Protocol for nodulation assay in pouches

 

1. Sterilize pouches by placing them between wet paper towels then wrapping them in aluminum foil wrap. If you do not autoclave them moist, then they become quite brittle.
2. Sterilize soybean seeds, and germinate 2-3 days on wet paper or water agar. Place 3-5 seedlings per pouch; place seeds in the trough formed in the paper wick (there is a germination paper inside of the pouch and there are holes within the trough). For soybean, given the size of their roots, it helps to use a sterile needle or spatula to make holes in the paper trough for the roots. Note it is useful to select the seedlings so that they have straight roots…just makes it easier to insert them and they tend to grow better in the pouches. We plant seedlings since it allows us to better select the plants and also to eliminate any that may have fungus, etc. Fungus is one of the primary problems you have in pouch grown plants. If you like, you can just plant the seeds in the paper trough and this can work fine but this gives you less of an opportunity to select the best seedlings.

If you do use seeds, then we usually put 5 seeds per pouch as at least 1 of them will not germinate properly

3. Add 8-10 ml sterile H₂O or ½ strength plant nutrient solution(c/o nitrogen) to the pouch, wrap it in alufoil (to keep in dark) and place them into growth chamber (soybean conditions) for 3 days/dark (if you use seeds in step 2) or straight into the light, if you use seedlings. When we do a lot of these, then we have some old 45 rpm record racks (probably impossible to find now) and place one pouch in each slot with cardboard on each side. This eliminates the need for the alufoil and keeps the roots dark. Again, for larger scale use, we also use sterile soda straws (the ones you buy individually wrapped are supposedly sterile) and place one down one side of each pouch. This allows you to water the plants from the bottom up without removing them from the record racks. Important to use this bottom up approach to keep from washing contamination into the pouches by watering from the top.
4. Inoculate 3-days old seedlings, with rhizobia of OD₆₀₀~ 0.05-0.1 by spraying or pipetting. We make every attempt to make sure that the inoculant is applied to the roots. Note if you use seedlings then inoculation can be done at the time of sowing, otherwise, when the seeds have germinated.

Note: if you used seeds, not seedlings, then remove seedlings that are not properly germinated or seem to have fungi. Also, if the root is not going towards inside of the pouch, make them fit into hole (within the trough of the paper wick) and ensure that the roots are facing the bottom of the pouch.

5. Cover the pouch (downwards from the troughto ensure that the roots are covered and are in dark). Place the pouches into pouch-rack (paper racks, come with the pouches) or, in our case, the record racks.
6. Never let the paper inside the pouch get dry but it is also important not to over water. You can water with either dH₂O or with ½ plant nutrient solution, sometimes we alternate just to be sure we do not get salt buildup. Normally, plants will grow nicely in the pouches from 14-21 days without any problem but usually tend to yellow if you extend beyond this period (likely associated with drop in Ph). However, we normally grow plants to 28 days in pouches. Depending on overall conditions, you can see nodule formation within 12 days or so, sometimes sooner, sometimes a bit later.

The biggest issues with the pouches is that the pH will normally drop to quite low unless you make efforts to prevent this. For example, using a well buffered plant nutrient solution for watering. In the olden days, we even added solid calcium carbonate to the pouches, which would dissolve overtime and maintain pH but of course changes other parameters, such as ionic strength. Hence, keep an eye on how damp the plants are and water as needed. Check on the medium every day, medium needs to be replenished approx. every other day but more often as plants get bigger.

7. Important to check the pouches every day, sometimes twice as day, since water usage will vary significantly based on the size of the plants and other growth conditions (chamber humidity, light intensity, temperature, etc).

