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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   

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Nature Communications
Apr 2017

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

  1. 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, GosselinTM, 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

  2. Plants
    Soybean (Glycine max Merr., cv. 29-I, Semillas Panorama, Colombia) plants of V2 (2nd trifoliate) stage and of comparable height were used

  3. 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)

  4. Molecular biology working kits
    1. MasterPureTM 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 )

  5. 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, InvitrogenTM, 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 )

  6. 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

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

Software

  1. SPSS (version 17.0)
  2. RStudio (version 0.98.1103)
  3. AxioVision software (Lite Edition 4.8.1, ZEISS)

Procedure

  1. Breeding and preparation of soybean plants
    1. Plant five soybean seeds per pot, in a total of 36 pots (180 seeds) and grow for seven days under greenhouse conditions (25 °C, 16:8 h light:dark cycle).
    2. Transfer plants to individual pots and grow for 21 days under greenhouse conditions.
    3. Select 36 plants of similar height and at developmental stage V2.
    4. Make a circular wound (5 mm diameter) in the central area of single leaflets of the first and second trifoliate leaves from each plant (Figure 1), using a robotic device that mimics insect herbivory damage (‘MecWorm’) (Mithöfer et al., 2005).
      Note: Alternatively, a cork borer, a paper hole punch or a sterile blade can be used for wounding.


      Figure 1. Schematic representation of the wounding and tissue sampling areas on a soybean plant. The scheme illustrates two wounded spots on the first and second trifoliate leaf (red regions), which had been inoculated at developmental stage V2 with B. gladioli Lv-StA in treated plants or sterile tap water in control plants. Black crosses indicate leaf areas later taken from developed soybean plants and used for FISH and quantitative PCR detection of B. gladioli Lv-StA. Note that wounding and infection were done at developmental stage V2 (only 2 fully developed trifoliate leaves present), while the scheme represents a plant at a later developmental stage, in which newer trifoliate leaves were also available for tissue sampling.

    5. Use sticky insect traps between plants to prevent potential contact with insects and thereby cross contamination between plants.
    6. Arrange plants randomly and shuffle every second day under controlled light management (Figure 2).


      Figure 2. Soybean plants organized in random order according to treatment and under controlled light conditions. The picture was acquired three weeks after inoculation.

    7. Water plants every second day with tap water.

  2. Calibration curve for determining bacterial cell concentration using optical density
    Note: Constructing a calibration curve is only necessary once for a specific spectrophotometer and bacterial strain, and should be done at least four days before initial plant inoculation considering the time required for CFU counting and preparation of the bacterial inoculum for infection.
    1. Culture symbiotic B. gladioli Lv-StA in a 50 ml centrifuge tube containing 20 ml King’s B liquid medium (see Recipes) at 30 °C and constant shaking (200 rpm) for 24 h.
    2. Centrifuge the bacterial culture at 2,400 x g for 5 min. Discard the supernatant and resuspend the bacterial pellet in sterile tap water.
    3. Prepare three dilution series from 10-1 to 10-7 in 1.5 ml reaction tubes using sterile tap water (i.e., three times seven dilutions).
    4. Measure optical density (OD600) of each sample three times, and plate 50 µl per sample on Petri dishes containing King’s B medium agar.
    5. Incubate the Petri dishes at 30 °C for 48-72 h.
    6. Count colony forming units (CFU) of 3-day old cultures on King’s B medium plates (only CFU counts between 20 and 200 colonies per plate were considered).
    7. Generate a calibration curve using a linear regression of OD600 in relation to CFU count.
    8. Calculate the OD600 corresponding to 105 CFUs µl-1.

  3. Bacterial inoculum preparation and plant infection
    Note: Bacterial inoculation was performed within two hours after wounding.
    1. Culture symbiotic B. gladioli Lv-StA in King’s B liquid medium at 30 °C and constant shaking (200 rpm) for 24 h (i.e., one day before plant wounding and infection).
    2. Centrifuge the bacterial culture at 2,400 x g for 5 min. Discard the supernatant and resuspend the bacterial pellet in sterile tap water.
    3. Measure the OD600 of the bacterial suspension and dilute to 105 CFUs μl-1 using sterilized tap water.
    4. Inoculate 18 plants with 10 µl of bacterial suspension each by placing a drop with a micropipette on the two wounded areas of the trifoliate leaves for each plant (Figure 1).
    5. Apply 10 µl of sterilized tap water on the two corresponding wounds of each of the 18 control plants (Figure 1).
    Note: The plant infection experiment was completely conducted in a biosafety level 2 laboratory given the assignment of Burkholderia gladioli to risk group 2 organisms according to German regulations (BioStoffV).

