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Oct 2020
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Tomato Stem Injection for the Precise Assessment of Ralstonia solanacearum Fitness in Planta
番茄茎杆注射法精确评价青枯菌在植物中的适应性   

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Abstract

Ralstonia solanacearum is a soil-borne pathogen with worldwide distribution that causes bacterial wilt disease in more than 250 plant species. R. solanacearum invades plants through the roots, reaches the vascular system, and colonizes the whole plant by moving through the xylem, where it eventually replicates rapidly, causing plant death. Usual assays to measure the virulence of R. solanacearum under laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a protocol to assess the replication of R. solanacearum following injection into tomato stems. This protocol includes four major steps: 1) growth of tomato plants; 2) R. solanacearum injection into tomato stems; 3) collection of tomato xylem samples and bacterial quantitation; and 4) data analysis and representation. This method bypasses the natural penetration process of the pathogen, thus minimizing variation associated with stochastic events during bacterial invasion, and provides a sensitive and accurate measurement of bacterial fitness inside xylem vessels.

Keywords: Ralstonia solanacearum (青枯病菌), Stem injection (干细胞注射), Tomato (西红柿), Virulence (毒性), Fitness (健身), Bacterial wilt (细菌性枯萎病)

Background

Ralstonia solanacearum is the causal agent of bacterial wilt disease in more than 250 plant species including important crops, such as tomato, potato, pepper, and eggplant, among others, and is considered one of the most dangerous plant pathogens in the world (Mansfield et al., 2012). R. solanacearum is a soil-borne pathogen that enters plants through the roots, using wounds, root tips, and secondary root emergence points as penetration sites; it then progresses via the root cortex, finally reaching the vascular system (Xue et al., 2020). In susceptible plants, R. solanacearum moves through the xylem vessels to colonize the whole plant; eventual replication may lead to populations up to 1010 bacteria per gram of plant tissue in diseased plants, which will block water and nutrient flow in the vascular system and eventually cause plant wilting and death (Peeters et al., 2013). Multiple experimental methods to assess R. solanacearum virulence in plants under laboratory conditions have been described previously (Morel et al., 2018). The most widely used assay relies on soil-drenching of plant roots with R. solanacearum suspensions, followed by observation of the resulting wilting symptoms over time (Morel et al., 2018). Other assays involve bypassing the root penetration process by introducing the bacterial suspensions directly into plant tissues (either leaves or stems) and provide more accurate assessments of bacterial replication (Macho et al., 2010; Morel et al., 2018; Yu and Macho, 2021).


In this protocol, we provide a detailed description of a method aimed at quantitating bacterial fitness in tomato xylem vessels by direct injection into the stem using a microsyringe; given the subsequent fast bacterial replication and plant colonization, we assume that a substantial proportion of this inoculum reaches the xylem vessels. At the desired time after inoculation, samples are taken from 2 cm above the inoculation site by collecting xylem sap from an excised stem section. Bacterial quantitation in this xylem sap provides a clean measurement of bacterial fitness in the plant vascular system. Like other Gram-negative bacterial pathogens, R. solanacearum requires a type III secretion system to inject type III effector (T3E) proteins into host cells and cause disease. Since T3E activities often contribute redundantly to the development of disease, it is generally difficult to determine their involvement in virulence; however, we have recently used this method to detect the virulence attenuation of different R. solanacearum knockout mutants lacking individual effectors (Xian et al., 2020; Yu et al., 2020) and to analyze bacterial pathogenicity following chemical treatment of plant tissues (Wang et al., 2021), revealing a simple, versatile, and powerful assay for the study of R. solanacearum-plant interactions.

Materials and Reagents

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

  2. Pipette tips

  3. Plastic Petri dishes (90 mm diameter)

  4. Jiffy pots (41 mm diameter) (Jiffy International, Kristiansand, Norway)

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

  6. Syringe filter (0.45 μm) (Millex, Millex®-HV, catalog number: SLHV033RB)

  7. Tomato seeds (Solanum lycopersicum cv. Moneymaker)

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

  9. Vermiculite (generic)

  10. Distilled sterile water

  11. 75% ethanol (Sinopharm Chemical Reagent Co, SCR®, catalog number: 801769610)

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

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

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

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

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

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

  18. Phi medium (see Recipes)

  19. 1% (W/V) TTC solution (see Recipes)

