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)

Abstract

Syncytial cells in soybean (Glycine max cultivar [cv.] Peking) roots infected by incompatible and compatible populations of soybean cyst nematode (SCN [Heterodera glycines]) were collected using laser capture microdissection (LCM). Gene transcript abundance was assayed using Affymetrix soybean GeneChips, each containing 37,744 probe sets. Our analyses identified differentially expressed genes in syncytial cells that are not differentially expressed in the whole root analyses. Therefore, our results show that the mass of transcriptional activity occurring in the whole root is obscuring identification of transcriptional events occurring within syncytial cells. In syncytial cells from incompatible roots at three dpi, genes encoding lipoxygenase (LOX), heat shock protein (HSP) 70, superoxidase dismutase (SOD) were elevated almost tenfold or more, while genes encoding several transcription factors and DNA binding proteins were also elevated, albeit at lower levels. In syncytial cells formed during the compatible interaction at three dpi, genes encoding prohibitin, the epsilon chain of ATP synthase, allene oxide cyclase and annexin were more abundant. By 8 days, several genes of unknown function and genes encoding a germin-like protein, peroxidase, LOX, GAPDH, 3-deoxy-D-arabino-heptolosonate 7-phosphate synthase, ATP synthase and a thioesterase were abundantly expressed. These observations suggest that gene expression is different in syncytial cells as compared to whole roots infected with nematodes. Our observations also show that gene expression is different between syncytial cells that were isolated from incompatible and compatible roots and that gene expression is changing over the course of syncytial cell development as it matures into a functional feeding site.

The use of laser capture microdissection to study the infection ofGlycine max(soybean) byHeterodera glycines(soybean cyst nematode)

“…8,10,15,16,21,22 We have used a bioinformatics approach to compare incompatible to compatible reactions at the whole root level using the G. max cultivar Peking as a common genetic background. 21 Peking was selected because bona fide incompatible and compatible reactions can be achieved by simply changing the race of nematodes used in those experiments.…”

“…In a second investigation, differential gene expression that was restricted locally to the syncytium during the onset of I or C was explored. 22 Right panel, the 847 genes representing the difference in gene number between the incompatible and compatible reaction are shown. The prominent contrasting profile represented genes that were suppressed at all time points during the compatible reaction (profile G, highlighted in pink).…”

“…25 It also allowed the isolation of homogeneous syncytium samples so that microarray analysis of syncytia undergoing a compatible or incompatible reaction could be performed. 22 Those analyses allowed for the direct comparison of the incompatible to the compatible syncytia at specific time points (Fig. 2).…”

“…Among those highlighted in the incompatible response at three dpi were those encoding lipoxygenase, heat shock protein 70 and superoxide dismutase. 22 …”

“…The whole root microarray analyses and syncytium microarray analyses generated huge amounts of both expression and derived data, and since our lab required unconventional data mining techniques to compare the responses in the different cell types, we developed an in-house database and custom data mining scripts. 21,22 One of our analyses compared whole root gene expression to syncytial gene expression during the compatible and incompatible responses. This comparison demonstrated that transcriptional activity (e.g., differential expression) within the syncytium is obscured by transcriptional activity in the whole root.…”