An improved procedure for isolation of high-quality RNA from nematode-infected Arabidopsis roots through laser capture microdissection
© Anjam et al. 2016
Received: 7 January 2016
Accepted: 19 April 2016
Published: 26 April 2016
Cyst nematodes are biotrophs that form specialized feeding structures in the roots of host plants, which consist of a syncytial fusion of hypertrophied cells. The formation of syncytium is accompanied by profound transcriptional changes and active metabolism in infected tissues. The challenge in gene expression studies for syncytium has always been the isolation of pure syncytial material and subsequent extraction of intact RNA. Root fragments containing syncytium had been used for microarray analyses. However, the inclusion of neighbouring cells dilutes the syncytium-specific mRNA population. Micro-sectioning coupled with laser capture microdissection (LCM) offers an opportunity for the isolation of feeding sites from heterogeneous cell populations. But recovery of intact RNA from syncytium dissected by LCM is complicated due to extended steps of fixation, tissue preparation, embedding and sectioning.
In the present study, we have optimized the procedure of sample preparation for LCM to isolate high quality of RNA from cyst nematode induced syncytia in Arabidopsis roots which can be used for transcriptomic studies. We investigated the effect of various sucrose concentrations as cryoprotectant on RNA quality and morphology of syncytial sections. We also compared various types of microscopic slides for strong adherence of sections while removing embedding material.
The use of optimal sucrose concentrations as cryoprotection plays a key role in RNA stability and morphology of sections. Treatment with higher sucrose concentrations minimizes the risk of RNA degradation, whereas longer incubation times help maintaining the morphology of tissue sections. Our method allows isolating high-quality RNA from nematode feeding sites that is suitable for downstream applications such as microarray experiments.
Plant-parasitic nematodes (PPNs) are obligate biotrophs that cause significant damage to almost every economically important crop . PPNs are classified based on their feeding habits as either ectoparasites or endoparasites. The endoparasitic root-knot (Meloidogyne spp.) and cyst nematodes (Globodera spp. and Heterodera spp.) are sedentary parasites of roots and the primary nematode pathogen of a wide range of crops. Infective stage juveniles (J2s) of cyst nematodes invade the host root and migrate intracellularly until they reach the vascular cylinder. There, these nematodes select an initial syncytial cell (ISC) and become sedentary. Within 24 h of the ISC selection the cells adjacent to the ISC appear hypertrophied and fused together due to local dissolution of the cell walls . Three days after ISC selection, the cells that have been incorporated into the syncytium are enlarged and exhibit the features of a typical syncytial cell such as condensed cytoplasm and enrichment of ribosomes, endoplasmic reticulum, mitochondria and plastids. The nuclei have become enlarged, and several smaller vacuoles are present in the cytoplasm. Moreover the outer cell walls thicken to withstand the high osmotic pressure within the syncytium . The syncytium serves as the sole source of nutrients and water for the rest of the nematode’s successive growth and development. A cocktail of secretions, which are synthesised in the oesophageal glands of the nematodes, are responsible for the modulation of the plant’s defence and the developmental pathways that lead to the formation of a syncytium [8, 9, 12, 29]. Therefore understanding the function of host genes that are required for syncytium development is crucial for identifying novel targets for nematode resistance.
Compatible interaction is best studied between cyst nematode Heterodera schachtii and the model plant Arabidopsis thaliana. In recent years a lot of work with this particular pathosystem has shown that the development of the syncytium is accompanied by massive transcriptomic, metabolomic and proteomic changes [11, 13, 31]. Nevertheless isolating pure syncytial material and extracting good quality RNA from Arabidopsis roots for downstream application, such as microarray analyses, qPCR and RNA-seq is challenging. Puthoff et al.  used the entire root systems of cyst nematode-infected Arabidopsis plants to perform the first transcriptomic studies. However including non-infected cells dilutes the feeding cell specific mRNA expression profile [10, 31]. In a step forward, Szakasits et al.  tried to overcome this limitation by collecting the pure cytoplasm of feeding cells through micro-aspiration. But producing a sufficient amount of material for analysis using this technique is laborious. Moreover it works better on larger, well-developed syncytial cells as compared to syncytial tissues during the early stages of development. An alternative and promising technique for collecting pure cells is laser capture microdissection (LCM).