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

Equipment

  1. Micro sample syringe 10 μl (Sangon Biotech, catalog number: E718LD0023)

  2. Sterile scalpel

  3. EasySpiral® Automatic Serial Diluter and Plater (optional) (Interscience, Easysprial®, catalog number: 412000)

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

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

  6. Centrifuge (Eppendorf, model: Centrifuge 5424)

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

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

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

  10. NanoDrop spectrophotometer (Thermo Scientific, NanoDrop 2000c)

  11. Water distiller/sterilizer (Millipore, model: Milli-Q integral 10L)

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

Software

  1. GraphPad Prism 7 (https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Growing tomato plants

    1. Germinate tomato seeds (Solanum lycopersicum cv. Moneymaker) in a 1:2 mixture of standard potting soil and vermiculite. Grow seedlings for one week in a growth chamber (25°C, 16 h light/8 h dark photoperiod, 130 mE m-2 s-1, 65% humidity) (Figure 1A).

    2. Soak Jiffy pots thoroughly with tap water without fertilizer (an excess of fertilizer may cause stress to the tomato seedlings) (see Note 1). Transfer tomato seedlings to individual water-soaked jiffy pots containing a pre-packed soil mixture (Figure 1B) (see Note 1). Grow tomato plants in the same growth chamber for another three weeks (Figure 1C) (see Note 2). Approximately 6-8 plants per treatment are recommended to obtain robust results.



      Figure 1. Growth of tomato seedlings in Jiffy pots. A. Germination of tomato seeds (Solanum lycopersicum cv. Moneymaker) in a 1:2 mixture of standard potting soil and vermiculite. B. Transferring tomato seedlings to individual water-soaked jiffy pots containing a pre-packed soil mixture. C. Four-week-old tomato plants ready for Ralstonia inoculation.


  2. Preparation of R. solanacearum inoculum

    1. Prepare solid and liquid phi medium (see Recipes) following standard procedures (Yu and Macho, 2021).

    2. Streak out R. solanacearum strains on solid phi medium plates containing TTC and glucose (see Recipes; Yu and Macho, 2021) and prepare a bacterial suspension as previously described (Yu and Macho, 2021).

    3. Dilute the final bacterial suspension in distilled water to an OD600 = 0.1, which corresponds to approximately 108 CFU/ml (Morel et al., 2018). Perform serial dilutions of the bacterial suspension using distilled water to reach 106 CFU/ml in a volume of 1 ml in a sterile 2-ml microcentrifuge tube (see Note 3) (Figure 2). Consider the possibility of testing the virulence of different doses of your specific strains under your specific experimental conditions. Under our conditions, we selected 106 CFU/ml as a suitable concentration to allow gradual but reproducible bacterial replication inside tomato stems during the first 3 days post-inoculation (dpi) without causing the development of necrotic symptoms during the experiment (Figure 3).



      Figure 2. Preparation of R. solanacearum inoculum


  3. Inoculation (stem injection) and sampling of xylem sap

    1. Using a 10-μl microsyringe, inject 5 μl bacterial suspension into the stems of 4-week-old tomato plants (see Note 4). The inoculation site should be approximately 2 cm below the cotyledon emergence site (Figure 3A and 3B). When performing stem injection, use an angle of approximately 30° between the syringe and the stem to facilitate injection of the bacterial suspension.

    2. Place the inoculated tomato plants in a growth chamber (28°C, 12 h light/12 h dark, 130 mE m-2 s-1, 75% humidity) for 36-72 h until subsequent sampling at the appropriate time points.

    3. Transect the stem with a sterile scalpel just below the cotyledon emergence site (Figure 3C).

    4. Using two fingers, gently squeeze the lower part of the tomato stem to allow the xylem sap to emerge and then collect 2.5 μl xylem sap using a 2.5-μl pipette (see Note 5) (Figure 3D).



      Figure 3. Stem injection and sampling of xylem sap. A. Four-week-old tomato plants in Jiffy pots. B. Using a 10-μl microsyringe, inject 5 μl bacterial suspension into the stems of 4-week-old tomato plants. C. Transect the tomato stems with a sterile scalpel just below the cotyledon emergence site. D. Collect 2.5 μl xylem sap at the cutting site using a micropipette.


