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Jul 2020
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A Fast and Easy Method to Study Ralstonia solanacearum Virulence upon Transient Gene Expression or Gene Silencing in Nicotiana benthamiana Leaves
利用本生烟叶片基因瞬时表达或沉默研究青枯菌毒力的快速简便方法   

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Abstract

Ralstonia solanacearum is a devastating soil-borne bacterial pathogen that causes disease in multiple host plants worldwide. Typical assays to measure virulence of R. solanacearum in laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a novel inoculation protocol to analyze the replication of R. solanacearum upon infiltration into the leaves of Nicotiana benthamiana, in which gene expression has been altered using Agrobacterium tumefaciens. The protocol includes five major steps: 1) growth of N. benthamiana plants; 2) infiltration of A. tumefaciens; 3) R. solanacearum inoculation; 4) sample collection and bacterial quantitation; 5) data analysis and representation. The transient gene expression or gene silencing prior to R. solanacearum inoculation provides a straightforward way to perform genetic analysis of plant functions involved in the interaction between pathogen and host, using the appropriate combination of A. tumefaciens and R. solanacearum strains, with high sensitivity and accuracy provided by the quantitation of bacterial numbers in plant tissues.

Keywords: Ralstonia solanacearum (青枯病菌), Nicotiana benthamiana (本氏烟草), Virulence (毒性), Infection (感染), Inoculation (接种), Infiltration (渗透)

Background

Ralstonia solanacearum is considered one of the most destructive bacterial pathogens in plants worldwide due to its aggressiveness and wide host range. R. solanacearum invades host plants through their roots and colonizes their xylem vessels, finally spreading through the whole plant and leading to bacteria wilt disease (Xue et al., 2020). R. solanacearum is able to infect more than 250 plant species and is particularly well-adapted to infecting crops from the Solanaceae family, including tomato, potato, tobacco, eggplant, and other economically important crops (Mansfield et al., 2012). Experimental assays to assess the virulence of R. solanacearum in Solanaceae plants under laboratory conditions mostly rely on soil-drenching inoculation with a bacterial suspension followed by observation of disease symptoms (Morel et al., 2018). Other methods are based on the injection or infiltration of the bacterial suspension directly into the stems or leaves, bypassing the root penetration process and allowing a more sensitive and accurate quantitation of bacterial numbers in plant tissues (Morel et al., 2018). These methods have been extensively used to compare the virulence of different R. solanacearum strains in Solanaceae plants; however, the analysis of different plant genotypes relies on the availability of relevant plant mutants or transgenic plants, or otherwise requires their generation, which is usually time-consuming. We recently developed a method to generate tomato plants with transgenic roots suitable for subsequent soil-drenching inoculation with R. solanacearum, which allows for the faster generation of transgenic root material to study root invasion and the subsequent generation of disease symptoms (Morcillo et al., 2020). However, this method does not allow for accurate quantitation of bacterial replication in plant tissues. Nicotiana benthamiana, a model plant from the Solanaceae family, is well known for its suitability for transient gene expression mediated by Agrobacterium tumefaciens (Li, 2011). Therefore, we envisioned a straightforward procedure to manipulate gene expression in N. benthamiana tissues using A. tumefaciens followed by the subsequent infiltration of R. solanacearum and quantitation of R. solanacearum replication over time. Such a method should overcome two major issues: (i) many R. solanacearum strains, including the reference GMI1000 strain, are not virulent in N. benthamiana due to the recognition of two effector proteins (AvrAA and RipP1) by the immune system of this plant species (Poueymiro et al., 2009); and (ii) the inoculated R. solanacearum strain should be differentiated from the A. tumefaciens population previously infiltrated into the same leaf tissues. To overcome the first issue, we used the R. solanacearum Y45 strain, which was originally isolated from tobacco plants (Li et al., 2011) and lacks both AvrAA and RipP1, allowing it to replicate effectively in N. benthamiana leaf tissue (Sang et al., 2020). To circumvent the second issue, we transformed R. solanacearum Y45 with a plasmid that confers resistance to the antibiotic tetracycline to allow the selection of R. solanacearum Y45 colonies in solid medium while avoiding growth of A. tumefaciens. This simple method can be used to accurately determine R. solanacearum virulence in N. benthamiana tissues expressing heterologous genes (Sang et al., 2020; Wei et al., 2020), overexpressing endogenous genes, or undergoing gene silencing mediated by RNAi. The versatility of this method allows the study of bacterial virulence factors in plant tissues by expressing them in plant cells prior to bacterial inoculation, as well as genetic analysis of the contribution of plant genes to disease resistance or susceptibility.

Materials and Reagents

  1. Conical centrifuge tubes (50 ml) (GeneBrick, catalog number: GP0-7500)

  2. Microcentrifuge tubes (1.5 ml) (BBI, catalog number: F600620-0001)

  3. Needleless syringes (1 ml)

  4. Plastic Petri dishes (90 mm diameter)

  5. Pipette tips (AIBIO, catalog number: T1040000)

  6. Paper towels

  7. Cork borer (Integra Miltex, model: 33-34-P/25)

  8. (Optional) Sprial counting grid (90 mm diameter) (Interscience, Easysprial®, catalog number: 413014)

  9. Metal beads

  10. Spectrophotometer plastic cuvettes (BRAND, catalog number: 759015)

  11. Syringe filters (0.45 μm) (Millex, Millex®-HV, catalog number: SLHV033RB)

  12. Distilled sterile water

  13. 75% ethanol (Sinopharm Chemical Reagent, SCR®, catalog number: 801769610)

  14. Triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, catalog number: T8877-5G)

  15. Glucose (Sinopharm Chemical Reagent, SCR®, catalog number: 63005518)

  16. Bacto peptone (BD, catalog number: 211677)

  17. Yeast extract (OXOID, catalog number: LP0021)

  18. Casein hydrolysate (Casamino acids) (Sigma-Aldrich, catalog number: 22090-500G)

  19. Agar (Sinopharm Chemical Reagent, SCR®, catalog number: 10000561)

  20. Tryptone (OXOID, catalog number: LP0042)

  21. Sodium chloride (NaCl) (Sinopharm Chemical Reagent, SCR®, catalog number: 10019318)

  22. 3’,5’-Dimethoxy-4’-hydroxyacetophenone (AS) (Sigma-Aldrich, catalog number: 2478-38-8)

  23. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sinopharm Chemical Reagent, SCR®, catalog number: 10012818)