LCM is becoming a popular tool among cell biologists because it allows isolation of RNA, DNA, proteins and metabolites from a heterogeneous cell population. Current LCM techniques are based on two principles. In the “touch-free” approach, cells identified under a microscope are excised by a laser beam and removed from their tissue context by gravity or by catapulting them away with a photonic pulse. Alternatively, cells are fused to a plastic membrane and are subsequently removed mechanically from the remaining tissue (reviewed in Ludwig and Hochholdinger ).
Successful LCM requires good morphological preservations of tissues. Furthermore, isolated nucleic acids (RNA or DNA), proteins or metabolites must be of high quality to ensure their utility for downstream applications. Therefore, tissue preparation is the crucial step towards successful RNA isolation of samples generated by LCM. Since LCM requires micro-sections of 2–20 µm, samples must be fixed and embedded prior to sectioning and mounting them on the slides. During this multistep procedure, RNA integrity remains at high risk of degradation due to the presence of RNases. Samples for immuno-histological analyses are generally fixed in paraformaldehyde and embedded in paraffin to ensure well-preserved tissue morphology. In contrast, ethanol:acetic acid (EAA) solution is preferred for fixation in experiments involving RNA extraction because paraformaldehyde cross links with RNA and thus makes its recovery difficult [14, 17, 18, 30]. After fixation the tissues are embedded either in paraffin at room temperature or in an optimum cutting temperature (OCT) medium at −20 °C (cryosectioning). While OCT-embedded samples are sectioned through a cryomicrotome at −20 °C, the paraffin-embedded samples are sectioned at room temperature. Cryosectioning is often preferred over sectioning of paraffin-embedded samples because processing the tissue at a low temperature reduces the activity of RNases and increase yield [1, 23]. Nevertheless keeping the RNA intact during the various steps of the cryosectioning for LCM poses a challenge and entails fixing and processing tissues. For example, plant cells contain big vacuoles for water storage, which may form crystals because of fast freezing of samples during cryosectioning leading to the destruction of cellular structures. Similarly, it is challenging to maintain the histological preservation during cryosectioning so that the target cells can be distinguished from others cell types. Therefore a number of tissue fixation and embedding procedures have been developed to ensure optimised handling of tissues with different physical rigidity .
Nakazono et al.  were the first to report application of LCM in plant samples by isolating the epidermis and vascular tissue cells of maize plants, which were subsequently used in microarray studies. Soon after, Kerk et al.  presented an RNA isolation technique using the paraffin-embedded sections of a variety of plant tissues. Further the LCM procedure has been applied to isolate syncytial material from soybean roots infected with cyst nematode H. glycines [15, 18, 19]. These authors applied paraffin embedding to process the syncytial samples and showed that a number of genes are differentially regulated in these plants upon nematode infection. But the utility of this protocol for nematode-infected Arabidopsis roots remains questionable as it may lead to poor quality RNA extraction. LCM was also used to capture giant cells and isolate RNA induced by root-knot nematodes in Arabidopsis, rice and tomato during different stages of infection [1, 3, 7, 16, 24, 26]. However no protocol describing the isolation of syncytial cells from Arabidopsis have been described so far. In the present study we optimised the LCM procedure for the isolation of syncytial cells from Arabidopsis roots in such a manner that their morphology can be preserved and high-quality RNA can be isolated for downstream applications such as microarray analyses.
To assess the potential of a previously described giant cell RNA isolation protocol in Arabidopsis for its utility for syncytial samples, we grew Arabidopsis plants in KNOP medium and infected them with J2 nematodes of H. schachtii. Root segments containing syncytia were cut at 5 days post inoculation (dpi). The infected root segments were subsequently fixed in EAA (3:1) and processed for LCM as described previously [1, 23, 24]. However, RNA was heavily degraded in these samples and was unusable for downstream applications (data not shown).
