In situ Hybridization (ISH) in Preparasitic and Parasitic Stages of the Plant-parasitic Nematode Meloidogyne spp.
植物寄生线虫属线虫寄生前及寄生阶段的原位杂交（ISH）
发布: 2018年03月20日第8卷第6期 DOI: 10.21769/BioProtoc.2766 浏览次数: 9753
评审: Eugenio LlorensAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Oct 2017
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Abstract
The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.
Keywords: Gene expression pattern (基因表达模式)
Plant pathogen (植物病原体)
Preparasitic and parasitic stages (寄生前和寄生阶段)
mRNAs (mRNAs)
Background
So far, the stable transformation of plant-parasitic nematode(s) has not been successful. ISH enables the analysis of spatio-temporal gene expression in vivo in whole-mount Meloidogyne spp. nematodes. These root-knot nematodes hatch in the soil as microscopic vermiform juveniles (J2) and infect host plant roots. J2s penetrate the root and migrate to the root vascular cylinder cells. The juveniles settle in the root and develop into J3 and J4 parasitic juveniles that induce the differentiation specialized feeding cells. The nematode eventually develops into a pear-shaped female that will release hundreds of eggs on the root surface. Here, we report a detailed protocol to detect single RNA molecules in preparasitic whole mount J2s and parasitic stages. ISH on parasitic stages requires an additional procedure the day before extraction of the nematodes from infected roots. We describe the detection of transcripts using digoxigenin (DIG)-labeled cDNA probes in nematode whole mount tissues.
Materials and Reagents
- Nitrile gloves
- RNase-free microcentrifuge tubes (1.5 ml) (e.g., Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: AM12450 )
- Sieve of 2.5 mm/250 µm/40 µm/10 µm
- 50 ml tubes (Corning, Falcon®, catalog number: 352070 )
- Paper towel
- Microscope slides and cover slip (e.g., Fisher Scientific, catalog number: 12-544-1 )
- Autoclaved razor blades
- RNase-free filtered tips, e.g.,
20 µl tips (Mettler-Toledo, Rainin, catalog number: 17007957 )
200 µl tips (Mettler-Toledo, Rainin, catalog number: 17002927 )
1,000 µl tips (Mettler-Toledo, Rainin, catalog number: 17014361 ) - Root-knot nematodes (e.g., M. incognita Morelos strain, M. enterolobii Godet strain) preparasitic J2s (10,000) or parasitic stages (50)
- DIG-labelled ISH probe(s)
Note: See Procedure A for probe synthesis and storage. - Forward and reverse primers designed to allow an amplicon size around 200 bp (e.g., SePOP Desalted oligos from Eurogentec)
- PCR-grade dNTPs (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297117 )
- Pfu DNA Polymerase (Promega, catalog number: M7741 )
- QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )
- QIAquick Gel extraction kit (QIAGEN, catalog number: 28704 )
- Digoxigenin (DIG) DNA Labeling Kit (Roche Diagnostics, catalog number: 11175025910 )
- TE buffer pH 8 (10 mM Tris-HCl, 1 mM EDTA) (e.g., Sigma-Aldrich, catalog number: 93283 )
- RNaseZap® RNase Decontamination Solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
- 70% ethanol
- Tap water
- Acetone (e.g., VWR, catalog number: 20065.293 )
- Methanol (e.g., VWR, catalog number: 20847.307 )
- Anti-Digoxigenin-AP-Fab fragments Labelling Mix (Roche Diagnostics, catalog number: 11093274910 )
- BCIP 5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine salt (Roche Diagnostics, catalog number: 11383221001 )
- NBT 4-Nitro blue tetrazolium chloride (Roche Diagnostics, catalog number: 11383213001 )
- Pectinex® (Sigma-Aldrich, catalog number: P2611 )
- Celluclast® (Sigma-Aldrich, catalog number: C2730 )
- Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
- Potassium phosphate, monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P9791 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Magnesium sulfate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 63138 )
- Sucrose (e.g., Sigma-Aldrich, catalog number: S9378 )
- 10x PBS (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9624 )
- 37% formaldehyde solution (Sigma-Aldrich, catalog number: F15587 )
Note: This product has been discontinued. - Proteinase K 20 mg/ml (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2546 )
- Formamide, deionized (Sigma-Aldrich, catalog number: F9037 )
- SSC buffer 20x (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9770 )
- Boehringer blocking reagent (Roche Diagnostics, catalog number: 11096176001 )
- SDS (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15553027 )
- Denhart’s solution (Sigma-Aldrich, catalog number: D2532 )
- EDTA pH 8 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9261 )
- Salmon sperm DNA (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15632011 )
- tRNA from baker’s yeast (Sigma-Aldrich, catalog number: R8759 , type X-SA)
- Maleic acid (Fisher Scientific, catalog number: 10348843)
Manufacturer: Acros Organics, catalog number: 125230051 . - Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Sodium hydroxide (NaOH) (VWR, catalog number: 28245.298 )
- Tris-base (e.g., Sigma-Aldrich, catalog number: T1503 )
- Concentrated HCl (VWR, catalog number: 20248.295 , type 35%)
- Deionized water
- Nuclease-free water (not DEPC-Treated; Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9938 )
- For nematode extraction (see Recipes)
- Pectinex® and Celluclast® mix
- M9-buffer pH 7
- 50% sucrose in M9 buffer
- RNase-free 1x PBS
- Pectinex® and Celluclast® mix
- For probe, fixation and hybridization, nuclease-free (see Recipes)
- Fixative buffer
- 1 mg/ml Proteinase K solution
- tRNA from Baker’s yeast
- Hybridization buffer (HB)
- Washing buffer 1 (4x SSC + 0.1% SDS)
- Washing buffer 2 (0.1x SSC + 0.1% SDS)
- 10% Boehringer blocking reagent
- Filtered maleic acid buffer, pH 7.5
- Tris-HCl pH 9.5
- Alkaline phosphatase detection buffer pH 9.5 (APB)
- Fixative buffer
Equipment
- Pipetmens (e.g., Gilson, models: P2 , P20 , P200 , P1000 )
- Thermocycler (e.g., Biometra, model: T3000 )
- NanoDrop (e.g., Agilent Technologies, model: Agilent 2100 Bioanalyser , catalog number: G2939)
- Microcentrifuge at room temperature (RT) (e.g., Hitachi Koki, model: CT15E ) with fixed-angle rotor (e.g., Hitachi Koki, model: T15A61 )
- Centrifuge for 50 ml tube (e.g., Eppendorf, model: 5804 R ) with swing-bucket rotor (e.g., Eppendorf, model: A-4-44 )
- 50 ml glass beaker (Corning, PYREX®)
- Fume hood
- Aquarium air pump (Rena Aquatic Supply, model: Model 301 )
- Glass plate
- Mini hybridization oven (Appligene, catalog number: 001050414 )
- Vortexer (e.g., Baxter, catalog number: S8223-1 )
- Orbital shaker (VWR)
- 37 °C water bath
- Fridge at 4 °C
- -20 °C freezer
- -80 °C freezer
- Autoclave
- Microscope (ZEISS, model: Axioplan 2 )
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jaouannet, M., Nguyen, C., Quentin, M., Jaubert-Possamai, S., Rosso, M. and Favery, B. (2018). In situ Hybridization (ISH) in Preparasitic and Parasitic Stages of the Plant-parasitic Nematode Meloidogyne spp.. Bio-protocol 8(6): e2766. DOI: 10.21769/BioProtoc.2766.
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分类
微生物学 > 微生物-宿主相互作用 > 线虫
植物科学 > 植物免疫 > 宿主-细菌相互作用
细胞生物学 > 细胞染色 > 核酸
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