  4. Bacterial quantitation

    1. Dissolve the xylem sap in 1 ml sterile water in a sterile 1.5-ml microcentrifuge tube (Figure 4A).

    2. Dilute and spread bacteria (50 μl) on phi medium plates using standard procedures, as previously described (Yu and Macho, 2021). Spreading can be performed manually using a sterile spreader, plastic beads, or the bottom of a 1.5-ml microcentrifuge tube, or using a spiral plater (Yu and Macho, 2021).

    3. Incubate the plates upright at 28°C for 2 days. Count the colonies and process the data (Figure 4B) (see Note 6).



      Figure 4. Preparation of serial dilutions and bacterial spreading. A. Dissolve the xylem sap containing Ralstonia in 1 ml sterile water in a sterile 1.5-ml microcentrifuge tube; dilute the suspensions by 10 to 104 times. B. Spread the diluted bacterial suspensions (we tried to spread 10-1 or 10-3) on phi medium plates using standard procedures, as previously described (Yu and Macho, 2021). Incubate the plates upright at 28°C for 2 days and count the colonies on solid phi medium plates. Photos in B show the usual colony pattern after plating using a spiral plater (Yu and Macho, 2021).

Data analysis

  1. Using the number of colonies on each readable plate, calculate bacterial numbers in the sampled xylem sap. If plating was performed manually, apply the following formula:

    R. solanacearum numbers in xylem sap (CFU/ml) = colony number × 20 × 103 × 10(dilution factor)/2.5

    Note: “Colony number”: number of colonies on the readable plate. “20” corresponds to the result of dividing 1000 μl final diluted bacteria between 50 μl bacterial solution spread on the plate. “103” corresponds to the conversion of CFU/μl to CFU/ml. “2.5” corresponds to the 2.5 μl xylem sap taken as a sample and dissolved in 1 ml water. “10(dilution factor)” corresponds to the additional serial dilution of the xylem sap.

  2. If plating was performed using a spiral plater, follow additional calculations as previously described (Yu and Macho, 2021).

  3. Repeat the experiment at least 3 times. Input the final data to GraphPad Prism 7 or a similar program for statistical analysis. To compare results individually, perform a Student’s t-test and report the mean value, SEM, and P value to indicate the statistical significance of the differences. Results are better displayed using a logarithmic scale and the individual data points together with the mean ± SEM (Figure 5).



    Figure 5. Representation of the growth of R. solanacearum GMI1000 wild-type, mutant, and complemented strain in tomato stems. Different colors represent different strains, and horizontal bars represent mean values (n = 6 plants per strain). Data are represented as the mean ± SEM. Asterisks indicate significant differences (***P < 0.0001, t-test). The graph was plotted using GraphPad Prism 7.

Notes

  1. If the tap water has a high mineral content or contains excessive impurities, purified water can also be used.

  2. Grow no more than 24 tomato plants in each tray (29 × 20 cm), and separate the plants to avoid intertwined roots among individual plants. Water the plants normally once every two days, and keep the jiffy pots wet.

  3. For serial dilution, use a 1:10 dilution for each step.

  4. Optionally, move the tomato plants to a growth chamber (28°C, 12 h light/12 h dark, 130 mE m-2 s-1, 75% humidity) 12 h before R. solanacearum injection to minimize interference caused by changing the growth chamber after inoculation. This does not make a difference under our conditions.

  5. For tomato sampling, remove the oozing sap gently with sterile paper, and clamp the stem top with a finger to push the xylem sap out for sampling.

  6. Waste disposal: Autoclave all the material used in this experiment before disposal to avoid releasing R. solanacearum into the environment. Clean the work bench and flow hood with 75% alcohol after the experiment.

Recipes

  1. Phi medium

    10 g Bacto peptone

    1 g Yeast extract

    1 g Casamino acids

    Add water to reach 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

    Note: Store 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 at 4°C.

    Note: Add 1% TTC and 20% glucose when preparing solid Phi medium plates to induce pink R. solanacearum colonies.

Acknowledgments

We thank Xinyu Jian for technical and administrative assistance during this work, in addition to 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.