  24. Potassium hydroxide (KOH) (Sinopharm Chemical Reagent, SCR®, catalog number: 10017018)

  25. MES free acid monohydrate (MES) (Amresco, catalog number: Amresco E169)

  26. Dimethyl sulfoxide (DMSO) (Diamond, catalog number: A100231-0500)

  27. Standard potting soil (Pindstrup, catalog number: 1034593214)

  28. Tetracycline hydrochloride (Sangon Biotech, catalog number: T0422)

  29. Vermiculite

  30. Phi medium (see Recipes)

  31. 1% (W/V) TTC solution (see Recipes)

  32. 20% (W/V) glucose solution (see Recipes)

  33. Agrobacterium infiltration buffer (see Recipes)

  34. LB medium (see Recipes)

  35. Tetracycline hydrochloride (optional, depending on the antibiotic resistance of the strains used) (see Recipes)

Equipment

  1. Tweezers

  2. Scissors

  3. Medium- and high-throughput tissue grinder (QIAGEN, model: Tissue Lyser II)

  4. NanoDrop spectrophotometer (Thermo Scientific, model: NanoDrop 2000c)

  5. Water distiller/sterilizer (Millipore, model: Mili-Q intergral 10L)

  6. Spiral plater (optional) (Interscience, Easysprial®, catalog number: 412000)

  7. Vortex (Scientific Industries, model: Vortex-Genie 2, catalog number: S1-0246)

  8. (Optional) Alcohol lamp

  9. Petri dish incubator at 28°C (Panasonic, model: MIR-262-PC)

  10. Autoclave (SANYO, model: MLS-3780)

  11. Flow hood (clean bench) (Shanghaishangjing, model: CA-1390-1)

  12. Plant growth chamber (Percival, model: I-36VL)

  13. Electronic balance (Sartorius, model: BSA224S)

  14. Centrifuge (Eppendorf, model: centrifuge 5424)

  15. pH meter (Sartorius, model: PB-10)

  16. Tube incubator (shaker) at 28°C (Eppendorf, New BrunswickTM, catalog number: m1324-0006)

Software

  1. GraphPad Prism 7 (GraphPad, http://www.graphpad.com), or equivalent program for statistical analysis

  2. Microsoft Excel (Microsoft), or equivalent spreadsheet calculation program

Procedure

  1. Growing Nicotiana benthamiana plants

    1. Sow N. benthamiana seeds in a 1:1 mix of potting soil and vermiculite and place in a growth room at 25°C and 65% relative humidity under a 16-h light/8-h dark photoperiod with a light intensity of 130 mE m-2·s-1.

    2. Transfer 9-day-old seedlings into individual pots and allow them to grow under the same growth conditions until they are 4-5 weeks old (Figure 1).



      Figure 1. Growth of Nicotiana benthamiana seedlings in soil. Red arrows indicate leaves suitable for infiltration.


  2. Transient expression using Agrobacterium tumefaciens (hereafter, Agrobacterium)

    Note: This is a standard procedure detailed in Li (2011).

    1. This part of the protocol is optional to perform transient gene expression or gene silencing prior to R. solanacearum inoculation. If this step is not needed, proceed to Section C.

    2. Prepare and autoclave LB medium with and without agar. Allow LB medium with agar to cool until the bottle can be touched with bare hands (but is still in liquid form), add the appropriate antibiotics, and pour the medium into Petri dishes under a sterile flow hood.

    3. Streak out the Agrobacterium strains carrying the desired plasmids (see Note 1) onto solid LB medium and incubate at 28°C for 24 h. Incubate for a longer time if bacterial colonies need to be isolated.

    4. Collect Agrobacterium biomass directly from the Petri dish using a sterile pipette tip and resuspend in 1 ml infiltration buffer. Dilute 10-fold and measure OD600 using a spectrophotometer.

    5. Prepare an Agrobacterium suspension with a final concentration of OD600 = 0.2-0.5 in infiltration buffer.

    6. Select fully expanded leaves of 4-5-week-old N. benthamiana plants for infiltration (Figure 2). Using a permanent marker pen, draw a circle on each half of the leaf to control the area for infiltration. On the abaxial side of the leaf, prick softly at the center of the circle with a needle (e.g., from a 1-ml syringe).

      Note: Infiltration time: it is better to inoculate R. solanacearum in the morning to obtain reproducible and robust infection results. Therefore, it is advised to infiltrate Agrobacterium in the morning and leave 24 hours as a suitable time for heterologous gene expression before R. solanacearum inoculation. For RNAi-mediated silencing, we leave 3-9 days between Agrobacterium and R. solanacearum inoculation, depending on the silencing efficiency of each construct (see Note 1).



      Figure 2. Illustration of the infiltration step


    7. Infiltrate the Agrobacterium suspension carefully into the marked area.

      1. To this end, place a finger on the adaxial side of the leaf and place the needleless syringe at the same spot on the abaxial side (avoid spots with excessive protuberant veins).

      2. Use a 90° angle to avoid excessive spillage of the Agrobacterium suspension, but press gently on the leaf to avoid tissue damage.

      3. Slowly press the plunger until the Agrobacterium suspension starts filling the plant tissue, which becomes visibly darker upon soaking. Agrobacterium containing the plasmid for gene expression/silencing and the control plasmid can be infiltrated side-by-side in the same leaf to avoid leaf-to-leaf variation (see notes).

      4. Wipe the wet surface of the leaf gently with tissue paper and correct the circles (if necessary) with the actual infiltrated area.

      Notes:

      1. Infiltration of samples/controls: the different Agrobacterium strains used for transient gene expression/silencing and the control strain can be infiltrated in each half of the same leaf to avoid leaf-to-leaf variation. In such cases, bacterial numbers can also be analyzed as a ratio of the bacterial growth in both halves of the leaf. However, it is important to note that, if the transiently expressed gene/protein induces systemic responses, these could also affect the other half of the leaf. Therefore, the choice should be made depending on the specific nature of the transiently expressed gene.

      2. Infiltration of N. benthamiana leaves works better in dry leaves without water droplets from condensation or contact with other leaves. Some practice is advised (choice of infiltrated area, position of hands, pressure over the leaf tissue and the plunger, etc.) to optimize leaf infiltration before the actual experiment.

    8. Place the plants back into the growth chamber at 25°C and 65% relative humidity under a 16-h light/8-h dark photoperiod with a light intensity of 130 mE m-2·s-1.


  3. Inoculation with Ralstonia solanacearum Y45 (Figure 3)

    1. Select a R. solanacearum strain that is pathogenic in N. benthamiana (e.g., Y45; [Li et al., 2011; Sang et al., 2020]). If Agrobacterium was previously infiltrated into plant tissues, the R. solanacearum strain used should be resistant to an antibiotic that allows the differentiation of Y45 from the existing Agrobacteria at the time of sampling (see Note 2).

    2. Prepare and autoclave phi medium with and without agar. Allow the phi medium with agar to cool until the bottle can be touched with bare hands (but is still in liquid form), then add 5 ml/L 1% TTC stock solution and 25 ml/L 20% glucose stock solution.

