Isolation of an Arabidopsis thaliana mutant in which the multiplication of both cucumber mosaic virus and turnip crinkle virus is affected

Abstract

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.

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 (M₆ generation) of RB663 (C) was quantitated for each region (Cau, R1, R2, and R3) as described by Yoshii et al. (43). Briefly, total-protein 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 F₁ plants. Thirteen individuals each of RB663 (M₆), RB568 (M₆), wild-type Col-0, and F₁ plants derived from the reciprocal crosses between Col-0 and RB663 (M₅) plants and 20 individuals of F₁ plants derived from the reciprocal crosses between RB568 (M₅) and RB663 (M₅) 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 M₆ plants of RB568 and RB663 were direct descendants of the M₅ plants of RB568 and RB663 used for the crosses.

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.

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 F₂ lines resulting from the self-pollination of F₁ 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.

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.

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 μg of CMV-Y RNA, 20 μg of TMV-Cg RNA, or 5 μg 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).