Co-infection with two strains of Brome mosaic bromovirus reveals common RNA recombination sites in different hosts

Abstract

We have previously reported intra-segmental crossovers in Brome mosaic virus (BMV) RNAs. In this work, we studied the homologous recombination of BMV RNA in three different hosts: barley (Hordeum vulgare), Chenopodium quinoa, and Nicotiana benthamiana that were co-infected with two strains of BMV: Russian (R) and Fescue (F). Our work aimed at (1) establishing the frequency of recombination, (2) mapping the recombination hot spots, and (3) addressing host effects. The F and R nucleotide sequences differ from each other at many translationally silent nucleotide substitutions. We exploited this natural variability to track the crossover sites. Sequencing of a large number of cDNA clones revealed multiple homologous crossovers in each BMV RNA segment, in both the whole plants and protoplasts. Some recombination hot spots mapped at similar locations in different hosts, suggesting a role for viral factors, but other sites depended on the host. Our results demonstrate the chimeric ('mosaic') nature of the BMV RNA genome.

Keywords: Brome mosaic bromovirus; RNA replication; homologous RNA recombination; host effects; recombination frequency; recombination hot spots.

Figure 1.

Agarose gel electrophoresis of the PCR-generated cDNA Fragments 2–3 from BMV RNA2, confirming co-infection by both F and R strains. The single cut observed for Mlu I digestion confirms the presence of R-BMV (marked by R), whereas the single cut with Pvu I confirms the presence of F-BMV (marked by F). Digestions of Fragments 2–3 from plants confirmed the BMV RNA2 mixture of both strains in the same host (B: Barley; C: C. quinoa; and N: N. benthamiana).

Figure 2.

Mapping of recombination regions between BMV RNAs 1 in three co-infected hosts. The locations of single-nucleotide differences (markers) on the RNA1 sequence (central gray thick line) are represented by short green vertical bars with the nt positions shown above. The RFN values were calculated as described in the text and their numbers plotted between the markers with the color-coded lines. The RFN values reflect the probability (percentage) of crossovers per nucleotide at the particular region. The RFNs for Ch. quinoa are denoted by green lines, N. benthamiana—by red lines, and for barley—by blue lines. The open reading frame (ORF) for protein 1a and the noncoding RNA1 regions are depicted below by thick and thin red lines, respectively, whereas boxed regions represent the two functional domains of the 1a protein: the NTPase and the helicase domains. The sequenced areas are represented by dashed lines, marking the two separately cloned fragments of the RNA1 molecule: Fragments 1 and 2. The total numbers of cDNA clones sequenced and the total fraction (%) of recombinants (RF) are shown by numbers at the bottom. The right-most column shows the total RF. The total RF values cannot be calculated for barley because of missing sequence data for Fragment 1.

Figure 3.

Mapping of recombination regions between BMV RNAs 2 in three co-infected hosts. The locations of nt markers alongside the BMV RNA2 sequence, the calculation, plotting, and color coding of RFN lines, and the 2a ORF are all marked as described in Fig. 2. The position of GDD motif, a characteristic feature of RdRp polymerases, is marked with a black vertical bar. Similar to Fig. 2, the areas of the separately cloned and sequenced Fragments 1, 2, and 3 are marked with dashed lines below. The numerals at the bottom show the total numbers of cDNA clones sequenced, and the total fraction (%) of recombinants that defines the RF for each fragment, followed by the total RF in the rightmost column.

Figure 4.

Mapping of recombination regions between BMV RNA3s in co-infected barley plants. The locations of markers on the RNA3 sequence, the calculation, plotting, and color coding of RFN lines, and the 3a and CP ORFs are all marked as described in Fig. 2. The mapped RNA3 sequences responsible for CP binding (PE site and B-box site) are marked with purple bars below. Similar to Fig. 2, the separately sequenced areas of RNA3 cDNA clones are depicted with dashed lines below. The numerals at the bottom show total amounts of cDNA clones sequenced and the total RF values (%).

Figure 5.

Mapping of recombination regions between BMV RNAs 2 (A) and RNAs 3 (B) in co-transfected barley protoplasts. The locations of nt differences (markers) on the BMV RNA sequence, the calculation, plotting, and depicting the ORFs all are as those in Figs 2 and 3. Two different ratios of F to R BMV RNA inoculums were tested for RNA2, and three ratios were tested for RNAs 3 inocula: 1F to 1R (blue line), 3F to 1R (yellow line), and 1F to 3R (black line). The separately sequenced areas of cDNA clones are depicted with dashed lines below. The numerals at the bottom show total numbers of cDNA clones sequenced and the total RF values (%).