In BiFC, we always coexpress two proteins of interest in one cell to investigate the interaction. And we usually use the Agrobacterium to infiltrate the tobacco cells.
How can researchers select which cells successfully gain the both plasmids (two different constructs expressing one of two proteins of interest respectively)? And how to control the copy numbers of the plasmids in tobacco cells? How many times can a tobacco cell be infected by Agrobacterium? Hope you can provide some reference for me.
When Agrobacterium is infiltrated into tobacco leaves, it can transform cells right away, or after 1 hour or after 10 hours or after 2 days etc... Agrobacterium will survive in the apoplast of the spongy parenchyma and continue to transform. A transformed plant cell can be super-transformed by the same or by another Agrobacterium. A transformed cell can first express and then be silenced. After 2 days, co-infiltrations usually are quite efficient, that means that the majority of transformed cells have been transformed with both strains of Agrobacterium. But if your BiFC constructs are the only markers on your T-DNA (half in one strain, the other half in another strain), you will only see fluorescence when the two are expressed in the same cell and BiFC is successful. Usually, the controls are inappropriate (for instance two BiFC halves expressed as fusions to proteins that reside in different cell compartments, and of course they won't interact. A good reviewer will spot this and reject the paper. BiFC controls have to be expressed in the same compartment as your test object, except that the two proteins don't interact. But you are right to worry about co-transformation, because a negative result can either mean lack of interaction, or lack of co-transformation. There is only one way in which you can tell that co-transformation has taken place, and that is to add further fluorescent markers on to your T-DNAs that can be distinguished from each other as well as from the BiFC signal. That's what people should do. Alternatively, place three genes onto a single T-DNA, the two BiFC constructs and another fluorescent marker that is different from the BiFC signal. This way 100% of the transformed cells have both BiFC halves, you can be sure of that.
Jürgen's answer is pretty comprehensive and accurate. We can't choose which cells or cell types are transformed, that's really a molecular decision made by the plant cell and the agrobacterium (e.g. mature Nicotiana guard cells are not transformed by agroinfiltration). To some degree, we can manipulate copy number by changing the agroinfiltration optical density, lower OD will have less agrobacterium in solution and reduce the chances of super-transformation. OD600 values such as 0.01 will lead to transformation and super-transformation of nearly all cells in a region. OD600 values of ~.0001 will provide isolated patches of cells. Agroinfiltration OD also affects the time to signal appearance and the apparent protein expression kinetics. High ODs decrease the amount of time between signal appearance and "overexpresion". BiFC and localization should be performed at the lowest OD possible to avoid artifacts.
The only way to ensure a cell receives equal concentrations of each expression cassette is to have them in the same plasmid. Note that equal expression cassette concentrations do not always equate with equal protein production. Some proteins are transcriptionally, translationally, or post-translationally regulated and maintained at a low level, which can lead to a weak BiFC signal even if the interacting partner is highly abundant. Staged infiltrations and using promoters of different strengths can help equilibrate protein concentrations.
BiFC is an imperfect system, and inherently flawed because of the self-assembly of the fragment pairs. I characterized of all the major BiFC methods currently used in metazoa and plants, and recently published a subset of this work in Plant Journal. In this work we developed the pDOE mVenus210 BiFC system which dramatically decrease the amount background signal due to self-assembly. The system allows allows expression of two test proteins and a transformation control from the same vector (as Jürgen described above). The 20 pDOE vector set allows for single vector BiFC, FRET, and co-localization experiments using 35S or Ubiquitin10 driven experiments.
DOI: 10.1111/tpj.12639
The article and the supplementary methods are open access, and both are posted here on ResearchGate. The vectors are available from TAIR/ABRC, but contact me if that is problematic.
Article Significant reduction of BiFC non-specific assembly facilita...
Typo revision:
The lower OD600 value for agroinfiltration should have been written as ~.001, lower values do work but dramatically increases the time spent searching for transformed cells.
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