Engineered Soybean Cyst Nematode Resistance

A variety of plant parasitic nematodes (PPNs), including the soybean cyst nematode (SCN), elicit the initiation, development and maintenance of a specialized nurse cell from which they derive their nutriment (Figure 1). Remarkably, during parasitism by the PPN, the nurse cell survives the apparently significant resource drain on the root cell that would be expect‐ ed to detrimentally impact normal physiological processes of the cell. This outcome indi‐ cates that the nematode has developed a well tuned apparatus to ensure that the root cell does not collapse and die during parasitism. In contrast, in the soybean-SCN pathosystem, the nurse cell and sometimes the surrounding cells are the sites of the defense response to the parasite (Ross, 1958; Endo, 1965). Therefore, plants have in place a mechanism to over‐ come the influence of the activities of the nematode. Identifying the factor(s) is of utmost im‐ portance in developing resistance to PPNs.


Introduction
A variety of plant parasitic nematodes (PPNs), including the soybean cyst nematode (SCN), elicit the initiation, development and maintenance of a specialized nurse cell from which they derive their nutriment ( Figure 1). Remarkably, during parasitism by the PPN, the nurse cell survives the apparently significant resource drain on the root cell that would be expected to detrimentally impact normal physiological processes of the cell. This outcome indicates that the nematode has developed a well tuned apparatus to ensure that the root cell does not collapse and die during parasitism. In contrast, in the soybean-SCN pathosystem, the nurse cell and sometimes the surrounding cells are the sites of the defense response to the parasite (Ross, 1958;Endo, 1965). Therefore, plants have in place a mechanism to overcome the influence of the activities of the nematode. Identifying the factor(s) is of utmost importance in developing resistance to PPNs.

