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)
Vincent P. Klink*, Nadim Alkharouf, Margaret MacDonald and Benjamin Matthews
United States Department of Agriculture, 10300, Baltimore Ave., Bldg. 006, Rm. 118, Beltsville, MD 20705-
2350, USA (*author for correspondence; e-mail firstname.lastname@example.org)
Received 26 February 2005; accepted in revised form 22 August 2005
Key words: aquaporin, Glycine max, Heterodera glycines (SCN), Laser capture microdissection (LCM),
pathogen, plant, soybean, tubulin
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), a-tubulin (GmTubA1), b-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 a-tubulin signal within the syncytium as compared to surrounding
tissue. However, a-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.
The soybean cyst nematode (SCN), Heterodera
glycines Ichinohe, is an obligate, sedentary endo-
parasite that is the major pathogen of soybean
(Glycine max, L. Merr.) and accounts for an
estimated 1 billion dollars in production losses
annually in the U.S. (Wrather et al., 2001). During
parasitism, the SCN penetrates roots and migrates
toward the vascular tissue, selects a pericycle or
endodermal cell, and initiates the formation of a
feeding cell. The nematode induces a variety of
changes in the cell it selects for its feeding site.
After 1 day, cellular hypertrophy occurs and cell
walls dissolve (Endo, 1964). By 42 h, cell wall
perforations are observed (Gipson et al., 1971)
and cytoplasm from adjoining pericycle, endoder-
mal and cortex cells merges to form a syncytium, a
structure that nourishes the SCN during its sed-
entary and reproductive phases of its life cycle.
The syncytium is very different, cytologically, from
the root cells it originates and undergoes a variety
of changes as it differentiates and becomes the
feeding site of the SCN (Endo, 1964, 1965, 1971;
Gipson et al., 1971; Jones and Northcote, 1972).
The cytoplasm of the developing syncytium
appears dense with numerous plastids and ER-
like material accumulating (Gipson et al., 1971).
Concomitantly, the large vacuole that is present in
the cell prior to feeding site selection becomes
Plant Molecular Biology (2005) 59:965–979 Ó Springer 2005