(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

1.      Excise root and leaf segments, wash with 100mM potassium
phosphate buffer (pH7.0) for 3 times.

2.      Immerse in the Gus substrate solution, 37C, dark, 12-24h

3.      Rinse in phosphate buffer

4.      Fix for 4h or longer

5.      Rinse in the same buffer

6.      Observe as whole specimens or as sections.

1M potassium phosphate buffer (pH7.0)

K2HPO4: 34.8g —>200ml

KH2PO4: 27.2g—->200ml

184.5ml K2HPO4+115.5ml KH2PO4—-> 300ml 1M potassium phosphate buffer
(pH7.0)

Gus substrate solution

Volume(500ml)

Final concentration

Potassium ferricyanide

82mg

0.5mM

Potassium ferrocyanide

105.6mg

0.5mM

Potassium phosphate buffer (pH7.0, 1M)

50ml

100mM

Store at -20C. Add 1mM x-gulc (MW521.8~~0.5mg/ml) freshly when use.

Fix solution

2.5% glutaraldehyde

200mM sodium cacodylate, pH7.2

1. Harvest tissue (can be stored at -70C)

2. Grind tissue in 100ul extraction buffer.

3. Spin 1min. Take supernatant, put on ice.

4A. 20ul for BCA assay (add 500ul assay reagent—-> 37C 30min
—–>562nm

4B. Add 0.05ul mercaptoethanol to the remnant 80ul, spin 10min.

5. Set up GUS assay:

In borosilicate tube (VWR 60825-550) combine:

Plant extract: 20ul

Assay mix: 80ul

Mix well. Assay mix is 70 ul extraction buffer and 10ul substrate
solution.

6. Incubate at 37C covered with parafilm for 1 hour exactly.

1.Stop FUS assay by adding 900ul 0.2M sodium carbonate solution.

2.Measure fluorescence in Sequoia-Turner fluorimeter using a MU standard
as a control.

Filters: 365nm/455nm

Fluorimeter must warm up for 15 minutes before use.

Gain 1 or Gain 10 should be sufficient.

Substrate solution: 1mM MUG in extraction buffer (22mg per 50ml)

Dissolve MUG in DMF first before adding to extraction buffer.

Store at 4C, but it is not stable for more than a few weeks.

Extraction buffer

50mM sodium phosphate pH7.0

10mM EDTA

0.1% laurylsarcosine

0.1% Triton-X-100

10mM beta-mecaptoethanol (can also try 1mM fresh DTT)

Stop solution

2.12g sodium carbonate in 100ml water.

BCA assay

Mix reagents A and B as indicated 50:1 just before use. Make a standard
of 0, 5, 10, 20ug BSA (in 1ml). Use 5, 10, 20ul protein solution per
assay and add 500ul assay reagent. Incubate for 30min at 37C, then read
Absorbance at 562nm.

MU standard

MU is not stable in solution and has to be prepared fresh, dissolved in
DMF and diluted to 1mM in extraction buffer, every time the standard is
measured.

9nmole MU in 1ml stop buffer will give a reading of 1559 at gain 1.

X=8.56 X 10(exp-3) X f/A

With

X=GUS activity in [pmole/min/ug protein]

F=fluorescence units

A=absorbance of BCA assay assuming that 20ul protein was assayed.

Preparation of Cells

1.      Inoculate 1l of L-broth with 1/100 volume of a fresh overnight culture

2.      37C, shaking, to ABS600 = 0.5-0.8

3.      Chill the flask on ice for 15-30min, centrifuge in a cold rotor 6000G, 10min

4.      Remove as much of the supernatant as possible, resuspend pellets in 1l 10% glycerol, repeat step 3

5.      Resuspend in 0.5 l 10% glycerol, repeat step 3

6.      Resuspend in 20ml ice cold 10% glycerol, repeat step 3

7.      Resuspend in 2-3ml 10% glycerol

8.      Frozen in aliquots in liquid N2, store at -80C.

Electroporation

1.      Chill electroporation cuvettes (use sterile ones, can be reused for 2-3 times) and white chamber slide

2.      Add 1-2ul plasmid/ligation product

3.      Add 40ul electroporation competent cells on the same spot where the plasmid was placed

4.      Agitate to mix, incubate for 1-2min

5.      Pulse (Setting: 1.8KV/200Ù/25uFD)

6.      Add 1ml LB in cuvette, transfer to eppendorf tube

7.      Shake for 1 hour

8.      Plate

I. Surface sterilization of rice seeds

·           De-hull rice seeds

·           Wash with 70% ethanol for 1 min in a Falcon tube

·           Replace 70% ethanol with 20% sodium hypochloride solution and incubate the seeds for 1 hour on a shaker in a slow motion

