Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies

Abstract

Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed "vector-enabled metagenomics" (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors (in this case, whiteflies) from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies (Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases.

Figure 1. Coverage of known virus genomes obtained from the whitefly viromes.

The reference genome from Genbank is shown in the top row of each panel, and the consensus represents the regions of the reference genome with coverage in the viromes. The numeric values represent the degree of genome coverage at each position. A & B) Near-complete coverage of Cucurbit leaf crumple virus DNA-A and DNA-B obtained from the Citra virome. C) Partial coverage of Sida golden mosaic virus DNA-A obtained from the Homestead virome. D) Complete coverage of the Tomato yellow leaf curl virus genome obtained from the Homestead virome.

Figure 2. Genomic and phylogenetic analysis of the virus genomes discovered by VEM.

The tree is midpoint rooted for clarity, all nodes with more than 75% bootstrap support are labeled, and new sequences are indicated by arrows. A) Maximum likelihood phylogeny of the entire DNA-A of the novel Chenopodium leaf curl virus - [USA:Florida:Citra: 2009], Chenopodium leaf curl virus - [USA:Florida:Citra: 2007:VEM] , and Sida golden mosaic Linguanea virus- [USA:Florida:Homestead:2009:VEM] with related begomovirus genome sequences. B) Maximum likelihood phylogeny of the entire DNA-B of the Sida golden mosaic virus - [USA:Florida:Citra:2007:VEM] and Whitefly VEM Begomovirus - [USA:Florida:Homestead:2009:VEM]. Begomovirus names are abbreviated according to convention. C & D) Genome characteristics and organization of the DNA-A, DNA-B and satellite DNA identified in the Citra and Homestead samples. The ORFs encode for: CP/AV1, coat protein; Rep/AC1, replication-associated protein; TrAP/AC2, transcription activator and/or silencing suppressor; REn/AC3, replication enhancer; MP/AV2, movement protein; AC4, AC4 protein; AC5, AC5 protein; MP/BC1, movement protein; NSP/BV1, nuclear shuttle protein. The positions of sequence fragments recovered in the virome, the conserved stem loop and nonanucleotide sequence are indicated.