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

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.

Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana.

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus ((TMV)MP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that (TMV)MP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of (TMV)MP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components diffuses in the ER membrane within the Pd driven by the concentration gradient between an infected cell and adjacent noninfected cells. The data also suggest that the virus replicase and MP function together in altering Pd conductivity.

beta-1,3-Glucanases: Plasmodesmal Gate Keepers for Intercellular Communication.

Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are not static, but show a dynamic nature and can be opened or closed. These controlled changes in Pd conductivity regulate plant symplasmic permeability and play a role both in development and defense processes. One of the mechanisms shown to produce these changes is the deposition and hydrolysis of callose by beta-1-3-synthase and glucanase, respectively. Recently we have identified the first beta-1,3-glucanase Arabidopsis enzyme that is associated to the macromolecular Pd complex, termed AtBG_pap. When fused to GFP, this previously identified GPI-anchored protein localizes to the ER and the plasma membrane where it appears in a punctuate pattern that colocalizes with callose present around Pd. In T-DNA insertion mutants that do not transcribe AtBG_pap, GFP cell-to-cell movement between epidermal cells is reduced and callose levels around Pd are elevated. In this addenda we review the plant developmental processes of symplasmic regulation that have been shown to include callose deposition and beta-1,3-glucanase activity, and suggest a role for AtBG_pap in these processes. Additionally, based on the ability of viral movement proteins (MPs) to interact with ankyrin repeat proteins, and together with our recent findings showing the involvement of viral particles in callose degradation, we also purpose a new model for the ability of viruses to overcome Pd-callose deposition, and mediate their cell-to-cell movement.

Class 1 reversibly glycosylated polypeptides are plasmodesmal-associated proteins delivered to plasmodesmata via the golgi apparatus.

SE-WAP41, a salt-extractable 41-kD wall-associated protein that is associated with walls of etiolated maize (Zea mays) seedlings and is recognized by an antiserum previously reported to label plasmodesmata and the Golgi, was cloned, sequenced, and found to be a class 1 reversibly glycosylated polypeptide ((C1)RGP). Protein gel blot analysis of cell fractions with an antiserum against recombinant SE-WAP41 showed it to be enriched in the wall fraction. RNA gel blot analysis along the mesocotyl developmental axis and during deetiolation demonstrates that high SE-WAP41 transcript levels correlate spatially and temporally with primary and secondary plasmodesmata (Pd) formation. All four of the Arabidopsis thaliana (C1)RGP proteins, when fused to green fluorescent protein (GFP) and transiently expressed in tobacco (Nicotiana tabacum) epidermal cells, display fluorescence patterns indicating they are Golgi- and plasmodesmal-associated proteins. Localization to the Golgi apparatus was verified by colocalization of transiently expressed AtRGP2 fused to cyan fluorescence protein together with a known Golgi marker, Golgi Nucleotide Sugar Transporter 1 fused to yellow fluorescent protein (GONST1:YFP). In transgenic tobacco, AtRGP2:GFP fluorescence is punctate, is present only in contact walls between cells, and colocalizes with aniline blue-stained callose present around Pd. In plasmolyzed cells, AtRGP2:GFP remains wall embedded, whereas GONST1:YFP cannot be found embedded in cell walls. This result implies that the targeting to Pd is not due to a default pathway for Golgi-localized fusion proteins but is specific to (C1)RGPs. Treatment with the Golgi disrupting drug Brefeldin A inhibits Pd labeling by AtRGP2:GFP. Integrating these data, we conclude that (C1)RGPs are plasmodesmal-associated proteins delivered to plasmodesmata via the Golgi apparatus.