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).
Roots of soybean, Glycine max cv. Kent L. Merr., plants susceptible to the soybean cyst nematode (SCN), Heterodera glycines Ichinohe, were inoculated and allowed to develop feeding sites (syncytia) for 8 days. Root samples enriched in syncytial cells were collected using laser capture microdissection (LCM). RNA was extracted and used to make a cDNA library and expressed sequence tags (ESTs) were produced and used for a Gene Ontology (GO) analysis. RT-PCR results indicated enhanced expression of an aquaporin (GmPIP2,2), alpha-tubulin (GmTubA1), beta-tubulin (GmTubB4) and several other genes in syncytium-enriched samples as compared to samples extracted from whole roots. While RT-PCR data showed increased transcript levels of GmPIP2,2 from LCM tissue enriched in syncytial cells, in situ hybridization showed prominent GmPIP2,2 hybridization to RNA in the parenchymal cells tightly juxtaposed to the syncytium. Immunolocalization indicated stronger alpha-tubulin signal within the syncytium as compared to surrounding tissue. However, alpha-tubulin labeling appeared diffuse or clumped. Thus, LCM allowed for the isolation of tissue enriched for syncytial cells, providing material suitable for a variety of molecular analyses.
Klink
Vincent P
VP
United States Department of Agriculture, 10300, Baltimore Ave., Bldg. 006, Rm. 118, Beltsville, MD, 20705-2350, USA. heartwood27@hotmail.com
Alkharouf
Nadim
N
MacDonald
Margaret
M
Matthews
Benjamin
B
D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D020224:Expressed Sequence Tags; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D005821:Genetic Techniques; D017403:In Situ Hybridization; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D008969:Molecular Sequence Data; D009348:Nematoda; D018517:Plant Roots / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology* / Q000378:metabolism* / Q000469:parasitology*; D014404:Tubulin / Q000378:metabolism
D020346:Aquaporins; D018076:DNA, Complementary; D014404:Tubulin; D012313:RNA