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Airbrush Infiltration Method for Pseudomonas syringae Infection Assays in Soybean
丁香假单胞菌感染大豆(喷枪渗透法)   

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Abstract

We developed this protocol to assay the extent of proliferation of Pseudomonas syringae pv. glycinea in soybean leaves. This method specifically enables accurate pathogenesis assays of soybean plants at V2/V3 (2nd/3rd trifoliate) or higher stages of growth. The leaves of soybean plants at these growth stages are not amenable to bacterial infiltration using routine needleless syringe infiltration due to the high number of trichomes on these mature leaves. This method enables efficient infiltration of bacteria into the epidermal cells of mature leaves using a pressure pump.

Materials and Reagents

  1. Plant material
    Soybean (Glycine max Merr.) plants of V2 (2nd trifoliate), V3 (3rd trifoliate), or higher stages of growth were used. Plants at VC (two leaf stage)/V1 (1st trifoliate) stages can also be used. Cultivars for various R loci: Rpg1-b (Harosoy), Rpg2 (Merit, Norchief), Rpg3 (Flambeau), Rpg4 (Flambeau), rpg (Essex).

  2. Bacterial strains
    1. P. syringae pv. glycinea expressing avr gene of interest via the broad host range plasmids pDSK519 or pDSK600.
    2. P. syringae pv. glycinea expressing empty pDSK519/600 plasmids as control.

  3. Media and buffers
    1. 10 mM MgCl2 (sterile)
    2. King’s B medium (plates and liquid media) (see Recipes)

  4. Other reagents
    1. Tryptone (Teknova, catalog number: T9012 )
    2. K2HPO4 (Fisher BioReagents, catalog number: BP363 )
    3. Glycerol (Affymetrix, catalog number: 16374 )
    4. Agar (Affymetrix, catalog number: 10654 )
    5. Silwet L-77 (Momentive, New Smyrna Beach)
    6. Antibiotics (Gold Biotechnology)
      1. Rifampicin (50 mg/ml) (R-120)
      2. Kanamycin (50 mg/ml) (K-120)
      3. Spectinomycin (100 mg/ml) (S-140)
      4. Sreptomycin (300 mg/ml) (S150)

Equipment

  1. High-speed floor centrifuge (Thermo Fisher Scientific, Sorvall RC 6 plus)
  2. Spectrophotometer (Thermo Fisher Scientific, model: BioMate 5 )
  3. Pressure pump (Gast Manufacturing, model: DOA-P704-AA )
  4. Airbrush (Badger Air-brush, model: 250-2 )
  5. Test tubes (Thermo Fisher Scientific)
  6. Microcentrifuge tubes (Thermo Fisher Scientific)
  7. Glass rods (Thermo Fisher Scientific)
  8. Pellet pestles (Sigma-Aldrich)
  9. Cork borer (1 cm diameter, Thermo Fisher Scientific)

Procedure

  1. Bacterial growth and inoculum preparation
    1. Streak -80 °C stock of bacterial culture on King’s B agar with required antibiotics (Rifampacin and Kanamycin for pDSK519 plasmid; Rifampacin, Spectinomycin and Streptomycin for pDSK600 plasmid). Incubate the plate at 29 °C till bacterial colonies grow (24-48 h).
    2. Inoculate a single colony of P. syringae in 10 ml King’s B medium with required antibiotics (Rifampacin and Kanamycin for pDSK519 plasmid; Rifampacin, Spectinomycin and Streptomycin for pDSK600 plasmid).
    3. Grow 12-16 h at 29 °C on rotary shaker at 200 rpm until the culture reaches OD (A600) 0.8-1.2. Do not use culture at OD600 <0.8, or >1.2.
    4. Centrifuge culture at 3000 rpm for 10 min. Discard the supernatant and resuspend the bacterial pellet in 10 ml, 10 mM MgCl2   by gentle pipetting (do not vortex).
    5. Measure OD (A600) of the bacterial suspension (this should be the same OD as your culture from King’s B medium, 0.8-1.2) and dilute to 105 CFU (colony forming units)/ml using 10 mM MgCl2 (1.0 OD measurement approximately corresponds to 2 x 108 CFU/ml).
    6. Add Silwett L-77 to a final concentration of 0.01% to the bacterial suspension and mix gently. Use bacterial suspensions within 1 h after preparation.