  4. Monitoring of plant fitness
    1. Monitor plant height, as well as time to flowering, every second day until day 38 after infection (a total of 20 measurement time points including the day of inoculation).
    2. Observe potential symptoms of leaf necrosis and chlorosis (Figure 3).


      Figure 3. Chlorosis of single soybean leaflets treated with Burkholderia gladioli Lv-StA in comparison to a water control. Leaflets wounded and treated with sterile water are shown close after treatment (A) and 38 days after treatment (B), as well as leaflets infected with bacterial suspension close after treatment (C) and 38 days after being treated (D).

    3. Measure the chlorophyll contents on three different spots of each wounded trifoliate leaflet every second day (triplicate measurements, not on the infection area) by using a chlorophyll meter SPAD-502Plus.
    4. Count the total number of seeds from all pods 38 days after infection.
    5. Also on day 38, dry the complete plant (above- and belowground parts) at 70 °C for 24 h and measure dry weight.
      Important: See Note at the beginning of Procedure E

  5. Quantification of infecting bacteria
    Note: Harvest two leaflets from each plant at the end of the experiment (at day 38 after inoculation) before measuring the dry weight of the complete plant (see Step D5).
    1. Cut pieces of leaflets (approximate area 1 x 1.5 cm) from different areas of each plant (see Figure 1) with sterile forceps and scissors and divide each sample into halves.
    2. Store half of each piece in sterile 1.5 ml tubes at -80 °C until further processing. Keep the other half of each piece for FISH analysis.
    3. Prepare sterile blunt 1 ml pipette tips as pestles. Flame the top of each tip shortly and then round it in 1.5 ml tubes containing DEPC treated H2O (Video 1).

      Video 1. Preparation of pestles from 1 ml sterile tips

    4. Weigh the stored leaflet pieces individually and then homogenize each sample in 1.5 ml tubes with the previously prepared pestles by pouring liquid nitrogen directly into each tube and crushing the tissue into powder (Video 2). If necessary for complete homogenization, add additional liquid nitrogen into the 1.5 ml tube and rapidly insert the pestle in the tube for grinding. While the pestle is not in use, you may partially close the tube (not tightly) to prevent powdered leaf tissue from spurting out of the tube.

      Video 2. Plant tissue homogenization using liquid nitrogen

    5. Extract nucleic acids using the MasterPureTM Complete DNA and RNA Purification Kit according to the manufacturer’s instructions. Perform centrifugation steps at 4 °C and resuspend DNA and RNA in Low TE-buffer (see Recipes).
    6. Perform reverse transcription on ice using the QuantiTect® Reverse Transcription Kit including a genomic DNA removal step and a corresponding control, following the manufacturer’s instructions.
    7. Conduct quantitative RT-PCR on the corresponding cDNA on a 167 bp fragment using primers Burk16S_StAG_F and Burk 3.1_R (Table 1) in a RotorGene®-Q cycler device following the protocol described for the Rotor-Gene SYBR Green PCR Kit.

      Table 1. Primers and probes used for amplification and fluorescence in situ hybridization


    8. Carry out a quantitative RT-PCR consisting of an initial denaturation step at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 sec, primer annealing at 65 °C for 30 sec and elongation at 72 °C for 20 sec. Include a melting curve with a temperature gradient from 60 °C to 99 °C within 4.25 min.
    Notes:
    1. DNA standards for quantification of the target fragments were obtained previously by PCR amplification and product recovery using the innuPREP Gel Extraction Kit. Purified DNA concentration was measured using a NanoDropTM 1000 spectrophotometer. Standards were used as a tenfold dilution series ranging from 10-1 to 10-8 ng µl-1.
    2. All solutions used for RNA extraction were prepared in DEPC (Diethylpyrocarbonate) treated H2O and RNaseZap® Decontamination Solution was used in order to avoid RNase contamination of equipment and workspaces.

