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Updated: Jun 25, 2026
Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types
Published on: May 10, 2016
Functional characterization of transcripts expressed in early-stage Meloidogyne javanica-induced giant cells isolated
John Fosu-Nyarko1, Michael G K Jones, Zhaohui Wang
1Plant Biotechnology Research Group, Western Australian State Agricultural Biotechnology Centre (SABC), School of Biological Sciences and Biotechnology, Murdoch University, Perth, WA6150, Australia.
Abstract:
The root-knot nematode Meloidogyne javanica induces giant cells and feeds from them during its development and reproduction. To study the cellular processes underlying the formation of giant cells, laser microdissection was used to isolate the contents of early-stage giant cells 4 and 7 days post-infection (dpi) from tomato, and cDNA libraries from both stages were generated with 87 [250 expressed sequence tag (EST) clones] and 54 (309 EST clones) individual transcripts identified, respectively. These transcripts have roles in metabolism, stress response, protein synthesis, cell division and morphogenesis, transport, signal transduction, protein modification and fate, and regulation of cellular processes. The expression of 25 selected transcripts was studied further by real-time quantitative reverse transcriptase-polymerase chain reaction. Among them, 13 showed continuous up-regulation in giant cells from 4 to 7 dpi. The expression of two transcripts was higher than in controls at 4 dpi and remained at the same level at 7 dpi; a further five transcripts were highly expressed only at 7 dpi. The Phi-1 protein gene, a cell cycle-related homologue in tobacco, was expressed 8.5 times more strongly in giant cells than in control cells at 4 dpi, but was reduced to 6.7 times at 7 dpi. Using in situ hybridization, the expression of the Phi-1 gene was preferentially localized in the cytoplasm of giant cells at 4 dpi, together with a pectinesterase U1 precursor gene. The identification of highly expressed transcripts in developing giant cells adds to the knowledge of the plant genes responsive to nematode infection, and may provide candidate genes for nematode control strategies.
Insights
Researchers identified key plant genes involved in tomato
Area of Science:
- Plant pathology
- Molecular biology
- Nematology
Background:
- Root-knot nematodes (Meloidogyne javanica) manipulate host cells to form giant cells for feeding.
- Understanding the molecular mechanisms of giant cell formation is crucial for developing crop protection strategies.
Purpose of the Study:
- To identify and characterize plant genes expressed during the early development of Meloidogyne javanica-induced giant cells in tomato.
- To explore potential molecular targets for nematode resistance.
Main Methods:
- Laser microdissection was employed to isolate cellular contents from early-stage giant cells (4 and 7 days post-infection).
- Expressed sequence tag (EST) libraries were constructed and analyzed to identify unique transcripts.
- Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and in situ hybridization were used to validate gene expression patterns.
Main Results:
- A total of 87 and 54 unique transcripts were identified at 4 and 7 days post-infection, respectively, involved in diverse cellular processes.
- Thirteen transcripts showed continuous up-regulation, while others exhibited stage-specific expression patterns.
- The Phi-1 gene, related to cell cycle regulation, was significantly upregulated in giant cells, with its expression localized in the cytoplasm.
Conclusions:
- The study identified novel plant genes responsive to nematode infection during giant cell development.
- These identified transcripts represent potential candidate genes for engineering nematode resistance in tomato and other crops.
