I transformed four different constructs into agro seperately (one of them is p19, silencing surpressor). Mix all four kinds of agro cultures with equal volume. (final OD=1) Inject the mixed culture in tobacco leaves. I saw many paper conducting this transient expression. And they successfully express their protein at the same tobacco leaf epidermal cells. But they don't use any selection methods or reporter gene to ensure the successful transfection of tobacco leaf cells. And even though tobacco cells are transfected by all four kinds of agro, are they transfected by same copy number between four different constructs? Why can this method be used so commonly and successfully with so many uncontrollable factors existing?
You are completely correct to question the common practice to simply mix different Agrobacterium strains when they have no markers (i.e. fluorescent reporters) as controls and hope that the majority of leaf epidermal cells are co-transformed with both (or more) and with comparable expression. It is complete chaos theory, there are many uncontrolled factors, and a lot of published results on co-transformation are not reproducible.
Even if some cells are co-transformed, they won't have the same copy number, the genes will integrate into different places thus having different expression due to position effect, and they don't transform at the same time, so the expression is again different. Agrobacterium stays alive in the apoplast, and can divide and transform at any time, either soon after infiltration, or 1-2 days later. The common idea that the single stranded T-DNA, coated with Vir proteins to keep it linear and type IV secretion competent, is mediating extrachromosomal transient expression is also unproven, and very very unlikely. The field is totally unaware of what happens between arrival of the single stranded protein coated T-DNA copy in the nucleoplasm and the final stable integration into the genome, and there is absolutely no evidence for the existence of a long lived double stranded extrachromosomal copy waiting to be integrated. Only double stranded DNA can be transcribed by RNA polymerase. It is very likely that heterologous expression only starts after integration. So one of the main reasons why different cells in an infiltrated leaf have different expression is because one cell had the T-DNA integrated within an hour after infiltration, and another cells perhaps 12 hours later, and yet again another cell 24 hours later, and perhaps another cell was transformed 2 hours after infiltration and supertransformed again 18 hours after infiltration, and finally some cells may have received the integrated copy a few hours before you go to the microscope and the transgene has not expressed yet. This is why the longer you wait, the more cells in the leaf are transformed or co-transformed. And then you can get silencing too if the gene product is harmful for the cell. It cannot be controlled.
So this is why we either put two genes to be co-transformed on the same T-DNA, or we add markers to the TDNA with the gene of interest to be able to tell that the cell is transformed with that T-DNA. The use of internal markers to guarantee co-transformation has been described in some of our recent papers:
Bottanelli, F., Gershlick, D.C., and Denecke, J. (2012). Traffic 13, 338-354.
Bottanelli, F., Foresti, O., Hanton, S., and Denecke, J. (2011). Plant Cell 23, 3007-3025.
We are currently working on triple and quadrouple expression systems to improve the work even further. Only when you put genes on the same T-DNA can you know for sure that they will be co-transformed, and expression correlates quite well, as demonstrated in Bottanelli 2012 supplemental data.
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