  6. Visualization of the infecting bacteria by FISH
    1. Cut and thinly slice the stem tissue of different plant regions (between 1st and 2nd trifoliates and above the 2nd trifoliate) with a razor blade in approximately 0.5 mm longitudinal sections.
    2. Fixate the sliced stem tissue and the half piece of leaflet recovered previously (see Step E1) at room temperature (RT) for 24 h in Carnoy’s fixative (see Recipes) and store at RT until further use.
    3. Wash samples at RT in 100% ethanol for 2 h and then bleach in 6% H2O2 (diluted in ethanol) for approximately 14 days. Change the bleach solution every third day.
    4. Wash the samples at RT in 100% ethanol (3 x 2 h), in 70% ethanol (2 x 1 h) and in PBS-Tx buffer (4 x 0.5 h, see Recipes).
    5. Hybridize the samples at 55 °C for 24 h in 1.5 ml tubes containing 270 µl hybridization buffer (see Recipes) with 15 µl (50 ng µl-1) of the Cy5-labeled general eubacterial probe EUB784, 15 µl (50 ng µl-1) of the Cy3-labeled Burkholderia specific probe Burk_16S (Table 1) and additionally 3 µl DAPI (0.5 mg ml-1) for host cell counterstaining.
    6. Wash samples at 55 °C in washing buffer (2 x 2 h, see Recipes) and then incubate samples in distilled H2O and a small amount (1-2 drops) of VECTASHIELD® for 2 h at room temperature.
    7. Transfer samples to a glass slide, cover with VECTASHIELD® and a cover slip, and observe samples using a fluorescence microscope (Figure 4).
    Notes:
    1. Step F3 is light sensitive and should be performed under dark or dim light conditions. We recommend covering the samples with aluminium foil or similar during incubation. Decolorization with bleaching solution is essential to avoid auto-fluorescence of the plant tissue.
    2. Steps F5-F7 involve the manipulation of fluorescent probes, therefore we recommend working under dark or dim light to minimize bleaching.
    3. During and until the final washing step with distilled H2O (Steps F5 and F6), it is essential that samples do not dry out completely, as the subsequent removal of unhybridized probe might be compromised.
    4. Some FISH probes designed for eubacteria might also hybridize to the plant chloroplasts (e.g., EUB388: 5’-GCTGCCTCCCGTAGGAGT-3’). The probe EUB784 was convenient in this sense, as no hybridization to chloroplasts was observed in silico nor in situ.


    Figure 4. Localization of Burkholderia gladioli cells in the soybean plant vascular system. Whole mount FISH on soybean leaf tissue showing an overlay image of hybridization with Burk_16S-Cy3 (red), EUB784-Cy5 (green) and host cell nuclei staining with DAPI (blue). The white arrow indicates Burkholderia cells in the vascular system of an infected soybean plant leaflet (A). No Burkholderia cells were observed in control plants (B). The scale bars represent 50 µm.

Data analysis

Statistical analyses were performed using SPSS (version 17.0). Seed output (number of seeds) and first flowering events were compared between B. gladioli infected plants and the uninfected controls using the Mann-Whitney U-test, given the non-normal distribution of the data. RStudio (version 0.98.1103) was used for linear mixed model (LMM) and generalized additive model (GAM) analysis using time as random factor and chlorophyll and treatment or height and treatment as fixed factors. The quantitative PCR data on Burkholderia abundance in different plant locations upon infection were analyzed using an ANOVA and Tukey’s post hoc tests after confirming the normal distribution of the data and homogeneity of variances. Results on seed output and Burkholderia quantification in plant tissues have been published previously (Flórez et al., 2017).

Recipes

  1. King’s B medium
    20 g peptone
    1.5 g K2HPO4
    10 ml glycerol
    15 g agar (only for solid media)
    1 L double distilled water
    Autoclave, then add 5 ml of sterile 1 M MgSO4
  2. Low TE-buffer
    10 mM Tris/HCl (in Milli-Q® water)
    0.1 mM EDTA (in Milli-Q® water)
  3. PBS-Tx buffer
    3 g Triton® X-100
    8 g NaCl
    0.2 g KCl
    1.4 g Na2HPO4
    0.3 g KH2PO4
    1 L double distilled H2O
  4. Carnoy’s fixative
    6 parts ethanol 100%
    3 parts chloroform 100%
    1 part acetic acid 100%
  5. Hybridization buffer
    900 mM NaCl
    0.01% sodium dodecyl sulfate (SDS)
    20 mM Tris/HCl pH 8.0
  6. Washing buffer
    5 mM EDTA
    100 mM NaCl
    0.1% SDS
    20 mM Tris/HCl pH 8.0

Acknowledgments

This protocol was adapted from (Flórez et al., 2017) and was carried out at the Max Planck Institute for Chemical Ecology (Jena, Germany). A specialized device for mechanical plant wounding previously developed by Dr. Axel Mithöfer et al. (2005) was used. We thank the Greenhouse team at the Max Planck Institute for Chemical Ecology for soybean cultivation and the Max-Planck-Society for funding. The authors have no conflict of interest or competing interests to declare.

