TITLE: Isolation of haustoria from stem rust Pgt 21-0 infected wheat seedlings
AUTHORS: Jamila Nasim, Ashley Jones, Benjamin Schwessinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Optimising haustoria isolation from infected wheat by reducing chloroplast density and increasing haustoria stability. Isolated haustoria will be used for high-molecular DNA extraction followed by long-read DNA sequencing.  </div></div>

[STEPS]
?. [Blender homogenisation]
Harvest 20–25 g of heavily infected tissue (leaf pieces of ~6 cm length), 6 days after infection (or 1 day before sporulation).
?. [Blender homogenisation]
To remove external contaminating organisms, wash with tap water several times.
?. [Blender homogenisation]
Using a blender, homogenise the infected plant material in 250 mL of homogenisation buffer at maximum speed for 25 s.
Nutribullet Select 1000 W: speed 2-3 for duration of 30 s-1 min.
?. [Filtration]
Filter the homogenate through a 100 μm nylon mesh by gravity ﬂow into Schott bottle using funnel. Forcibly squeeze out as much residual homogenate from the leaf debris as possible, maximising cell capture.
?. [Filtration]
Distribute the ﬁltrate evenly across eight chilled 50 mL Falcon tubes.
?. [Percoll 1: cell fractionation by density]
Bring each pellet to 5 mL with 1x IB, carefully resuspending with a transfer pipette.
?. [Percoll 1: cell fractionation by density]
Centrifuge at 25,000 rcf and 4°C for 30 min without braking.
11824 rpm on  Avanti J-E (Beckman Coulter) Centrifuge.
?. [Percoll 1: cell fractionation by density]
Carefully remove the tubes from the centrifuge, and draw off the top two layers (approx 7-10 ml) from each tube very slowly using a transfer pipette.
Haustoria from different fungal species exhibit slightly different density characteristics. It is highly recommended to establish the density level at which the majority of haustoria resolve. Small-scale density tests can be performed by following the protocol above until step 8. Once the tubes are removed from the centrifuge, aliquots of 1 ml are taken carefully with a wide-bore pipette tip, and the number of haustoria can be counted in each aliquot using a hemocytometer.
?. [Percoll 1: cell fractionation by density]
Centrifuge at 1,080 rcf and 4°C for 15 min.
?. [Percoll 2: cell fractionation by density (optional)]
Prepare a 33% Percoll solution (15 mL per sample).   ABC1ReagentPer sample (15 mL)For 9 (includes 1 dead volume) (135 mL)2Percoll5 mL45 mL310x MM1.5 mL13.5 mL4MQW8.5 mL76.5 mL
ABC1ReagentPer sample (15 mL)For 9 (includes 1 dead volume) (135 mL)2Percoll5 mL45 mL310x MM1.5 mL13.5 mL4MQW8.5 mL76.5 mL
Later, 15 mL of Percoll solution will be added to 5 mL of sample (20 mL total)After combining with the sample, final Percoll concentration will be 25%.Will have approximately 12 tubes. Make one extra sample solution for dead volume. This protocol increases the Percoll to sample ratio, providing better separation of layers.
?. [Percoll 2: cell fractionation by density (optional)]
Centrifuge at 25,000 rcf and 4°C for 30 min without braking.
?. [Percoll 2: cell fractionation by density (optional)]
Centrifuge at 1,080 rcf and 4°C for 15 min.
?. [If proceeding to flow cytometry]
Resuspend the pellets in 1× IB to a ﬁnal volume of 4 mL, and transfer the suspension to a glass vial with plastic lid, 6 ml capacity.
?. [If proceeding to flow cytometry]
Add 200 μl of 1 mg/ml Con A-Alexa 488 (Invitrogen), cover the container with foil, and mix gently on a rotary mixer for 20 min at room temperature (or for 45 min at 4 °C).
?. [If proceeding to flow cytometry]
Use 1× PBS as sheath ﬂuid, run cells using the lowest sample pressure with a resulting sample rate of 5,000–10,000 events per second. Collect sorted cells in 5 mL glass tubes containing 1× PBS, keeping them at 4 °C, and agitating them periodically at 300 rpm to prevent settling.
