Materials and Reagents

Plant material
Soybean (Glycine max Merr.) plants of V2 (2^nd trifoliate), V3 (3^rd trifoliate), or higher stages of growth were used. Plants at VC (two leaf stage)/V1 (1^st trifoliate) stages can also be used. Cultivars for various R loci: Rpg1-b (Harosoy), Rpg2 (Merit, Norchief), Rpg3 (Flambeau), Rpg4 (Flambeau), rpg (Essex).
Bacterial strains
1. P. syringae pv. glycinea expressing avr gene of interest via the broad host range plasmids pDSK519 or pDSK600.
2. P. syringae pv. glycinea expressing empty pDSK519/600 plasmids as control.
Media and buffers
1. 10 mM MgCl₂ (sterile)
2. King’s B medium (plates and liquid media) (see Recipes)
Other reagents
1. Tryptone (Teknova, catalog number: T9012 )
2. K₂HPO₄ (Fisher BioReagents, catalog number: BP363 )
3. Glycerol (Affymetrix, catalog number: 16374 )
4. Agar (Affymetrix, catalog number: 10654 )
5. Silwet L-77 (Momentive, New Smyrna Beach)
6. Antibiotics (Gold Biotechnology)
  1. Rifampicin (50 mg/ml) (R-120)
  2. Kanamycin (50 mg/ml) (K-120)
  3. Spectinomycin (100 mg/ml) (S-140)
  4. Sreptomycin (300 mg/ml) (S150)

Equipment

High-speed floor centrifuge (Thermo Fisher Scientific, Sorvall RC 6 plus)
Spectrophotometer (Thermo Fisher Scientific, model: BioMate 5 )
Pressure pump (Gast Manufacturing, model: DOA-P704-AA )
Airbrush (Badger Air-brush, model: 250-2 )
Test tubes (Thermo Fisher Scientific)
Microcentrifuge tubes (Thermo Fisher Scientific)
Glass rods (Thermo Fisher Scientific)
Pellet pestles (Sigma-Aldrich)
Cork borer (1 cm diameter, Thermo Fisher Scientific)

Procedure

Bacterial growth and inoculum preparation
1. Streak -80 °C stock of bacterial culture on King’s B agar with required antibiotics (Rifampacin and Kanamycin for pDSK519 plasmid; Rifampacin, Spectinomycin and Streptomycin for pDSK600 plasmid). Incubate the plate at 29 °C till bacterial colonies grow (24-48 h).
2. Inoculate a single colony of P. syringae in 10 ml King’s B medium with required antibiotics (Rifampacin and Kanamycin for pDSK519 plasmid; Rifampacin, Spectinomycin and Streptomycin for pDSK600 plasmid).
3. Grow 12-16 h at 29 °C on rotary shaker at 200 rpm until the culture reaches OD (A₆₀₀) 0.8-1.2. Do not use culture at OD₆₀₀ <0.8, or >1.2.
4. Centrifuge culture at 3000 rpm for 10 min. Discard the supernatant and resuspend the bacterial pellet in 10 ml, 10 mM MgCl₂ by gentle pipetting (do not vortex).
5. Measure OD (A₆₀₀) of the bacterial suspension (this should be the same OD as your culture from King’s B medium, 0.8-1.2) and dilute to 10⁵ CFU (colony forming units)/ml using 10 mM MgCl₂ (1.0 OD measurement approximately corresponds to 2 x 10⁸ CFU/ml).
6. Add Silwett L-77 to a final concentration of 0.01% to the bacterial suspension and mix gently. Use bacterial suspensions within 1 h after preparation.
Soybean infiltration
1. Attach appropriate ports of airbrush (Figure 1) to the pressure pump (40 psi or less, Figure 2) and beaker containing bacterial suspension using rubber tubing.
2. Infiltrate soybean plants at V2 stage on the abaxial surface of the trifoliate leaves using the airbrush, while holding the leaf against a flat surface (such as a Petri plate) ensuring that the pressure does not damage the leaf during infiltration (see Video 1).
3. Inoculate one trifoliate per plant, and at least 5 plants per bacterial strain. 5-8 ml of bacterial inoculum is sufficient to completely infiltrate one trifoliate.
4. Mock inoculations should be done similarly 10 mM MgCl₂ + 0.01% Silwett L-77 instead of the bacterial suspension.
  