References

  1. Flórez, L. V. and Kaltenpoth, M. (2017). Symbiont dynamics and strain diversity in the defensive mutualism between Lagria beetles and Burkholderia. Environ Microbiol 19(9): 3674-3688.
  2. Flórez, L. V., Scherlach, K., Gaube, P., Ross, C., Sitte, E., Hermes, C., Rodrigues, A., Hertweck, C. and Kaltenpoth, M. (2017). Antibiotic-producing symbionts dynamically transition between plant pathogenicity and insect-defensive mutualism. Nat Commun 8: 15172.
  3. Frago, E., Dicke, M. and Godfray, H. C. (2012). Insect symbionts as hidden players in insect-plant interactions. Trends Ecol Evol 27(12): 705-711.
  4. Frank, S. A. (1997). Models of symbiosis. Am Nat 150 Suppl 1: S80-99.
  5. Gilbert, S. F., Sapp, J. and Tauber, A. I. (2012). A symbiotic view of life: we have never been individuals. Q Rev Biol 87(4): 325-341.
  6. Kaltenpoth, M., Yildirim, E., Gurbuz, M. F., Herzner, G. and Strohm, E. (2012). Refining the roots of the beewolf-Streptomyces symbiosis: antennal symbionts in the rare genus Philanthinus (Hymenoptera, Crabronidae). Appl Environ Microbiol 78(3): 822-827.
  7. Mithöfer, A., Wanner, G. and Boland, W. (2005). Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission. Plant Physiol 137(3): 1160-1168.
  8. Opelt, K., Berg, C., Schonmann, S., Eberl, L. and Berg, G. (2007). High specificity but contrasting biodiversity of Sphagnum-associated bacterial and plant communities in bog ecosystems independent of the geographical region. ISME J 1(6): 502-516.
  9. Salles, J. F., De Souza, F. A. and van Elsas, J. D. (2002). Molecular method to assess the diversity of Burkholderia species in environmental samples. Appl Environ Microbiol 68(4): 1595-1603.
  10. Stammer, H. J. (1929). Die Symbiose der Lagriiden (Coleoptera). Zoomorphology 15(1/2): 1-34.

简介

为了研究宿主 - 微生物相互作用的建立和后果,开发适用于每个系统的受控感染测定法以及评估成功感染及其相关作用的适当方法是重要的。在这里,我们描述了大豆植物的细菌接种程序,然后评估全身感染和对植物健康的影响。使用模拟昆虫食草动物的装置对大豆(Glycine max)幼苗进行机械性伤害,并用先前从昆虫宿主分离的已知细胞数目的伯克霍尔德氏菌(B.coli)进行接种。通过监测植物发育过程中身高,开花时间和叶绿素含量的变化以及通过与用无菌水接种的植物相比量化种子产量来评估对植物的影响。使用定量PCR和荧光原位杂交(FISH)在来自发育植物的组织中检查细菌感染的存在和增殖。

微生物与不同的真核生物建立共生关系,对宿主的适应性有着深远的影响,从有益到不利(Frank,1997)。在许多情况下,这些协会是直接或间接的影响与其他生物,如潜在的替代主机相互作用。举例来说,植物,微生物和昆虫之间有许多三方相互作用,其中微生物共生体在不同宿主之间传播并影响相关生物体的生理或生态(Frago等人)。 ,2012; Gilbert et al。,2012)。在植物性的Lagriinae甲虫中,与来自植物致病性分支的细菌(Burkholderia gladioli)建立了共生伙伴关系,表明这种关联在三方相互作用的情况下发展。先前已经证明了从 Lagria villosa 甲虫到大豆植物的共生体的水平传递,表明了宿主之间动态转换的可能性(Flórezet al。,2017)。此外,这些共生体是从甲虫卵(Stammer,1929)的母体到子代垂直传播的,它们抑制病原真菌的生长(Flórezet al。,2017)。符合这种共生的动态性,至少有三种不同的共生菌株B.剑兰存在于 L中。只有一种在体外培养成功(Flórez和Kaltenpoth,2017)。该协议已被开发用于评估可培养的B的能力。唐菖蒲细菌感染大豆植物,这是一种常见的食物来源。毛茸茸甲虫,并评估感染对植物健康的影响。

关键字:细菌植物感染, 植物适应性测定, 微生物 - 宿主相互作用, 全包埋FISH, 唐菖蒲伯克霍尔德菌, 大豆

材料和试剂

  1. 物料
    1. 直径6厘米的植物盆栽(容积120毫升)
    2. 直径10厘米和16厘米的植物碟(分别为下部和上部直径)
    3. 离心管50ml(Corning,Falcon ,目录号:352070)
    4. 细木棍30厘米(Schreiber-Online-Handel,SplittstäbeNaturweide,目录号:231979935696)
    5. 剃刀刀片(Schreiber,11-0100)
    6. 反应管1.5毫升(SARSTEDT,目录号:72.690.001)
    7. 培养皿直径9毫米(Carl Roth,Gosselin TM ,目录号:ALA5.1)
    8. 1毫升枪头(SARSTEDT,目录号:70.762.010)
    9. 聚赖氨酸载玻片(Thermo Fisher Scientific,Menzel-Gläser,目录号:J2800AMNZ)
    10. 显微镜盖玻片24 x 60毫米#1(Thermo Fisher Scientific,Menzel-Gläser,目录号:CS2460100)
    11. 铝箔