?. [If proceeding to flow cytometry]
Set ﬂuorescence and scatter parameters. Identify and exclude chlorophyll-containing particles based on chlorophyll autoﬂuorescence. Identify and select the population of Con A-positive particles by Alexa 488 ﬂuorescence. Use forward and side scatter information to identify and exclude doublets by comparing the forward scatter height and width and the side scatter height and width. To optimise the forward scatter signal, use a neutral density 2 ﬁlter in front of the forward scatter detector.
?. [If proceeding to flow cytometry]
Conﬁrm sort purity by running a small amount of the sorted cells to determine the percentage of positive events for the sort parameters (see Note).
Typically, 9 × 10 5 –1.5 × 10 6  puriﬁed haustorial cells can be puriﬁed from 15 to 20 g of wheat tissue heavily infected with P. striiformis (in our case it is P. graminis).Vital tests should be applied at this stage to verify the viability of haustoria post-sorting. A high percentage of viable cells could be very important for some subsequent applications as isolation of intact RNA or proteins. In this protocol the author used CellTracker™ Orange CMRA (Invitrogen, Catalog number C34551) successfully following the manufacturer’s instructions, and obtained ≥98 % viable haustoria post-sorting.
?. [If proceeding to flow cytometry]
Pool the samples from FACS puriﬁcation and centrifuge them at 1,080 ×  g  for 15 min at 4 °C. Discard the supernatant and snap-freeze the ﬁnal pellet in liquid nitrogen, and store it at −80 °C until required.
?. [If proceeding to flow cytometry]
Pellet the haustoria at 4,000 ×  g  in a benchtop centrifuge for 5 min at 4 °C. Remove the supernatant and wash the pellet twice with 1× IB. Resuspend in a ﬁnal volume of 4 ml of 1× IB; keep sample on ice in the dark and proceed directly to FACS sorting.
?. [If proceeding to flow cytometry]
Set up and optimise the cell sorter. For fungal haustoria (5–20 μm in diameter): 100 μM nozzle, 20 psi sheath pressure.
The procedure of setting up a ﬂow cytometer varies depending on the machine and needs to be performed by appropriately trained personnel.
?. [If proceeding to flow cytometry]
This section describes the use of ﬂow cytometry to sort haustoria based their ﬂuorescence after labeling with Con A-Alexa 488. The Alexa 488 ﬂ uor is excited using a 13 mW 488 nm solid-state laser (Sapphire, Coherent Inc. Santa Clara, CA) and detected using a 502 nM longpass as well as a 530/30 nM bandpass ﬁlter. Chlorophyll autoﬂuorescence is excited with the same 488 nm laser and detected using a 655 longpass and 695/40 bandpass ﬁlter.
?. [If proceeding to flow cytometry]
Dilute the haustorial sample 1:10 with chilled 1× PBS to adjust the concentration of the chloroplast–fungal cell mixture to approximately 2.5–5.0 × 10 6  particles/ml.
?. [Blender homogenisation]
Incubate the tissue for 1 min in chilled 70% ethanol. Wash tissue with chilled MilliQ water several times to remove the alcohol. Dry the tissue as much as possible with Kimwipes (Kimtech).
?. [Filtration]
Centrifuge at 1,080 rcf and 4°C for 15 min, using a centrifuge with swing out rotor.
?. [Percoll 1: cell fractionation by density]
Dilute the pooled haustorial fraction 1:10 with 1× IB (for 70 mL, add 630 mL 1x IB). Mix well and distribute the mixture into four chilled Nalgene tubes (250 mL)
The volumes may differ depending on how much grams of infected leaves were used.
?. [Percoll 2: cell fractionation by density (optional)]
Dilute the pooled haustorial fraction 1:10 with 1× IB (for 70 mL, add 630 mL 1x IB). Mix well and distribute the mixture into chilled Nalgene tubes (250 mL)
?. [Filtration]
Filter the remaining homogenate through the 100 μm nylon mesh, combining with the first homogenate.
?. [Hypotonic lysis 1 (optional) ]
Resuspend each pellet in 20 mL pre-chilled hypotonic lysis buffer by gentle mixing or pipetting with a transfer pipette.
?. [Hypotonic lysis 1 (optional) ]
Centrifuge at 1,080 rcf and 4°C for 15 min, using a centrifuge with swing out rotor.