·           In plant-hood, wash the seeds with sterile distilled water for 5-6 times (After the final wash, the seeds can be left over-night in dark in sterile distilled water on a shaker)

II. Callus induction

·           Transfer surface sterilized seeds, on by one, to the callus induction medium -ND2 (see Appendix I) in 9cm petri-dishes (30ml of the medium per plate and place 26 seeds per plate). Seal the plates with parafilm. Incubate the plates in the dark at 26 C. Ten days later, transfer the calli induced from the scutellar region to fresh ND2 medium. The calli should be subcultured every two weeks.

III. Pre-culture

·           After 2-3 2-week subculture cycles, transfer the looslely attached small globular calli (1-3mm in diameter) to ND2 medium for 4-day-pre-culture. Conditions for culturing the calli are same as above.

IV. Osmotic treatment

·           Prepare plates (9cm petri-dishes) containing ND2MS medium (see Appendix II). Arrange the precultured calli in a circle ( the same size as a quarter) on agar surface 4 hours before transformation.

V. Biolistic transformation using the PDS-1000 particle gun

·           Microcarrier (gold) preparation

1.          Place 30 mg of gold particles (Bio-Rad) in a 1.5 mL microtube

2.          Add 0.5 mL 100% ethanol and vortex for 1-2 min

3.    Centrifuge the microtube at 10,000 rpm for 1 min. Remove supernatant. Repeat steps 2 and 3 twice

4.    Add 0.5 mL sterile distilled water, resuspend, centrifuge, and remove supernatant. Repeat 3 times

5.       Resuspend microcarriers in 0.5 ml sterile distilled water.

6.       Store at 4 C

·           Preparing CaCl₂ and Spermidine

1.      CaCl₂: 2.5 M, autoclaved, store at ¨C20 C

2.      Spermidine: 0.1 M, filter, store at ¨C20 C for no more than 1 month

·           Sterilization of sonsumables and assemble

1.   Soak macrocarrier holders, macrocarries, stopping screen and rupture disks (1100 Psi) in 70% ethanol for at least 15 min

2.      Dry in the hood on sterile filter paper

3.     Rinse other metallic parts (acceleration tube, rupture sisk retaining cap, and microcarrier launch assembly) in 70% ethanol

4.      Dry theses inside the hood

5.      Clean the components of PDS eith 70% ethanol. Allow to dry

·           DNA precipitation

1.   Under continuous vortexing, add to a 50 ul gold aliquot the following in order: 5ul DNA (DNA concentration=1ug mL^-1), 50ul 2.5 M CaCl₂, 20ul 0.1 M spermidine

2.        Continue vortexing for 3 min

3.        Incubate the mixture at room temperature for 10 min

4.        Spin at 10,000 rpm for 5-10 sec.

5.        Remove supernatant

6.        Add 250ul 100% ethanol

7.        Vortex for 2 min

8.        Centrifuge at 13,000 rpm for 1 min

9.        Remove supernatant

10. Resuspend microcarriers in 60ul 100% ethanol (for 5 bombardments)

11.    Pipet 10ul of the DNA-coated microcarriers onto the center of each macrocarrier

12.    Dry for about 1 min

·           PDS operation

1.        Open the Helium valve, adjust the pressure to 1300 Psi

2.        Switch PDS on

3.        Switch vacuum pump on

4.        Adjust the gap between rupture dish and macrocarier to 9 cm

5.        Load the rupture disk

6.        Install macrocarriers into macrocarrier holder

7.        Load the microcarrier launch assembly

8.        Position the sample for bombardment at the desired lever

9.        Close the chamber door

10.    Ture the VACUUM switch on to the VAC position

11.    Press and hold the FIRE switch until the disk ruptures at 26-28 inHg

12.    Release the FIRE switch immediately after the disk ruptures

13.    Place the VACUUM switch to the VENT position

14.    Remove the sample and treat accordingly

15.    Discard the macrocarrier, stopping screen, and rupture disk

VI. Selection and regeneration

·           The bombarded samples were incubated in the dark for 16-20 h at 26 C. Later,  transfer calli in rows in petri-dishes containing ND2H25 medium (for HPT gene) or ND2G25  medium( for NPTII gene). Seal the plates with parafilm. Incubate the plates in dark at 26 C.