  2. 植物
    使用V2(2-tri-trifoliate)阶段并具有可比较的高度的大豆(Glycine max Merr。,cv.29-I,Semillas Panorama,Colombia) >
  3. 细菌株
    在80℃的30%甘油中储存的,先前从女性附属腺分离的剑麻Burkholderia gladioli Lv-StA细菌培养物。 villosa 甲虫(Flórez et al 。,2017)

  4. 分子生物学工作包
    1. MasterPure TM完整DNA和RNA纯化试剂盒(Epicentre,目录号:MC85200)
    2. QuantiTect®逆转录试剂盒(QIAGEN,目录号:205311)
    3. SYBR 绿色转子基因PCR试剂盒(QIAGEN,目录号:204074)
    4. InnuPREP凝胶提取试剂盒(Analytik Jena,产品目录号:845-KS-5030050)

  5. 其他试剂
    1. 无菌自来水(高压灭菌)
    2. 用DEPC(焦碳酸二乙酯)(Carl Roth,目录号:K028.1)处理过的H 2 O 2。
    3. 液氮
    4. RNaseZap去污溶液(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9782)
    5. 乙醇100%(卡尔罗斯,目录号:5054.5)
    6. 在100%乙醇(Carl Roth,目录号:9681.4)中的H 2 O 2:6%
    7. DAPI(4',6-二脒基-2-苯基吲哚二盐酸盐)(Carl Roth,目录号:6335.1)
    8. 乙醇70%
    9. Milli-Q 水
    10. VECTASHIELD(Vector Laboratories,目录号:H-1000)
    11. 大豆蛋白胨(AppliChem,目录号:A2206)
    12. 磷酸二氢钾(K 2 HPO 4)(Carl Roth,目录号:P749.1)
    13. 甘油(Carl Roth,Rotipuran ,目录号:3783.1)
    14. 琼脂神户我(卡尔罗斯,目录编号:5210.2)
    15. 硫酸镁水合物(MgSO 4•xH 2 O)(Carl Roth,目录号:0261.1)
    16. Tris / HCl(Carl Roth,Pufferan ,目录号:4855.2)
    17. EDTA溶液pH 8.0(EUROCLONE,目录号:EMR034500)
    18. Triton X-100(Carl Roth,目录号:3051.3)
    19. 氯化钠(NaCl)(Carl Roth,目录号:3957.1)
    20. 氯化钾(KCl)(Carl Roth,目录号:6781.3)
    21. 磷酸二氢钠(Na 2 HPO 4)(Carl Roth,目录号:P030.1)
    22. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:3904.2)
    23. 氯仿(Carl Roth,目录号:7331.2)
    24. 乙酸100%(卡尔罗斯,目录号:6755.1)
    25. 十二烷基硫酸钠(SDS)(Carl Roth,目录号:4360.1)

  6. 媒体和缓冲区(见食谱)
    1. King's B液体和固体培养基
    2. 低TE缓冲
    3. PBS-Tx缓冲区
    4. 卡诺伊的固定剂
    5. 杂交缓冲液
    6. 清洗缓冲液

设备

  1. 粘虫带(CONRAD,GLUPAC,型号:GB011)
  2. II级生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:Safe 2020,1.2)
  3. 玻璃接种撒播机
  4. 剪刀
  5. 镊子
  6. 烘箱(Memmert,型号:INB 200)
  7. 涡旋(Scilogex,型号:MX-S,目录号:821200049999)
  8. 微量移液管1ml和10μl(Eppendorf,型号:Research plus Plus,产品目录号:3120000062和3120000020)
  9. 叶绿素计(柯尼卡美能达,型号:SPAD-502Plus)
  10. 分光光度计UV / VIS(Eppendorf,型号:BioPhotometer 6131,目录号:6131 000.012)
  11. 加热器混合块(Biozym,Bioer Technology,型号:MB-102)
  12. 微型离心机(Eppendorf,型号:5418,目录号:5418000017)
  13. 实时PCR循环仪(QIAGEN,型号:Rotor-Gene Q)
  14. NanoDrop TM 1000分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:ND TM-1000)
  15. 荧光显微镜(ZEISS,型号:AxioImager Z1)
  16. 彩色相机(蔡司,型号:AxioCam MRm)

软件

  1. SPSS(版本17.0)
  2. RStudio(版本0.98.1103)
  3. AxioVision软件(精简版4.8.1,蔡司)

程序

  1. 大豆植物的选育和制备
    1. 在温室条件下(25℃,16:8小时光照:黑暗周期),每盆种植5个大豆种子,共36盆(180粒种子),并种植7天。

    2. 在温室条件下将植物转移到单独的盆中并生长21天
    3. 选择36个相似高度的植物,在发育阶段V2。
    4. 使用模拟昆虫食草损害的机器人设备('MecWorm')(Mithöferet al。,2001)在每个植物的第一和第二枳叶的单个小叶的中心区域中制作圆形伤口(直径5mm) et al。,2005)。
      注意:另外,也可以使用软木塞钻孔机,纸孔打孔机或无菌刀片进行伤害。


