A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.
In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.
1. Plant material
2. Delivery and expression of functional viral genome components to plant cells by agroinfiltration
3. Purification of BMV virions
Note: Based on RNA content, the extinction coefficient for BMV is 54.
4. Preparation of capsid protein subunits for in vitro assembly
5. In vitro assembly of RNA containing virions
6. Fabrication of Optical Viral Ghosts (OVGs)
7. Representative Results
Figure A. TEM image of negatively stained BMV virions purified from N. benthamiana plants agroinfiltrated with a mixture of all three wild type BMV agroconstructs (scale bar = 100 nm).
Figure B. Pellet of in vitro assembled ICG containing OVGs following high-speed centrifugation.
Figure C. TEM image of negatively stained ICG containing OVGs (scale bar = 100 nm).
Figure D. Absorbance (Top) and fluorescence (bottom) spectra of OVGs. The excitation wavelength used for OVG emission was 620 nm.
Agroinfiltration approach presented here can widely be applicable to a wide range of plant viruses. A hallmark feature of this approach is synchronized delivery of multiple agroconstruct to the same cell-a major drawback commonly associated with routinely used mechanical inoculation of plant viruses. In vivo and in vitro assembly studies using brome mosaic virus as a model can be conducted efficiently by following few tips.(i)For successful infiltration of N. benthamiana leaves, do not water the plants for 24 hours before infiltration; (ii) Agrobacterium suspension culture would spread into intracellular spaces with ease, if infiltration were performed between 4-5 PM; (iii) The optical density of the culture should not exceed 1.0 at OD600. Infiltration of agrocultures with higher cell density is known to induce cell toxicity and leaf senescence9,10 that could severely affect viral replication and subsequent virion formation; (iv) During infiltration gentle pressure should be applied on the abaxial side of the leaf to prevent extensive damage to the leaf, which might trigger immune response in plant; (v) Sucrose density gradient from 10% to 40% can be made in a tube by making 25% of sucrose in BMV suspension buffer (by adding the highest concentration and lowest concentration and dividing it by 2 i.e., 10%+ 40% / 2 = 25%) and immediately freeze at -80 °C for 2-4 hr and further allowing the sucrose to thaw slowly by keeping it overnight at 4 °C in an straight position.
The authors have nothing to disclose.
The authors would like to thank several members of the lab for their valuable suggestions in the development of agroinfiltration and in vitro assembly assays. This work was funded by a grant from University of California. This study was supported in parts by a grant from the National Science Foundation (CBET-1144237).
Name of the reagent | Company | Catalog number |
MES Sodium salts | Sigma-aldrich | M2993 |
Indocyanine green | Sigma-aldrich | I2633 |
Beckman ultracentrifuge | Beckman Coulter | Model: L8-70M |
Centricon-100 column | Millipore | YM-100 |
Spectrophotometer | Cary 50, Varian Inc. | Part number 10068900 |
Spectrofluorometer | Fluorolog 3, Jobin-Yvon. | Part number FL3-21 |