The use of laser capture microdissection to study the infection of Glycine max (soybean) by Heterodera glycines (soybean cyst nematode)

Abstract

The soybean cyst nematode (Heterodera glycines) is an obligate parasite of soybean (Glycine max). It is the most destructive pathogen of G. max, accounting for approximately 0.46–0.82 billion dollars in crop losses, annually, in the U.S. Part of the infection process involves H. glycines establishing feeding sites (syncytia) that it derives its nourishment from throughout its lifecycle. Microscopic methods (i.e., laser capture microdissection [LCM]) that faithfully dissect out those feeding sites are important improvements to the study of this significant plant pathogen. Our isolation of developing feeding sites during an incompatible or a compatible reaction is providing new ways by which this important plant-pathogen interaction can be studied. We have used these methods to create cDNA libraries, clone genes and perform microarray analyses. Importantly, it is providing insight not only into how the root is responding at the organ level to H. glycines, but also how the syncytium is responding during its maturation into a functional feeding site.

Introduction

Parasitic nematodes are poorly understood and pose a major threat to the agricultural industry.^1–4 Among the most costly is the interaction between Glycine max and Heterodera glycines. Since the first report of H. glycines in the U.S.,⁵ it has become the most significant pathogen of G. max and now accounts for an estimated 0.46 to 0.82 billion dollars in production losses, annually, in the U.S.⁶ The G. max-H. glycines system is a powerful one for studying this type of interaction because knowledge derived from gene expression experiments can be translated directly to improve resistance in an agriculturally relevant plant. Our lab has been using a genomics approach to study this significant soybean pathogen at both the whole root (organ) and syncytium (cellular) level.

Infection at the Organ Level

During the infection process (Fig. 1), second stage juvenile (J2) H. glycines invade roots. After penetration of the root, the infective J2 (iJ2) migrates through the cortex. After 24 hours post infection (hpi) the nematodes reach the stele where they select and establish their feeding sites.^7–16 Our lab developed in-house microarrays to study the interaction between G. max and H. glycines.¹⁷ This initial investigation studied the interaction at a single time point during a compatible reaction.¹⁷ That study was then expanded to include time points prior to nematode feeding site selection (6 and 12 hours post infection [hpi]) and time points afterwards (1,2,4,6,8 days post infection [dpi]).¹⁸ This study found the activation of various defense responses during a compatible reaction. However, those defense responses ultimately fail as the nematodes become established within those roots. It was clear from these early studies that G. max roots were responding to the presence of H. glycines before they establish their feeding sites (i.e., at times before 24 hpi and as early as 6 hpi).¹⁸ Thus, from these studies, a model could be inferred that the root cells are remodeling their transcriptional program as the iJ2s burrow through the root. In light of this observation, it can also be inferred that all of the new transcriptional activity that occurs during infection is not limited to the syncytium because they have not even formed by the 6 or 12 hpi time points.¹⁸ Based upon these exciting results, it was clear that repeating these experiments in resistant cultivars would provide additional insights into the infection process.

Figure 1.

Life cycle of H. glycines. (A), second stage juveniles (J2) (gray) hatch and migrate toward the root. (B), The iJ2 nematodes (dark gray) burrow into the root and migrate toward the root stele (green) (C), feeding site selection (yellow). (D), J2 nematodes molt into J3 then J4. The female is shown here in red. During this time, the original feeding site (yellow) is incorporating adjacent cells (deep purple) via cell wall degradation and fusion events. Meanwhile, the male discontinues feeding at the end of its J3 stage. (E) The male and female J4 nematodes mature into adults. By this time, the feeding site (light blue) has matured into a syncytium where the female is actively feeding. The vermiform male (purple) migrates toward the female (red) to copulate. (F) After ∼30 days, the female is clearly visible externally because its body emerges from the root tissue. (G) The cyst, ultimately, can lie dormant in the soil for years.