      图1.大豆植物上的伤口和组织取样区域的示意图该方案显示在第一和第二枳叶(红色区域)上的两个受伤部位,其在发育阶段V2与 B。剑兰在处理的植物中的Lv-StA或在对照植物中的无菌自来水。黑色杂交表示之后从开发的大豆植物取得的叶子区域,并用于FISH和定量PCR检测B.剑兰 Lv-StA。注意伤害和感染在发育阶段V2完成(仅存在2个完全发育的枳叶),而该方案代表在后期发育阶段的植物,其中新的枳叶也可用于组织取样。

    5. 在植物之间使用粘性的昆虫陷阱,以防止与昆虫的潜在接触,从而在植物间交叉污染。

    6. 在受控的照明管理下,每隔一天随机排列植物并洗牌(图2)

      图2.根据处理和在受控光照条件下按随机顺序组织的大豆植物接种后三周获得图片。
    7. 每隔一天用自来水浇水。

  2. 使用光密度测定细菌细胞浓度的校准曲线
    注意:对于特定的分光光度计和细菌菌株,构建校准曲线只需要一次,考虑到CFU计数和制备用于感染的细菌接种物所需的时间,应该在植物初始接种前至少四天进行。 / em>
    1. 文化共生 B。在含有20ml King's B液体培养基(参见配方)的50ml离心管中,在30℃和恒定摇动(200rpm)下培养24小时。
    2. 将细菌培养物在2400×g下离心5分钟。丢弃上清,并用无菌自来水重悬细菌沉淀。
    3. 在1.5ml反应管中,使用无菌自来水(em-ie,三次七次稀释)制备三个稀释系列,从10 -1到10 -7 )。
    4. 测量每个样品的光密度(OD 600),三次,每个样品在含有King's B中琼脂的培养皿上平板50μl。

    5. 在30°C孵育培养皿48-72小时。
    6. 在King's B培养基平板上计数3日龄培养物的菌落形成单位(CFU)(只考虑每个菌落平均20到200个菌落的CFU)。
    7. 使用OD <600>与CFU计数相关的线性回归生成校准曲线。
    8. 计算对应于10 5 CFUsμl<-1> 的OD <600>。

  3. 细菌接种准备和植物感染
    注意:创伤后2小时内进行细菌接种
    1. 文化共生 B。在King's B液体培养基中于30℃下恒定摇动(200rpm)24小时(即在植物伤害和感染前一天)进行Lv-StA培养。 >
    2. 将细菌培养物在2400×g下离心5分钟。丢弃上清,并用无菌自来水重悬细菌沉淀。
    3. 测量细菌悬浮液的OD 600并用无菌自来水稀释至10 5 CFUμl-1。

    4. 接种18株植物与10μL的细菌悬液,每个植物在枳叶的两个创伤区域放一滴微量移液器(图1)。
    5. 在18个对照植物的两个对应的伤口上涂抹10μl无菌自来水(图1)。
    注:植物感染试验完全在生物安全2级实验室进行,根据德国法规(BioStoffV),将剑杆菌分配到危险的2族生物体中。 />
  4. 植物健康监测
    1. 监测株高和开花时间,每隔一天到感染后第38天(共20个测量时间点,包括接种当天)。
    2. 观察叶片坏死和萎黄的潜在症状(图3)。


      图3.用剑杆菌Lv-StA处理的单一大豆叶片与水对照相比的叶绿体萎缩处理后显示受伤和用无菌水处理的小叶(A )和治疗后38天(B),以及治疗后感染细菌悬液(C)和治疗后38天(D)感染的小叶。


    3. 使用SPAD-502Plus叶绿素计,每隔一天测量每个受伤tri鱼小叶的三个不同点上的叶绿素含量(一式三份,不在感染面积上)。
    4. 计数感染后38天所有豆荚中的种子总数。
    5. 同样在第38天,在70°C干燥整个植物(地上部分和地下部分)24h,测量干重。
      重要提示:请参阅程序E开始时的注意

  5. 量化感染细菌
    注意:在测量完整植物的干重之前(见步骤D5),在实验结束时(在接种后第38天)从每个植物收获两个小叶。
    1. 用无菌镊子和剪刀从每个植物的不同区域切下小片(约1×1.5厘米)(见图1),并将每个样品分成两半。
    2. 将每个样品的一半存放在-80℃的无菌1.5ml试管中,直到进一步处理。保留每件的另一半进行FISH分析。
    3. 准备无菌钝1毫升枪头杵。每个尖端的顶部很快燃烧,然后在含有DEPC处理的H 2 O(视频1)的1.5ml试管中圆形化。