?. [Percoll 2: cell fractionation by density (optional)]
Carefully remove the tubes from the centrifuge, and draw off the top two layers (approx 7-10 ml) from each tube very slowly using a transfer pipette.
?. [Blender homogenisation]
Wash with tap water three times or until sodium hypochlorite is completely removed.
?. [Blender homogenisation]
Incubate for 3 min in 2% sodium hypochlorite.
?. [Filtration]
Recover the solid particles retained on the mesh and return them to the blender, add the remaining homogenisation buffer and blend for 15 s.
?. [Filtration]
Filter homogenate through 1 layer of Miracloth using a funnel and 1 L Schott bottle. Gently squeeze residual homogenate through the Miracloth.
Miracloth 22-25 um. The pore size of the meshes is critical for reducing contaminating particles before FACS. Different fungal species display a diverse range of haustoria size, thus small-scale isolations to test different pore sizes and microscopic analyses are necessary to optimize the ﬁltration step.
?. [Percoll 2: cell fractionation by density (optional)]
Bring each pellet to 5 mL with 1x IB.
?. [Percoll 2: cell fractionation by density (optional)]
Add 15 mL of the 33% Percoll solution to each sample, bring the final volume to 20 mL. Mix gently by inverting and swirling.
?. [Filtration]
Repeat Filtration through 2 layers of Miracloth (using a new Schott bottle).
?. [Filtration]
Carefully discard the supernatant, being careful not to dislodge the pellet.
?. [Hypotonic lysis 1 (optional) ]
Incubate at 4°C for 1 h, with gentle rotation, ~20 rpm. Can place a rotator in a cold room.
?. [Hypotonic lysis 1 (optional) ]
Carefully discard the supernatant, being careful not to dislodge the pellet.
?. [Percoll 1: cell fractionation by density]
Prepare a 40% Percoll solution (15 mL per sample).   ABC1ReagentPer sample (15 mL)For 13 (includes 1 dead volume) (195 mL)2Percoll6 mL78 mL310x MM1.5 mL19.5 mL4MQW7.5 mL97.5 mL
ABC1ReagentPer sample (15 mL)For 13 (includes 1 dead volume) (195 mL)2Percoll6 mL78 mL310x MM1.5 mL19.5 mL4MQW7.5 mL97.5 mL
Later, 15 mL of Percoll solution will be added to 5 mL of sample (20 mL total)After combining with the sample, final Percoll concentration will be 30%.Will have approximately 12 tubes. Make one extra sample solution for dead volume. This protocol increases the Percoll to sample ratio, providing better separation of layers.
?. [Percoll 1: cell fractionation by density]
Add 15 mL of the 40% Percoll solution to each sample, bring the final volume to 20 mL. Mix gently by inverting and swirling.
?. [Percoll 1: cell fractionation by density]
Carefully discard the supernatant, being careful not to dislodge the pellet.
?. [Hypotonic lysis 2 (optional)]
Resuspend each pellet in 20 mL pre-chilled hypotonic lysis buffer by gentle mixing or pipetting with a transfer pipette.
?. [Hypotonic lysis 2 (optional)]
Incubate at 4°C for 1 h, with gentle rotation, ~20 rpm. Can place a rotator in a cold room.
?. [Hypotonic lysis 2 (optional)]
Centrifuge at 1,080 rcf and 4°C for 15 min, using a centrifuge with swing out rotor.
?. [Hypotonic lysis 2 (optional)]
Carefully discard the supernatant, being careful not to dislodge the pellet.
?. [Percoll 2: cell fractionation by density (optional)]
Carefully discard the supernatant, being careful not to dislodge the pellet.
?. [If extracting DNA from the raw pellet]
Add 1.5 mL of 1x IB to the pellet and gently resuspend with a transfer pipette.
?. [If extracting DNA from the raw pellet]
Transfer to a 2 mL Eppendorf tube. Save a 40 μL aliquot for microscopy confirmation of haustoria, storing in fridge at 4°C.
?. [If extracting DNA from the raw pellet]
Centrifuge at 1,080 rcf and 4°C for 15 min.
?. [If extracting DNA from the raw pellet]
Carefully discard the supernatant, being careful not to dislodge the pellet.
?. [If extracting DNA from the raw pellet]
Store the pellet at -80°C until DNA extraction.