用昆虫细菌共生体唐菖蒲伯克霍尔德菌感染大豆植株及植物适应性评价

Paul Gaube; Martin Kaltenpoth; Laura V. Flórez

doi:10.21769/BioProtoc.2663

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Infection of Soybean Plants with the Insect Bacterial Symbiont Burkholderia gladioli and Evaluation of Plant Fitness

用昆虫细菌共生体唐菖蒲伯克霍尔德菌感染大豆植株及植物适应性评价

Paul Gaube

MK Martin Kaltenpoth

Laura V. Flórez email

发布: 2017年12月20日第7卷第24期 DOI: 10.21769/BioProtoc.2663 浏览次数: 6995

评审: Andrea PuharDaniel F. CaddellAnonymous reviewer(s)

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Abstract

To investigate the establishment and consequences of host-microbe interactions, it is important to develop controlled infection assays suitable for each system, as well as appropriate methods to evaluate successful infection and its associated effects. Here, we describe a procedure for bacterial inoculation of soybean plants, followed by the assessment of systemic infection and impact on plant fitness. Soybean (Glycine max) seedlings were mechanically wounded using a device that mimics insect herbivory and inoculated with known cell numbers of Burkholderia gladioli bacteria previously isolated from an insect host. The impact on the plants was evaluated by monitoring changes in height, time to flowering and chlorophyll content during plant development, and by quantifying seed production in comparison to plants inoculated with sterile water. The presence and proliferation of bacterial infection were examined in tissues from developed plants using quantitative PCR and fluorescence in situ hybridization (FISH).

Keywords: Bacterial plant infection (细菌植物感染)

Plant fitness assay (植物适应性测定)

Microbe-host interactions (微生物 - 宿主相互作用)

Whole-mount FISH (全包埋FISH)

Burkholderia gladioli (唐菖蒲伯克霍尔德菌)

Soybean (Glycine max) (大豆)

Background

Microbes establish symbiotic associations with diverse eukaryotic organisms and can have profound effects on host fitness, ranging from beneficial to detrimental (Frank, 1997). In many cases, these associations are directly or indirectly influenced by interactions with additional organisms, like potential alternative hosts. As an example, there are numerous three-way interactions between plants, microbes and insects, in which microbial symbionts are transmitted between the different hosts and affect the physiology or ecology of the organisms involved (Frago et al., 2012; Gilbert et al., 2012). In phytophagous Lagriinae beetles, a symbiotic partnership has been established with bacteria from a plant pathogenic clade, Burkholderia gladioli, suggesting that this association evolved in the context of a tripartite interaction. Horizontal transmission of the symbionts from Lagria villosa beetles to soybean plants was previously demonstrated, indicating the possibility for dynamic transitions between hosts (Flórez et al., 2017). Furthermore, the symbionts are transmitted vertically, from mother to offspring on the surface of the beetle eggs (Stammer, 1929), where they inhibit the growth of pathogenic fungi (Flórez et al., 2017). In line with the dynamic nature of this symbiosis, at least three different strains of symbiotic B. gladioli are present in L. villosa, of which only one has been successfully cultured in vitro so far (Flórez and Kaltenpoth, 2017). This protocol has been developed to assess the ability of culturable B. gladioli bacteria to infect soybean plants, a common food source for L. villosa beetles, and evaluate the impact of infection on plant fitness.