    3. Streak out Y45 onto solid phi medium and incubate at 28°C for 2 days.

    4. Select pink colonies and pick using a sterile pipette tip to inoculate 10 ml phi liquid medium in a 50-ml conical centrifuge tube. Incubate the cultures on a shaker at 28°C overnight.

    5. Centrifuge the suspensions for 5 min at 4,000 × g. Remove the supernatant and resuspend the bacterial pellet in 10 ml sterile distilled water. Measure OD600 using a spectrophotometer. A suspension in distilled water with an OD600 = 1 contains approximately 109 cfu/ml (Morel et al., 2018).

      Note: In our experience, resuspending the bacteria in sterile distilled water (or other solution, such as MgCl2) does not affect the viability or virulence of R. solanacearum.



      Figure 3. Ralstonia solanacearum inoculation and collection. A. Bacteria growing on solid phi medium with TTC. B. Collection of bacterial biomass from pink colonies using a pipette tip. C. Bacterial culture after a 14-h overnight incubation (OD600 = 1.08). D. Bacterial suspension in sterile distilled water (left).


    6. Prepare a suspension of Y45 at the final desired concentration, depending on your requirements, by performing serial dilutions using sterile distilled water. Consider the possibility of testing the virulence of different doses of your specific strain under your specific experimental conditions. Under our conditions, we selected 105 cfu/ml as a suitable concentration that allows reproducible bacterial replication without causing the development of necrotic symptoms during the experimental period (Figure 4).



      Figure 4. Symptoms of N. benthamiana tissues inoculated with different doses of Y45 at the indicated days post-inoculation (dpi) with R. solanacearum. Upper images show the adaxial sides, and lower images show the abaxial sides.


    7. Select fully expanded leaves of 4-5-week-old N. benthamiana plants for infiltration (Figure 1). If Agrobacterium was previously infiltrated (Section B; see note at the Step B6 regarding infiltration time), infiltrate Y45 into the marked circles using a 1-ml needleless syringe as previously performed for Agrobacterium (Figure 2). Dry the surface of the leaves using tissue paper to remove residual bacterial inoculum from the leaf surface and place the inoculated plants in the growth chamber until sample collection.

      Note: After Y45 infiltration, we usually place plants in a different growth chamber at 27°C and 75% relative humidity since R. solanacearum infection is more efficient and reproducible at a higher temperature and humidity; however, a specific test experiment could be conducted to determine the efficiency and reproducibility of infection depending on the availability of growth chambers under these conditions.


  4. Sample collection

    1. Prepare 1.5-ml microcentrifuge tubes and measure their weight using an electronic balance.

    2. Cut the inoculated leaves using scissors and place them in a clean Petri dish containing sterile distilled water. Immerse both sides of the leaf in water for 5 s to wash the leaf surface and remove the surface water by placing the leaf on clean tissue paper.

    3. Collect 4 leaf discs (4 mm diameter) from each infiltrated area (avoiding areas with potential damage from the infiltration step) using a sterile cork borer and transfer the discs to 1.5-ml microcentrifuge tubes (Figure 5). Measure the weight of the tubes again.



      Figure 5. Collection of samples. A. Four leaf discs collected in 1.5-ml tubes using a 4-mm diameter cork borer. B. Homogenized plant tissue in 1 ml sterile water.


    4. Add 3 small metal beads to each tube using tweezers, followed by 100 μl sterile distilled water. Grind plant tissue using a tissue grinder at a frequency of 25 oscillations/s for 2 min.

    5. Add 900 μl sterile distilled water to the homogenized tissue and mix well using a vortex (Figure 5). Perform serial dilutions of the homogenized tissue by placing 100 μl in 900 μl sterile distilled water. Remember to change pipette tips between each dilution to avoid carryover of bacteria from higher dilutions.

    6. Spread 50 μl appropriate dilutions onto solid phi medium plates containing the appropriate antibiotic to differentiate Y45 colonies from the existing Agrobacteria in the homogenized tissue (see Notes). See the legend of Figure 6 for an estimation of the dilutions plated at each time point. Spreading onto plates can be performed manually using a sterile spreader, sterile plastic beads, or the bottom of a sterile 1.5-ml microcentrifuge tube, or using a spiral plater (optional, see notes) (Figure 6).

      Notes:

      1. Spreading colonies onto plates: disinfect hands with 75% alcohol before plating, and work under a sterile flow hood. An alcohol lamp can be used to avoid contamination if working on a bench. If plates are too wet after spreading the sample dilutions, they can be subsequently dried under a flow hood to obtain clear single colonies.

      2. Spiral plater: if available, a spiral plating machine can be used to spread bacterial dilutions. This is particularly useful for large-scale experiments, where numerous samples are required. There is no significant difference in the results obtained by plating manually and those obtained using the machine, as shown in Figure 6A and 6B.



      Figure 6. Ralstonia solanacearum Y45 growth. A. Colonies obtained on solid phi medium plates using the same bacterial dilution, by manual spreading or using the spiral plating machine. B. Comparison of cfu numbers using manual spreading or the spiral plating machine. C. Time-course experiment to determine Y45 growth. N. benthamiana leaves were first inoculated with Agrobacterium to express GFP (used as a control protein in our assays); one day later, a 105 cfu/ml inoculum of Y45 was infiltrated into the same leaves. Samples were then collected at 1, 2, 3, and 4 days post-inoculation (dpi) with Y45, as indicated. To count these numbers, 100 (undiluted) and 10-1 dilutions were plated 1 dpi, 10-1, and 10-3 dilutions were plated 2 dpi, 10-3 and 10-4 dilutions were plated 3 dpi, and 10-4 and 10-5 dilutions were plated 4 dpi.


    7. Incubate the plates upright (see Notes) at 28°C for 2 days. Count the colonies and process the data.

      Note: Incubation of R. solanacearum after sampling: colonies of R. solanacearum are very mucoid; therefore, plates should be incubated upright to avoid bacterial secretions dripping.

Data analysis

To calculate the number of bacteria by plant fresh weight, import the tube weight, full weight, dilution factor, and number of cfu (colony-forming units) into an Excel spreadsheet. Enter the sector of the plate when counting colonies plated using a spiral plater. Table 1 shows examples of the calculations performed when plating using the spiral plater and manually.


Table 1. Calculation of bacterial numbers in plant tissues



Calculation steps:

These calculations correspond to cfu numbers obtained by manual plating. If plating using a spiral plater, follow the manufacturer’s instructions to calculate bacterial numbers.

  1. Obtain the fresh weight of the plant tissue using the formula =(C8-B8)*1,000 and change the units from g to mg for subsequent calculation.