      视频1
    4. 称量储存的小叶片,然后将1.5 ml试管中的每个样品与先前制备好的杵通过将液氮直接倒入每个试管中并将组织粉碎成粉末(视频2)均质化。如果需要完成均质化,向1.5毫升管中加入额外的液氮,并迅速将研磨棒插入管中进行研磨。虽然杵没有被使用,你可能会部分关闭管(不紧),以防止粉状叶组织喷出管。

      视频2
    5. 使用MasterPure TM完整DNA和RNA纯化试剂盒按照制造商的说明书提取核酸。在4°C进行离心步骤,并在低TE缓冲液中重悬DNA和RNA(见食谱)。
    6. 按照制造商的说明,使用QuantiTect®逆转录试剂盒(包括基因组DNA去除步骤和相应的对照)在冰上进行逆转录。
    7. 使用引物Burk16S_StAG_F和Burk3.1_R(表1),在RotorGene™-Q循环仪中按照针对Rotor-Gene SYBR所述的方案对167bp片段上的相应cDNA进行定量RT-PCR绿色PCR试剂盒。

      表1.在原位杂交中用于扩增和荧光的引物和探针


    8. 进行由95℃10分钟的初始变性步骤,95℃变性10秒,65℃引物退火30秒和72℃延伸的45个循环组成的定量RT-PCR持续20秒。
      在4.25分钟内包含一个温度梯度从60°C到99°C的解链曲线
    注意:
    1. 先前通过PCR扩增和使用innuPREP凝胶提取试剂盒进行产物回收来获得用于定量目标片段的DNA标准品。使用NanoDrop TM 1000分光光度计测量纯化的DNA浓度。标准物被用作十倍稀释系列,其范围从10到1/8到10到10,
    2. 所有用于RNA提取的溶液都是在DEPC(二乙基焦碳酸酯)处理过的H 2 O 2和RNaseZap 为了避免RNase污染设备和工作空间,使用去污溶液。

  6. 通过FISH可视化感染细菌
    1. 用剃刀切割并薄切不同植物区域的茎组织(第1和第2和第3片之间以及第2和第3片之间)刀片在大约0.5毫米纵向部分。
    2. 在室温(RT)固定切片的茎组织和半片叶片(见步骤E1)在Carnoy固色剂中(参见食谱)24小时,并保存在室温直到进一步使用。
    3. 将样品在室温下在100%乙醇中洗涤2小时,然后在6%H 2 O 2(在乙醇中稀释)中漂洗大约14天。
      每三天更换一次漂白液
    4. 在室温下在100%乙醇(3×2小时),70%乙醇(2×1小时)和PBS-Tx缓冲液(4×0.5小时,参见食谱)中洗涤样品。
    5. 在含有270μl杂交缓冲液(参见配方)的1.5ml试管中,在55℃下将样品与15μl(50ngμl-1)Cy5标记的普通真空细菌探针EUB784杂交24小时, Cy3标记的Burkholderia特异性探针Burk_16S(表1)和另外3μlDAPI(0.5mg ml -1)的15μl(50ngμl-1) )用于宿主细胞复染。
    6. 在洗涤缓冲液(2×2小时,见配方)中于55℃清洗样品,然后将样品在蒸馏水中和少量(1-2滴)的VECTASHIELD 在室温下2小时。
    7. 将样品转移到载玻片上,用VECTASHIELD®和盖玻片盖住,用荧光显微镜观察样品(图4)。
    注意:
    1. 步骤F3是光敏感的,应该在黑暗或昏暗的光线条件下进行。我们建议在孵化期间用铝箔或类似物覆盖样品。漂白液脱色对于避免植物组织的自发荧光至关重要。
    2. 步骤F5-F7涉及荧光探针的操作,因此我们建议在黑暗或昏暗的光线下工作,以尽量减少漂白。
    3. 在直至用蒸馏水(步骤F5和F6)进行最后的洗涤步骤期间和直到样品不需要样品完全干燥,因为随后去除未杂交的探针可能会受到损害。
    4. 设计用于真细菌的一些FISH探针也可以与植物叶绿体杂交(例如,EUB388:5'-GCTGCCTCCCGTAGGAGT-3')。在这个意义上,探针EUB784是方便的,因为在硅片上或在原位没有观察到与叶绿体的杂交。


    图4.大豆植物血管系统中的剑杆菌细胞的定位在大豆叶组织上的整个FISH显示与Burk_16S-Cy3(红色)杂交的重叠图像, EUB784-Cy5(绿色)和用DAPI(蓝色)染色的宿主细胞核。白色箭头表示感染的大豆植物小叶(A)的血管系统中的伯克霍尔德氏菌(Burkholderia)细胞。在对照植物(B)中没有观察到伯克霍尔德氏菌(Burkholderia)细胞。比例尺代表50微米。