It is possible to study an incompatible and compatible reaction in G. max because well-defined H. glycines populations are available that confer both reactions.^19,20 It is interesting to note that resistant varieties of G. max also undergo an infection process that is often similar to the compatible response during the early stages of infection.^{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. We selected three time points for the analysis. The first time point was 12 hpi, a time when the iJ2 nematodes were still burrowing through the root, but have not yet selected feeding sites. The second time point was three dpi. This is a time when the nematodes have selected and started making feeding sites in both the incompatible and compatible reaction. Finally, an eight dpi time point was selected. The eight dpi time point represents a period when the incompatible roots have overcome the infection while, in the compatible reaction, the nematodes are still actively feeding and growing. Those analyses showed that the root is reacting differently to the compatible and incompatible races by the 12 hpi time point. Therefore, it appeared that the root was engaging transcriptional activity, possibly throughout the root, during infection. Thus, new transcriptional activity specific to the compatible and incompatible reaction is not limited to the syncytium (Fig. 2). Importantly, contrasting gene expression occurring between the compatible and compatible reactions is widespread during these two reactions and involves genes that belong to important defense pathways (Fig. 2). Thus, the resistance reaction may not be limited to gene expression within the syncytium and neighboring cells.

Figure 2.

Gene expression during infection. Left panel, similar profile.²¹ Incompatible (N=965 genes) and compatible (N=118 genes) profiles for genes that are suppressed at the 12 hpi time point and subsequently, those genes are induced at the three and eight dpi time points in both reactions. The differences can be expressed as incompatibility ratios (I:C). Thus, in this profile, 965/118, the I:C = 8.18. The difference in gene number (N=847) for those roots undergoing a compatible reaction meant that those genes were behaving differently during the compatible reaction. Below, whole root analysis. A cartoon depicts the experimental samples used in the whole root (12 hpi, 3 and 8 dpi) and syncytium (3 and 8 dpi) analyses. Two models of infection were presented for the whole root experiment.²¹ From those experiments it appeared that differences in gene expression characterizing incompatible (I) (dark blue) and compatible C) (red) are present before the formation of the syncytium. These changes in gene expression begin early on (by 12 hpi) as I (pink) and C (green) nematodes burrow through the root. These root transcriptional changes that characterize I and C continue on as nematodes establish syncytia by three dpi. Syncytia in I white arrow) or C (black arrow) roots appear similar anatomically at three dpi. By eight dpi, the I syncytium white arrow) collapses. Meanwhile C syncytia (black arrow) continue to develop. 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).

Infection at the Cellular Level

It is clear from historical observations that gene expression within the syncytium is different from its neighboring cells and the whole root.² Thus, methods to isolate syncytia, such as laser capture micro-dissection (LCM), may provide additional insights into their biology. LCM is a precise and accurate alternative to hand dissections for isolating homogeneous cell populations that are otherwise recalcitrant to their isolation.^23,24 Thus, LCM provided an opportunity to study the syncytium as an emergent property of the development of infection between G. max and H. glycines, by allowing the construction of cDNA libraries, cloning of genes and expression analyses.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). It also allowed for the comparison of syncytia at different time points during a compatible reaction (Fig. 2). This resulted in the identification of many differentially expressed genes that were specific to the incompatible or compatible response within syncytium samples. Among those highlighted in the incompatible response at three dpi were those encoding lipoxygenase, heat shock protein 70 and superoxide dismutase.²²

Bioinformatics

The purpose of the microarray experiments described in previous sections was to determine transcriptional differences during a compatible and incompatible response at both the organ (whole root) and cellular (syncytium) level in the H. glycines-G. max system. 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. The use of LCM helped to de-obfuscate gene expression occurring in the syncytium from that occurring in the surrounding cell types (i.e., cortex, root stele), thus showing that it is an effective and desirable technique for studying plant-pathogen interactions.

Abbreviations

hpi

hours post inoculation

dpi

days post inoculation

SCN

soybean cyst nematode

J2

second stage juvenile

LCM

laser capture microdissection

Addendum to: A comparative microarray analysis of an incompatible and compatible disease response by soybean (Glycine max) to soybean cyst nematode (Heterodera glycines) infection. Klink VP, Overall CC, Alkharouf N, MacDonald MH, Matthews BF. Planta. 2007 In press.

and

Addendum to: Laser capture microdissection (LCM) and comparative microarray expression analysis of syncytial cells isolated from incompatible and compatible soybean roots infected by soybean cyst nematode (Heterodera glycines) Klink VP, Overall CC, Alkharouf N, MacDonald MH, Matthews BF. Planta. 2007 In press.