  2. Calculate bacterial numbers (cfu/ml) using the formula (F8/H8)*1,000, and change the volume units from μl to ml.

  3. Column E indicates the dilution series. For example, E8 indicates that bacteria were counted after two serial dilutions; therefore, the dilution factor (J8 = 10^E8) is 1,000.

  4. To refer bacterial numbers to plant fresh weight, the weight units can be changed again from mg to g, and the final product can be calculated using the following formula: K8 = PRODUCT(I8, 1,000, J8, 1/D8), resulting in 1316923077 cfu/gFW. This value can be converted for representation in a logarithmic scale on the base of 10 (row L).

  5. Input the resulting final data into GraphPad Prism7 or a similar program for statistical analysis. To compare results individually, preform a Student’s t-test and present the mean value, standard error, and p value to indicate the statistical significance of the differences.

Notes

  1. Choice of Agrobacterium strains: we usually use the Agrobacterium GV3101 strain carrying plasmids from the pGWB series (Nakagawa et al., 2007a) to express tagged proteins in N. benthamiana. When GFP fusion proteins are used, we use free GFP as a control protein (Nakagawa et al., 2007b) [as in Figure 2B of Sang et al. (2020)]. To perform transient gene silencing, we use an RNAi approach with pK7GWIWG2-II vectors (Karimi et al., 2002). Gene fragments targeting specific genes using RNAi can be easily designed using diverse online tools (https://vigs.solgenomics.net/; http://plantgrn.noble.org/pssRNAit/). We use Agrobacterium carrying an empty vector as a negative control. It is important to always verify gene overexpression as well as silencing efficiency by performing RNA extraction and RT-qPCR.

  2. Choice of R. solanacearum strain: to allow for the differentiation of Y45 from the existing Agrobacteria at the time of sampling, we transformed the Y45 wild type strain with a pRCT-derivative plasmid (Monteiro et al., 2012) using natural transformation (Coupat et al., 2008). This plasmid confers resistance to tetracycline; therefore, we add tetracycline to the phi medium used to plate bacteria after sampling.

  3. Preparation of plates: after pouring the medium into Petri dishes, keep the plates uncovered under the flow hood for approximately 40 min when plating using the spiral plater or for around 20 min when plating manually. The spiral plater requires the plates to be drier in order to prevent the different circles from contacting each other; on the other hand, manual plating is more uniform if the plates are more humid. Sterile water can be added to the plates before manual plating if they are too dry.

  4. Data representation: representation of the bacterial numbers (or cfu) can be performed relative to the fresh weight (as indicated in our protocol) or relative to leaf area, using the size of the cork borer as a reference. In the latter case, there is no need to measure the weight of the tubes before and after sampling.

  5. Waste disposal: autoclave all the material used in this experiment before disposal to avoid release of R. solanacearum into the environment. Clean the work bench and flow hood with 75% alcohol after finishing the experiment.

Recipes

  1. Phi medium

    10 g Bacto peptone

    1 g Yeast extract

    1 g Casamino acids

    Add water to 1 L.

    To prepare solid medium, add 15 g agar to 1 L medium and autoclave.

  2. 1% (W/V) TTC solution

    Dissolve TTC in distilled water and filter the solution with a 0.45-μm syringe filter to avoid contamination.

    Note: Store the 1% TTC solution at room temperature or 4°C in a dark environment.

  3. 20% (W/V) glucose solution

    Glucose should be dissolved in distilled water and autoclaved. Store this stock solution at 4°C.

  4. Agrobacterium infiltration buffer

    1 M MES 100 μl

    1 M MgCl2 100 μl

    150 mM AS 10 μl

    Add 9.890 ml water to reach 10 ml.

    Adjust MES to pH 5.7 with KOH.

    Dissolve MES and MgCl2 in sterile water and filter the solutions with a 0.45-μm syringe filter to avoid contamination.

    Store the solution at 4°C.

    Dissolve AS in DMSO and store the stock solution at -20°C.

  5. LB medium

    10 g Tryptone

    5 g NaCl

    5 g Yeast extract

    Adjust pH to 7.5 with KOH, and add water to 1 L.

    To prepare solid medium, add 15 g agar to 1 L medium and autoclave.

  6. Tetracycline (optional, depending on the resistance of the strains used)

    Prepare a stock solution of tetracycline hydrochloride (10 mg/ml) in sterile distilled water (working in a flow hood) and store at -20°C. The final concentration in the medium should be 10 μg/ml.

Acknowledgments

We thank Wei Ding for the original donation of the Y45 strain, Gang Yu for critical reading of this manuscript, Xinyu Jian for technical and administrative assistance during this work, and all the members of the Macho laboratory for the constant improvement of this protocol and helpful discussions. Work in the Macho laboratory is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB27040204), the Chinese 1000 Talents Program, and the Shanghai Center for Plant Stress Biology (Chinese Academy of Sciences).

Competing interests

The authors have no competing interests to declare.

Ethics

No human or animal subjects are used in this protocol.

References

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  14. Xue, H., Lozano-Duran, R., and Macho, A.P. (2020). Insights into the root invasion by the plant pathogenic bacterium Ralstonia solanacearum. Plants (Basel) 9(4): 516.

简介

[摘要]青枯病菌是一种毁灭性的土传细菌病原体,可在全球多种寄主植物中引起疾病​​。在实验室条件下测量青枯菌毒力的典型检测方法依赖于浸透土壤的接种,然后对疾病症状进行观察和评分。这里,我们描述了一种新接种原COL来分析所述的复制青枯菌后渗入所述的叶片本塞姆氏烟草,在WH ICH基因表达使用已经改变的根癌农杆菌 . 该协议包括五个主要步骤:1)本氏烟草植物的生长;2)根癌农杆菌的浸润;3)青枯菌接种;4) 样品采集和细菌定量;5) 数据分析与表示。瞬时基因表达或基因之前沉默青枯雷尔氏菌接种提供了一种简单的方法执行中涉及的病原体和宿主之间的相互作用植物功能基因分析,使用的适当组合根瘤农杆菌和青枯雷尔氏菌STR AINS,具有高植物组织中细菌数量的定量提供了灵敏度和准确性。

