[Arabidopsis] RE:Ion Leakage

[Holt, Ben F. III]

I haven't done this assay in a long time, but you can look at Figure 2C in Cell 108, 743-754 for an example measuring HR and/or disease related cell death after bacterial infiltration. If my history is correct, I think C.J. Baker's lab originally reported this in the early 90's and we started using it in the Dangl lab at UNC-Chapel Hill about 10 years later. Since then a number of other papers have reported the use of conductance/ion leakage as a proxy for dell death measurement. You will have to test it in your own system, but I can say that cell death and disease symptoms do proceed ex vivo. In fact, the curves of increasing ion conductivity in water containing these infiltrated leaf disks would look pretty much identical to a "curve" of visual symptoms for HR or disease related cell death symptoms. The general protocol is straightforward:


 1.  Infiltrate leaf with bacteria (and don't be going crazy with the syringe - if you can't infiltrate 1/2 the leaf by touching the leaf and infiltrating in ONE spot, you need to practice that technique first)
 2.  Perhaps wait 1/2 hour to hour
 3.  Cut out uniform leaf disks with a sharp cork borer - avoid syringe damaged area if they exist and try to take samples in the same area on each leaf
 4.  Wash in distilled water in weigh boats to remove ions released by cutting the disks - I think we originally did 1/2 hour wash, but that's probably overkill
 5.  While the disks are washing, measure out identical, small volumes of distilled water into tubes (conductivity probe must fit!) or weigh boats. I can't remember the ratios, but I want to say roughly 6-8 disks in 20ml water - play with it.
 6.  Take a zero measurement of the water to make sure your water and tubes are ion free upfront
 7.  Moving quickly, move disks to paper towels to remove wash water (very gently dab with with kimwipe) and place in prepared tubes
 8.  Measure conductivity changes over time as a proxy for ion leakage/cell death

Notes: If you use tubes, be careful not to touch (crush) leaf disks when inserting the probe to make measurements. Also, as with all proxy measurements, at a minimum, you had best be sure that the ion leakage measurements correlate with known visual symptoms. Finally, don't expect the leaf disks to show typical HR symptoms (visually) while floating in the water, but if you remove them from the water (and compare to controls), they will show real obvious HR collapse as soon as they dry up a bit.

If you want to know the latest method and/or corrections for ion leaks in my memory, try writing Jeff Dangl (UNC) or Dave Mackey (OSU).

Good luck

Ben

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Ben Holt
Assistant Professor
University of Oklahoma
Department of Botany and Microbiology
GLCH Rm 219
770 Van Vleet Oval
Norman, OK  73019
Phone (405)325-9018
FAX   (405)325-7619
http://www.ou.edu/cas/botany-micro/faculty/holt.html