If the genes you're trying to overexpress are too similar to the Argonaute N. benth genes you'll get silencing rather than overexpression. You should infiltrate a silencing inhibitor at the same time with your constructs of interest. We always mix 1:1 agro expressing our gene of interest and agro expressing p15.

Agro-infiltration is famously straight-forward and easy, except when it isn't!

My first question would be, do you obtain adequate signal in control transformations using the empty vector (N-terminal mCherry::mcs). No control signal could indicate:

1) Your confocal parameters are not optimized for mCherry. Recheck all of your settings, one-by-one, e.g. laser power, amplifier, pinhole, filter/detector pathway, detector gain, etc. Remember that some microscopes have additional apertures/switches that are manually controlled and their status may not show up in your confocal software.

2) Your plants have been stressed and not too happy about participating in your experiments. Previous droughts and stresses can shut down transformation and/or expression. The leaves of very young plants (5-6 leaves) are very soft and small (2.5-3.5 cm wide), but usually produce large amounts of protein very quickly. Untagged 35:mVenus etc can easily produce signal at 24-30 hours post-infiltration.

3) Your proteins are not stable in this system. Some proteins are immediately degraded and signal never accrues, while other proteins have distinct windows of visualization. Two proteins I previously worked with had a short window of stability: signal appeared ~40 hrs after infiltration, peaked ~48 hours in their correct locations, and were completely degraded by 60 hours. One of these proteins could be stabilized by adding a binding partner and could be seen even at 96 hours, the other could not. One small soluble protein I worked could be faintly observed at 36 hours post-infiltration, but would never accrue to a "good" signal no matter how long I waited.

4) Your infiltration OD is quite high, we usually infiltrate at OD values of 0.008-0.01, or perhaps 0.02 depending on the infiltration purpose. You could be initiating a signaling cascade that affects cell dynamics or even whole cell viability. One of my colleagues proteins can be localized at very low OD, but causes visual leaf death at even slightly higher values. For another example, infiltration experiments using PLD1 are tricky as the toxic effects of lipase overexpression have to be balanced against protein complex visualization. While focused primarily on BiFC and localization, or our Plant Journal paper has some infiltration OD, post-infiltration timing, and plant health examples you might find interesting (Gookin and Assmann, 2014, doi: 10.1111/tpj.12639).

Best of luck!

Tim

Thank you a lot Tim. 

I've also search some varibles which could decrease the efficiency of my transformations. Yes, my positive controls work (mCherry). However, it didn't work like the last time. Taking your very good suggestions into account, I really thing it was or my unhealthy plants or the high OD. My plants were a little bit older then usual. 

I will also do a little day-post infection test to see if I can observe a difference between treatments.

Thank you very much Tim.

I wish you all the best

If the genes you're trying to overexpress are too similar to the Argonaute N. benth genes you'll get silencing rather than overexpression. You should infiltrate a silencing inhibitor at the same time with your constructs of interest. We always mix 1:1 agro expressing our gene of interest and agro expressing p15.

I agree with all the previous comments. We also realised that using fresh transformed Agro cells strongly increases the signal. Here the protocol:

·         The Agrobacterium tumefaciens strain GV3101 pMP90 is transformed with the silencing suppressor p19 (Voinnet et al., 2003). (Rif, Gent, Kan)

·         Make competent cells of this strain

·         Transform GV3101 pMP90 p19 with expression clones and culture on YEB medium plates (Rif, Gent, Kan and the antibiotic for your construct). Select positive colonies by PCR

·         Make an overnight culture of the select colony (2x5 ml Rif, Gent, Kan, selected antibiotic) (28 degrees, rotate)

·         Take 4 ml from overnight culture 16 ml fresh medium

·         Incubate for 2-4 h at 28 degrees (rotate)

·         Pellet by centrifugation (4000 rpm 20 min) and resuspend in 8 ml infiltration medium

·         Measure OD600 (of a 1:10 dilution)

·         Adjust with infiltration medium to reach an OD600 of 0.8 (if you mix two different constructs each should have an OD600 of 0.4)

·         Leave on ice for at least one hour

·         Infiltrate the underside of the leaves

·         Place a plastic cover over the plants for one night, remove after that

·         2-3 days after infiltration (apparently up to 1 week but I never tried that) protein production can be induced by brushing (spraying) a solution of 20µM ß-Estradiol + 0,1% TWEEN20 (in H2O) (for our constructs) on the underside of the transformed leaves. Place a plastic cover on the plants again after that.

·         Confocal microscopy after 2-20 hours, depending on the construct. I normally induced in the morning to look at the plant in the afternoon.

·         Good Luck! 

Infiltration medium:

o   5 % Saccharose

o   1 spatula (just a bit) Glucose

o   1 spatula (just a bit) MgSO4

o   450 µM Acetosyringone

o   0,01 % Silwett 

Tips:

·         In the morning of the day on which you want to perform the infiltration you should water the plants and place them under a plastic cover in order for them to open the stomata

·         You need at least 2 ml to infiltrate 1 leaf, at the beginning it might be good to prepare 5 ml per construct to be sure. The medium should completely enter into the leaf, if you just get discs the infiltration is not working properly and it might be impossible to see any signal.

·         The developmental stage of the tobacco leaf is very important for a successful infiltration. Use young leaves not fully developed from a plant with 6-4 true leaves

Thank you so much guys. I really appreciate the help. I will follow your suggestions. 

best.

Have you considered doing a western to check if it's an expression issue or a confocal issue?

Ok so, there Are Many Parameters that may affect agroinfiltration and protocols vary. I have tried many and worked with this for a few years now, especially with low expressing proteins. the most important one (according to my studies) is to use young plants (like 4 weeks old) and NON flowering plants (maybe check my publications of you feel like it, there is a lot about agroinfiltration)

Use GFP fused to mCherry as a control (in case your tag is easily cleaved) You should be able to see both expressing, especially as GFP is an extremely stable protein. If you see your GFPprotein, you can start improving your protocol. As for all organisms, the agro bacteria should be in log growth phase, OD= around 0.7, when spinning down to adjust OD for infiltration (OD=0.5-1 is perfectly fine) If they are over the log growth phase many bacteria will be dead and their lysed proteins will trigger plant immunity and reduce agro-transformation. I usually grow agrobacterium for 2 days to stationary phase and dilute 1:20 for o/n and they are in approximately the right growth phase.

Standard infiltration buffer is 10uM MES pH 5.7 (as the apoplastic pH-maybe your biggest mistake) 10uM mgCL2 and 100-200 uM acetosyringone. Was once in this buffer and then dilute in this buffer to the approbiate OD (0.5-1 is standard as I said) Don't use sugars or cold treatment, but maybe keep them shaking for a bit in the buffer at 20C to RT (agrobacterium is a soil bacterium and likes the cold, but ice is way too cold) to get them activated by the Acetosyringone-didn't think it was massively critical, but it helps.

USE young non flowering plants

Hello Isabel, 

THank you so much for your advise. 

I was talking with some of my collegues and she told me that in N. benthamiana agroinfiltration the MES doesn' t make any difference if you infiltrate just with MgCl2. According to your experiment with low expressing proteins, did you see any difference in these two buffers, MES+MgCL2+Aceto and MgCl2 + Aceto

Thank you again