【背景】青枯病菌因其侵袭性和广泛的寄主范围而被认为是全球植物中最具破坏性的细菌病原体之一。青枯菌通过根部侵入寄主植物并定植于木质部血管,最终蔓延至全株并导致青枯病(Xue et al. , 2020 )。R. solanacearum能够感染超过 250 种植物,特别适合感染茄科作物,包括番茄、马铃薯、烟草、茄子和其他重要经济作物(Mansfield等,2012 )。实验测定以评估的毒力青枯雷尔氏菌在茄科实验室条件下的植物大多依靠土壤浸润接种细菌悬浮液,随后对疾病症状的观察(莫瑞尔等人,2018 )。其他方法基于将细菌悬浮液直接注入或渗透到茎或叶中,绕过根部渗透过程,并允许对植物组织中的细菌数量进行更灵敏和准确的定量(Morel等人,2018 年)。这些方法已被广泛用于比较不同青枯菌菌株在茄科植物中的毒力;然而,不同植物基因型的分析依赖于相关植物突变体或转基因植物的可用性,或者需要它们的生成,这通常是耗时的。我们最近开发以产生番茄与适合于与随后土壤浸润接种转基因根的植物的方法青枯菌,其允许所述以研究根入侵和随后产生的疾病症状的更快代转基因根材料的(莫尔西略等。, 2020 ) 。然而,日是方法不允许在植物组织中细菌复制的准确定量。烟草本塞姆氏,从模式植物茄科家庭,是众所周知的,其适用性通过介导的瞬时基因表达根癌农杆菌(李,2011 )。因此,我们设想一个简单的程序来操纵基因表达的本塞姆氏烟草使用组织根癌农杆菌随后的后续浸润青枯雷尔氏菌和孔定量TA和灰青枯雷尔氏菌的复制随着时间的推移。这样一个方法应该克服两个主要问题:(ⅰ)中号任何青枯菌株,包括参考GMI1000应变,不在毒性本塞姆氏烟草由于由免疫系统识别2个效应蛋白(AvrAA和RipP1)的这种植物物种(Poueymiro等人,2009 年);和(ii)所述接种青枯从菌株应有所区别根瘤农杆菌群体先前渗入同一叶组织。为了解决第一个问题,我们我们编的青枯菌Y45菌株,它最初是从烟草植物中分离(李等人,2011 )并缺失AvrAA和RipP1,允许它在有效复制本塞姆氏叶组织(桑等人,2020 年)。为了避免第二个问题,我们用赋予抗生素四环素抗性的质粒转化青枯菌Y45 ,以便在固体培养基中选择青枯菌 Y45 菌落,同时避免根癌农杆菌的生长。这种简单的方法可用于准确测定表达异源基因的本氏烟草组织中青枯菌的毒力(Sang等人,2006)。 , 2020; 魏等人。, 2020 ) 、过表达内源基因或经历由 RNAi 介导的基因沉默。这种方法的多功能性允许通过在细菌接种之前在植物细胞中表达它们来研究植物组织中的细菌毒力因子,以及对植物基因对疾病抗性或易感性的贡献的遗传分析。

关键字:青枯病菌, 本氏烟草, 毒性, 感染, 接种, 渗透

材料和试剂
 
1.锥形离心管(50 ml)(GeneBrick,目录号:GP0-7500)      
2.微量离心管(1.5 ml)(BBI,目录号:F600620-0001)      
3.无针注射器小号(1ml)中      
4.塑料P ETRI培养皿(直径90mm)      
5.移液器吸头(AIBIO,目录号:T1040000)      
6.纸巾      
7.软木钻孔机(Integra Miltex,型号:33-34-P/25 )      
8. (可选)Sprial计数网格(90毫米直径)(INTERSCIENCE,ê asysprial ® ,目录号:413014 )      
9.金属珠      
10.分光光度计塑料比色皿(BRAND,目录号:759015)   
11.注射器过滤小号(0.45微米)(的Millex,的Millex ® -HV,目录号:SLHV033RB)   
12.蒸馏无菌水   
13. 75%的乙醇(国药化学试剂,SCR ® ,目录号:801769610 )   
14.氯化三苯基四唑鎓(TTC)(Sigma-Aldrich,目录号:T8877-5G)   
15.葡萄糖(国药化学试剂,SCR ® ,目录号:63005518 )   
16. Bacto蛋白胨(BD ,目录号:211677)   
17.酵母提取物(OXOID,目录号:LP0021)   
18.酪蛋白ħ ydrolysate(酪蛋白氨基酸)(Sigma-Aldrich公司,目录号:22090-500G)   
19.琼脂(国药化学试剂,SCR ® ,目录号:10000561 )   
20.胰蛋白胨(OXOID,目录号:LP0042)   
21.氯化钠(NaCl)(国药化学试剂,SCR ® ,目录号:10019318 )   
22. 3',5'-二甲氧基-4'-羟基苯乙酮(AS)(Sigma-Aldrich,目录号:2478-38-8)   
23.六水氯化镁(MgCl 2    · 6H 2 O)(国药化学试剂,SCR ® ,目录号:10012818 )
24.氢氧化钾(KOH)(国药化学试剂,SCR ® ,目录号:10017018 )   
25. MES游离酸一水合物(MES)(Amresco,目录号:Amresco E169)   
26.二甲基亚砜(DMSO)(钻石,目录号:A100231-0500)   
27.标准盆栽土(Pindstrup,目录号:1034593214)   
28.四环素盐酸盐(Sangon Biotech,目录号:T0422)   
29.蛭石   
30.披MEDI微米(参见ř ecipes)   
31. 1%(W / V)TTC溶液(见ř ecipes)   
32. 20%(W / V)的葡萄糖溶液(见ř ecipes)   
33.农杆菌浸润缓冲器(见ř ecipes)   
34. LB培养基(参见ř ecipes)   
35.四环素盐酸盐(任选的,取决于荷兰国际集团所使用的菌株的抗生素抗性)(见ř ecipes)   
 
设备
 
镊子
剪刀
介质-和高-通量组织研磨器(QIAGEN ,型号:组织Lyser II )
纳米d ROP分光光度计(Thermo Scientific的,型号:纳米滴2000C)
水蒸馏器/消毒器(Millipore,型号:Mili-Q intergral 10L)
螺旋电镀(可选)(Interscience,E asysprial ® ,目录号:412000 )
Vortex(科学工业,型号:Vortex-Genie 2,目录号:S1-0246)
(可选)酒精灯
28 °C培养皿培养箱(松下,型号:MIR-262-PC)
高压釜(SANYO,型号:MLS-3780)
流罩(洁净台)(上海尚晶,型号:CA-1390-1)
植物生长室(Percival,型号:I-36VL)
电子天平(Sartorius,型号:BSA224S)
离心机(Eppendorf,型号:离心机 5424)
pH计(Sartorius,型号:PB-10)
28 °C管式培养箱(振荡器)(Eppendorf,New Brunswick TM ,目录号:m1324-0006)
 
软件
 
Graph P ad Prism 7(Graph P ad,http://www.graphpad.com),或用于统计分析的等效程序
Microsoft Excel (Microsoft) 或等效的电子表格计算程序
 