History
Documented accounts reveal that soybean has been in cultivation for thousands of years (Hymowitz et al. 1970), beginning in Asia perhaps as early as 3,500 B.C. (Liu et al. 1997). While the natural range of soybean is East Asia, after thousands of years of cultivation a true understanding of its native range is complicated at best. However, the extensive range of wild soybean and obvious differences in its growth habit indicates that while environmental cues may be responsible for changes in soybean and plant growth habit in general (Garner Allard 1930; Chapin III et al. 1985; Day et al. 1999), genetic variation that exists in wild populations is of significant benefit to agriculture for production purposes and developing resistance to its many pathogens. This assessment is particularly true for soybean and its most significant pathogen, SCN, as many ecological collections have resulted in the identification of naturally occurring resistance (Ross and Brim, 1957;Ross, 1958  While knowledge of soybean's cultivation is long and extensive, scientific information on its dominant pathogen, the SCN, began with its description (Ichinohe, 1952). However, reports going back as early as the 1880's (Noel 1992) and late 1930s (Ichinohe, 1961) reveal knowledge of the nematode and appreciation of its pathogenic capacity. The SCN is a devastating pathogen that causes approximately 7-10% production loss, worldwide, annually and suppresses seed yield more than any other single soybean pathogen (Wrather et al. 1995(Wrather et al. , 2001a(Wrather et al. , b, 2003(Wrather et al. , 2006Pratt and Wrather, 1998). In contrast, in some fields, as much as a 15% loss in yield has been observed with no visible signs of disease on soybean (Wang et al. 2003). Observations such as these could complicate SCN management since the disease can occur without knowledge of it being present in a particular field.
The near perfect overlap of the agricultural range of soybean with the distribution of SCN, infection creates a scenario where there is a high probability of a widespread and significant effect on yield. Conservative reports have shown that there is approximately 1.1-1.5 billion dollars in agronomic losses, annually, worldwide (Wrather et al. 2001). The overlapping distribution of SCN with that of soybean production was not always the case. Historically, SCN was not found in the U.S., or likely even North America or the New World. That situation changed when the SCN was first identified in the U.S. in North Carolina in 1954 by Winstead et al. and published a year later (Winstead et al. 1955). Unfortunately for agriculture, SCN is readily transmissible as evidenced by its identification in localities as far away as Mississippi only a few years later by 1957 (Spears, 1957). The SCN now is a registered invasive species in the U.S. Notably, in the U.S., SCN causes more agricultural loss to soybean than the rest of its pathogens combined (Wrather et al. 2001(Wrather et al. , 2006. Making the problem worse for agriculture is the genetic diversity of the SCN (Golden et al. 1970; Riggs and Schmidt 1988;Niblack et al. 2002;Bekal et al. 2008). SCN research has determined that the nematode is a species complex originally subdivided into four races (Golden et al. 1970) which later was expanded into 16 races Schmidt 1988, 1991) that have been reorganized, further subdivided and reclassified into distinct populations (Niblack et al. 2002;Niblack and Riggs, 2004). The term population was designated since genetically pure clones are impossible to obtain in the sexually reproducing SCN system (Niblack et al. 2002). The classification scheme of Niblack et al. (2002) is based on the varying ability of SCN populations to infect a panel of 7 soybean genotypes that can resist infection to varying levels. It is noted that some of these designated populations are "strains" that are maintained in the greenhouse and genetically purified through hundreds of generations of single cyst descent (Niblack et al. 1993). Therefore, the genetic background in these "strains" may not resemble the original field-extracted population since allelic forms of the parasitism genes would likely be lost through this purification process.
The genetic diversity found in SCN (Golden et al. 1970; Riggs and Schmidt 1988;Niblack et al. 2002;Bekal et al. 2008) likely aids in its ability to infect and reproduce on plants other than soybean. Thus, from an ecological standpoint, SCN could pose a threat to plants that grow outside of production areas. This potential problem would be exasperated if those plant species are listed as endangered or threatened species or are a significant component of the ecological community. A number of studies have shown that the SCN reproduces on at least, but certainly is not limited to, 97 legume and 63 non-legume hosts (Epps and Chambers, 1958; Hamblen, 1962, 1966a, b) and new SCN hosts are determined on a regular basis (Creech et al. 2006). It has been many years since species range tests have been performed for SCN so it is likely that these lists of hosts are not comprehensive. This virulence capability of SCN poses a problem in terms of its management since SCN populations could be maintained by weedy plants that grow or overwinter in fallow fields or along the boundaries of acreage that is in production (Creech et al. 2006). In addition to these problems, SCN does not even have to reproduce in the plant to still cause damage to the plant. While the genetic diversity of both soybean and SCN may appear to complicate an understanding of the process of infection and the development of resistant cultivars, the natural variation in both the germplasm of soybean (Doyle et al. 1999) and SCN (Bekal et al. 2003;) presents many opportunities to understand the basic machinery of infection of the SCN and the genotype-specific nuances that regulate both susceptibility and defense. These features make the soybean-SCN pathosystem an extremely valuable experimental model (Barker et al. 1993;Opperman and Bird, 1998;Niblack et al. 2006;Klink et al. 2010a).

Methods to control SCN infection
Historically, SCN has been managed through a combination of chemical control, cropping systems, biological control and the identification and use of resistant germplasm. Chemical control for pathogens using methyl bromide first occurred in France (Schneider et al. 2003;Rosskopf et al. 2005) and had subsequently been used for decades for both pre-and post plant nematode control. However, methyl bromide is a chemical that has been phased out of use in 2005 in the U.S. (Schneider et al. 2003;Rosskopf et al. 2005) because it has been classified as a Class 1 (Group VI) stratospheric ozone depletor by the Environmental Protection Agency. Because of the loss of this major control agent for the SCN, even in developing countries by the year 2015 (Rosskopf et al. 2005), it was important to identify other strategies that could be included in the SCN management plan. Biocontrol measures that include bacteria, fungi or even their proteins are feasible (Chen and Dickson, 1996;Riggs, 1991, 1995;Liu and Chen, 2001;Meyer and Huettel, 1996;Timper et al., 1999). Other control methods that had already been used extensively for decades include the time honored crop rotation strategy. This strategy has reduced SCN populations below damaging levels (Francl and Dropkin, 1986;Sasser and Uzzell, 1991;Koenning et al. 1993). Rotating with nonhosts over a 2-3 year period mitigated the undesirable levels of SCN in the field (Ross, 1962;Francl and Dropkin, 1986). Other cropping systems that have had success in SCN control are the use of blending, resistant cultivars and cropping sequence, among others (Niblack and Chen, 2004). While successful, a problem with cropping strategies is that the interval is not long enough to compete with the 9 year cycle that cysts can remain viable, but dormant in production fields (Inagake and Tsutsumi, 1971). With these strategies in place it is possible to develop a tightly managed regime, incorporating some or all of these technologies and principles to mitigate SCN damage. Lastly, genetic engineering has begun to take root with potential as a method to generate resistance (Steeves et al. 2006;McLean et al. 2007;Klink et al. 2009a;Matsye et al. 2012). However, for genetic engineering to be successful, it is first required that candidate genes be identified. The identification of these genes has happened through a series of RNA gene expression studies, employing soybean germplasm that exhibits resistance to SCN.