Pouches – cyg seed germination pouches (Mega-International of Minneapolis)

https://mega-international.com/

Straw – plastic drinking straws sanitary wrapped; 7¾” jumbo

Fahraeus medium (nothing special about this…you can use other nutrient solutions…indeed, something that is better buffered than Fahraeus is probably better)

• Prepare Fahraeus medium(Fahraeus, 1957) from concentrated stock solutions (autoclaved and stored at 4°C as follows):
• 5 mM MgSO4·H₂O
• 7 mM KH₂PO₄
• 8 mM Na₂HPO₄·2H₂O
• 50 μM Fe-EDTA [prepared using FeSO₄and disodium EDTA; alternatively use 20 mM ferric citrate (stored in the dark) to 50 μM final concentration]
• 1 μg/liter MnSO₄
• 1 μg/liter CuSO₄
• 1 μg/liter ZnSO₄
• 1 μg/liter H₃BO₃
• 1 μg/liter Na₂MoO₄
• Milli-Q-purified H₂O up to 1 liter
• Adjust pH to ∼5
• Autoclave

1. DNase treatment of total RNAs

On ice, mix the following:

*         RNAs                                        10ug (not more than 29.5 ul)

*         10 * RTase buffer                       5ul

*         RQ1 DNase                               2ul

*         H2O                                           up to 36.5ul

Mix well, spin down and incubate at 37C for 15min.

Inactive the DNase at 65C for 10min, then transfer the tubes on ice.

2. RTase (Synthesis of the first strand cDNAs)

On ice, add the following to the DNase-treated RNAs:

*         0.1M DTT                                5ul

*         10mM dNTPs                         5ul

*         0.5ug/ul oligo-dT                    2ul

*         MMLV-RTase                        1ul

*         RNase-inhibitor                      0.5ul

Incubate the tubes 1 hour at 37C then 2min at 92C.

For long storage, store the cDNAs at -80C, otherwise at -20C.

3. PCR

Perform the PCR by using 1 ul of cDNAs for a 50 ul PCR reaction.

Based on Inoue et al (1990), Gene, 96:23-28, with modifications.

1. Culture cells (DH5a in my case) on LB agar plate at 37oC overnight.
2. Pick up 10 -12 large colonies and culture in 250ml SOB in a 1L flask, 19oC with vigorous shaking to OD600=0.5 (normally it takes 24-36hrs)
3. Place the flask in ice for 10 min.
4. Pelleting the cell by spining at 4000rpm for 10 min at 4oC.
5. Gently resuspend the cell in 80ml ice-cold TB and store on ice for 10 min.
6. Spin at 4000rpm for 10 min at 4oC
7. Gently resuspend the pellet in 20ml ice-cold TB and 1.4ml DMSO (the DMSO needs to be stored at -20oC o/n before use).
8. Aliquote the cell to 50 to 500ul for transformation or store at  -70oC

   *         Note: The E. coli cells prepared this way are normally 100 to 1000 times more efficient than normal calcium method, so do not plate too dense!

• SOB solution:

  *         0.5% yeast extract
  *         2% tryptone
  *         10mM NaCl
  *         2.5mM KCl
  *         10mM MgCl2
  *         10mM MgSO4
  *         Dissolve in nanopure water and autoclave to sterilize.

• TB solution:
  *         10mM PIPES
  *         15mM CaCl2
  *         250mM KCl
  *         Dissolve in nanopure water and adjust pH to 6.7 with KOH or
  HCl and then add MnCl2 to 55mM, and adjust to final volume. Sterilize by
  filtration with 0.45um filter and store at 4oC

1.      50-100mg leaf tissue in 1.5 ml eppendorf tube (1 cotyledon for
PCR only)

2.      Prepare fresh microprep buffer, RT

3.      Add 200ul buffer, grind tissue (rinse pestle with water between
samples). Add another 55ul buffer, shake entire rack by hand.

4.      65C, 30-120min

5.      Fill the tube with chloroform, mix well (shaking up and down
50-100 times)

6.      10000 rpm, 5min

7.      Pipet off aqueous phase (~0.5ml), add 1X volume of cold
isopropanol, invert tube repeatedly until DNA precipitates.