程序
 
种植本氏烟草植物
在盆栽土和蛭石的 1:1 混合物中播种本氏烟草种子,并放置在 25 °C和 65% 相对湿度的生长室中,在 16 小时光照/8 小时黑暗光周期下,光照强度为 130 mE米-2 ·秒-1 。
转让9日龄幼苗到单独的盆中,并允许他们以成长下相同的生长条件,直到他们4-5周小号老(图1)。
 
 
图1的生长本塞姆氏烟草幼苗我Ñ土壤。红色箭头表示适合渗透的叶子。
 
使用根癌农杆菌(以下简称农杆菌)的瞬时表达
注:这是Li ( 2011) 中详述的标准程序。
协议的这一部分是可选的,用于在青枯菌接种之前进行瞬时基因表达或基因沉默。如果不需要此步骤中,进行到小号挠度C.
准备和高压灭菌 LB 培养基有和没有琼脂。允许用琼脂的LB培养基中,以冷却,直至瓶可以与用手直接接触(但仍是液体形式),添加适当的抗生素,然后倒在介质进入P ETRI菜肴下无菌流罩。
将携带所需质粒(见注 1 )的农杆菌菌株划线到固体 LB 培养基上,并在 28 °C下孵育24 小时。如果需要分离细菌菌落,则孵育更长时间。
收集农杆菌直接从生物质P ETRI培养皿用无菌移液管尖端和重新悬浮在1ml缓冲液浸润。使用分光光度计稀释 10 倍并测量 OD 600 。
制备农杆菌悬浮液与OD的最终浓度600 = 0.2 -在渗透缓冲0.5。
选择4-5的完全展开的叶片-周大的烟草本塞姆氏的渗透植物(图2 )。使用永久性记号笔,在叶子的每一半上画一个圆圈,以控制渗透区域。在叶的远轴侧,在用针的圆的中心轻轻扎(例如,从1 -毫升注射器)。
ñ OTE :渗透时间:最好是接种青枯菌在上午获得可重复性和强大的感染导致。因此,建议渗透土壤杆菌在早晨第二离开24小时内青枯雷尔氏菌接种前用合适的时间用于异源基因表达。对于 RNAi介导的沉默,我们在农杆菌和青枯菌接种之间留出 3-9 天的时间,这取决于每个构建体的沉默效率(参见注释 1)。
 
 
图 2. 渗透步骤图示
 
将农杆菌悬浮液小心地渗入标记区域。
为此,将手指放在叶子的正面,将无针注射器放在背面的同一位置(避免静脉过多突出的位置)。
使用 90 °角以避免农杆菌悬浮液过度溢出,但轻轻按压叶子以避免组织损伤。
慢慢按下柱塞,直到农杆菌悬浮液开始填充植物组织,浸泡后植物组织明显变暗。含有用于基因表达/沉默的质粒的农杆菌和对照质粒可以并排渗入同一片叶子中,以避免叶间变异(见注释)。
用纸巾轻轻擦拭叶子的湿表面,并用实际渗透区域校正圆圈(如有必要)。
              ñ OTE小号:
的样品/对照浸润:用于瞬时基因表达/沉默和对照菌株的不同农杆菌菌株中可以渗入每个HAL ˚F同一叶的,以避免叶到叶的变化。在这种情况下小号,细菌数目也可以被分析为一个在叶的两半的细菌生长的比率。然而,重要的是要注意,如果瞬时表达的基因/蛋白质诱导全身反应,这些也可能影响叶子的另一半。因此,应根据瞬时表达基因的具体性质进行选择。
本氏烟草叶子的渗透在干燥叶子中效果更好,没有因冷凝或与其他叶子接触而产生的水滴。一些实践建议(选择渗透面积,手的位置,在叶组织和柱塞压力等。实际实验之前),以优化叶的渗透。
将植物放回25°C 和 65% 相对湿度的生长室中,在 16 小时光照/8 小时黑暗光周期下,光照强度为 130 mE m -2 ·s -1 。
 
接种青枯病菌 Y45 (图 3)
选择在本氏烟草中具有致病性的青枯菌菌株(例如,Y45;[ Li, et al ., 2011 ; Sang et al. , 2020 ] )。如果农杆菌先前已渗入植物组织,则所使用的青枯菌菌株应能耐受抗生素,以便在采样时将 Y45 从现有农杆菌中分化出来(见注 2 )。
准备和高压灭菌 phi 培养基有和没有琼脂。让带有琼脂的phi 培养基冷却,直到可以赤手触摸瓶子(但仍为液体形式),然后加入 5 ml/L 1% TTC 储备液和 25 ml/L 20% 葡萄糖储备液。
将 Y45 划线到固体 phi 培养基上,并在 28 °C下孵育2 天。
选择粉红色菌落并使用无菌移液管尖端来接种10毫升挑披液体培养基,在50 -毫升锥形离心管中。在 28 °c的振动筛上孵育培养物过夜。
将悬浮液以 4,000 × g离心 5 分钟。去除上清液并将细菌沉淀重悬在 10 ml 无菌蒸馏水中。使用分光光度计测量 OD 600 。在蒸馏水中的悬浮液用OD 600 = 1含有约10 9 CFU /毫升(莫瑞尔等人。,2018)。
注:我ñ我们的经验,再悬浮的细菌在无菌蒸馏水(或其他解决方案,比如氯化镁2 )不影响该青枯菌的活力或毒力。
 
 
图 3.青枯病菌接种和收集。A. 在含有 TTC 的固体 phi 培养基上生长的细菌。B.使用移液器吸头从粉红色菌落中收集细菌生物量。一个14之后C.细菌培养- ħ孵育过夜(OD 600 = 1.08)。D. 无菌蒸馏水中的细菌悬浮液(左)。
 
在最终所需浓度制备悬浮液Y45的,根据您的要求,通过进行使用无菌蒸馏水连续稀释。考虑在特定实验条件下测试不同剂量特定菌株毒力的可能性。在我们的条件下,我们选择了 10 5 cfu/ml 作为合适的浓度,它允许可重复的细菌复制,而不会在实验期间引起坏死症状的发展(图 4)。
 
 
图4的症状本生烟草在所指示的天用不同剂量的Y45的接种组织接种后(DPI)以青枯雷尔氏菌。上图显示正面,下图显示背面。
 
选择4-5的完全展开的叶片-周大的烟草本塞姆氏的渗透植物(图1)。如果农杆菌以前浸润(小号挠度乙;见注在小号关于浸润时间TEP B6 ),浸润Y45到标出的圆形,使用1 -毫升需要乐少如先前注射器执行用于农杆菌(图2)。用纸巾擦干叶子表面,去除叶子表面残留的细菌接种物,然后将接种的植物放在生长室中,直到收集样品。
注意:甲压脚提升Y45浸润,我们通常将在不同的生长室的植物在27 ℃下和75%相对湿度,因为青枯感染是在更有效的和可再现的一个较高的温度和湿度; 然而,根据这些条件下生长室的可用性,可以进行特定的测试实验以确定感染的效率和再现性。
 
