High-frequency reversion of geminivirus replication protein mutants during infection

Abstract

The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.

FIG. 1.

L148 mutants are impaired for TGMV AL1 replication. (A) Schematic of the TGMV AL1 protein. Solid boxes mark the locations of the three motifs conserved among rolling-circle replication initiator proteins, the oval indicates a predicted pair of α-helices, and the stippled box shows the location of the ATP binding motif. Helix 4 residues (E146 and L148) that were mutated are indicated. (B) The sequence between TGMV AL1 amino acids 144 and 154 (helix 4) is shown. E146 and L148 substitutions are shown for the five AL1 mutants below the sequence. The codons specifying residues E, A, and L of helix 4 and the mutations introduced into the three codons are shown on the right (modified nucleotides are indicated by lowercase type). Mutants L148V and L148V* differ only in the third position of the codon. (C) Replication of TGMV AL1 mutants was analyzed in tobacco protoplasts by agarose gel blot hybridization. Lanes 1 to 6 are transfections with TGMV A replicons with either wild-type (wt) (lane 1) or mutant AL1 genes corresponding to E146A L148A (lane 2), L148G (lane 3), L148V (lane 4), E146A L148V (lane 5), and L148V* (lane 6). The positions of double-stranded (ds) and single-stranded (ss) forms of TGMV A DNA are marked on the left. An overexposed image (magnification, ×20) of lanes 4 to 6 is shown on the right. The levels of replication of the different mutants relative to wild-type TGMV (100) are indicated at the bottom.

FIG. 2.

Symptom appearance is delayed in plants infected with TGMV L148 valine mutants. N. benthamiana plants cobombarded with either wild-type or mutant TGMV A and wild-type TGMV B replicons were examined daily for the appearance of symptoms in new growth. The dpi when plants displayed unequivocal symptoms (yellow veins and leaf curling) are plotted for each construct. The symbols represent when individual plants displayed symptoms for wild type (×), L148V (○), L148V* (□), and E146A L148V (†). The total number of plants was 12 for the wild type, 8 for L148V, 10 for L148V*, and 9 for E146A L148V. The arrows indicate the average time of symptom appearance for the plants infected with each construct. The data summarize results from two independent experiments.

FIG. 3.

Viral DNA accumulation is delayed in plants infected with TGMV L148 valine mutants. N. benthamiana plants were bombarded with DNA corresponding to TGMV A and B replicons. The AL1 gene either was the wild type (wt) (lanes 1 and 2) or carried the L148V (lanes 3 to 7), L148V* (lanes 8 to 12), or E146A L148V (lanes 13 to 17) mutation. For each construct, total DNA was isolated from young leaves of the same five plants at 7, 14, and 19 dpi and analyzed by agarose gel blot hybridization. The positions of single-stranded (ss) and double-stranded (ds) forms of TGMV A DNA are marked on the left. ND, not determined.

FIG. 4.

TGMV L148 valine mutants revert at high frequency. Total DNA was isolated from symptomatic leaves of N. benthamiana plants infected with mutant TGMV A and wild-type TGMV B replicons at 19 dpi. The AL1 coding region between amino acids 120 and 180 was amplified from individual plants and sequenced directly. (A) Modifications recovered at codon 148 for L148V, L148V*, and E146A L148V mutants. The original mutations are designated by lowercase type, and the nucleotide changes in the revertants are shown by uppercase, boldface type. The resulting amino acid, the number of plants, and time of symptom appearance (dpi) are shown on the right for each type of revertant. The dagger corresponds to the second-site revertants in B. The total number of plants analyzed for each TGMV mutant is indicated. (B) The TGMV AL1 sequence between amino acids 115 and 156 is shown, with the locations of the predicted α-helices and a conserved sequence marked. Mutations in the second-site revertants are listed on the left, and the amino acid changes are shown below the corresponding positions.

FIG. 5.