  2. Soybean infiltration
    1. Attach appropriate ports of airbrush (Figure 1) to the pressure pump (40 psi or less, Figure 2) and beaker containing bacterial suspension using rubber tubing.
    2. Infiltrate soybean plants at V2 stage on the abaxial surface of the trifoliate leaves using the airbrush, while holding the leaf against a flat surface (such as a Petri plate) ensuring that the pressure does not damage the leaf during infiltration (see Video 1).
    3. Inoculate one trifoliate per plant, and at least 5 plants per bacterial strain. 5-8 ml of bacterial inoculum is sufficient to completely infiltrate one trifoliate.
    4. Mock inoculations should be done similarly 10 mM MgCl2 + 0.01% Silwett L-77 instead of the bacterial suspension.


      Figure 1. Image of airbrush used for bacterial infiltration


      Figure 2. Image of pressure pump used for bacterial infiltration

  3. Monitor bacterial proliferation
    1. Collect 3 leaf discs from inoculated leaves at 1 h post infiltration. The 1 h wait prevents erroneous bacterial numbers from any excess inoculum on the leaf surface. By 1 h post infiltration, the infiltrated leaves should no longer appear wet. Collect leaf discs using 1 cm cork borer from each plant. This is your 0 dpi sample.
    2. Homogenize leaf discs in 0.3 ml, 10 mM MgCl2 in a microcentrifuge tube manually using pellet pestle (Figure 3). For reproducibility and accurate bacterial count homogenize until no leaf pieces are visible. (Figure 4). Increase final volume to 1 ml after complete homogenization.
    3. Dilute 10x using 10 mM MgCl2 and plate 100 μl on King’s B medium (one sample per plate) using a glass spreader. Incubate plates at 29 °C till colonies grow (usually 24-48 h). Plate minimum 3 technical replicates per bacterial strain for every genotype. Technical repeats indicate independent leaf extracts from the same set of plants infected with the same bacterial culture.
    4. Count the number of bacterial colonies on entire plate, and adjust for dilution factor to determine total bacterial number. Plot bacterial counts as LOG10 values of CFU/unit leaf disc. It is important to adjust the dilution factor to obtain colonies in a countable range (10-300) depending upon the genotype of the plant. It is recommended to repeat the experiment using lower dilutions for plating if colony numbers are less than 10 or using higher dilutions when colony numbers are more than 300 per plate.
    5. Repeat steps 1-4 at 3, 4, 6 dpi (or other desired time points). Dilute homogenized tissue 5,000-10,000 times dilution for 3 dpi and later samples. Use minimum 4-5 technical replicates for these time points. Use 3-4 biological replicates (indicates independent infections using independent bacterial culture and set of plants) per bacterial strain and per plant genotype.
    6. Hypersensitive reaction related symptoms are visually detectable by 6-7 dpi (Figure 5).


      Figure 3. Image of pellet pestle used for homogenization of leaf material


      Figure 4. Image of homogenate of infected leaf material


      Figure 5. Morphological symptoms associated with the hypersensitive response of P. syringae pv. glycinea infected soybean leaves

Representative data

  1. Video 1. Airbrush infiltration

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  2. The protocol has high reproducibility (>90%) in our hands. We normally use at least four biological replicates per genotype for each experiment, and each infection experiment is conducted with a minimum three technical replicates. The standard error for colony count ranges between LOG10 value of 0.01-0.2.
  3. See Selote and Kachroo (2010); Selote et al. (2013) and (2014); Wang et al. (2014) for typical bacterial counts observed on various cultivars.

Notes

  1. Care should be taken to avoid damaging the leaves during the inoculation process.
  2. It is best to use fully expanded leaves for infection and to inoculate all leaves of one trifoliate per plant.
  3. Spraying the bacterial suspension over the leaf surface is generally sufficient. It is not necessary to saturate leaves with the bacterial suspension as excessive pressure during infiltration can result in wilting of the leaf.

Recipes

  1. King’s B medium (plates and liquid media)
    20 g tryptone
    1.5 g K2HPO4
    10 ml glycerol
    15 g agar, make up volume to 1 L with water
    Autoclave, then add 5 ml of sterile MgSO4 (1 M)

Acknowledgments

This work was supported by funding from the United Soybean Board (project #1291) and the Kentucky Soybean Promotion Board. This protocol is modified from a previous method used by Keen et al. (1990).