·           2-3 weeks later, transfer the small protuberances that grew from the calli onto ND2H50 medium or ND2G50 medium.

·           2 weeks later, transfer the resistant calli onto fresh ND2H50 medium or ND2G50 medium for 2 weeks propagation.

·           Then, transfer the resistant calli onto ND2HS medium or ND2GS medium.

·           Finally, resistant calli were regenerated on NN.2B2H medium or NN.2B2G medium under continuous light at 26 C.

 

Appendix

I.                     N6 medium

Bottle	Component	Stock solution (g L-1)	Amount to take per liter of preparation (mL)	Final concentration (mg L-1)
A	KNO3	141.50	20	2830.0
B	MgSO4	18.5	10	185.0
	MnSO4.4H2O	0.44		4.4
	ZnSO4.7H2O	0.15		1.5
	(NH4)2SO4	46.3		463.0
C	KH2PO4	40.0	10	400.0
	KI	0.08		0.8
	HBO3	0.16		1.6
D	CaCl2.2H2O	16.6	10	166.0
E	FeSO4.7H2O	2.78	10	27.8
	Na2EDTA	3.73		37.7
		mg 100 mL-1
Vatamins	Nicotinic acid	50	1	0.5
	Glycine	200		2.0
	Thiamine HCl	100		1.0
	Pyridoxine HCl	50		0.5
Sucrose				30000.0
Agar				8000.0
PH	5.8

II.                  ND2 medium

N6 + proline 500 mg L^-1, casamino acid 300 mg L^-1, 2,4-D 2 mg L^-1

III.                ND2MS medium

ND2 + mannitol 30 g L^-1, sorbitol 30 gL^-1

IV.                ND2H25/ ND2G25/ ND2H50/ ND2G50 media

ND2 + Hygromycin 25 mg L^-1/ G 418 25 mg L^-1/ Hygromycin 50 mg L^-1/ G 418 50 mg L^-1

V.                   NN.2B2H/NN.2B2G media

N6-Agar + casamino acid 300 mg L^-1, NAA 0.2 mg L^-1, 6-BA 2 mg L^-1, phytogel 2.5 g L^-1, Hygromycin 50 mg L^-1/ G 418 50 mg L^–

1.        Grow Bradyrhizobium strain in HM medium (Nieuwkoop et al., 1987) containing only arabinose as carbon source to an OD600nm= 0.4 to 0.8. Grow E. coli strain(s) in LB to approximately the same OD.
2.        Pellet 1 ml of both strains in a sterile microcentrifuge tube at 10,000 rpm for 3 min. If either stain was grown with antibiotics, then wash twice with 1 ml of HM medium and pellet again. For triparental mating, do the same to the strain with the helper plasmid.
3.        Decant the supernatant and resuspend the cell pellet in 50-100 µl of HM broth by pipetting (no vortexing).
4.        Mix the strain suspensions together and transfer the mixture onto a sterile 0. 45µM filter and place the filter (cell side up) onto a HM agar plate. Let the dry to remove any excess moisture and then incubate at 30oC for two days. Using a day-old HM plate helps to reduce the moisture. Set up controls with filters containing both donor and recipient alone.
5.        After incubation, lift the filter from the plate and place into a 1.5 ml microcentrifuge tube. Add 0.5-1.0 ml HM medium and vortex to suspend cells.
6.        Spread 100-200 µl of the cell suspension onto RDY agar plates (So et al., 1987) containing the appropriate antibiotics. The same is done for controls. Incubate plates at 30oC. Colonies usually appear in approximately 6-10 days.

References:

Nieuwkoop, A.J., Z. Banfalvi, N. Deshman, D. Gerhold, M.G. Schell, K. Sirotkin, and G. Stacey. 1987. A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum. J. Bacteriol. 169: 2631-2638.

So, J.-S., A.L.M. Hodgson, R. Haugland, M. Leavitt, Z. Banfalvi, A.J. Nieuwkoop, and G. Stacey. 1987. Mol. Gen. Genet. 207: 15-23.