Overview of In Planta Sub-Cellular Localization by Agroinfiltration

“Where is my protein of interest located?” is the first question you ask yourself when you want to study the function or characterize your protein of interest. As a plant biologist, it can take months to grow a stably transformed transgenic plant, even with the fastest plant model.

Good news, though! Agroinfiltration of tobacco leaves has come to the rescue for studying sub-cellular localization; no more months-long waiting period with the what-if-it-doesn’t-work nightmares.

Time savior: Agroinfiltration

Agroinfiltration is a method for the transient expression of your protein of interest in a plant system. You can use it for the production of recombinant proteins or simply to determine the sub-cellular localization of your protein.

As the name suggests, you use the soil bacterium, Agrobacterium tumefaciens, to transfer your gene into the plant cell. It is a rapid and very simple technique. All you need to do is put your gene of interest into a suitable binary vector and transform it into Agrobacterium tumefaciens; later you will inject it into leaves. You only have to wait for 2-3 days for the results. I have had good experience using tobacco plants (Nicotiana benthamiana) as a plant model for the protein localization.

It emits and you see: Fluorescence proteins

How do you locate your protein in planta? First of all, you have to make sure that you “see” your protein, i.e. you need to make it detectable. You can use markers that not only make your gene detectable, but also give you the opportunity to quantify the expression.

Fluorescent proteins (FP) are the best option as they allow you to monitor cellular processes in the living system with the help of wide field fluorescence and confocal microscopy. This is advantageous to other markers which may require you to add a substrate for detection that might interfere with cellular function (e.g. GUS gene encoded beta-glucuronidase).

Many of the fluorescent proteins are the genetic mutant of Green Fluorescent Protein (GFP) derived from jelly fish Aequorea victoria. You can choose from a wide range of commercially available FPs, e.g. YFP, mCherry, dsRED, BFP etc.

Everything in Frame: Fusion proteins

To make your protein visible, you need to tag it with one of the markers we just discussed above. You can add the fluorescent protein to the c-terminal end of your protein so that the produced fusion protein will be located wherever your protein normally resides.

Once you have chosen the marker, you need to make sure that it expresses well. Remember to remove the stop codon from your gene of interest and make sure that it is in the right reading frame with the fluorescent protein. You can always design overhang primers to get rid of the stop codon from your gene.

What do I see: Selection of positive and negative controls

Positive controls

As you know, controls (positive and negative) are always helpful in experiments. If you just use a marker for your gene of interest, of course you see the fluorescence; but what if you had another protein of known location in addition to your protein, wouldn’t it be great to compare? You can choose two different fluorescent proteins that emit in non-overlapping wavelengths (e.g. YFP and RFP) for the control and your protein of interest. Sometimes, it’s even helpful to use more than one positive control.

Negative controls

Sometimes, it is very useful to have an ATG-less fluorescence protein sequence to make sure what you see is certainly the fluorescence from the fusion protein. In that case you can also use an empty vector as a negative control. Many such constructs are commercially available, but if you are not a big fan of buying them, why don’t you DIY?

Carrier of your interest: Binary vectors and Agrobacterium sp.

Agrobacterium sp. is the vehicle of choice for transferring DNA into plant cells where the integration of the gene into the plant genome will occur. Use Agrobacterium tumefaciens (causes crown-gall disease) for this purpose. These bacteria use conjugative transfer of the tumor inducing DNA (T-DNA) from the Ti plasmid into the plant cell. To use this, exchange the T-DNA region with your gene of interest keeping the 25bp long region known as the left and right border in the T-DNA. This flanking region allows the transfer of DNA fragments from any plasmid into the plant nucleus.

You will also need to choose a binary vector that can easily propagate not only in E.coli, but also in Agrobacterium. Generally the 35S promoter of Cauliflower mosaic virus is a good choice as a promoter. Don’t forget to test and confirm a clone before transforming the final construct into Agrobacterium.

Now-a-days, we get everything ready-made; there are plenty of “disarmed” Agrobacterium tumefaciens strains available which were engineered to have a shorter Ti plasmid for the ease of cloning. Likewise, many binary vectors are already available.

Helper to enhance: Suppressor of post-transcriptional gene silencing

The expression level of your gene peaks usually around 60-72 hours after you infiltrate the leaves. After that, it declines rapidly due to post-transcriptional gene silencing in plant. To increase gene expression, co-express a suppressor of post-transcriptional gene silencing along with your control and protein of interest. p19 protein of tomato bushy stunt virus is well known for this purpose, and I personally have had good experience using this.

Put it inside: Agrofiltration of tobacco leaves

In a later article I will cover agrofiltration in detail, but here are some general tips:

• Use healthy 2-4 week old tobacco plants (Nicotiana benthamiana).
• You can combine the strains containing your control, protein of interest and p19 all together.
• Usually you grow the Agrobacterium culture to a final OD₆₀₀ of 0.1 to 1 prior to infiltration; however you might have to optimize the culture for efficient expression of your protein.
• Before infiltration of the tobacco leaves, make sure you incubate the culture in infiltration buffer (MES, MgCl₂and Acetosyringone) for a few hours, this allows the activation of the Agrobacterium tumefaciens.
• You can use needle-less small syringes to vacuum infiltrate. Simply press the mouth of the syringe on the bottom part of the leaves while exerting an optimal pressure on the other side with your finger.
• Once done, wait for 2-3 days and then check for fluorescence.

You will need to spend some time standardizing your protocol, but once you are successful, it will seem like a piece of cake! And after a long time of work, you will finally know where your protein is located. Bliss isn’t it?