拟南芥叶质外体中丁香假单胞菌种群的表面接种和定量

Cristián  Jacob; Shweta  Panchal; Maeli  Melotto

doi:10.21769/BioProtoc.2167

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Peer-reviewed

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

拟南芥叶质外体中丁香假单胞菌种群的表面接种和定量

CJ Cristián Jacob ^* email

Shweta Panchal ^* email

Maeli Melotto email

(*contributed equally to this work) 发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2167 浏览次数: 13547

评审: Jyotiska ChaudhuriAnonymous reviewer(s)

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Abstract

Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

Keywords: Leaf inoculation (叶接种)

Stomatal defense (气孔防御)

Pseudomonas syringae (丁香假单胞菌)

Foliar internalization (叶片内化)

Apoplastic bacterial population (质外体细菌种群)

Background

Plant pathogenic bacteria causing foliar diseases may penetrate the leaf epidermis through wounds and natural openings such as stomata. Stomata are microscopic pores that mediate the regulation of transpiration and the exchange of gases between the plant and the atmosphere. Interestingly, we have demonstrated that bacteria can induce stomatal closure. This phenomenon is now recognized as stomatal defense, which hampers bacterial internalization into the leaf decreasing disease development (reviewed by Melotto et al., 2017, in press). Here, we describe a method adapted from Katagiri et al. (2002) and Panchal et al. (2016a and 2016b) to measure the total endophytic bacterial population of Pseudomonas syringae within Arabidopsis leaf tissue after surface inoculation. This procedure is useful to estimate bacterial penetration of leaves through stomata in a laboratory setting.

Materials and Reagents

3.5-inch square pots with holes (Hummert International, catalog number: 12-1300-1 )
Soil mix, SunGro Sunshine^® #1 Mix (Crop Production Services, catalog number: 1000590701 ) or equivalent
Fine vermiculite (Growers Solution, catalog number: Vermiculite4cf )
48 inch x 100 ft Charcoal fiberglass screen (The Home Depot, catalog number: 3000016 )
15 ml and 50 ml centrifuge tubes
Plastic domes (Hummert International, catalog number: 65-6964-1 )
Sharpie markers, paper towels, Kimwipes, disposable gloves, rubber bands, forceps
Square Petri dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 240835 )
Micropipettes (Rainin Pipet-Lite^TM)
1.5 ml microfuge tubes
Plastic pestles that fit 1.5 ml microfuge tubes (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: 19923-0001 )
Plastic trays without holes (Hummert International, catalog number: 65-6963-1 )
Microbreathe face mask (VWR, catalog number: 10833-224 )
Arabidopsis thaliana (L. Heyhn.) ecotype Columbia (Col-0, ABRC stock CS60000). Seeds can be stored at 4 °C and are viable for 3-4 years
Pseudomonas syringae bacterial culture (stored in 25% glycerol at -80 °C)
Gnatrol (Hummert International, catalog number: 01-2035-1 )
Arabidopsis controlled release fertilizer (LEHLE SEEDS, catalog number: PM-11 )
Appropriate antibiotic (e.g., Rifampicin)
MgCl₂ solution
Glycerol (MP Biomedicals, catalog number: 151194 )
Silwet L-77 (LEHLE SEEDS, catalog number: VIS-30 )
Reagent alcohol (Sigma-Aldrich, catalog number: 793183 )
Sterile distilled water
Agarose (VWR, catalog number: 97062-250 )
Tryptone (IBI Scientific, catalog number: 41116105 )
Yeast extract (U.S.Biotech Sources, catalog number: Y01PD-500 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-500 )
Bacteriological agar (IBI Scientific, catalog number: IB49171 )
0.1% agarose (see Recipes)
Low-sodium Luria Bertani medium (see Recipes)

Equipment

1 ml micropipette
Refrigerator or a cold room
Plant growth chamber (Caron Products & Services, model: 6341-2 )
Shaker incubator (VWR, catalog number: 12620-946 )
Spectrophotometer (Thermo Fisher Scientific, model: Spectronic 20D+ or equivalent)
Centrifuge (Eppendorf, model: 5810 )
Handheld electric drill (BLACK + DECKER, catalog number: LDX120C )
Cork borer No. 2 (Cole-Parmer, catalog number: EW-06298-98 )
Digital hygrometer (VWR, catalog number: 35519-047 )
Quantum meter (Apogee, catalog number: BQM )
Vortex (BioExpress, GeneMate, catalog number: S-3200-1 )
Autoclave
laminar flow hood

Procedure

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Jacob, C., Panchal, S. and Melotto, M. (2017). Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast. Bio-protocol 7(5): e2167. DOI: 10.21769/BioProtoc.2167.