References

  1. Macho, A. P., Guidot, A., Barberis, P., Beuzon, C. R. and Genin, S. (2010). A competitive index assay identifies several Ralstonia solanacearum type III effector mutant strains with reduced fitness in host plants. Mol Plant Microbe Interact 23(9): 1197-1205.
  2. Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, S. V., Machado, M. A., Toth, I., Salmond, G. and Foster, G. D. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13(6): 614-629.
  3. Morel, A., Peeters, N., Vailleau, F., Barberis, P., Jiang, G., Berthome, R. and Guidot, A. (2018). Plant Pathogenicity Phenotyping of Ralstonia solanacearum Strains. Methods Mol Biol 1734: 223-239.
  4. Peeters, N., Guidot, A., Vailleau, F. and Valls, M. (2013). Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era. Mol Plant Pathol 14(7): 651-662.
  5. Wang, Y., Zhao, A., Morcillo, R.J.L., Yu, G., Xue, H., Rufian, J.S., Sang, Y., and Macho, A.P. (2021) A bacterial effector uncovers a novel pathway involved in tolerance to bacterial wilt disease. Molecular Plant 30:S1674-2052(21)00159-3.
  6. Xian, L., Yu, G., Wei, Y., Rufian, J. S., Li, Y., Zhuang, H., Xue, H., Morcillo, R. J. L. and Macho, A. P. (2020). A Bacterial Effector Protein Hijacks Plant Metabolism to Support Pathogen Nutrition. Cell Host Microbe 28(4): 548-557 e547.
  7. Xue, H., Lozano-Durán, R., and Macho, A.P. (2020). Insights into the root invasion by the plant pathogenic bacterium Ralstonia solanacearum. Plants (Basel) 9(4): 516.
  8. Yu, G., Xian, L., Xue, H., Yu, W., Rufian, J. S., Sang, Y., Morcillo, R. J. L., Wang, Y. and Macho, A. P. (2020). A bacterial effector protein prevents MAPK-mediated phosphorylation of SGT1 to suppress plant immunity. PLoS Pathog 16(9): e1008933.
  9. 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.

简介

[摘要]青枯病菌是一种全球分布的土传病原菌,可引起250多种植物青枯病。青枯菌侵入植物通过根部,达到血管系统,并定居于全厂通过通过木质部,在移动它最终迅速复制,造成植株死亡。通常的试验来测量的毒力青枯雷尔氏菌在实验室条件下依靠土壤淋接种之后的疾病症状的观察和评分。在这里,我们描述了一个协议来评估R. solanacearum在注入番茄茎后的复制。该协议包括四个主要步骤:1)番茄植株的生长;2)青枯菌注射到番茄茎中;3)的番茄木质部样品和收集细菌孔定量吨通货膨胀; 和4)数据分析和表示。这种方法绕过了病原体的自然渗透过程,从而最大限度地减少了细菌入侵过程中与随机事件相关的变化,并提供了对木质部血管内细菌适应性的灵敏而准确的测量。

[背景]青枯病菌是250多种植物青枯病的病原体,包括番茄、马铃薯、辣椒、茄子等重要作物,被认为是世界上最危险的植物病原体之一。曼斯菲尔德等人,2012 年)。青枯雷尔氏菌是一种土壤传播的病原体即通过根进入植物,使用伤口,根尖,和次生根EMERG EnCE的作为渗透部位点; 然后通过根皮层前进,最终到达血管系统(Xue et al ., 2020)。在易感植物中, R. solanacearum 通过木质部血管移动到整株植物中;最终的复制可能导致患病植物中每克植物组织中多达 10 10个细菌的数量,这将阻止维管系统中的水分和养分流动,最终导致植物枯萎和死亡(Peeters等,2013)。之前已经描述了在实验室条件下评估青枯菌在植物中毒力的多种实验方法(Morel等,2018)。Ť他最广泛使用的测定依赖于土壤浸润植物的根与青枯菌悬浮液,随后所得到的萎蔫症状观察随时间(莫瑞尔等人。,2018)。其它测定法涉及通过直接引入细菌悬液我绕过根渗透过程n要的植物组织(或者叶子或茎),并提供细菌复制的更准确的评估(马霍等人。,2010;莫瑞尔等人。,2018 ; Yu和猛男,2021 年)。