样品采集
制备1.5 -毫升微量离心管中,并测量它们的重量用电子天平。
切接种叶用剪刀并将它们放置在一个干净的P ETRI菜含有无菌蒸馏水中。将叶子的两侧浸入水中 5 s 以清洗叶子表面, 并将叶子放在干净的纸巾上去除表面水。
收集来自各渗透区使用无菌木塞打孔器和盘转移到1.5(避免与从所述渗透步骤的潜在损害的区域)4个的叶盘(直径4毫米)-毫升微量离心管中(图5)。再次测量管子的重量。
 
 
图 5. 样本收集。A.四叶收集在1.5光盘-用4毫升管-毫米直径木塞穿孔。B. 在 1 ml 无菌水中均质的植物组织。
 
使用镊子将 3 个小金属珠添加到每个管中,然后加入100 μl 无菌蒸馏水。使用组织研磨机以25 次振荡/s的频率研磨植物组织2 分钟。
将900 μl 无菌蒸馏水添加到均质组织中,并使用涡流充分混合(图 5)。将 100 μl 放入 900 μl 无菌蒸馏水中,对匀浆组织进行连续稀释。请记住在每次稀释之间更换移液器吸头,以避免更高稀释度中的细菌残留。
扩散上50μl的适当稀释到固体披培养基平板含有合适的抗生素,以从现有的土壤杆菌在匀浆组织分化Y45菌落(见Ñ OTES)。看到的图6的图例镀稀释的估计在每个时间点。传播ING上到可使用手动无菌涂布器,无菌塑料珠来执行板,或无菌1.5底部- ml离心管,或使用旋转接种仪(可选的,见注)(图6)。
注意事项:
将菌落传播到平板上:电镀前用 75% 的酒精对手进行消毒,并在无菌流动罩下工作。如果在工作台上工作,可以使用酒精灯来避免污染。如果在铺开样品稀释液后板太湿,它们可以随后在流动罩下干燥以获得清晰的单菌落。
螺旋式开发平台ER :如果有的话,螺旋电镀机可以被用来传播细菌稀释。这对于需要大量样本的大规模实验特别有用。没有显著差异我N乘手动电镀获得的结果和那些获得使用机器,如图6A和6B所示。
 
 
图 6.青枯病菌 Y45 生长。A.菌落获得ö使用相同的细菌稀释Ñ固体披培养基平板,通过手动扩频或使用螺旋电镀机。B. 使用手动涂布或螺旋电镀机比较 cfu 数。C.时间-课程的实验,以确定Y45增长。本塞姆氏叶先INOC农杆菌ulated表达GFP(作为一个在我们的实验控制蛋白); 一天后,将 10 5 cfu/ml 的 Y45 接种物渗入相同的叶子中。如图所示,然后在用 Y45 接种后 (dpi) 1、2、3 和 4 天收集样品。计数这些数字,10 0 (未稀释)和10个-1稀释度铺板1个DPI,10 -1 ,和10个-3铺板稀释2 dpi的10 -3和10 -4稀utions铺板3 dpi的,并10 -4和10 -5稀释液接种4 dpi。
 
孵育所述板直立(见Ñ OTES)在28°C 2 天。计数的殖民地和处理的数据。
注:采样后青枯菌的培养:青枯菌菌落非常黏膜ID ; 因此,板应当孵育直立以避免细菌分泌物博士我p平。
 
数据分析
 
由植物鲜重计算细菌的数量,导入了在管的重量,全部重量,稀释因子,和CFU数(菌落形成单位)到Excel电子表格。计数使用螺旋镀板的菌落时,输入板的扇区。表1示出实施例的利用螺旋接种仪和手动电镀时的计算来执行。
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说明: logonew                                                                
 
表 1. 植物组织中细菌数量的计算
 
      
计算步骤:
这些计算对应于通过手动电镀获得的 cfu 数。如果使用螺旋板进行电镀,请按照制造商的说明计算细菌数量。
1.使用公式 =(C8-B8)* 获得植物组织的鲜重       1,000并将单位从 g 更改为 mg 以进行后续计算。
2.使用公式(F8/H8)*1,000 计算细菌数(cfu/ml),并将体积单位从μl 更改为ml。      
3. Ç olumn E表示稀释系列。例如,E8 表示细菌在两次连续稀释后计数;因此,稀释系数 (J8 = 10^E8) 为 1,000。      
4.将细菌数与植物鲜重相关联,重量单位可以再次从mg变为g,最终产品可以使用以下公式计算:K8 = PRODUCT(I8, 1,000, J8, 1/D8),结果为 1316923077 cfu/gFW。该值可以转换为以 10(L 行)为底的对数刻度表示。      
5.输入得到的最终数据在到的Gr aphPad Prism7或用于统计分析一个类似的方案。要单独比较的结果,大跳学生牛逼-检验和展示的平均值,标准误差和p值来表示的差异有统计学意义。      
 
笔记
 
农杆菌菌株的选择:我们通常使用携带 pGWB 系列(Nakagawa等,2007a )质粒的农杆菌 GV3101 菌株在本氏烟草中表达标记蛋白。当使用GFP融合蛋白,我们使用GFP自由作为一个对照蛋白(中川等人,2007年b )[如在图2B的桑等人。(2020 年)] 。为了进行瞬时基因沉默,我们使用带有pK7GWIWG2-II 载体的n RNAi 方法(Karimi等,2002)。基因片段靶向荷兰国际集团利用RNAi可使用不同的在线工具可以很容易地设计特定基因(https://vigs.solgenomics.net/ ; http://plantgrn.noble.org/pssRNAit/)。我们使用携带空载体的农杆菌作为阴性对照。通过执行 RNA 提取和 RT- q PCR始终验证基因过表达和沉默效率非常重要。
的选择青枯菌菌株:允许用于Y45的从现有的农杆菌在取样时间的分化,我们转化有一个PRCT衍生质粒中的Y45野生型菌株(蒙泰罗等人,2012 )使用天然转化(Coupat等人,2008 年)。该质粒赋予对四环素的抗性;因此,我们在取样后将四环素添加到用于平板细菌的phi 培养基中。
板的制备:浇注培养基后到培养皿,保持该未覆盖板下的流罩约40分钟时使用螺旋接种仪或大约20分钟电镀时高原荷兰国际集团手动。螺旋接种仪需要将板是干燥器,以便防止不同圆从接触荷兰国际集团彼此; 另一方面,如果板更潮湿,则手工电镀更均匀。如果板太干,可以在手动电镀之前将无菌水添加到板中。
数据表示:在细菌数目(或CFU)的表示可以执行相对于所述鲜重(如在我们的协议指示的)或使用软木钻孔作为基准的大小相对于叶面积,。在后一种情况下,无需在采样前后测量试管的重量。
垃圾处理:蒸压都在这个实验中使用dispos前的材料人到避免释放青枯菌在环境。完成实验后,用 75% 的酒精清洁工作台和流罩。
 
