**1.1 Plant nematodes**

Plant parasitic nematodes cause severe damage to plants and are responsible for billions of dollars of losses worldwide (Koenning et al. 2007). Soybean cyst nematode (SCN; *Hederodera glycines*; Fig. 1a ) and root-knot nematode (RKN; *Meloidogyne* spp.; Fig. 1b) are sedentary obligate parasites of plants. SCN is the major pest of soybean and causes an estimated one billion dollars in losses annually in the US (Wrather & Koenning 2006). RKN is a major pest of vegetables and can become a serious problem on soybean, especially on edible soybean planted in areas used to grow vegetables (Adegbite & Adesiyan 2005). The genera *Meloidogyne* is widespread and is considered, economically and agriculturally, as a very important group of plant pathogens. The host range of *Meloidogyne* is very wide as it attacks almost all plant species (Sasser 1980). Both SCN and RKN are sedentary endoparasites and they cause dramatic morphological and physiological changes in plant cells while inflicting severe decreases on yield. Chemical methods of nematode control are costly and can damage the environment, especially with contamination of ground water. Therefore, the preferred method of nematode control is the use of resistant or tolerant varieties, when available. Unfortunately, a plant with resistance to one population of nematode is often susceptible to a different population due to the wide genetic variation of nematode populations.

When a plant parasitic nematode infects a plant root, the nematode and the plant enters an intricate interactive relationship with the host that is attempting to inhibit nematode development, while the nematode's goal is to develop and reproduce. The life cycle of SCN and cellular responses of soybean to SCN infection have been documented and reviewed extensively (Bird & Koltai 2000; Endo 1964; Endo, 1965; Endo, 1992; Goverse *et al*. 2000; Lilley *et al*. 2005; Mitchum & Baum 2008; Niblack *et al*. 2006; Williamson & Gleason 2003; Abad & Williamson 2010; Klink *et al.* 2011a). The SCN egg can be found in soil and within the mature female. The second stage juvenile (J2) hatches from the egg, searches for a root of a plant host, penetrates the root epidermis, and migrates intracellularly, using its stylet and enzyme secretions to disrupt cells and force its way toward the vascular tissue.

Changes in the Expression of Genes in Soybean Roots Infected by Nematodes 79

plant cell morphology and metabolism to form a feeding structure, called the syncytium in the case of SCN or giant cell in the case of RKN. More than 60 genes have been identified that are expressed in the esophageal glands of SCN, many of which have no known function (Gao *et al*. 2001, 2003; Williamson & Gleason 2003; Davis *et al.,* 2004; Davis & Mitchum, 2005). Some of the genes encoding these proteins are similar to microbial genes or genes of animal-parasitic nematodes. Knowledge about these secreted proteins from the nematode and their interactions with targets within the plant cell during infection provides a better

During the establishment of their feeding sites, nematodes secrete into the plant cell several different proteins and enzymes made in the esophageal gland (Davis *et al*. 2004; Gao *et al*. 2001, 2003). The SCN esophageal glands produce -1,4-endoglucanase and pectate lyase to degrade the plant cell wall (Smant *et al*., 1998; Hang *et al*., 2003). Some enzymatic reactions of these nematode proteins on the cell wall may produce compounds that interact with signal transduction receptors on the plant host cells (Davis *et al*. 2004; Davis & Mitchum 2005: Mitchum & Baum, 2008). A model of a potential secretomes from plant parasitic nematode has been proposed by Davis et al. (2004) and shows involvement of cell wall remodeling proteins, such as endoglucanases, and expansions. Plant parasitic nematodes also produce proteins that may mimic plant proteins, such as chorismate mutase (Doyle & Lambert, 2003; Bekal *et al*. 2003; Lambert *et al.* 1999) and CLAVATA (Wang *et al*. 2005; Wang *et al.,* 2010; Replogle *et al*. 2010 ). Some of the secreted proteins contain a peptide sequence that targets the protein to the nucleus, while other proteins remain in the cytoplasm of the plant cell

Gene expression has been examined in both compatible and incompatible interactions of SCN with soybean roots using Affimetrix microarrays containing approximately 37,000 set of probes (Klink et al. 2007 a; 2009a, 2010, 2011b) (Ithal et al. 2007a,b). The identification of gene expression occurring specifically within the syncytium was first reported by Klink et al. (2005). The experiments provided a means for examining expression at the genomic scale. Also, changes in gene expression in the cells at the feeding site of the nematode have been examined using microarrays (Klink et al. 2007b, 2009a, 2010a, 2011b; Ibrahim et al. 2011). In all of these studies approximately two to ten per cent of the genes represented on the microarray changed in expression by over 1.5-fold. Through microarray studies, many genes were identified that are involved in metabolism, energy, defense and other areas, which provided new insights into plant-pathogen interactions. At the first phase of parasitism, which is prior to feeding or at 12 h after infection (dai), gene expression patterns in the root were found to be similar in both the susceptible and resistant reaction, when the nematode first attempts to establish itself in the host. Gene expression during the second phase depends on the defense response of the host plant (Klink *et al.*, 2007a). If the host is resistant or displays an incompatible interaction to the nematode, then gene expression patterns are different than if the host is susceptible or if the host displays a compatible reaction with the nematode, although there are some commonalities (Klink et al. 2007b, 2009a, 2010b). In either case a syncytium is formed. However, in the incompatible interaction, the syncytium degrades, whereas the syncytium is maintained and expands in the compatible interaction. During the formation of the nematode feeding sites, many pathways are involved in the induction of syncytia. For example, solidifying and lignifying

understanding of the interaction between the host cells and the parasite.

