transmitting-plant-viruses-using-whiteflies

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several ...

University of Florida 

Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of approximately 10(-4) substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10,000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered.

Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias.

A new system for inoculation of plants with begomoviral DNA without cloning or the use insect vectors is described. Total DNA extracted from begomovirus-infected plants was amplified by rolling circle amplification (RCA) using the bacteriophage phi29 DNA polymerase, and inoculated to plants by particle bombardment. Infection rates of up to 100% were obtained using this technique. This technique successfully inoculated all the begomoviruses evaluated: five bipartite (Bean golden yellow mosaic virus, Cabbage leaf curl virus, Squash leaf curl virus, Tomato mottle virus, Watermelon chlorotic stunt virus) as well as one monopartite (Tomato yellow leaf curl virus). The success of the technique was not dependent upon plant species. Four species from three plant families [Phaseolus vulgaris (bean), Solanum lycopersicum (tomato), Cucurbita pepo (squash), and Citrullus lanatus (watermelon)], could all be inoculated by this technique. The success of the method was not dependent upon either the type or the age of the source of virus. Infectious DNA was obtained successfully from fresh, freeze-dried or desiccated plant material, from squashes of plant leaves on FTA cards, as well as from the insect vector. Plant material collected and dried as long as 25 years ago yielded infectious DNA by this method. In summary, this method can be used to obtain infectious DNA of single-stranded circular DNA viruses that can be activated for purposes of completing Koch's postulates, for preservation of pure virus cultures, and for many other applications where infectious DNA is required.

Inoculation of plants with begomoviruses by particle bombardment without cloning: Using rolling circle amplification of total DNA from infected plants and whiteflies.

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.

Article	Year
Experimental observations of rapid Maize streak virus evolution reveal a strand-specific nucleotide substitution bias. Virology journal\| PubMed ID: 18816368	2008
Inoculation of plants with begomoviruses by particle bombardment without cloning: Using rolling circle amplification of total DNA from infected plants and whiteflies. Journal of virological methods\| PubMed ID: 20447420	2010
Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies. PloS one\| PubMed ID: 21544196	2011

Jane E. Polston

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