在这个协议中,我们提供了一个旨在孔定量的方法的详细描述吨在番茄木质部导管荷兰国际集团细菌健身通过直接注入离子在以使用微量杆; 鉴于随后的快速细菌复制和植物定植,我们假设该接种物的很大一部分到达木质部血管。在接种后的期望的时间,取出样品从2厘米接种部位上面通过从切除茎段收集木质部汁液。细菌孔定量吨通货膨胀在该木质部汁液提供在植物维管系统细菌健身的清洁测量。像其他ģ冲压阴性细菌病原体,青枯雷尔氏菌需要一个类型III分泌系统注入III型效应(T3E)中的蛋白质,以宿主细胞中并引起疾病。由于T3E活动往往对疾病的发展作出贡献冗余,所以一般很难确定其参与的毒力; 然而,我们最近已经用这种方法来检测不同的毒力衰减青枯缺乏个体效应敲除突变体(冼等人,2020;于等人,2020) ,并分析细菌致病以下植物组织的化学处理( Wang等人,202 1 ),揭示了一种用于研究青枯菌与植物相互作用的简单、通用和强大的分析方法。

关键字:青枯病菌, 干细胞注射, 西红柿, 毒性, 健身, 细菌性枯萎病

材料和试剂
 
1.微量离心管(1.5 ml和2 ml )(BBI,目录号:F600620-0001)      
2.移液器吸头      
3.塑料P ETRI培养皿(直径90mm)      
4. Jiffy pots(直径41 mm)(Jiffy International,克里斯蒂安桑,挪威)      
5.分光光度计塑料比色皿(BRAND,目录号:759015)      
6.针筒式滤器(0.45微米)(的Millex,的Millex ® -HV,目录号:SLHV033RB)      
7.番茄种子(Solanum lycopersicum cv . Moneymaker )      
8.标准盆栽土(Pindstrup,目录号:1034593214)      
9.蛭石(通用)      
10.蒸馏无菌水   
11. 75%的乙醇(国药化学试剂有限公司,SCR ® ,目录号:801769610 )   
12.氯化三苯基四唑鎓(TTC)(Sigma-Aldrich,目录号:T8877-5G)   
13.葡萄糖(国药化学试剂有限公司,SCR ® ,目录号:63005518 )   
14. Bacto蛋白胨(BD ,目录号:211677)   
15.酵母提取物(OXOID,目录号:LP0021)   
16.酪蛋白ħ ydrolysate(酪蛋白氨基酸)(Sigma-Aldrich公司,目录号:22090-500G)   
17.琼脂(国药化学试剂有限公司,SCR ® ,目录号:10000561 )   
18. Phi 培养基(见食谱)   
19. 1% (W/V) TTC 溶液(见配方)   
20. 20% (W/V) 葡萄糖溶液(见配方)   
 
设备
 
微量样品注射器10μl (Sangon Biotech,目录号:E718LD0023)
无菌手术刀
EasySpiral ®自动连续稀释器和平板(可选)(Interscience,E asysprial ® ,目录号:412000 )
植物生长室(Percival,型号:I-36VL)
高压釜(SANYO,型号:MLS-3780)
离心机(Eppendorf,型号:C entrifuge 5424)
pH计(Sartorius,型号:PB-10)
28 °C培养皿培养箱(松下,型号:MIR-262-PC)
28 °C管式培养箱(振荡器)(Eppendorf,New Brunswick TM ,目录号:m1324-0006)
纳米d ROP分光光度计(Thermo Scientific的,纳米滴2000C)
蒸馏水/消毒器(Millipore公司,型号:祢升升I-Q可积10L)
Vortex(科学工业,型号:Vortex-Genie 2,目录号:S1-0246)
 
软件
 
GraphPad Prism 7 ( https://www.graphpad.com/scientific-software/prism/ )
 
程序
 
种植番茄植物
在标准盆栽土壤和蛭石的 1:2 混合物中发芽番茄种子(Solanum lycopersicum cv. Moneymaker)。在生长室(25°C,16 小时光照/8 小时黑暗光周期,130 mE m -2 s -1 ,65% 湿度)中生长一周的幼苗(图1A)。
用自来水彻底浸泡的Jiffy盆无肥料(过量的肥料的可能引起应力的番茄幼苗)(小号EE注1)。将番茄幼苗转移到含有预包装土壤混合物(图 1B)(见注 1)的单个浸水瞬间盆中。在同一个生长室中再种植番茄植物三周(图 1C)(见注 2)。建议每次处理大约 6-8 株植物以获得稳健的结果。
 
 
 
 
图 1. Jiffy 盆中番茄幼苗的生长。A.番茄种子(Solanum lycopersicum cv. Moneymaker)在标准盆栽土和蛭石的 1:2 混合物中发芽。B.转移番茄幼苗到个人水渍瞬间盆含有预包装的土壤混合物。C. 4 周龄的番茄植株准备接种Ralstonia 。
 
