Streamlined generation of plant virus infectious clones using the pLX mini binary vectors.

Recent metagenomic surveys have provided unprecedented amounts of data that have revolutionized our understanding of virus evolution and diversity. Infectious clones are powerful tools to aid the biological characterization of viruses. We recently described the pLX vectors, a set of mini binary T-DNA vectors (∼3 kb) that includes strong bacterial terminators and a minimal replicon from the broad-host-range plasmid pBBR1, which replicate autonomously in both Escherichia coli and Agrobacterium. In this study, a workflow that encompassed pLX binary vectors, overlap-based assembly strategies, and sequencing-by-synthesis verification steps is described and applied for the streamlined generation of infectious clones suitable for Agrobacterium-mediated delivery. The pLX-based vectors herein assembled include the first infectious clone of Wasabi mottle virus, a crucifer-infecting tobamovirus, as well as binary vectors of positive-single-stranded RNA and single- and double-stranded DNA viruses from the Potyviridae, Geminiviridae and Caulimoviridae families, respectively. Finally, the clones generated were used to agro-inoculate the model plant Arabidopsis thaliana and infections were confirmed by a multiplex RT-PCR assay. This workflow facilitated the rapid generation of infectious clones which, together with agro-infection scalability, would allow the pursuit of systematic insights into virus biology and physiology of plant infections and the design of novel biotechnological applications.

Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins.

Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vectors, a set of mini binary T-DNA plasmids suitable for Type IIS restriction endonuclease- and overlap-based assembly methods. pLX vectors include replicons from compatible broad host range plasmids. Simultaneous usage of pBBR1- and RK2-based pLX vectors in a two-plasmid/one-Agrobacterium strain strategy allowed multigene delivery to plants. Adoption of pLX vectors will facilitate routine plant transformations and targeted mutagenesis, as well as complex part and circuit characterization.

Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis.

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

Stability of Tobacco etch virus infectious clones in plasmid vectors.

Tobacco etch virus (TEV) has been traditionally used as a model to research many aspects of the molecular biology of plant RNA virus and, more recently, experimental evolution. However, the only plasmid of this virus species with an infectious clone that has been commonly available to research (pTEV7DA) is rather unstable when propagated in the bacterium Escherichia coli. Here, the TEV infectious clone contained in pTEV7DA is used to construct three new plasmids that allowed infecting the host plants from RNA transcripts synthesized in vitro (pMTEV), directly from plasmid DNA (p35TEV) and by agroinoculation (pGTEV). To increase stability of the three constructed plasmids in E. coli, superfluous vector sequences were removed and the virus expression cassettes were inserted between the plasmid replication origins and antibiotic selection markers in reverse orientation to the latter gene. Although the TEV cDNA in these three new plasmids is not interrupted by any exogenous sequence, they are more stable than the parental pTEV7DA during propagation in E. coli, indicating a major contribution of the plasmid context in virus cDNA stability. Using the different inocula produced from the three new plasmids the TEV infectivity was also compared. The results showed that agroinoculation is the most effective inoculation method and is where symptoms unfold earlier.