Replication analysis of TGMV revertants. (A) Replication of TGMV A replicons encoding AL1 revertants was analyzed in tobacco protoplasts by agarose gel blot hybridization. Lanes 1 to 6 are transfections with TGMV A replicons with either wild-type (wt) (lanes 1 and 3) or mutant AL1 genes corresponding to L148M (lane 2), L148I (lane 4), C128W L148V (lane 5), and R125G L148V I155L (lane 6). The position of the double-stranded (ds) TGMV A DNA is marked on the left, and levels of replication relative to wild-type TGMV (100) at each exposure are indicated at the bottom of each lane. (B) N. benthamiana plants were cobombarded with wild-type or mutant TGMV A and wild-type TGMV B DNA. At 7 dpi, total DNA was isolated from three individual plants infected with TGMV B and either wild-type TGMV A (lanes 1 to 3) or mutant replicons carrying the L148M (lanes 4 to 6), L148I (lanes 7 to 9), C128W L148V (lanes 10 to 12), or R125G L148V I155L (lanes 13 to 15) mutations. DNA accumulation was monitored by agarose gel blot hybridization. The positions of single-stranded (ss) and double-stranded forms of TGMV A are marked on the left.

FIG. 6.

Reversion of the CaLCuV L145A mutation in N. benthamiana. (A) The sequence of wild-type CaLCuV AL1 from amino acids 141 to 177 is shown. The location of helix 4 and the L145A mutation is indicated. The † symbol marks the amino acid modified in the second-site revertant L145A I167L in D and E. (B) Comparison of the heights of mock-, CaLCuV (wild-type [wt])-, or L145A (mutant)-inoculated N. benthamiana plants at 25 dpi. Each point represents an individual plant, with the mean height for each treatment shown at the top. The means of the three treatments were statistically different (P < 0.01 in a two-tailed Student's t test). (C) N. benthamiana plants were cobombarded with a wild-type CaLCuV B replicon and wild-type CaLCuV A (lanes 1 and 2) or an L145A mutant replicon (lanes 3 to 17). Total DNA from the same five plants at 7, 16, and 25 dpi was analyzed by DNA blot hybridization. The positions of single-stranded (ss) and double-stranded (ds) forms of CaLCuV A DNA are marked on the left. Viral DNA was detected only in plants displaying symptoms. (D) The AL1 coding region between amino acids 132 and 349 was amplified from individual plants and sequenced directly. The original mutation is designated by lowercase type, and the nucleotide changes in the revertants are shown by uppercase, boldface type. Numbers of plants are shown on the right for each type of revertant for bombardment (gun) and agroinoculation (agro) experiments. The total number of plants analyzed for each inoculation protocol is indicated below. (E) Replication of CaLCuV A mutants in tobacco protoplasts was analyzed by agarose gel blot hybridization. Lanes 1 to 6 correspond to transfections with CaLCuV A replicons with either wild-type (lane 1 and 3) or mutant AL1 genes corresponding to L145A (lane 2), L145V (lane 4), L145A I167L (lane 5), and L145T (lane 6). The relative accumulation of viral DNAs is given at the bottom of each lane, with the wild type set at 100 for each exposure.

FIG. 7.

Reversion of the CaLCuV L145A mutation in Arabidopsis plants. A. thaliana plants agroinoculated with a wild-type CaLCuV B replicon and wild-type CaLCuV A or an L145A mutant replicon are shown. (A) Mock (left), wild-type (middle), and L145A (right) symptoms at 20 dpi. (B) L145A symptoms at 29 dpi. (C) The AL1 coding region between amino acids 132 and 349 was amplified from individual plants and sequenced directly. The original mutations are designated by lowercase type, and the nucleotide changes in the revertants are shown by uppercase, boldface type. The altered amino acid and the numbers of plants are shown on the right for each type of revertant. The total number of plants analyzed is indicated below. (D) Total DNA was isolated from plants at 29 dpi and analyzed by agarose gel blot hybridization. The reversion at L145A is indicated at the top of each lane. The position of double-stranded (ds) CaLCuV A DNA is marked on the left.