References

  1. Fu, D. Q., Ghabrial, S. and Kachroo, A. (2009). GmRAR1 and GmSGT1 are required for basal, R gene-mediated and systemic acquired resistance in soybean. Mol Plant Microbe Interact 22(1): 86-95.
  2. Kachroo, A., Fu, D. Q., Havens, W., Navarre, D., Kachroo, P. and Ghabrial, S. A. (2008). An oleic acid-mediated pathway induces constitutive defense signaling and enhanced resistance to multiple pathogens in soybean. Mol Plant Microbe Interact 21(5): 564-575.
  3. Keen, N. T., Tamaki, S., Kobayashi, D., Gerhold, D., Stayton, M., Shen, H., Gold, S., Lorang, J., Thordal-Christensen, H., Dahlbeck, D. and Staskawicz, B. (1990). Bacteria expressing avirulence gene D produce a specific elicitor of the soybean hypersensitive reaction. Mol Plant Microbe Interact 3(2): 112–121.
  4. Selote, D. and Kachroo, A. (2010). RPG1-B-derived resistance to AvrB-expressing Pseudomonas syringae requires RIN4-like proteins in soybean. Plant Physiol 153(3): 1199-1211.
  5. Selote, D., Robin, G. P. and Kachroo, A. (2013). GmRIN4 protein family members function nonredundantly in soybean race-specific resistance against Pseudomonas syringae. New Phytol 197(4): 1225-1235.
  6. Selote, D., Shine, M. B., Robin, G. P. and Kachroo, A. (2014). Soybean NDR1-like proteins bind pathogen effectors and regulate resistance signaling. New Phytol 202(2): 485-498.
  7. Singh, A. K., Fu, D. Q., El-Habbak, M., Navarre, D., Ghabrial, S. and Kachroo, A. (2011). Silencing genes encoding omega-3 fatty acid desaturase alters seed size and accumulation of Bean pod mottle virus in soybean. Mol Plant Microbe Interact 24(4): 506-515.
  8. Wang, J., Shine, M. B., Gao, Q. M., Navarre, D., Jiang, W., Liu, C., Chen, Q., Hu, G. and Kachroo, A. (2014). Enhanced disease susceptibility1 mediates pathogen resistance and virulence function of a bacterial effector in soybean. Plant Physiol 165(3): 1269-1284.

简介

我们开发了该方案来测定丁香假单胞菌pv的增殖程度。 在大豆叶中。 该方法特异性地能够在V2/V3(2期/3期/3期/3期/3期/3期)或更高生长阶段进行大豆植物的准确发病机理测定。 由于这些成熟叶上的大量毛状体,大豆植物在这些生长阶段的叶子不适于使用常规无针注射器浸润的细菌浸润。 该方法使用压力泵能够有效地将细菌浸润到成熟叶的表皮细胞中。

材料和试剂

  1. 植物材料
    大豆(大豆(Malcine max)Merr。)植物的V2(2倍三叶),V3(3倍三叶)或更高的生长阶段 用过的。 也可以使用VC(两叶期)/V1(1期三叶期)阶段的植物。 用于各种 R 位点的栽培品种:Rpg1-b (Harosoy),Rpg2 (Merit,Norchief), Rpg3 Flambeau),Rpg4 (Flambeau), rpg (Essex)。

  2. 细菌菌株
    1. p。 丁香树 pv。 通过广泛的宿主范围质粒pDSK519或pDSK600表达 avr 基因。
    2. p。 丁香树 pv。 表达空pDSK519/600质粒作为对照。

  3. 媒体和缓冲区
    1. 10mM MgCl 2(无菌)
    2. King's B培养基(培养基和液体培养基)(参见配方)

  4. 其他试剂
    1. 胰蛋白胨(Teknova,目录号:T9012)
    2. (Fisher BioReagents,目录号:BP363)
    3. 甘油(Affymetrix,目录号:16374)
    4. 琼脂(Affymetrix,目录号:10654)
    5. Silwet L-77(迈图,新士麦那海滩)
    6. 抗生素(Gold Biotechnology)
      1. 利福平(50mg/ml)(R-120)
      2. 卡那霉素(50mg/ml)(K-120)
      3. 壮观霉素(100mg/ml)(S-140)
      4. 链霉素(300mg/ml)(S150)