8.      Immediately spin at 10000rpm for 5min (No more)

9.      Wash pellet with 70% ethanol

10.  Dry

11.  Resuspend in 50ul TE at 65C for 15min

12.  Spin 10min at 10000rpm, store at -20C.

13.  1ul for PCR, 15-25ul for southern blot (5-10ug DNA, 15-20 units
enzyme) (If 1 cotyledon was used, 5ul for PCR)

DNA extraction buffer(pH 7.5)

50ml

100ml

Final concentration

Sorbitol (MW 182.2)

3.19g

6.38g

0.35M

Tris-base (1M)

5ml

10ml

0.1M

EDTA (0.5M)

0.5ml

1ml

5mM

Nuclei lysis buffer:

15ml

30ml

Final concentration

Tris (1M)

10ml

20ml

0.2M

EDTA (0.5M)

5ml

10ml

0.05M

NaCl

5.84g

11.68g

2M

CTAB

1g

2g

2%

Sarkosyl: 5% (w/v)

Microprep buffer:

DNA extraction buffer

25ml

15ml

10ml

Nuclei lysis buffer

25ml

15ml

10ml

5% sarkosyl

10ml

6ml

4ml

Sodium bisulfite

0.2g

0.12g

0.08g

1.        Stock solution

(1)    5×MOPS

ddH2O                                          400ml

3M NaOAc (pH7.0)                      6.67ml

0.5M EDTA (pH8.0)                         5ml

MOPS                                           10.4g

NaOH                                           ~0.875g to pH7.0

Add ddH2O to 500ml, add 500ul DEPC

Set overnight, autoclave for 15min.

(2)    0.5M Na3PO4 (pH7.2)

ddH2O                                            1800ml

NaH2PO4·H2O                               43.6g

Na2HPO4·7H2O                         183.3g

Add ddH2O to 2l, autoclave for 15min.

(3)    10×SSC

ddH2O                                              1800ml

NaCl                                                  175g

Trisodium citrate                                88.2g

Adjust pH to 7.0 with HCl, add ddH2O to 2l, add 2ml DEPC

Set overnight, autoclave for 15min.

(4)    DEPC ddH2O

(5)    14% SDS

2.        Electrophoresis

(1)    Formaldehyde gel ( Pretreat comb and plate with ethanol and Rnase away)

50ml             100ml               200ml           250ml

Agarose             0.5g              1.0g                 2.0g               2.5g

5×MOPS          10ml             20ml                 40ml              50ml

DEPC H2O       38ml             77.5ml              155ml           194ml

Melt

65C H2O bath 10min

Add 37% Formaldehyde  2.7ml           5.4ml                10.8ml          13.5ml

Pour gently

Set 15-20min

(2)    Running buffer (1×MOPS )

250ml             600ml             1000ml

DEPC H2O        200ml             480ml              800ml

5×MOPS             50ml             120ml              200ml

(3)    Sample preparation (30ul)

Formamide                                               15ul

5×MOPS                                                    6ul

formaldehyde                                            4.8ul

RNA                                                          10ug ( less than 4.2ul)

DEPC H2O                                                 add to 30ul

65C 10min, put on ice, add 3ul 10×loading buffer.

(4)    Electrophoresis ( Pretreat tank and cylinder with Sparkleen)

Pre- electrophoresis gel at 5V/cm for 5min

Load samples

Run gel at 4V/cm for 1-2h.

3.        Transfer of RNA from Gel to Membrane

Wash gel with DEPC H2O  3 times ( 20min RT H2O, 10min 65C H2O, 20min RT H2O)

Wash with 10×SSC for 30min

Vacuum blot 90min at 5in pressure, using 10×SSC as transfer buffer

Rinse with 2×SSC for 5min

80C bake 30min.

4.        Hybridization

(1)    (Pre)hybridization solution       50ml ( 30ml for prehybridization, 20ml for hybridization)

0.5M Na3PO4 (pH7.2)                 25ml

14% SDS                                     25ml

(2)    Probe labeling

Probe                                              100ng

ddH2O                                             add to 30ul

boil 5min, put on ice

5×labeling mix                                10ul

d(C,T,G)TP mix (1.5mM)                 2ul

BSA(10mg/ml)                                  2ul

Klenow(5u/ul)                                   1ul

P32-dATP                                         5ul

RT 2hrs, add 2ul 0.5M EDTA, boil 10min, G-50 micro column clean, use for hybridization.

(3)    Prehybridization (65C) for more than 2 hrs( while probe labeling), hybridization (65C) overnight.

(4)    Wash

14% SDS                                               89ml

Na3PO4                                                  10ml

ddH2O                                                    151ml

wash for 2 times at 65C, use 125ml wash solution each time.