An overview of influence of tissue fixation and embedding on RNA quality
Cryoprotection in sucrose
Modification of cryoprotection steps yielded good quality RNA
Incubation in higher sucrose concentrations affected tissue morphology
Poly-l-lysine-coated glass slides strongly adhere to micro-sections
Different types of glass slides with diverse features are available. In this study, four types of slides were tested to obtain a strong tissue adherence: (a) polyethylene teraphthalate (PET)-membrane-coated slides, (b) polyethylene napthalate (PEN)-membrane-coated slides, (c) Superfrost® positively charged slides, and (d) poly-l-lysine-coated glass slides. We mounted 10 µm sections on PET- and PEN-membrane-coated glass slides and washed them with PBS buffer and 70 % ethanol to remove the OCT medium. The complete removal of the OCT medium is necessary for laser cutting. However during washing, all sections were washed away from the slides. We pre-treated the slides with UV light for 30 min to 4 h to enhance adherence, but the majority of the sections was still washed off during the washing step. Next we used Superfrost® positively-charged glass slides, on which tissue retention was greatly improved, while the use of poly-l-lysine-coated glass slides proved robust in holding the tissues during the washing step. Hence, in all subsequent experiments, we used poly-l-lysine-coated glass slides, and the laser capture machine was also calibrated to glass slides.
In vitro amplification of cDNA
After the successful use of LCM on animal and human tissues, it has become increasingly popular among plant scientists because of its ability to isolate a single cell-type from a heterogeneous population. The collected material can be used for a broad range of downstream molecular studies, for example, qPCR, RNAseq, microarray and proteomics. In contrast to animal cells the presence of a cell wall and large vacuole in plants necessitates modifications to preserve cell morphology and macromolecules, such as RNA and proteins. As the use of LCM in plant sciences expands, a variety of optimized protocols for specific cell-type harvest have been established. However in contrast to a typical plant cell with a large central vacuole, the cyst-nematode-induced syncytium is a distinctive structure surrounded by a thick cell wall that contains several smaller vacuoles. Therefore performing LCM on syncytial samples may necessitate certain modifications in existing protocols.
In this study we used a previously described protocol for LCM on giant cells induced by root-knot nematodes in Arabidopsis as a starting point . However when we incubated the samples in various sucrose concentrations, the RNA in the samples was highly degraded (Fig. 1a). In contrast incubating samples directly in a 34 % sucrose solution preserved the RNA integrity, indicating that RNase activity may not completely stop at lower sucrose concentrations. A higher concentration of sugar minimise the availability of water in the tissues, which may reduce enzymatic activities including that of RNase. The cryoprotective use of higher sucrose concentrations (20 or 30 %) has already been reported in animal tissues when performing LCM and immunohistochemistry [6, 20, 21]. The use of an RNase inhibitor, for example, RNase later®, maintains high-quality RNA; however RNase later® severely compromised the morphology of the sections in some reports .
Although the 34 % sucrose concentration significantly increased RNA quality, the morphology of the sections was strongly compromised. We reasoned that this may be due to direct tissue incubation in a higher concentration sucrose solution (34 %), which may result in a rapid loss of water and thus shrunk tissues. Indeed tissue morphology was maintained through longer vacuum infiltration and an overnight incubation in a 34 % sucrose solution (see “Methods”). The slow intake of solution might have helped the cells maintain their shape.
After the morphology of the sections was satisfying, the embedding medium was removed completely by washing which is a perquisite for the laser beam to function properly. The commercially available OCT medium is water soluble and can be removed easily by washing with PBS buffer. However the root sections can also be washed away unless they adhere strongly to the slides. We tried membrane-covered slides (PET and PEN), which permit very fine dissection of target cells, and the targeted area can be catapulted as a single intact piece, but they proved very poor in holding root sections during the washing step. In contrast, Ramsay et al.,  used PEN slides for the LCM of giant cells from tomato roots, and Barcala et al.  used Superfrost® positively-charged glass slides for giant cells in Arabidopsis roots. However we found that poly-l-lysine-coated glass slides robustly anchored the frozen sections of 5-day-old syncytia.