设备

  1. 高速地面离心机(Thermo Fisher Scientific,Sorvall RC 6 plus)
  2. 分光光度计(Thermo Fisher Scientific,型号:BioMate 5)
  3. 压力泵(Gast Manufacturing,型号:DOA-P704-AA)
  4. 喷枪(Badger空气刷,型号:250-2)
  5. 试管(Thermo Fisher Scientific)
  6. 微量离心管(Thermo Fisher Scientific)
  7. 玻璃棒(Thermo Fisher Scientific)
  8. 丸状杵(Sigma-Aldrich)
  9. 软木钻孔器(直径1cm,Thermo Fisher Scientific)

程序

  1. 细菌生长和接种物准备
    1. 条纹-80°C细菌培养物在King's B琼脂上,需要 抗生素(Rifampacin和卡那霉素用于pDSK519质粒; Rifampacin, 用于pDSK600质粒的壮观霉素和链霉素)。 孵育平板 在29℃下直至菌落生长(24-48小时)。
    2. 接种a 单个菌落的丁香假单胞菌在10 ml King's B培养基中培养 抗生素(Rifampacin和卡那霉素用于pDSK519质粒; Rifampacin, 用于pDSK600质粒的壮观霉素和链霉素)。
    3. 增长 在29℃下在200rpm的旋转振荡器上12-16小时,直到培养物达到 OD(A 600)0.8-1.2。 不要在OD 600 <0.8或> 1.2使用培养物。
    4. 以3000rpm离心培养10分钟。 弃去上清液   将细菌沉淀重悬于10ml,10mM MgCl 2, 温柔 移液(不要涡旋)。
    5. 测量细菌的OD(A 600) 悬浮(这应该是与你的文化相同的国王的B 培养基,0.8-1.2)并稀释至10 5 CFU(集落形成单位)/ml,使用 10mM MgCl 2(1.0OD测量大约对应于2×10 8 CFU/ml)。
    6. 加入Silwett L-77至终浓度为0.01% 细菌悬浮液轻轻混匀。 使用细菌悬浮液 在制备后1小时内

  2. 大豆侵入
    1. 将喷枪的适当端口(图1)连接到压力泵(40 psi或更低,图2)和含有细菌悬浮液的烧杯   橡胶管。
    2. 在V2阶段浸润大豆植物 背面的三叶叶使用喷枪,而 将叶保持在平坦表面(例如培养皿)上   在渗透过程中压力不会损坏叶片(见 视频1)。
    3. 每株接种一株三叶草,至少5株 植物。 5-8ml的细菌接种物就足够了 以完全渗透一个三叶
    4. 应该类似地进行模拟接种10mM MgCl 2 + 0.01%Silwett L-77,而不是细菌悬浮液。


      图1.用于细菌浸润的喷枪的图像


      图2.用于细菌浸润的压力泵的图像

  3. 监测细菌增殖
    1. 在浸润后1小时从接种的叶子收集3片叶片。 1小时等待防止错误的细菌数量从任何过量 接种在叶表面。 浸润后1小时,浸润 叶不应再出现湿。 收集叶盘使用1厘米软木 螟虫。 这是您的0 dpi样本。
    2. 均质化 叶盘在0.3ml,10mM MgCl 2中的微量离心管中 使用颗粒杵(图3)。重现性和准确性 细菌计数匀浆直到没有叶片可见。 (图4)。  完全匀浆后,将终体积增至1ml
    3. 使用10mM MgCl 2稀释10倍,并在King's B培养基(一个 样品/板)。孵育板在29°C,直到 菌落生长(通常24-48小时)。板最少3次技术重复 每个细菌菌株的每个基因型。技术重复说明 独立的叶提取物从相同组的植物感染 相同的细菌培养
    4. 计数细菌菌落的数量 在整个板上,并调整稀释因子以确定总量 细菌数。将细菌计数作为CFU /单位叶的LOG <10>值  盘。重要的是调整稀释因子以获得菌落 在可计数范围(10-300),取决于植物的基因型。 建议使用较低稀释倍数重复实验 如果菌落数小于10或使用更高的稀释度,则为电镀 当菌落数大于每板300时。
    5. 重复步骤 1-4(在3,4,6dpi(或其他期望的时间点))。 稀释均匀 组织5,000-10,000倍稀释,用于3dpi和更晚的样品。 使用 这些时间点最少4-5次技术重复。 使用3-4 生物复制(表示使用的独立感染 独立细菌培养物和植物组) 和每株植物基因型
    6. 过敏反应相关症状可通过6-7dpi视觉检测(图5)。