(5)    Strip

10×SSC                                                    10ml

14% SDS                                                    35ml

ddH2O                                                     955ml

wash for 2 times in boiling solution, use 500ml solution each time.

(All microscopy, including sectioning, silver enhancement, and immunolocalization was performed by Ms. Susan Fink and Dr. John Dunlap, The University of Tennessee, Center for Electron Microscopy).

 

 

Tissue Preparation for Immunogold Labeling

 

Root and stem tissues were obtained from uninoculated and BradyrhizobiumjaponicumUSDA110 inoculated plants at time points ranging from 2 days to 15 days post-inoculation.  Root tissue was obtained from the root meristem, as well as the region of the root corresponding to the zone of the first emergent root hairs.  Stem tissues were harvested from the shoot meristematic region of 15-day-old G. soja plants.  Secondary, lateral roots were also collected at time points ranging from 2 days to 15 days post-inoculation.

Tissue samples were fixed in 1% paraformaldehyde, 1%glutaraldehyde, 0.1M sodium cacodylate buffer (pH 7.0) for 1 hour at room temperature, then washed in 1X SSC buffer (0.15M NaCl, 0.015M Na-citrate, pH 7.0) three times, 20 minutes each.  Samples were then dehydrated with an initial series of ethanol (10%, 20%) for 10 minutes each at 20°C.  Samples were further dehydrated in ethanol (30%, 50%, 75%, 90%, 100%) for 1 hour each at -20°C.  Dehydrated samples were gradually infiltrated with LR (London Resin Co., Bershire, England):ethanol : LR White (2:1, 1:1, 1:2, 1 hour each) at -20°C, and pure LR White resin overnight at -20°C.  Semi-thin sections (1mm) for silver enhancement and thin sections (100nm) for electron microscopy were prepared using a Reichert OMU3 ultramicrotome (Reichert, Vienna, Austria).

Conventional fixation of soybean nodules was performed as follows: Nodules from G. sojaplants 15 days post-inoculation were fixed in 3% glutaraldehyde in 0.5 M phosphate buffer (pH 6.8) for 1 hour at room temperature.  Samples were then fixed in 2% OsO₄ in 0.05M phosphate buffer (pH 6.8) for 1 hour at room temperature.  Following fixation, samples were dehydrated in an acetone series (25%, 50%, 75%, 95%, 100%) for 30 minutes each at room temperature.  Dehydrated samples were gradually infiltrated with Spurr resin (Electron Microscopy Sciences, Fort Washington, PA): acetone: Spurr’s (2:1) for one hour at room temperature, followed by infiltration with acetone: Spurr’s (1:2, pure Spurr’s) overnight each at room temperature.  Samples were polymerized in fresh Spurr resin at 75°C for 16 hours.  Following polymerization, thin sections were prepared as described above.

 

Silver Enhancement Microscopy

 

Thin sections of stem, root, and nodules were obtained from the LR White and Spurr embedded blocks using glass knives on gelatin-coated glass slides.  Sections were incubated in serum blocking solution (SBS) (10% non-immune serum, goat) for 15 minutes at room temperature.  A 1:3 (SBS:antibody) dilution of Protein A-purified anti-GS50 or anti-GS52 IgG antibody was added to the sample and incubated at 4°C for 1.5 hours.  Slides were then washed three times with 1X TTBS Buffer (20 mM Tris, 500 mM NaCl, 0.5 % Tween-20, pH 7.0) twice for two minutes each, followed by two-2 minute washes in TBS (20 mM Tris, 500 mM NaCl, pH 7.0).   Samples were then incubated with 1:10 diluted secondary antibody, goat anti-rabbit IgG gold conjugates (GAR-gold 15 nm, ER Laboratory, San Mateo, CA) for 1 hour at room temperature.  Pre-immune controls were included for all tissue samples.  Slides were washed with 1X TTBS, followed by TBS as described above.  Air-dried, gold-labeled sections were treated with silver enhancer solution A and B (1:1) (Sigma Chemical, St. Louis, MO) for 8-10 minutes and washed with distilled water.  Samples were fixed in 2.5% sodium thiosulfate for 2-3 minutes.  Sections were observed under an Epifluorescent microscope with a polarizing filter (Nikon Inc., Melville, NY).