(Elling *et al*., 2007).

**2. Gene expression in soybean** 

Fig. 1. (*Left*) Female cyst of the soybean cyst nematode at 21 days after infection(dai). (*Right*) Gall formed by the root-knot nematode at 14 dai. The RKN appears red after staining with acid fuchsin.

There nematode selects a feeding cell adjacent to the vascular tissue, pierces the cell wall and injects material from its esophageal gland. The proteins injected by SCN alter the physiology and metabolism of the plant cell and surrounding cells so a syncytium is formed by dissolution of the walls of surrounding cells and the fusion of those adjacent cells The nematode becomes sedentary, feeds and molt three times to reach maturity. The anterior portion of the female SCN remains inside the root while the posterior portion breaks through the epidermis of the root at approximately 12 to 14 dai. At maturity, the outer integument of the mature female SCN hardens to protect eggs within its body, while some eggs are extruded in a gelatinous mass. SCN can complete its life cycle in three to four weeks with one female producing 200 to 600 eggs (Young, 1992). Thus, SCN can complete numerous life cycles during the soybean growing season and infest a field rapidly.

The RKN follows a similar pattern of development to that of SCN. The RKN also goes through five different developmental stages starting with the J1 which molts once inside the egg. After hatching, the motile J2 immediately searches for a plant host and infects immediately behind the root tip and migrates between the plant cells. RKN does not feed during this stage; instead it uses its lipid reserves in the gut (Eisenback & Triantaphyllou, 1991). When the RKN J2 reaches the vascular cylinder, it becomes sedentary and establishes its permanent feeding site by injection of proteins into selected parenchymal adjacent to the vascular system to form giant cells (Caillaud *et al.*, 2001). The giant cells expand and undergo multiple rounds of mitosis without cell division. After feeding for only 24 hours, the RKN molts three times to reach the adult stage (Eisenbach & Triantaphyllou, 1991). The entire body of the RKN remains within the root and infection of roots by RKN can be easily recognized by the "knots" or "galls" formed where they feed and develop (Caillaud *et al.*, 2001). The mature adult female deposits its eggs in a gelatinous mass, which remain attached to the end of the female's body and can be observed on the gall surface. One adult female can lay hundreds to thousands of eggs in three months.

It is important to reiterate that the SCN and RKN puncture the plant cell wall with its stylet to inject secretions from its esophageal glands. These secretions are important to altering the plant cell morphology and metabolism to form a feeding structure, called the syncytium in the case of SCN or giant cell in the case of RKN. More than 60 genes have been identified that are expressed in the esophageal glands of SCN, many of which have no known function (Gao *et al*. 2001, 2003; Williamson & Gleason 2003; Davis *et al.,* 2004; Davis & Mitchum, 2005). Some of the genes encoding these proteins are similar to microbial genes or genes of animal-parasitic nematodes. Knowledge about these secreted proteins from the nematode and their interactions with targets within the plant cell during infection provides a better understanding of the interaction between the host cells and the parasite.

During the establishment of their feeding sites, nematodes secrete into the plant cell several different proteins and enzymes made in the esophageal gland (Davis *et al*. 2004; Gao *et al*. 2001, 2003). The SCN esophageal glands produce -1,4-endoglucanase and pectate lyase to degrade the plant cell wall (Smant *et al*., 1998; Hang *et al*., 2003). Some enzymatic reactions of these nematode proteins on the cell wall may produce compounds that interact with signal transduction receptors on the plant host cells (Davis *et al*. 2004; Davis & Mitchum 2005: Mitchum & Baum, 2008). A model of a potential secretomes from plant parasitic nematode has been proposed by Davis et al. (2004) and shows involvement of cell wall remodeling proteins, such as endoglucanases, and expansions. Plant parasitic nematodes also produce proteins that may mimic plant proteins, such as chorismate mutase (Doyle & Lambert, 2003; Bekal *et al*. 2003; Lambert *et al.* 1999) and CLAVATA (Wang *et al*. 2005; Wang *et al.,* 2010; Replogle *et al*. 2010 ). Some of the secreted proteins contain a peptide sequence that targets the protein to the nucleus, while other proteins remain in the cytoplasm of the plant cell (Elling *et al*., 2007).