青枯菌接种物的制备
制备固体和液体披培养基(参见ř ecipes)按照标准程序(Yu和马霍,2021) 。
条纹出青枯雷尔氏菌在含有TTC和葡萄糖固体披培养基平板株(见ř ecipes;于和马霍,2021 ),并如先前所述(制备细菌悬浮液于和马霍,2021 )。
稀释在蒸馏水中的最终细菌悬浮液至OD 600 = 0.1,其对应于约10 8 CFU / ml的(莫瑞尔等人,2018)。执行使用蒸馏水细菌悬浮液的系列稀释液以达到10 6 CFU / ml的在在无菌2的1ml体积- ml离心管(见注3)(图2) 。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 6 CFU/ml 作为合适的浓度,以允许在接种后的前 3 天 (dpi) 内番茄茎内逐渐但可重复的细菌复制,而不会在实验过程中引起坏死症状的发展(图 3) .
 
 
图 2.青枯菌接种物的制备
 
接种(茎注射)和木质部汁液取样
使用10 -微升微量,注入5μl的细菌悬浮液到茎的4周龄的番茄植株小号(见注4)。接种部位应该是低于约2厘米子叶EMERG ENCE站点(图3A和3B)。进行茎注射时,注射器和茎之间使用大约30°的角度,以促进细菌悬浮液的注射。
代替接种番茄植株在生长室中(28℃,12小时光照/ 12小时黑暗,130 ME米-2小号-1 ,75%湿度)36 - 72小时,直到随后在适当的时间点取样。
横切杆用无菌解剖刀只是子叶EMERG下面ENCE位点(图3C)。
使用两个手指,轻轻挤压的下部番茄茎以允许木质部汁液出现,然后收集2.5微升木质部汁液用一个2.5 -微升移液管(见注5)(图3D) 。
 
 
图 3. 木质部汁液的茎注射和取样。A. 4周龄的番茄植株的Jiffy锅小号。B.使用10 -微升微量,注入5μl的细菌悬浮液入的4周龄的番茄植物的茎小号。C.断面的番茄干小号用无菌手术刀就在子叶下方EMERG ENCE网站。D.使用微量移液器在切割部位收集 2.5 μl 木质部汁液。
 
细菌孔定量Ť通货膨胀
将木质部汁液溶解在无菌 1.5 - ml 微量离心管中的 1 ml 无菌水中(图 4A)。
稀和传播细菌(50微升)ø N使用标准方法披培养基平板,先前所描述的(Yu和马霍,2021)。扩展可以使用无菌涂布器,手动执行塑料珠,或1.5的底部- ml离心管,或使用旋转接种仪(于和马霍,2021)。
孵育所述板直立在28°C 2 天。计数的菌落并处理的数据(图4B)(见注6)。
 
 
图4.连续稀释和细菌传播的制备ING 。A.溶解含有木质部汁液青枯在1ml无菌水中在无菌1.5 - ml离心管; 将悬浮液稀释 10 至 10 4倍。B.扩散的稀释细菌升悬浮液(我们试图传播10 -1或10 -3 )ö使用标准程序Ñ披培养基平板,如先前所描述(于和马霍,2021)。孵育的在直立板28 ℃,2天,并计数的菌落直径:Ñ固体披培养基平板。B 中的照片显示了使用螺旋平板进行平板接种后的常见菌落模式(Yu 和 Macho,2021 年)。
 
数据分析
 
使用菌落数ø n各自可读板,计算在采样木质部汁液细菌数。如果电镀是手动进行的,请应用以下公式:
青枯菌在木质部汁液中的数量(CFU/ml)=菌落数×20×10 3 ×10 (稀释因子)/2.5
注:“菌落数”:可读板上的菌落数。“20”对应于将1000 μl 最终稀释的细菌在 50 μl 细菌溶液涂在板上的结果。“10 3 ”对应于CFU/ μl到CFU/ml的转换。“2.5”对应于2.5微升木质部汁液作为一个样品,并溶于1ml水中。“10 (稀释因子)”对应于木质部汁液的额外系列稀释。
如果使用螺旋电镀机进行电镀,请按照前面描述的额外计算(Yu 和 Macho,2021 年)。
重复实验至少 3 次。将最终数据输入到 Graph P ad Prism 7 或类似程序进行统计分析。要单独比较结果,执行一个学生的牛逼-测试和报告的平均值,SEM和p值来表示的差异有统计学意义。使用对数刻度和单个数据点以及平均值± SE M可以更好地显示结果(图 5)。
 