In conclusion, we have shown that fixation with EAA and a direct infiltration with 34 % sucrose, prior to embedding in OCT medium generates syncytial samples with well-preserved morphology that are suitable to distinguish syncytial cells from uninfected surrounding tissue during LCM. Moreover, high quality RNA was extracted from LCM-processed syncytial samples, which was suitable for downstream applications such as microarrays analysis and RT-PCR. Importantly, we were able to analyse the RNA quality prior to amplification, which has not been shown previously . Our protocol will help analysing gene expression during early stages of cyst nematode infection, which may help understanding genes and pathways that are important for nematode development. In addition, development of analytical tools such as syncytium-specific promoters will be greatly facilitated by application of this methodology.
Tissue preparation and sample collection
10-day-old Arabidopsis thaliana plants were grown on KNOP medium and inoculated with 60–70 juveniles of Heterodera schachtii as described previously . Subsequently, 5-day-old infected root samples were hand-dissected and vacuum infiltrated in EAA solution (3:1) for 20 min. Afterwards, EAA solution was replaced with fresh solution and samples were incubated at 70 rpm on an undulating shaker at 4 °C for 1 h.
The fixative solution was replaced with a 34 % sucrose solution prepared in 0.01 M PBS buffer (treated with 0.1 % DEPC), pH 7.4. Safranin-O (0.01 % in water; Cat. No. S2255. Sigma-Aldrich, USA) was added to visualise the otherwise transparent tissues. Moreover, to facilitate solution penetration, a vacuum was applied for 45 min at 4 °C. Subsequently, samples were incubated in an undulating shaker at 4 °C for 24 h.
The tissues were horizontally embedded in a tissue freezing medium, OCT (Polyfreeze®, Cat No. 25112. Poly Sciences, Germany) using Cryomold Tissue-Tek® (Cat No. E62534. EMS, USA). The embedded tissues were frozen by touching the back of cryomolds to liquid nitrogen. The embedded samples (OCT blocks) were stored at −80 °C until cryosectioning.
Cryosectioning and laser capture microdissection
The OCT blocks containing tissues were left inside a cryomicrotome (CM 1850 UV, Leica, Germany) pre-cooled to −20 °C for 15 min for acclimatisation before making sections. Longitudinal sections of 10 µm were mounted on poly-l-lysine-coated glass slides (Cat no. J2800AMNZ. Manzel Glaser, Germany). After collecting 10–15 sections on the cryomicrotome platform, a slide at RT was suddenly placed over the sections to adhere them. The slides with sections were stored in 70 % ethanol, which was already pre-chilled in the cryomicrotome.
Primer sequences used in this study
Beta tubulin 4
MSA and YL performed experiments. MSA, SS and FMWG designed the research and wrote the paper. FH, CM, and MI provided technical support and helped editing the manuscript. All authors read and approved the final manuscript.
We thank Jennifer Topping and Keith Lindsey for technical training of Muhammad Shahzad Anjam.
The authors declare that they have no competing interests.
Muhammad Shahzad Anjam was supported by a grant from German Exchange Service (DAAD).
