Illuminating an Ecological Blackbox: Using High Throughput Sequencing to Characterize the Plant Virome Across Scales

Abstract

The ecology of plant viruses began to be explored at the end of the 19th century. Since then, major advances have revealed mechanisms of virus-host-vector interactions in various environments. These advances have been accelerated by new technlogies for virus detection and characterization, most recently including high throughput sequencing (HTS). HTS allows investigators, for the first time, to characterize all or nearly all viruses in a sample without a priori information about which viruses might be present. This powerful approach has spurred new investigation of the viral metagenome (virome). The rich virome datasets accumulated illuminate important ecological phenomena such as virus spread among host reservoirs (wild and domestic), effects of ecosystem simplification caused by human activities (and agriculture) on the biodiversity and the emergence of new viruses in crops. To be effective, however, HTS-based virome studies must successfully navigate challenges and pitfalls at each procedural step, from plant sampling to library preparation and bioinformatic analyses. This review summarizes major advances in plant virus ecology associated with technological developments, and then presents important considerations and best practices for HTS use in virome studies.

Keywords: high throughput sequencing; historical advances; opportunities and challenges; plant virome; virus ecology and evolution.

FIGURE 1

Time line of the major advances in plant virus ecology, linked to the evolution of the detection methods.

FIGURE 2

Methods for characterizing the viromes of plant communities. Various factors may potentially impact HTS-based viral ecology studies, at the different steps of these studies: plant sampling at days post-infection (DPI), field sampling, pooling strategies, sample and library preparation methods, selection of the sequencing platforms and bioinformatic analyses. Biological data on the viruses, their host plants and vectors (if known) could also be useful for viral ecology studies.

FIGURE 3

Sample preparation for virus ecological studies using HTS. Samples taken from plants, vectors or other sources (waters, animals, etc.) may contain various viral sequences including DNA viruses (blue), RNA viruses (green), viroids (yellow) or viral sequences integrated in the host genome (red). Variants can be produced by mutations (white) and recombinations (black) during the infection. The most frequently used protocols to analyze viral sequences by HTS are based on the extraction of total RNA, dsRNA, small RNA, and Virion-Associated Nucleic Acids (VANA). By using sequencing platforms, sets of reads are obtained and have then to be processed by bioinformatic pipelines to reassemble them in order to detect, identify and quantify the virus and viroids present. Dotted figures correspond to viral sequences sometimes detected with the preparation method described.