 

Immunogold Labeling for Electron Microscopy

 

Ultra-thin sections (100nm) were treated with SBS as described above for 15 minutes.  Protein A-purified GS50 or GS52 antibodies were diluted  1:5 in 1X TTBS + 5% normal goat serum and added to the samples.   Thin sections were incubated for 1.5 hours at room temperature, followed by 4 washes with PBS.  Samples were incubated with 1:50 diluted secondary antibody[goat anti-rabbit IgG gold conjugates (GAR-gold 15 nm)] for 1 hour at room temperature.    Samples were washed 4 times in PBS, 10 minutes each.  Samples were stained for 45minutes with 2% aqueous uranyl acetate and for 5 minutes with lead citrate.  Sections were viewed under 75kV with a Hitachi 600 transmission electron microscope.

Medium:

20 g            Dextrose

1 pkg         Murashige’s and Skoog’s plant salt mixture

1 l               ddH2O

adjust pH to 5.7 with 10 N KOH (~ 250 µl)

autoclave for 15 min. When finished, remove promptly from autoclave.

1. Turn on hood and UV for ~ 30 min before start.

2. Wipe bench with EtOH.

3. Transfer less than 1000 seeds to 1.5 ml Eppendorf tube.

4. Add 200 ul 75% EtOH.

5. Immediately add 600ul sterile water, pipette 600 ul liquid off .

6. Repeat 5 for 3-4 times.

7. Add 200 ul bleach, mix well, set for 5min.

8. Add 600 ul sterile water, pipette 600 ul liquid off.

9. Repeat 8 for 5-6 times.

10. Cold treat for 2 days (this is day 1).

11. Transfer seeds into 50 ml Falcon tube with 10ml liquid medium at day 3.

12. Shake under lights. Seeds will germinate after a few days.

13. Change fresh liquid medium at day 13.

14. Add chitin at day 14.

* You can sterilize as many as 1000 seeds in one 1.5 ml Eppendorf tube. But after cold treatment, they have to be seperated into different falcon tubes (50 seeds/tube, 10ml liquid medium).

For me, I calculate the number of samples I will need for chitin treatment first. Say, if you want to do 5 treatments for the seeds from the same line, you will need (5+1)*50=300 seeds (See below for the reason I add 1). I count the seed number, it should be final number*120%, that is 360 seeds, because you will lose some seeds during sterilization. These seeds can be sterilized in a single 1.5 ml Eppendorf tube. After cold treatment, I transfer the seeds

into a 15ml falcon tube, add liquid medium to 6ml. Prepare 5 50ml falcon tubes, preadd 9ml liquid medium. Mix the seeds well by pippett the solution and transfer them to the 50ml falcon tube, 1ml/tube. It will be much easier to control the seed number you get by add 1 more sample and use 6ml instead of 5ml. (if you have 10 samples you will need to prepare seeds for 12 samples at the beginning)

Medium:

20 g            Dextrose

1 pkg         Murashige’s and Skoog’s plant salt mixture

1 l               ddH2O

adjust pH to 5.7 with 10 N KOH (~ 250 µl)

autoclave for 15 min. When finished, remove promptly from autoclave.

1. Turn on hood and UV for ~ 30 min before start.

2. Wipe bench with EtOH.

3. Transfer about 50 seeds to 1.5 ml Eppendorf tube.

4. Add 200 ul 75% EtOH.

5. Immediately add 600ul sterile water, pipette 600 ul liquid off.

6. Repeat 5 for 3-4 times.

7. Add 200 ul bleach, mix well, set for 5min.

8. Add 600 ul sterile water, pipette 600 ul liquid off.

9. Repeat 8 for 5-6 times.

10. Cold treat for 2 days.

11. Transfer seeds into 50 ml Falcon tube with 10ml liquid medium.

12. Shake under lights. Seeds will germinate after a few days.   Cultures grow up in two weeks.

13. Change to fresh liquid medium before chitin treatment.

SSG Preparation

1.  Soybean seedlings were germinated in the dark for 5-6 days.  Note:  the seeds should be germinated under
conditions where just enough water is added for the 5 day germination period.  As a yard stick, add enough water to make sure that the paper towels are wet, leaving about an addition 10-15 ml in the pans.

