Oct 02, 2020

Public workspacePurification of Haustoria from Arabidopsis in Response to Infection by E. cichoracearum

DOI
dx.doi.org/10.17504/protocols.io.tqkemuw
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DOI: dx.doi.org/10.17504/protocols.io.tqkemuw
Protocol Citation: Harley King 2020. Purification of Haustoria from Arabidopsis in Response to Infection by E. cichoracearum. protocols.io https://dx.doi.org/10.17504/protocols.io.tqkemuw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: September 18, 2018
Last Modified: October 02, 2020
Protocol Integer ID: 15852
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Abstract
This procedure describes steps for the purification of Arabidopthis thaliana haustoria in response to powdery mildew infection by fungus Golovinomyces cichoracearum.


Grind Plants Containing Haustoria
Grind Plants Containing Haustoria
  1. Weigh out 5-10g frozen Arabidopsis leaves. Set aside 2-3 leaves for quantifying haustoria with a confocal microscope before purification.
  2. Grind leaves in ice-cold Amount8 mL 1x PBS buffer pH 7.4 for 1 min in kitchen blender in cold room.
  3. Filter grindate through 100um Nylon mesh. Haustoria are about 10-20um in diameter.
  4. With a glass rod, remove unfiltered debris to blender. PBS buffer can be used to wash the membrane with a transfer pipette into the blender.
  5. Add Amount5 mL 1x PBS buffer and grind for another 45 seconds.
  6. Filter through 100um Nylon mesh.
  7. Filter the collected filtrated from steps 3 and 6 and pass through a 40um mesh. A vacuum and modified filter assembly can be used to expedite the purification. Wash the membrane with PBS buffer into the trash.
  8. Pass filtrate through 40um mesh again.
  9. Transfer ~13-15mL to a 15mL concial tube. Pellet filtrate at 1000g for 5 min.
  10. Remove supernatant.
  11. Resuspend pellete in 2mL 1x PBS buffer. Set asside 40ul for quantifying haustoria.
  12. Investigate haustoria with confocal microscope and a hemacytometer before commiting to the the next steps.

Note
Col-O plants should be processed concurrently as a negative control especially if haustoria will be filtered by FACS.

Purify Haustoria Using Percoll Cushion
Purify Haustoria Using Percoll Cushion
Using Percoll, make a 60% solution and a 40% solution with 1x PBS.
Layer 4mL of the 60% solution on the bottom of a 15mL conical tube.
Layer 6mL of the 40% solution on the top of the 60% solution.
Gently add 1mL of resuspended pellet containing the haustoria to the top 40% Percoll solution.
Centrifuge at 2500g for 10 minutes.
The haustoria will penetrate the 40% layer but not the 60% layer.
Remove the 40% layer. Mix, then pellet at 1000g for 5 min.
Resuspend in 2mL. Removed 40ul aliquot for quantifying haustoria.
If sufficient, proceed to next step.
Incubate Haustoria with anti-GFP (Chromotek) Conjugated to Dynabeads
Incubate Haustoria with anti-GFP (Chromotek) Conjugated to Dynabeads
  1. Conjugate anti-GFP Trap antibody to Dynabeads following manufacturer's recommended protcol.
  2. Incubate beads with haustoria with end-over-end rotation overnight.
  3. Wash beads 5x with 1x PBS pH7.4.
  4. Disrupt haustoria-GFP Trap binding following manufacturer's protocol.
  5. Proceed to FACS purification


Follow previous experiments to use as a baseline in purifying haustoria with FACS. Use col-o plants as negative control.