食谱
 
Phi 介质
10 克 Bacto 蛋白胨
1 克酵母提取物
1 克酪蛋白氨基酸
加水至1升。
到prepar ë固体培养基,15g琼脂添加到1升介质和高压釜。
1% (W/V) TTC 解决方案
溶解TTC在蒸馏水中,并过滤以0.45的溶液-微米注射器式过滤器,以避免污染。
注意:将 1% TTC 溶液储存在室温或 4°C 的黑暗环境中。
20% (W/V) 葡萄糖溶液
葡萄糖应溶解在蒸馏水中并高压灭菌。将此储备溶液储存在 4°C。
农杆菌浸润缓冲液
1 M MES 100 μl
1 M 氯化镁2 100 微升
150 mM AS 10 μl
添加 9.890 毫升水至 10 毫升。
用 KOH将MES 调整到 pH 5.7 。
溶解MES和的MgCl 2在无菌水中,并用0.45微米的滤器的解决方案-微米注射器式过滤器,以避免污染。
将溶液储存在 4°C 。
将AS溶解在 DMSO 中,并将原液储存在 -20°C 。
LB培养基
10 克胰蛋白胨
5 克氯化钠
5 克 酵母提取物
调节pH至7.5,用KOH,并加水至1升。
到prepar ë固体培养基,15g琼脂添加到1升介质和高压釜。
四环素(可选,取决于所用菌株的抗性)
制备的储备溶液吨etracycline盐酸盐在无菌蒸馏水(在流罩的工作),并存储(10毫克/毫升)在-20℃下。的F培养基中伊纳勒浓度应为10微克/毫升。
 
致谢
 
我们感谢魏丁对 Y45 菌株的原始捐赠,感谢刚玉对这份手稿的批判性阅读,感谢简新玉在这项工作中提供的技术和行政协助,感谢 Macho 实验室的所有成员对本协议的不断改进和帮助讨论。Macho实验室的工作得到了中国科学院战略重点研究计划(XDB27040204)、中国千人计划和上海植物胁迫生物学中心(中国科学院)的支持。
 
利益争夺
 
作者没有要声明的竞争利益。
 
伦理
 
本协议中不使用人类或动物受试者。
 
参考
 
1. Coupat, B.、Chaumeille-Dole, F., Fall, S.、Prior, P.、Simonet, P.、Nesme, X. 和 Bertolla, F. (2008)。       Ralstonia solanacearum物种复合体中的自然转化:可以转移的 DNA 的数量和大小。FEMS 微生物生态系统。66:14-24。
2. Karimi, M.、Inzé, D. 和 Depicker, A. (2002)。用于农杆菌介导的植物转化的GATEWAY TM载体。       趋势植物科学7(5): 193-195。
3. Li, X. (2011)。          Nicotiana bethamiana 协议的渗透以通过农杆菌进行瞬时表达。 生物协议e95。
4. Li, Z., Wu, S., Bai, X., Liu, Y., Lu, J., Liu, Y., Xiao, B., Lu, X., and Fan, L. (2011) . 烟草青枯病病原体青枯病菌基因组序列。J Bacteriol 193:6088-6089。         
5. Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, SV, Machado, MA, Toth, I.、Salmond, G. 和 Foster, GD (2012)。分子植物病理学中的10大植物病原菌。Mol Plant Pathol 13( 6) : 614-629。         
6. Monteiro, F.、Solé, M.、van Dijk, I. 和 Valls, M.(2012 年)。用于青枯病菌启动子探测、突变互补和致病性研究的染色体插入工具箱。Mol 植物-微生物相互作用25(4): 557-568。         
7. Morcillo, RJ, Zhao, A., Tamayo-Navarrete, MI, Garcia-Garrido, JM 和 Macho, AP (2020)。          牛逼随后接种omato根变换青枯小号olanacearum对青枯病直白的遗传分析。J Vis Exp (157)。
8. Morel, A.、Peeters, N.、Vaileau, F.、Barberis, P.、Jiang, G.、Berthomé, R. 和 Guidot, A.(2018 年)。P兰特的致病性表型青枯雷尔氏菌菌株。分子生物学方法 1734:223-239。         
9. Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007a)。开发一系列网关二元载体pGWB,实现植物转化融合基因的高效构建。J Biosci Bioeng 。104(1):34-41。         
10. Nakagawa, T., Suzuki, T., Murata, S., Nakamura, S., Hino, T., Maeo, K., Tabata, R., Kawai, T., Tanaka, K., Niwa, Y ., Watanabe, Y., Nakamura, K., Kimura, T. 和 Ishiguro, S. (2007b)。改进的网关二元载体:用于在植物转基因分析中创建融合构建体的高性能载体。Biosci Biotechnol Biochem 71(8): 2095-2100。     
11. Poueymiro, M.、Cunnac, S.、Barberis, P.、Deslandes, L.、Peeters, N.、Cazale-Noel, AC、Boucher, C. 和 Genin, S. (2009)。两个III型分泌系统效应由青枯雷尔氏菌GMI1000确定烟草宿主范围的特异性。Mol 植物-微生物相互作用。22(5):538-550。     
12. Sang, Y., Yu, W., Zhuang, H., Wei, Y., Derevnina, L., Yu, G., Luo, J. 和 Macho, AP (2020)。我NTRA应变诱导和植物免疫抑制通过青枯雷尔氏菌III型效应器在本塞姆氏烟草。植物社区1:100025。     
13. Wei, Y.、Balaceanu, A.、Rufian, JS、Segonzac, C.、Zhao, A.、Morcillo、RJL 和 Macho, AP(2020 年)。大豆中进化出的免疫受体复合物可以感知多态性细菌鞭毛蛋白。纳特。社区11 (1): 3763。     
14. Xue, H.、Lozano-Duran, R. 和 Macho, AP (2020)。深入了解植物病原菌青枯病菌对根系的入侵。植物(巴塞尔)9(4):516。     
 
 
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引用:Yu, W. and Macho, A. P. (2021). A Fast and Easy Method to Study Ralstonia solanacearum Virulence upon Transient Gene Expression or Gene Silencing in Nicotiana benthamiana Leaves. Bio-protocol 11(15): e4116. DOI: 10.21769/BioProtoc.4116.
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