Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing

Abstract

Tomato is a major vegetable crop that has tremendous popularity. However, viral disease is still a major factor limiting tomato production. Here, we report the tomato virome identified through sequencing small RNAs of 170 field-grown samples collected in China. A total of 22 viruses were identified, including both well-documented and newly detected viruses. The tomato viral community is dominated by a few species, and they exhibit polymorphisms and recombination in the genomes with cold spots and hot spots. Most samples were coinfected by multiple viruses, and the majority of identified viruses are positive-sense single-stranded RNA viruses. Evolutionary analysis of one of the most dominant tomato viruses, Tomato yellow leaf curl virus (TYLCV), predicts its origin and the time back to its most recent common ancestor. The broadly sampled data have enabled us to identify several unreported viruses in tomato, including a completely new virus, which has a genome of ∼13.4 kb and groups with aphid-transmitted viruses in the genus Cytorhabdovirus Although both DNA and RNA viruses can trigger the biogenesis of virus-derived small interfering RNAs (vsiRNAs), we show that features such as length distribution, paired distance, and base selection bias of vsiRNA sequences reflect different plant Dicer-like proteins and Argonautes involved in vsiRNA biogenesis. Collectively, this study offers insights into host-virus interaction in tomato and provides valuable information to facilitate the management of viral diseases.IMPORTANCE Tomato is an important source of micronutrients in the human diet and is extensively consumed around the world. Virus is among the major constraints on tomato production. Categorizing virus species that are capable of infecting tomato and understanding their diversity and evolution are challenging due to difficulties in detecting such fast-evolving biological entities. Here, we report the landscape of the tomato virome in China, the leading country in tomato production. We identified dozens of viruses present in tomato, including both well-documented and completely new viruses. Some newly emerged viruses in tomato were found to spread fast, and therefore, prompt attention is needed to control them. Moreover, we show that the virus genomes exhibit considerable degree of polymorphisms and recombination, and the virus-derived small interfering RNA (vsiRNA) sequences indicate distinct vsiRNA biogenesis mechanisms for different viruses. The Chinese tomato virome that we developed provides valuable information to facilitate the management of tomato viral diseases.

Keywords: small RNA sequencing; tomato virome; viral diversity and evolution.

FIG 1

Virus detection in field-grown tomato samples in China using sRNA sequencing. (A) Distribution of the sampling regions in China. Color and size of the circles indicate the sampling size. Regions are shown by acronyms, and their full names are listed in Table S1 in the supplemental material. (B) Read size distribution in collected samples. (C) Classification of viruses detected in the tomato samples. (D) Viral discovery curve to assess the saturation and estimate the richness (number of viral species). The solid green line is the rarefaction curve, the dashed green line indicates the Chao2 estimation of asymptotic richness by sample number, and shading shows 95% confidence intervals. Current sampling size and estimated size for 97% sampling completeness are marked. (E) Summary of samples by the number of detected viruses per sample.

FIG 2

Viral distribution pattern in field-grown tomato samples from China. Regions of samples are clustered according to virus occurrence profiles and displayed on the top. The full information for the region acronyms is listed in Table S1 in the supplemental material. Viruses that were first reported in tomato are in blue text.

FIG 3

RT-PCR validation of a subset of identified tomato viruses.

FIG 4

Population diversity of tomato viruses in China. Genomes of the seven most prevalent tomato viruses are shown. SNP positions in the genome of each virus are indicated as black lines in the outer circle of each plot. Colored segments close to the outer circle are coding regions. Depth for vsiRNA reads mapped on the positive strand (red) or negative strand (green) is shown in the inner circle. Orange and gray lines link the recombination sites from positive-positive and positive-negative strands, respectively. Recombination frequencies in the samples are indicated by the line width.

FIG 5

Phylogeny of TYLCV in China. Genome sequences of TYLCV strains identified in this study and those from China and elsewhere downloaded from GenBank (accession numbers are shown in the figure) were used for the phylogenetic analysis. A maximum likelihood (ML) tree was constructed using PhyML with the best-fitted model GTR +Γ4 and 100 bootstraps. Branches in red indicate the highly diverged strains collected from Shandong province.

FIG 6

Maximum clade creditability tree of TYLCV coat proteins. The time scale below the tree corresponds to the mean posterior estimate of the age in years. Bars represent 95% highest posterior density (HPD) intervals for the estimated divergence time. Nodes in the tree with posterior probabilities greater than 0.5 are labeled with red circles, and those with probabilities smaller than 0.5 are labeled with green circles. Accession number, region, and sampling year for each TYLCV are included in its identifier, and samples from China are shaded with pink.

FIG 7

Genome and phylogeny of tomato yellow mottle-associated virus (TYMaV). (A) Genome structure of two representative plant viruses in the genus Cytorhabdovirus and TYMaV. sRNA read distribution on the genome of TYMaV is shown at the bottom. (B) Symptoms of TYMaV-infected tomato plants. The image in the inset is the magnified tomato leaf from the indicated yellow-circled region. (C) Sequence features of the TYMaV genome. The upper panel shows the near-complementary structure of the 5′ and 3′ termini in the genome. The lower panel shows the conserved motif sequences in the intergenic regions. (D) Phylogenic tree of 11 available genomes of members in the genus Cytorhabdovirus constructed with five conserved proteins (N, P, M, G, and L).

FIG 8

Characteristics of tomato virus siRNA sequences. (A) Length distribution, polarity profiles, and paired distance (21- and 22-nt reads) of siRNA reads mapped to viral genomes. Columns labeled with stars are positions of 2 nt for the 3′ overhang and 19 nt (for 21-nt vsiRNA) or 20 nt (for 22-nt vsiRNA) for the 5′ overhang, respectively. (B) Paired vsiRNA distance of siRNA reads in ToMV. (C) Examples of three scenarios (perfectly matched, 5′ overhang, and 3′ overhang) of paired vsiRNAs and normalized nucleotide percentage at each position of siRNAs. Normalized nucleotide percentage is calculated by dividing the base percentage at each position with the mean percentage of the respective base between positions 2 and 18. Only paired reads with a 2-nt 3′ overhang were used for calculation. (D) Clusters of tomato viruses based on the correlation coefficient of the normalized nucleotide percentage. Viruses were grouped into the same clusters if their correlation coefficient was ≥0.6. The base composition at the 5′-most position on vsiRNA reads for each cluster is indicated.