from the protoplasts were prepared: one set was hybridized with probes that detect either CMV-Y-related RNAs, TMV-Cg-related RNAs, or TCV-B-related RNAs, and the other set was hybridized with a probe that detect UBQ5 mrna. The positions of bands for CMV-Y RNAs 1, 2, 3, and 4, TMV-Cg genomic RNA, 30,000-molecular-weight protein and CP subgenomic mrnas, TCV-B genomic RNA, or UBQ5 mrna are indicated to the right of each panel. (B) Graphic representation of the time course of viral RNA accumulation in protoplasts. The intensity of bands for CMV-Y RNA 4, TMV-Cg genomic RNA, TCV-B genomic RNA, and UBQ5 mrna on Northern blots was quantified. Boxes and error bars show means and standard deviations in three independent experiments of relative viral RNA accumulation normalized by the intensity of UBQ5 mrna bands in corresponding lanes (intensity of UBQ5 mrna 1). of CMV-related RNAs within infected cells but, rather, affects the spreading from cell to cell within the inoculated leaf. At present, however, another possibility, i.e., that the host response to repress CMV multiplication is more strongly induced in RB663 plants, cannot be excluded. Furthermore, in addition to the less efficient spreading of CMV within an inoculated leaves, it is possible that long-distance movement of CMV is also affected in RB663 plants. Comparison of the size exclusion limit of plasmodesmata in leaf tissue cells in the presence of CMV 3a movement protein (MP) (either microinjected or transgenically expressed) between wild-type and RB663 plants or determination of the localization of CMV MP within infected tissue may provide us with further information on the mechanisms of inhibition of CMV multiplication by the cum2-1 mutation. In RB663 plants, TCV multiplication was also delayed in either inoculated or systemically infected leaves (Fig. 4 and 5). Furthermore, in RB663 protoplasts, the multiplication of TCV was similar to that in wild-type protoplasts (Fig. 5). These results suggest that the cell-to-cell movement of TCV was affected in RB663 plants, as observed with CMV. As far as we examined, the phenotypes of delayed accumulation of CMV and TCV cosegregated without exception. Therefore, it is likely that the cum2-1 mutation affects the multiplication of TCV as well as that of CMV, although, at present, the remote possibility that an independent mutation affecting TCV multiplication is present near the cum2 locus cannot be excluded. Molecular cloning of the wild-type CUM2 locus and introduction of a minimal DNA segment complementing the cum2-1 mutation into RB663 plants will test these possibilities. Provided that the cum2-1 mutation affects the multiplication of TCV, this is a unique characteristic, since most resistance genes of plants against viruses are specific to each virus. As far as we are aware, this is the first report of an A. thaliana mutation affecting two distinct viruses. For the cell-to-cell movement of CMV, two virus-encoded proteins, 3a MP and CP, are necessary (4, 19, 37). 3a MP is known to localize at plasmodesmata (40), where it is able to enlarge the size exclusion limit (8, 39) and cooperatively bind single-stranded nucleic acids in a non-sequence-specific manner in vitro (23). The genome of TCV encodes two nonstructural proteins, p8 and p9, which are both necessary for the

7 VOL. 72, 1998 A. THALIANA MUTANTS WITH DELAYED CMV AND TCV SPREADING 8737 cell-to-cell movement of TCV (13, 22, 24). TCV CP is necessary for the cell-to-cell movement in Nicotiana benthaniana but is not necessary in A. thaliana, Chenopodium amaranticolor, or Brassica campestris (13, 22, 24). TCV p8 is reported to have an RNA binding activity like that of CMV MP (41), but any other functional or structural similarity between the MPs of CMV and TCV has not been pointed out so far. Despite the apparent difference in the organization of the MPs between CMV and TCV, our results may suggest a common aspect to the mechanisms underlying the cell-to-cell movement of these two viruses, mediated by the CUM2 factor. At the same time, however, it is also possible that the CUM2 factor has nonhomologous functions during the cell-to-cell movement of CMV and TCV. ACKNOWLEDGMENTS We are grateful to D. Bartlem, E. Nambara, and T. Yamanaka for insightful discussion and to K. 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8 JOURNAL OF VIROLOGY, July 2003, p Vol. 77, No X/03/$ DOI: /JVI AUTHOR S CORRECTION Isolation of an Arabidopsis thaliana Mutant in Which the Multiplication of Both Cucumber Mosaic Virus and Turnip Crinkle Virus Is Affected Motoyasu Yoshii, Norimichi Yoshioka, Masayuki Ishikawa, and Satoshi Naito Department of Applied Bioscience, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo , Japan Volume 72, no. 11, p , The cum2 mutant protoplasts used in this study were found to be heavily contaminated with wild-type or heterozygous protoplasts. Therefore, the data obtained by using the cum2 mutant protoplasts (Fig. 6 and the related parts of the text) are incorrect. We regret any inconvenience that this may have caused. 8178