数据分析

统计分析使用SPSS(版本17.0)进行。 B之间比较种子产量(种子数量)和第一次开花事件。唐菖蒲感染的植物和未感染的对照,使用Mann-Whitney U-检验,给出数据的非正态分布。 RStudio(版本0.98.1103)用于线性混合模型(LMM)和广义加性模型(GAM)分析,以时间为随机因子,叶绿素和处理或高度和处理为固定因子。在确认数据的正态分布和方差的均匀性之后,使用ANOVA和Tukey's post hoc测试分析感染后不同植物位置的伯克霍尔德氏菌(Burkholderia)丰度的定量PCR数据。以前已经公布了种子产量和植物组织中的伯克霍尔德氏菌定量结果(Flórez等人,2017)。

食谱

  1. 国王B媒体
    20克蛋白胨
    1.5克K 2 HPO 4 4克/升 10毫升甘油
    15克琼脂(只适用于固体培养基)
    1升双蒸馏水
    高压灭菌,然后加入5毫升无菌1M MgSO 4
  2. 低TE缓冲
    10mM Tris / HCl(在Milli-Q水中)
    0.1mM EDTA(在Milli-Q水中)
  3. PBS-Tx缓冲区
    3克Triton®X-100
    8克NaCl
    0.2克KCl
    1.4克Na 2 HPO 4 4/2 0.3克KH 2 PO 4 4克/克 1 L双蒸H 2 O
  4. 卡诺伊的固定剂
    6份乙醇100%
    3份氯仿100%
    1份乙酸100%
  5. 杂交缓冲液
    900 mM NaCl
    0.01%十二烷基硫酸钠(SDS)
    20 mM Tris / HCl pH 8.0
  6. 清洗缓冲液
    5 mM EDTA
    100 mM NaCl
    0.1%SDS
    20 mM Tris / HCl pH 8.0

致谢

该协议是从(Flórez等人,2017年)改编的,并在马克斯•普朗克化学生态研究所(德国耶拿)进行。由AxelMithöfer博士等人开发的机械植物创伤专用设备 。 (2005)被使用。我们感谢马克斯•普朗克大学化学生态学研究所的大豆种植研究所和Max-Planck-Society的资助。作者没有利益冲突或竞争利益申报。

参考

  1. Flórez,L. V.和Kaltenpoth,M.(2017)。 拉格里亚甲虫和之间防御互惠共生的共生动态和应变多样性 Burkholderia 。 Environ Microbiol 19(9):3674-3688。
  2. Flórez,L. V.,Scherlach,K.,Gaube,P.,Ross,C.,Sitte,E.,Hermes,C.,Rodrigues,A.,Hertweck,C.和Kaltenpoth,M.(2017)。 抗生素生产共生体在植物致病性和防虫共生之间动态过渡。 > Nat Commun 8:15172.
  3. Frago,E.,Dicke,M.和Godfray,H.C。(2012)。 昆虫共生体作为昆虫与植物相互作用中的隐性参与者 Trends Ecol Evol 27(12):705-711。
  4. Frank,S.A。(1997)。 共生模式 Am Nat 150 Suppl 1: S80-99。
  5. Gilbert,S.F。,Sapp,J。和Tauber,A.I。(2012)。 生命共生的观点:我们从来都不是个人。 Q Rev生物学87(4):325-341。
  6. Kaltenpoth,M.,Yildirim,E.,Gurbuz,M.F。,Herzner,G。和Strohm,E。(2012)。 精炼beewolf-链霉菌的根源共生:触角中的共生体稀有属 Philanthinus (膜翅目昆虫,Crabronidae)。 Appl Environ Microbiol 78(3):822-827。
  7. Mithöfer,A.,Wanner,G.和Boland,W.(2005)。 喂食斜纹夜蛾对利马豆叶的影响。 II。持续的类似于昆虫摄食的机械伤害足以引起与草食有关的挥发性排放。植物生理学137(3):1160-1168。
  8. Opelt,K.,Berg,C.,Schonmann,S.,Eberl,L.和Berg,G。(2007)。 高特异性但与之相反的水生植物的生物多样性相关的细菌和植物群落沼泽生态系统独立于地理区域。 ISME J 1(6):502-516。
  9. Salles,J.F.,De Souza,F.A。和van Elsas,J.D。(2002)。 评估环境样本中伯克霍尔德氏菌物种多样性的分子方法 Appl Environ Microbiol 68(4):1595-1603。
  10. Stammer,H. J.(1929)。 死亡共生体(鞘翅目) 变形术 15(1/2),1-34。
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引用: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.
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