Gene expression in early stages of giant cells induced by meloidogyne javanica using Laser Capture Microdissection
Ramsay, K., Wang, Z. and Jones, M.G.K. (2005) Gene expression in early stages of giant cells induced by meloidogyne javanica using Laser Capture Microdissection. In: 44th Annual Meeting of the Society of Nematologists, 9 - 13 July 2005, Fort Lauderdale, Florida.
Root-knot nematodes (Meloidogyne spp.) are economically important plant pests that establish a specific relationship with host roots, and induce the formation of feeding cells known as ‘giant cells’. These provide nutrients, and support nematode growth and reproduction. The induction of the feeding cells involves a series of developmental and biochemical changes, reflecting alterations in expression of many endogenous plant genes in response to nematode stimuli. Identification of key genes/products in the interaction is a major challenge in understanding how root development is modified during giant cell formation. We have used a micropipette system to obtain pure giant cell cytoplasm for molecular analysis, but it is difficult to obtain pure cytoplasm from early stages of the interaction. Laser Capture Microdissection (LCM) is a technique that was developed to isolate specific cells from heterogeneous tissues, and in this study LCM has been used to access the cytoplasm of 4 days post inoculation giant cells in tomato roots, from paraffin-embedded sections. Total RNA was isolated from laser dissected sections, and used in RT-PCR to investigate expression of some cell cycle genes (cyclins) in giant cells, and to confirm that full length cDNA fragments were present. Two D-type cyclin genes, LeCycD3;2 and LeCycD3;3, were expressed at higher levels in giant cells compared to other cell-cycle-related cyclin genes, suggesting that the induction of the G1 phase of the cell cycle may be triggered in response to stimulation by the infecting nematode. The isolated RNA was then amplified using in vitro transcription by T7 RNA polymerase. After two rounds of amplification, the amplified RNA was used to construct a giant cell cDNA library.
|Publication Type:||Conference Item|
|Murdoch Affiliation:||Western Australian State Agricultural Biotechnology Centre|
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