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, Plant pathogen, Preparasitic and parasitic stages, 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
Equipment
Procedure
Data analysis
Nematodes are examined with differential interference contrast microscopy. The signal observed should be compared to the images obtained with the control sense probe (negative control) (Figure 3). A positive control should be added. We usually used the MiPG gene encoding a polygalacturonase expressed in the two sub ventral glands (Jaubert et al., 2002a). We recommend taking pictures of at least 40 stained nematodes, analyzing the cellular localization of detected RNA spots, and to count the number of stained nematodes. Each ISH experiment should be reproduced independently. ISH could be performed after siRNA gene silencing of the target gene to ensure specificity and/or to evaluate the silencing effect. Moreover, control experiments using a different set of probes hybridizing the same target gene could be done to ensure the specificity of the probes to the target gene. Figure 3. Tissue expression of M. incognita EFFECTOR1 (MiEFF1) in preparasitic J2 and parasitic juveniles. A-B. Transcripts from MiEFF1 (Minc17998) were localized in the salivary dorsal gland (arrowhead) of preparasitic J2s. C. The sense MiEFF1 control probe yielded no labeling of nematode tissues. MiEFF1 tissue expression in a parasitic juvenile was presented in Jaouannet et al., 2012. Scale bars = 50 µm.
Notes
Recipes
For nematode extraction
Acknowledgments
This protocol has been adapted from de Boer et al. (1998) and Rosso et al. (1999). It has made possible the study of the expression profile of several dozens of genes (Jaubert et al., 2002a and 2002b; Neveu et al., 2003; Jaubert et al., 2004; Ledger et al., 2006; Dubreuil et al., 2007; Jaouannet et al., 2012; Jaouannet et al., 2013; Danchin et al., 2013; Nguyen et al., 2017). These works were funded by INRA, CNRS, University of Nice Sophia Antipolis, the Region Provence-Alpes-Côte d’Azur, the French Government, the European Union and NATO Collaborative Research Grant. C-N.N. was supported by USTH fellowships, 911-USTH program, from the Ministry of Education and Training of The Socialist Republic of Vietnam. The authors have no conflict of interest or competing interests to declare.
References
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Dear Dr. Favery,Thanks very much for your help and suggestion on the in situ hybridization!Lei