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Peer-reviewed

In planta Transcriptome Analysis of Pseudomonas syringae

丁香假单胞菌的植物转录组分析

TN Tatsuya Nobori
KT Kenichi Tsuda

发布: 2018年09月05日第8卷第17期 DOI: 10.21769/BioProtoc.2987 浏览次数: 8357

评审: Zhibing LaiChao-Jan LiaoReza Sohrabi

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Abstract

Profiling bacterial transcriptome in planta is challenging due to the low abundance of bacterial RNA in infected plant tissues. Here, we describe a protocol to profile transcriptome of a foliar bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, in the leaves of Arabidopsis thaliana at an early stage of infection using RNA sequencing (RNA-Seq). Bacterial cells are first physically isolated from infected leaves, followed by RNA extraction, plant rRNA depletion, cDNA library synthesis, and RNA-Seq. This protocol is likely applicable not only to the A. thaliana–P. syringae pathosystem but also to different plant-bacterial combinations.

Keywords: RNA-Seq (转录组测序技术) search Bacterial pathogen (细菌性病原体) search Arabidopsis (拟南芥) search Transcriptome (转录组) search Pseudomonas (假单胞菌) search

Background

Plants have evolved innate immune systems to fend off pathogen attack. Molecular mechanisms of pathogen recognition and immune signaling pathways have been intensively studied over the past decades. However, how plant immunity affects pathogen metabolism to inhibit pathogen growth is scarcely understood often because of the difficulty in profiling pathogen responses in planta. In case of bacterial pathogens, transcriptome profiling inside plant leaves is difficult to study because the amount of bacterial mRNA is much lower than that of plants; and it is particularly challenging at an early stage of infection due to the low population density of bacteria in plants. To overcome this limitation, we established a method for isolating bacteria from the infected plant leaves and profiling bacterial transcriptome with RNA-Seq. This method has been successfully used for profiling the transcriptome of the model bacterial pathogen Pseudomonas syringae pv. tomato DC3000 in the model plant Arabidopsis thaliana in various conditions (Nobori et al., 2018).

Materials and Reagents

  1. 50 ml Falcon tubes (Corning, Falcon®, catalog number: 352070 )
  2. 1.5 ml Eppendorf Safe-Lock tubes (Eppendorf, catalog number: 0030120086 )
  3. Pipette tips, 1,000 μl and 10 μl volumes (Corning, DeckWorksTM, catalog numbers: 4124 and 4120 )
  4. Sterile Serological Pipet, 25 ml (Corning, catalog number: 4251 )
  5. 1 ml needleless syringe (BD, Luer-LokTM, catalog number: 303172 )
  6. 4 mm stainless steel balls (Mühlmeier)
  7. 6 μm filter mesh (Bückmann, catalog number: 20000796 )
  8. 30-32 days old Arabidopsis thaliana plants
  9. Pseudomonas syringae pv. tomato DC3000 (Pto)
  10. 100% ethanol (VWR International, catalog number: 20821.321 )
  11. Chloroform (Carl Roth, catalog number: 4432 )
  12. peqGOLD TriFast (VWR, PeqLab, catalog number: 30-2010 )
  13. Phenol solution pH 8.0 (Sigma-Aldrich, catalog number: P4557 )
  14. Nuclease-Free Water (not DEPC-Treated) (Thermo Fisher Scientific, InvitrogenTM, Ambion®, catalog number: AM9932 )
  15. Tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich, catalog number: C4706 )
  16. RNAqueous®-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, InvitrogenTM, Ambion®, catalog number: AM1931 )
  17. TURBO DNA-free Kit (Thermo Fisher Scientific, InvitrogenTM, Ambion®, catalog number: AM1907 )
  18. Ribo-Zero rRNA Removal Kit (Plant) (Illumina, catalog number: MRZPL1224 )
  19. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
  20. Ovation® Complete Prokaryotic RNA-Seq DR Multiplex Systems (NuGEN Technologies, catalog number: 0326-32 )
  21. QIAGEN-tip 500 (QIAGEN, catalog number: 10063 )
  22. Liquid nitrogen
  23. NaOH (Carl Roth, catalog number: 6771 )
  24. Bacterial isolation buffer (see Recipes)

Equipment

  1. Eppendorf Reference Pipette (Eppendorf, 100-1,000 μl)
  2. Gilson PIPETMAN Classic pipette (Gilson, model: P20, catalog number: F123600 )
  3. Electric pipette (BrandTech Scientific, model: accu-jet® pro, catalog number: 26330 )
  4. Refrigerated centrifuge (Eppendorf, models: 5417 R and 5810 R )
  5. Roller mixer SRT1 (Stuart Scientific, model: SRT1 )
  6. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
  7. Shaker
  8. Thermal cycler (DNA Engine Tetrad 2 Peltier Thermal Cycler, Bio-Rad, model: Tetrad 2 )
  9. NanoDropTM One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM OneC , catalog number: ND-ONE-W)
  10. -80 °C freezer
  11. Autoclave
  12. Fume hood
  13. Forceps (Fine Science Tools, model: Dumont #5, catalog number: 11251-10 )
  14. Plastic cutter
  15. Scissors
  16. Illumina HiSeq-3000 sequencer

Software

  1. BowTie2
  2. Python package HTSeq

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Nobori, T. and Tsuda, K. (2018). In planta Transcriptome Analysis of Pseudomonas syringae. Bio-protocol 8(17): e2987. DOI: 10.21769/BioProtoc.2987.

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分类

植物科学 > 植物分子生物学 > RNA

微生物学 > 微生物-宿主相互作用 > 细菌

分子生物学 > RNA > RNA 测序

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