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- Barcala M, Garcia A, Cabrera J, Casson S, Lindsey K, Favery B, Garcia-Casado G, Solano R, Fenoll C, Escobar C. Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells. Plant J. 2010;61:698–712.View ArticlePubMedGoogle Scholar
- Bevilacqua C, Makhzami S, Helbling JC, Defrenaix P, Martin P. Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by laser capture microdissection. BMC Cell Biol. 2010;11:95.View ArticlePubMedPubMed CentralGoogle Scholar
- Fosu-Nyarko J, Jones MGK, Wang Z. Functional characterization of transcripts expressed in early-stage Meloidogyne javanica-induced giant cells isolated by laser microdissection. Mol Plant Pathol. 2009;10:237–48.View ArticlePubMedGoogle Scholar
- Gautam V, Sarkar AK. Laser assisted microdissection, an efficient technique to understand tissue specific gene expression patterns and functional genomics in plants. Mol Biotechnol. 2015;57:299–308.View ArticlePubMedGoogle Scholar
- Golinowski W, Grundler FMW, Sobczak M. Changes in the structure of Arabidopsis thaliana during female development of the plant-parasitic nematode Heterodera schachtii. Protoplasma. 1996;194:103–16.View ArticleGoogle Scholar
- Haynes T, Luz-Madrigal A, Reis ES, Ruiz NPE, Grajales-Esquivel E, Tzekou A, Tsonis PA, Lambris JD, Del Rio-Tsonis K. Complement anaphylatoxin C3a is a potent inducer of embryonic chick retina regeneration. Nat Commun. 2013;4:2312.View ArticlePubMedPubMed CentralGoogle Scholar
- He B, Magill C, Starr JL. Laser capture microdissection and real-time PCR for measuring mRNA in giant cells induced by Meloidogyne javanica. J Nematol. 2005;37:308–12.PubMedPubMed CentralGoogle Scholar
- Hewezi T, Baum TJ. Manipulation of plant cells by cyst and root-knot nematode effectors. Mol Plant Microbe Interact. 2013;26:9–16.View ArticlePubMedGoogle Scholar
- Hewezi T, Juvale PS, Piya S, Maier TR, Rambani A, Rice JH, Mitchum MG, Davis EL, Hussey RS, Baum TJ. The cyst nematode effector protein 10A07 targets and recruits host posttranslational machinery to mediate its nuclear trafficking and to promote parasitism in Arabidopsis. Plant Cell. 2015;27:891–907.View ArticlePubMedPubMed CentralGoogle Scholar
- Hofmann J, Hess PH, Szakasits D, Blöchl A, Wieczorek K, Daxböck-Horvarth S, Bohlmann H, van Bel AJE, Grundler FMW. Diversity and activity of sugar transporters in nematode-induced root syncytia. J Exp Bot. 2009;60:3085–95.View ArticlePubMedPubMed CentralGoogle Scholar
- Hofmann J, El Ashry A, Anwar S, Erban A, Kopka J, Grundler F. Metabolic profiling reveals local and systemic responses of host plants to nematode parasitism. Plant J. 2010;62:1058–71.PubMedPubMed CentralGoogle Scholar
- Holbein J, Grundler FMW, Siddique S. Plant basal defence to nematodes: an update. J Exp Bot. 2016. doi:10.1093/jxb/erw005.PubMedGoogle Scholar
- Hütten M, Geukes M, Misas-Villamil JC, van der Hoorn RAL, Grundler FMW, Siddique S. Activity profiling reveals changes in the diversity and activity of proteins in Arabidopsis roots in response to nematode infection. Plant Physiol Biochem. 2015;14:36–43.View ArticleGoogle Scholar
- Inada N, Wildermuth MC. Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf. Planta. 2005;221:9–16.View ArticlePubMedGoogle Scholar
- Ithal N, Recknor J, Nettleton D, Maier T, Baum TJ, Mitchum MG. Developmental transcript profiling of cyst nematode feeding cells in soybean roots. Mol Plant Microbe Interact. 2007;20:510–25.View ArticlePubMedGoogle Scholar
- Ji HL, Gheysen G, Denil S, Lindsey K, Topping JF, Nahar K, Haegeman A, De Vos WH, Trooskens G, Van Criekinge W, De Meyer T, Kyndt T. Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots. J Exp Bot. 2013;64:3885–98.View ArticlePubMedPubMed CentralGoogle Scholar
