TITLE: Purification of Haustoria from Arabidopsis in Response to Infection by E. cichoracearum
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This procedure describes steps for the purification of </span><span style = "font-style:italic;">Arabidopthis thaliana</span><span> haustoria in response to powdery mildew infection by fungus </span><span style = "font-style:italic;">Golovinomyces cichoracearum</span><span>. </span></div></div>

[STEPS]
?. [Grind Plants Containing Haustoria]
Weigh out 5-10g frozen Arabidopsis leaves. Set aside 2-3 leaves for quantifying haustoria with a confocal microscope before purification.Grind leaves in ice-cold  1x PBS buffer pH 7.4 for 1 min in kitchen blender in cold room.Filter grindate through 100um Nylon mesh. Haustoria are about 10-20um in diameter.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.Add  1x PBS buffer and grind for another 45 seconds.Filter through 100um Nylon mesh.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.Pass filtrate through 40um mesh again.Transfer ~13-15mL to a 15mL concial tube. Pellet filtrate at 1000g for 5 min. Remove supernatant.Resuspend pellete in 2mL 1x PBS buffer. Set asside 40ul for quantifying haustoria.Investigate haustoria with confocal microscope and a hemacytometer before commiting to the the next steps.Grind leaves in ice-cold 1x PBS buffer pH 7.4 for 1 min in kitchen blender in cold room.Filter grindate through 100um Nylon mesh. Haustoria are about 10-20um in diameter.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.Add  1x PBS buffer and grind for another 45 seconds.Filter through 100um Nylon mesh.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.Pass filtrate through 40um mesh again.Transfer ~13-15mL to a 15mL concial tube. Pellet filtrate at 1000g for 5 min. Remove supernatant.Resuspend pellete in 2mL 1x PBS buffer. Set asside 40ul for quantifying haustoria.Investigate haustoria with confocal microscope and a hemacytometer before commiting to the the next steps.Add 1x PBS buffer and grind for another 45 seconds.Filter through 100um Nylon mesh.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.Pass filtrate through 40um mesh again.Transfer ~13-15mL to a 15mL concial tube. Pellet filtrate at 1000g for 5 min. Remove supernatant.Resuspend pellete in 2mL 1x PBS buffer. Set asside 40ul for quantifying haustoria.Investigate haustoria with confocal microscope and a hemacytometer before commiting to the the next steps.
8 mL
5 mL
Col-O plants should be processed concurrently as a negative control especially if haustoria will be filtered by FACS.
?. [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]
Conjugate anti-GFP Trap antibody to Dynabeads following manufacturer's recommended protcol.Incubate beads with haustoria with end-over-end rotation overnight.Wash beads 5x with 1x PBS pH7.4.Disrupt haustoria-GFP Trap binding following manufacturer's protocol. Proceed to FACS purification
?. [Incubate Haustoria with anti-GFP (Chromotek) Conjugated to Dynabeads]
Follow previous experiments to use as a baseline in purifying haustoria with FACS. Use col-o plants as negative control.