  Figure 1. Image of airbrush used for bacterial infiltration
  
  Figure 2. Image of pressure pump used for bacterial infiltration
Monitor bacterial proliferation
1. Collect 3 leaf discs from inoculated leaves at 1 h post infiltration. The 1 h wait prevents erroneous bacterial numbers from any excess inoculum on the leaf surface. By 1 h post infiltration, the infiltrated leaves should no longer appear wet. Collect leaf discs using 1 cm cork borer from each plant. This is your 0 dpi sample.
2. Homogenize leaf discs in 0.3 ml, 10 mM MgCl₂ in a microcentrifuge tube manually using pellet pestle (Figure 3). For reproducibility and accurate bacterial count homogenize until no leaf pieces are visible. (Figure 4). Increase final volume to 1 ml after complete homogenization.
3. Dilute 10x using 10 mM MgCl₂ and plate 100 μl on King’s B medium (one sample per plate) using a glass spreader. Incubate plates at 29 °C till colonies grow (usually 24-48 h). Plate minimum 3 technical replicates per bacterial strain for every genotype. Technical repeats indicate independent leaf extracts from the same set of plants infected with the same bacterial culture.
4. Count the number of bacterial colonies on entire plate, and adjust for dilution factor to determine total bacterial number. Plot bacterial counts as LOG₁₀ values of CFU/unit leaf disc. It is important to adjust the dilution factor to obtain colonies in a countable range (10-300) depending upon the genotype of the plant. It is recommended to repeat the experiment using lower dilutions for plating if colony numbers are less than 10 or using higher dilutions when colony numbers are more than 300 per plate.
5. Repeat steps 1-4 at 3, 4, 6 dpi (or other desired time points). Dilute homogenized tissue 5,000-10,000 times dilution for 3 dpi and later samples. Use minimum 4-5 technical replicates for these time points. Use 3-4 biological replicates (indicates independent infections using independent bacterial culture and set of plants) per bacterial strain and per plant genotype.
6. Hypersensitive reaction related symptoms are visually detectable by 6-7 dpi (Figure 5).
  
  Figure 3. Image of pellet pestle used for homogenization of leaf material
  
  Figure 4. Image of homogenate of infected leaf material
  
  Figure 5. Morphological symptoms associated with the hypersensitive response of P. syringae pv. glycinea infected soybean leaves

Representative data

Video 1. Airbrush infiltration

The protocol has high reproducibility (>90%) in our hands. We normally use at least four biological replicates per genotype for each experiment, and each infection experiment is conducted with a minimum three technical replicates. The standard error for colony count ranges between LOG₁₀ value of 0.01-0.2.
See Selote and Kachroo (2010); Selote et al. (2013) and (2014); Wang et al. (2014) for typical bacterial counts observed on various cultivars.

Notes

Care should be taken to avoid damaging the leaves during the inoculation process.
It is best to use fully expanded leaves for infection and to inoculate all leaves of one trifoliate per plant.
Spraying the bacterial suspension over the leaf surface is generally sufficient. It is not necessary to saturate leaves with the bacterial suspension as excessive pressure during infiltration can result in wilting of the leaf.

Recipes

King’s B medium (plates and liquid media)
20 g tryptone
1.5 g K₂HPO₄
10 ml glycerol
15 g agar, make up volume to 1 L with water
Autoclave, then add 5 ml of sterile MgSO₄ (1 M)

Acknowledgments

This work was supported by funding from the United Soybean Board (project #1291) and the Kentucky Soybean Promotion Board. This protocol is modified from a previous method used by Keen et al. (1990).

References

Fu, D. Q., Ghabrial, S. and Kachroo, A. (2009). GmRAR1 and GmSGT1 are required for basal, R gene-mediated and systemic acquired resistance in soybean. Mol Plant Microbe Interact 22(1): 86-95.
Kachroo, A., Fu, D. Q., Havens, W., Navarre, D., Kachroo, P. and Ghabrial, S. A. (2008). An oleic acid-mediated pathway induces constitutive defense signaling and enhanced resistance to multiple pathogens in soybean. Mol Plant Microbe Interact 21(5): 564-575.
Keen, N. T., Tamaki, S., Kobayashi, D., Gerhold, D., Stayton, M., Shen, H., Gold, S., Lorang, J., Thordal-Christensen, H., Dahlbeck, D. and Staskawicz, B. (1990). Bacteria expressing avirulence gene D produce a specific elicitor of the soybean hypersensitive reaction. Mol Plant Microbe Interact 3(2): 112–121.
Selote, D. and Kachroo, A. (2010). RPG1-B-derived resistance to AvrB-expressing Pseudomonas syringae requires RIN4-like proteins in soybean. Plant Physiol 153(3): 1199-1211.
Selote, D., Robin, G. P. and Kachroo, A. (2013). GmRIN4 protein family members function nonredundantly in soybean race-specific resistance against Pseudomonas syringae. New Phytol 197(4): 1225-1235.
Selote, D., Shine, M. B., Robin, G. P. and Kachroo, A. (2014). Soybean NDR1-like proteins bind pathogen effectors and regulate resistance signaling. New Phytol 202(2): 485-498.
Singh, A. K., Fu, D. Q., El-Habbak, M., Navarre, D., Ghabrial, S. and Kachroo, A. (2011). Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean. Mol Plant Microbe Interact 24(4): 506-515.
Wang, J., Shine, M. B., Gao, Q. M., Navarre, D., Jiang, W., Liu, C., Chen, Q., Hu, G. and Kachroo, A. (2014). Enhanced disease susceptibility1 mediates pathogen resistance and virulence function of a bacterial effector in soybean. Plant Physiol 165(3): 1269-1284.