Materials and Reagents

Materials
1. Plant pots 6 cm diameter (Volume 120 ml)
2. Plant saucers Ø 10 cm and 16 cm (lower and upper diameter, respectively)
3. Centrifuge tubes 50 ml (Corning, Falcon^®, catalog number: 352070 )
4. Thin wood sticks 30 cm (Schreiber-Online-Handel, Splittstäbe Naturweide, catalog number: 231979935696 )
5. Razor blade (Schreiber, 11-0100)
6. Reaction tubes 1.5 ml (SARSTEDT, catalog number: 72.690.001 )
7. Petri dishes Ø 9 mm (Carl Roth, Gosselin^TM, catalog number: ALA5.1 )
8. 1 ml pipette tips (SARSTEDT, catalog number: 70.762.010 )
9. Polysine^® glass slides (Thermo Fisher Scientific, Menzel-Gläser, catalog number: J2800AMNZ )
10. Microscope cover slips 24 x 60 mm #1 (Thermo Fisher Scientific, Menzel-Gläser, catalog number: CS2460100 )
11. Aluminium foil
Plants
Soybean (Glycine max Merr., cv. 29-I, Semillas Panorama, Colombia) plants of V2 (2^nd trifoliate) stage and of comparable height were used
Bacterial strain
Burkholderia gladioli Lv-StA bacterial culture stored in 30% glycerol at -80 °C, previously isolated from accessory glands of a female L. villosa beetle (Flórez et al., 2017)
Molecular biology working kits
1. MasterPure^TM Complete DNA and RNA Purification Kit (Epicentre, catalog number: MC85200 )
2. QuantiTect^® Reverse Transcription Kit (QIAGEN, catalog number: 205311 )
3. SYBR^® Green Rotor-Gene PCR Kit (QIAGEN, catalog number: 204074 )
4. InnuPREP Gel Extraction Kit (Analytik Jena, catalog number: 845-KS-5030050 )
Other reagents
1. Sterile tap water (autoclaved)
2. H₂O treated with DEPC (Diethylpyrocarbonate) (Carl Roth, catalog number: K028.1 )
3. Liquid nitrogen
4. RNaseZap^® Decontamination Solution (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9782 )
5. Ethanol 100% (Carl Roth, catalog number: 5054.5 )
6. H₂O₂ 6% in 100% ethanol (Carl Roth, catalog number: 9681.4 )
7. DAPI (4',6-Diamidino-2-phenylindol Dihydrochlorid) (Carl Roth, catalog number: 6335.1 )
8. Ethanol 70%
9. Milli-Q^® water
10. VECTASHIELD^® (Vector Laboratories, catalog number: H-1000 )
11. Peptone from soybean (AppliChem, catalog number: A2206 )
12. Potassium phosphate dibasic (K₂HPO₄) (Carl Roth, catalog number: P749.1 )
13. Glycerol (Carl Roth, Rotipuran^®, catalog number: 3783.1 )
14. Agar-Agar Kobe I (Carl Roth, catalog number: 5210.2 )
15. Magnesium sulfate hydrate (MgSO₄·xH₂O) (Carl Roth, catalog number: 0261.1 )
16. Tris/HCl (Carl Roth, Pufferan^®, catalog number: 4855.2 )
17. EDTA solution pH 8.0 (EUROCLONE, catalog number: EMR034500 )
18. Triton^® X-100 (Carl Roth, catalog number: 3051.3 )
19. Sodium chloride (NaCl) (Carl Roth, catalog number: 3957.1 )
20. Potassium chloride (KCl) (Carl Roth, catalog number: 6781.3 )
21. Sodium phosphate dibasic (Na₂HPO₄) (Carl Roth, catalog number: P030.1 )
22. Potassium phosphate monobasic (KH₂PO₄) (Carl Roth, catalog number: 3904.2 )
23. Chloroform (Carl Roth, catalog number: 7331.2 )
24. Acetic acid 100% (Carl Roth, catalog number: 6755.1 )
25. Sodium dodecyl sulfate (SDS) (Carl Roth, catalog number: 4360.1 )
Media and buffers (see Recipes)
1. King’s B liquid and solid medium
2. Low TE-buffer
3. PBS-Tx buffer
4. Carnoy’s fixative
5. Hybridization buffer
6. Washing buffer

Equipment

Sticky insect tape (CONRAD, GLUPAC, model: GB011 )
Class II biological safety cabinet (Thermo Fisher Scientific, Thermo Scientific^TM, model: Safe 2020 , 1.2)
Glass inoculation spreader
Scissors
Forceps
Drying oven (Memmert, model: INB 200 )
Vortex (Scilogex, model: MX-S , catalog number: 821200049999)
Micropipettes 1 ml and 10 µl (Eppendorf, model: Research^® plus, catalog numbers: 3120000062 and 3120000020 )
Chlorophyll meter (Konica Minolta, model: SPAD-502Plus )
Spectrophotometer UV/VIS (Eppendorf, model: BioPhotometer 6131 , catalog number: 6131 000.012)
Heater mixing block (Biozym, Bioer Technology, model: MB-102 )
Micro-centrifuge (Eppendorf, model: 5418 , catalog number: 5418000017)
Real-time PCR cycler (QIAGEN, model: Rotor-Gene Q )
NanoDrop^TM 1000 spectrophotometer (Thermo Fisher Scientific, Thermo Scientific^TM, model: ND^TM-1000 )
Fluorescent microscope (ZEISS, model: AxioImager Z1 )
Colour camera (ZEISS, model: AxioCam MRm )

Software

SPSS (version 17.0)
RStudio (version 0.98.1103)
AxioVision software (Lite Edition 4.8.1, ZEISS)

Procedure

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Gaube, P., Kaltenpoth, M. and Flórez, L. V. (2017). Infection of Soybean Plants with the Insect Bacterial Symbiont Burkholderia gladioli and Evaluation of Plant Fitness. Bio-protocol 7(24): e2663. DOI: 10.21769/BioProtoc.2663.