      图3.用于叶材料均质化的颗粒杵的图像


      图4.感染叶材料匀浆的图像


      图5.与 P的过敏反应相关的形态学症状。 丁香 pv。 glycinea 感染的大豆叶

代表数据

  1. 视频1.喷枪浸润
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  2. 该方案在我们手中具有高可重复性(> 90%)。 对于每个实验,我们通常每个基因型使用至少四个生物学重复,并且每个感染实验以至少三个技术重复进行。 菌落计数的标准误差范围在10log-10的值0.01-0.2之间。
  3. 见Selote和Kachroo(2010); Selote (2013)和(2014); Wang等人(2014)对于在各种品种上观察到的典型细菌计数

笔记

  1. 应注意避免在接种过程中损伤叶子。
  2. 最好使用完全展开的叶子进行感染,并接种每株植物一片三叶草的所有叶片
  3. 将细菌悬浮液喷洒在叶表面上通常是足够的。 不需要用细菌悬浮液使叶子饱和,因为渗透过程中的过度压力可能导致叶枯萎。

食谱

  1. King's B培养基(培养基和液体培养基)
    20克胰蛋白酶
    1.5克K sub 2 HPO 4
    10ml甘油 15克琼脂,用水补足体积至1升 高压灭菌,然后加入5ml无菌MgSO 4(1M)

致谢

这项工作得到了联合大豆董事会(项目#1291)和肯塔基大豆促进委员会的资助。 该协议根据Keen等人(1990)使用的先前方法修改。

参考文献

  1. Fu,D.Q.,Ghabrial,S。和Kachroo,A。(2009)。 GmRAR1和GmSGT1是大豆中基础,R基因介导的和系统性获得性抗性所必需的。 a> Mol Plant Microbe Interact 22(1):86-95
  2. Kachroo,A.,Fu,D.Q.,Havens,W.,Navarre,D.,Kachroo,P.and Ghabrial,S.A。(2008)。 油酸介导的途径在大豆中诱导组成型防御信号传导和增强对多种病原体的抗性。 a> Mol Plant Microbe Interact 21(5):564-575。
  3. Keenay,D。,Stayton,M.,Shen,H.,Gold,S.,Lorang,J.,Thordal-Christensen,H.,Dahlbeck,D。和Staskawicz,B。(1990)。 表达无毒基因D的细菌产生大豆过敏反应的特异性激发剂。 Mol Plant Microbe Interact 3(2):112-121。
  4. Selote,D。和Kachroo,A。(2010)。 RPG1-B衍生的对表达AvrB的丁香假单胞菌的抗性需要RIN4 like proteins in soybean。 Plant Physiol 153(3):1199-1211。
  5. Selote,D.,Robin,G.P.和Kachroo,A。(2013)。 GmRIN4蛋白家族成员在针对假单胞菌(Pseudomonas syringae)的大豆种族特异性抗性中非常规地起作用 New Phytol 197(4):1225-1235。
  6. Selote,D.,Shine,M.B.,Robin,G.P.和Kachroo,A。(2014)。 大豆NDR1样蛋白结合病原体效应物并调节抗性信号传导。新的Phytol 202(2):485-498
  7. Singh,A.K.,Fu,D.Q.,El-Habbak,M.,Navarre,D.,Ghabrial,S.and Kachroo,A。(2011)。 编码ω-3脂肪酸去饱和酶的基因沉默 种子大小和豆荚斑驳病毒在大豆中的积累。 Mol Plant Microbe Interact 24(4):506-515。
  8. Wang,J.,Shine,M.B.,Gao,Q.M.,Navarre,D.,Jiang,W.,Liu,C.,Chen,Q.,Hu,G.and Kachroo,A.(2014)。 增强的疾病易感性1介导大豆中细菌效应物的病原体抗性和毒力功能。 em> Plant Physiol 165(3):1269-1284
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引用:Shine, M., Fu, D. and Kachroo, A. (2015). Airbrush Infiltration Method for Pseudomonas syringae Infection Assays in Soybean . Bio-protocol 5(6): e1427. DOI: 10.21769/BioProtoc.1427.
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