- Kerk NM, Ceserani T, Tausta SL, Sussex IM, Nelson TM. Laser capture microdissection of cells from plant tissues. Plant Physiol. 2003;132:27–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Klink VP, Alkharouf N, MacDonald M, Matthews B. Laser capture microdissection (LCM) and expression analyses of Glycine max (soybean) syncytium containing root regions formed by the plant pathogen Heterodera glycines (soybean cyst nematode). Plant Mol Biol. 2005;59:965–79.View ArticlePubMedGoogle Scholar
- Klink VP, Overall CC, Alkharouf NW, MacDonald MH, Matthews BF. Laser capture microdissection (LCM) and comparative microarray expression analysis of syncytial cells isolated from incompatible and compatible soybean (Glycine max) roots infected by the soybean cyst nematode (Heterodera glycines). Planta. 2007;226:1389–409.View ArticlePubMedGoogle Scholar
- Krol J, Busskamp V, Markiewicz I, Stadler MB, Ribi S, Richter J, Duebel J, Bicker S, Fehling HJ, Schubeler D, Oertner TG, Schratt G, Bibel M, Roska B, Filipowicz W. Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell. 2010;141:618–31.View ArticlePubMedGoogle Scholar
- Kwon B, Houpt TA. A combined method of laser capture microdissection and X-Gal histology to analyze gene expression in c-Fos-specific neurons. J Neurosci Methods. 2010;186:155–64.View ArticlePubMedPubMed CentralGoogle Scholar
- Ludwig Y, Hochholdinger F. Laser microdissection of plant Cells. In: Plant cell morphogenesis: methods and protocols. Methods Mol Biol. 2014;1080, 249–258.Google Scholar
- Nakazono M, Qiu F, Borsuk LA, Schnable PS. Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell. 2003;15:583–96.View ArticlePubMedPubMed CentralGoogle Scholar
- Portillo M, Lindsey K, Casson S, Garcia-Casado G, Solano R, Fenoll C, Escobar C. Isolation of RNA from laser-capture-microdissected giant cells at early differentiation stages suitable for differential transcriptome analysis. Mol Plant Pathol. 2009;10:523–35.View ArticlePubMedGoogle Scholar
- Puthoff DP, Nettleton D, Rodermel SR, Baum TJ. Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles. Plant J. 2003;33:911–21.View ArticlePubMedGoogle Scholar
- Ramsay K, Wang ZH, Jones MGK. Using laser capture microdissection to study gene expression in early stages of giant cells induced by root-knot nematodes. Mol Plant Pathol. 2004;5:587–92.View ArticlePubMedGoogle Scholar
- Sasser JN, Freckman DW. A world perspective on nematology—the role of the society. J Nematol. 1986;18:596.Google Scholar
- Siddique S, Matera C, Radakovic ZS, Hasan MS, Gutbrod P, Rozanska E, Sobczak M, Torres MA, Grundler FMW. Parasitic worms stimulate host NADPH oxidases to produce reactive oxygen species that limit plant cell death and promote infection. Sci Signal. 2014;7:ra33.View ArticlePubMedGoogle Scholar
- Siddique S, Radakovic ZS, De La Torre CM, Chronis D, Novak O, Ramireddy E, Holbein J, Matera C, Hutten M, Gutbrod P, Anjam MS, Rozanska E, Habash S, Elashry A, Sobczak M, Kakimoto T, Strnad M, Schmulling T, Mitchum MG, Grundler FMW. A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants. Proc Natl Acad Sci USA. 2015;112:12669–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Srinivasan M, Sedmak D, Jewell S. Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol. 2002;161:1961–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Szakasits D, Heinen P, Wieczorek K, Hofmann J, Wagner F, Kreil DP, Sykacek P, Grundler FMW, Bohlmann H. The transcriptome of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots. Plant J. 2009;57:771–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Wyss U, Grundler FMW. Heterodera schachtii and Arabidopsis thaliana, a model host-parasite interaction. Nematologica. 1992;38:488–93.View ArticleGoogle Scholar