2. Cut of roots and freeze in liquid nitrogen.

3. To extract inducer, thaw the root tissue, and add Ethanol (Optima grade; Fisher Scientific) in the following ratio:  2 ml Ethanol for every gram of tissue.  Typically 100-150 g of tissue is used.

4.  Grind tissue in waring blendor, and place mixture in clean glass flask (500 ml) and shake 2 h at RT.  The mixture is left overnight in the cold room.

5. Spin mixture JA17 (40000 rpm) to remove root debris.  Keep supernatant.

6.  Rotary vap supernatant to concentrate 10 fold.  Starting with 100 gram of tissue, this usually results in about
10-15 ml of concentrated SSG.  Store at -20 C.  Use about 5 ul of sample in assays with the nolA-lacZ fusion.  Increasing the amount of SSG does not increase activity.

Isolation of Inducer

1.  Prepare Sep-pak as per manufacturers instructions.  This entails first washing the column sequentially with100% MeOH, and water.  Add 2 ml of concentrated SSG to the sep-pak, reapplying flow through 3 times.  Keep flow through.

2. The Sep-pak column is then washed with 3 x with water, 3 x with 60% MeOH and then eluted with 3 x 100% MeOH.

3.  Eluted sample is concentrated by “air-drying” with the “house”-air.  Prior to HPLC, the sample is resuspended in 5 ml 60% MeOH.  Note:  the sample is very concentrated at this juncture, and this fraction is usually diluted 4-5 fold (ie. 500 ul of sample into 5 ml 60% MeOH) and applied to the HPLC.

4.  The column used is:  C18, phenomenex Jupiter, 250 x 4.6 mm, 5 microns.

5.  HPLC conditions:  load sample 60% MeOH/40% water.

1 min: 60% MeOH

40 min 100% MeOH

hold 15 min at 100% MeOH

return (5 min) to 60% MeOH.

6.  Active fractions are highlighted in the attached.  Peak No. 4 is chitinase sensitive.  Peaks,12, 12a and 13 are active on the nolA fusion but are not chitinase sensitive.

XAD column Protocol

1. As an alternative means to boost inducer isolation, the following protocol was utilized to enrich for inducer.

2. XAD column matrix was prepared as directed by the manufacturer.  (Typically 5 g of matrix was used for each XAD based purification).

3. XAD beads were transferred to a 500 ml beaker and MeOH added to cover the resin.  The beads were soaked for 15 min in MeOH.

4. Methanol was replaced, and the beads were washed with distilled water.

5. Washing with distilled water was repeated 3 times to remove all residual MeOH, and the beads allowed to sit in water for 10 min.

6.  The slurry was then removed and added to 5 ml of SSG extract (the SSG extract can be diluted at this stage with water to make up a total volume of about 15 ml).

7.  This SSG-XAD slurry was incubated overnight with gentle rocking at RT in the dark in 20 ml vials containing PFTE caps.

8.  Following incubation, the suspension was allowed to sit and the unbound SSG removed.  The beads were washed with water 5 times, and eluted first with 100% MeOH (4 times on a rotary shaker at 150 rpm).  The MeOH fractions were pooled.

9.  The remaining bound material was then eluted with acetone.

10. Both MeOH and acetone eluted fractions were air dried separately, resuspended separately in water before being applied to the sep-pak column as described above.  Following sep-pak purification, the samples were analyzed by HPLC as previously described.

Preparation of CDF

1.                  Bacteria cultures were grown up in Minimal medium to
late log phase (OD600 = 2.0).

2.                  Equal volume of ethyl acetate (Optima Grade, Fisher
Scientific) was then added and incubated, with shaking at 200 rpm, for 1
h.

3.                  The flasks were then allowed to sit for 1 h, or as
long as it takes to separate the aqueous phase from the ethyl acetate
phase.  Should the layers fail to separate, additional ethyl acetate is
added and step 3 is repeated.

4.                  Remove the top layer containing the ethyl acetate
using a glass pipette, and store in glass container.  The ethyl acetate
is removed by blow-drying, using the house air-line.

5.                  The concentrated extract, containing CDF, is then
stored at -20 C.

HPLC analyses of CDF