A single amino acid change in turnip crinkle virus movement protein p8 affects RNA binding and virulence on Arabidopsis thaliana

Abstract

Comparison of the symptoms caused by turnip crinkle virus strain M (TCV-M) and TCV-B infection of a resistant Arabidopsis thaliana line termed Di-17 demonstrates that TCV-B has a greater ability to spread in planta. This ability is due to a single amino acid change in the viral movement protein p8 and inversely correlates with p8 RNA binding affinity.

FIG. 1

Structure of TCV and chimeric genomes and their effects on lesion size and virulence. (A) Genetic organization of TCV. The positions and functions of the TCV open reading frames are diagrammed. The boxes below represent TCV-M and TCV-B sequences. The vertical bars between the boxes denote nucleotide changes that are silent at the protein level, while the asterisks represent nucleotide changes that result in different amino acids in the proteins. The locations of the restriction enzyme sites used to produce the chimeric genomic clones are shown. The sizes of the fragments produced by these enzymes are noted at the bottom of panel A. (B) Sizes of lesions produced by chimeric genomes. The genomes are represented by a three-letter code wherein the first letter represents the origin of the replicase region, TCV-B (B) or TCV-M (M), the second letter represents the origin of the p8 and p9 regions, and the third letter represents the origin of the coat and 3′ end. The lesions produced by all possible combinations of the three TCV regions were measured with verniers at 3 (hatched bars) and 6 (solid bars) days p.i. Each bar on the graph represents the average of 20 lesions produced by each chimeric genome on several plants. The standard deviations (error bars) are noted on the graph. As the lesions are not completely symmetrical, the smallest axis was chosen for measurement. (C) Virulence of different chimeric genomes. Plants inoculated with the chimeric genomes were observed for 3 weeks p.i. and scored for the presence of disease symptoms in uninoculated portions. For each experiment, the genome causing the highest percentage of plants with systemic disease symptoms was given the value of 100%, and the remaining genomes were expressed as relative percentages. This experiment was performed six times, and the total relative percentage was averaged over each of the six independent experiments. This procedure was necessary due to variability from experiment to experiment in the absolute numbers of plants that developed symptoms due to environmental effects. Standard deviations (error bars) are noted on the graph.

FIG. 2

Fine map of the region encoding the movement proteins. The 0.42-kb EcoRI-to-BglI movement region is represented by the open box, with the single nucleotide change noted by the asterisk. The open reading frames (ORFs) or portions of open reading frames contained within this region are delineated by the solid boxes and labeled. The amino acid (aa) sequence of p8 immediately surrounding the amino acid difference between strains M and B at residue 25 is diagrammed, along with the corresponding difference in net charge of this region.

FIG. 3

Cross-linking of His-p8M and His-p8B to RNA. A uniformly labeled 5′ 229-nucleotide fragment of the TCV genome was incubated with either His-p8B or His-p8M in the presence of the indicated salt concentrations. The reaction mixtures were then irradiated with UV light, treated with RNase A, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 15% gel. (A) Autoradiogram. The numbers above each lane indicate the molar NaCl concentration in each reaction mixture. The image was obtained with a digital camera. (B) Quantitation of cross-linking data. The data are the averages of results of four independent experiments. Dashed line, binding by His-p8B; solid line, binding by His-p8M. Error bars denote standard deviations. Quantitation was done by densitometry of the X-ray film.