 
图5. ř的epresentation的生长青枯雷尔氏菌GMI1000野生型,突变体在番茄茎,和补偿菌株小号。不同的颜色代表不同的菌株,水平条代表平均值(每个菌株n = 6 株植物)。数据被表示为在平均值± SE中号。星号表示显着差异(*** P < 0.0001,t检验)。ģ拍摄和使用格拉夫绘制P广告棱镜7。
 
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
加水达到1大号。到prepar ë固体培养基,琼脂添加15克至1种大号介质和高压釜
1%(W / V)TTC搜索解决方案Ñ
将TTC溶解在蒸馏水中
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。STO再在4℃下。
注:加1%TTC和20%克lucose当prepar ING固体披介质板小号诱导粉红青枯结肠IES 。
笔记
 
如果该自来水具有一个高矿物质含量或包含过多的杂质,也可以使用纯净水。
生长不超过24番茄植物中的每个托盘(29 × 20cm)中,并分离的植物,以避免单个植物中缠结根。通常每两天给植物浇一次水,并保持花盆湿润。
对于连续稀释,使用一个1 :10稀释度的每一个步骤。
可选地,移动的番茄植株的生长室(28℃,12小时光照/ 12小时黑暗,130 ME米-2小号-1 ,75%湿度)12小时前青枯菌注射以减少所造成的改变干扰接种后的生长室。在我们的条件下,这并没有什么不同。
番茄取样时,用无菌纸轻轻取出渗出的汁液,用手指夹住茎顶部,将木质部汁液推出取样。
垃圾处理:一utoclave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。实验后用 75% 酒精清洁工作台和流罩。
 
致谢
 
除了Macho 实验室的所有成员不断改进本协议和有益的讨论外,我们还感谢 Xinyu Jian 在这项工作中提供的技术和行政协助。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。
 
参考
 
Macho, AP, Guidot, A., Barberis, P., Beuzon, CR 和 Genin, S. (2010)。竞争指数测定确定了几种在宿主植物中适应性降低的青枯病菌 III 型效应突变株。Mol 植物微生物相互作用23(9): 1197-1205。
Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariariyanum, 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。
Morel, A., Peeters, N., Vailleau, F., Barberis, P., Jiang, G., Berthome, R. 和 Guidot, A. (2018)。青枯病菌菌株的植物致病性表型分析。分子生物学方法1734:223-239。
Peeters, N.、Guidot, A.、Vaileau, F. 和 Valls, M.(2013 年)。Ralstonia solanacearum ,后基因组时代广泛存在的细菌性植物病原体。Mol Plant Pathol 14(7): 651-662。
Wang , Y., Zhao , A., Morcillo , RJL, Yu , G., Xue , H., Rufian, JS, Sang , Y., and Macho , AP (2021)一种细菌效应揭示了一种与耐受有关的新途径为青枯病。分子植物30:S1674-2052(21)00159-3。
Xian, L., Yu, G., Wei, Y., Rufian, JS, Li, Y., Zhuang, H., Xue, H., Morcillo, RJL 和 Macho, AP (2020)。细菌效应蛋白劫持植物代谢以支持病原体营养。细胞宿主微生物28(4):548-557 e547。
Xue, H.、Lozano-Durán, R. 和 Macho, AP (2020)。洞察由植物病原细菌的入侵根青枯雷尔氏菌。植物(巴塞尔) 9 (4): 516。
Yu, G., Xian, L., Xue, H., Yu, W., Rufian, JS, Sang, Y., Morcillo, RJL, Wang, Y. 和 Macho, AP (2020)。细菌效应蛋白阻止 MAPK 介导的 SGT1 磷酸化以抑制植物免疫。PLoS 病原体16(9):e1008933。
Yu, W. 和 Macho, AP (202 1 )。一种在本氏烟草叶片中研究青枯病菌对瞬时基因表达或基因沉默的毒力的快速简便方法。生物方案11 ( 15 ) :e4116。
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引用:Wang, Y., Xian, L., Yu, G. and Macho, A. P. (2021). Tomato Stem Injection for the Precise Assessment of Ralstonia solanacearum Fitness in Planta. Bio-protocol 11(16): e4134. DOI: 10.21769/BioProtoc.4134.
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