Begomovirus-Associated Satellite DNA Diversity Captured Through Vector-Enabled Metagenomic (VEM) Surveys Using Whiteflies (Aleyrodidae)

Abstract

Monopartite begomoviruses (Geminiviridae), which are whitefly-transmitted single-stranded DNA viruses known for causing devastating crop diseases, are often associated with satellite DNAs. Since begomovirus acquisition or exchange of satellite DNAs may lead to adaptation to new plant hosts and emergence of new disease complexes, it is important to investigate the diversity and distribution of these molecules. This study reports begomovirus-associated satellite DNAs identified during a vector-enabled metagenomic (VEM) survey of begomoviruses using whiteflies collected in various locations (California (USA), Guatemala, Israel, Puerto Rico, and Spain). Protein-encoding satellite DNAs, including alphasatellites and betasatellites, were identified in Israel, Puerto Rico, and Guatemala. Novel alphasatellites were detected in samples from Guatemala and Puerto Rico, resulting in the description of a phylogenetic clade (DNA-3-type alphasatellites) dominated by New World sequences. In addition, a diversity of small (~640-750 nucleotides) satellite DNAs similar to satellites associated with begomoviruses infecting Ipomoea spp. were detected in Puerto Rico and Spain. A third class of satellite molecules, named gammasatellites, is proposed to encompass the increasing number of reported small (<1 kilobase), non-coding begomovirus-associated satellite DNAs. This VEM-based survey indicates that, although recently recovered begomovirus genomes are variations of known genetic themes, satellite DNAs hold unexplored genetic diversity.

Keywords: Ipomoea satellites; alphasatellites; begomovirus; begomovirus-associated satellites; betasatellites; gammasatellites; metagenomics; ssDNA; vector; whitefly.

Figure 1

Schematics depicting general genome organization and features observed in novel alphasatellites (A) and Ipomoea satellites (B) detected during this study. All genomes are characterized by a putative origin of replication (ori) marked by a stem-loop structure containing a conserved nonanucleotide motif. Alphasatellites encode a replication-associated protein (Rep) and exhibit an adenine-rich (A-rich) region. Ipomoea satellites do not exhibit any coding regions; however, these gammasatellites contain an A-rich region as well as a conserved region ~100 nt long shared among various gammasatellites (γSCR). The γSCR contains a 23 nt stretch where all gammasatellites reported to date share high similarities with betasatellites, named here the satellite common region (SCR) core. Some Ipomoea satellites from Puerto Rico share high identities with a 66 nt stretch (highlighted in black) found within the rep gene of sweet potato leaf curl virus (SPLCV) genomes detected in the same region.

Figure 2

Midpoint-rooted maximum likelihood phylogenetic tree of predicted alphasatellite and nanovirus replication-associated protein sequences. Alphasatellites detected during this study are highlighted with a red star. Three groups of alphasatellites based on amino acid pairwise-identities are highlighted in shades of grey, including DNA-1-type alphasatellites from the Old World, DNA-2-type, and DNA-3-type alphasatellites mainly identified in the New World. Branches exhibiting less than 70% branch support were collapsed. Branches with approximate likelihood ratio test (aLRT) support > 91% are indicated with black circles, whereas branches exhibiting 85%–90% support are marked with a white circle. A list of sequences used for phylogenetic analysis and Rep pairwise identities are provided in Supplemental File S2.

Figure 3

(A) Two-dimensional color-coded matrix depicting genome-wide pairwise identities observed among gammasatellites and (B) consensus sequences observed within a conserved satellite common region shared among various gammasatellites (γSCR). Mixed bases in the consensus sequences include K (G/T), R (A/G), W (A/T), and H (A/C/T). The γSCR was observed in Ipomoea satellites (Ipomoea sats) as well as tomato leaf curl virus sat (ToLCV sat) and malvastrum leaf curl satellite (MLC sat). A conserved 23 nt stretch (positions 663–686; labelled SCR core) found within the γSCR (positions 613–711) shares high pairwise identities with betasatellites and all gammasatellites reported to date, including novel New World satellites (NW sats) identified in Cuba and satellites identified in whiteflies from Florida, USA (VEM sats). Nucleotide positions are based on VEM Ipomoea begomovirus satellite 1. A list of sequences used for calculating identity scores among gammasatellites as well as betasatellites used for investigating the SCR core is provided in Supplemental File S3.