Available resistant germplasm
Once SCN was identified in the U.S. (Winstead et al. 1955), a very large need existed to determine if soybean germplasm existed that could resist infection. The vast and expansive range of soybean (Morse, 1927) and visually obvious variations in growth form in its various ecological habitats provided the possibility that germplasm that was resistant to SCN existed in its wild populations. Established in 1898, the development of a substantial and publically available seed bank was initiated that is maintained by the USDA-National Plant Germplasm System (USDA-NPGS) (Morse, 1927;Bernard et al. 1987). It now contains approximately 20,000 varieties (accessions) with each accession classified as a plant introduction (PI) through a numbering system. Many of the 7,867 PIs that were already available by 1944, just 10 years before the identification of SCN in the U.S., had been collected in trips to China, Japan, India and Korea in a small window of time between 1924and 1932(Bernard et al. 1987. The public availability of the germplasm allowed it to be used in a series of trials to determine if any of the available accessions was resistant to SCN. A number of accessions were determined to be resistant to SCN through two large trials that studied about 5,700 accessions (Ross and Brim, 1957;Ross, 1958;Epps and Hartwig, 1972). Research on these accessions has resulted in the identification of approximately 118 sources of resistance (Concibido et al. 2004). However, only approximately seven sources are used for cultivar development in the U.S. (Shannon et al. 2004). These accessions include the G. max PIs known as Peking (G. max [Peking] ) and G. max [PI 88788] . Currently, G. max [Peking] and G. max [PI 88788] resistance germplasm is present in >97% of all commercial cultivars in the U.S. (Concibido et al. 2004). In addition to these PIs, hundreds of additional accessions that can resist SCN infection have been identified in China (Ma et al. 2006;Li et al. 2011). These banks of germplasm provide an important and substantial genetic resource for understanding the process of parasitism in soybean at the cellular level. This is important to understand because the infection of soybean involves very specific cell types that react in very specific ways to SCN parasitism.

Cytological reaction during resistance
The SCN can remain viable in the soil in eggs ensheathed within the carcass of the dead mother (cyst wall) for up to 9 years (Inagake and Tsutsumi, 1971). However, the devastating interaction of the SCN with soybean begins when it burrows into the root through the epidermal and cortical cells. This has been shown both by cytological studies and gene expression studies of time points collected before the formation of syncytia (Alkharouf et al. 2006;Klink et al. 2009b). The interaction continues through the initiation and subsequent formation a multinucleate nurse cell known as a syncytium from pericycle or neighboring cells (Ross, 1958;Endo, 1964Endo, , 1992. The formation of the syncytium is likely to be a very coordinated process, occurring through the injection and subsequent activity of nematode parasitism proteins (Atkinson and Harris, 1989; Lambert et al. 1999;De Boer et al. 1999, 2002Wang et al. 1999Wang et al. , 2001Wang et al. , 2003 Cytological studies of the SCN infection process ( Figure 1) have shown that the cellular response of soybean to SCN infection can be divided into an earlier phase (phase 1) and a later phase (phase 2) (Ross, 1958;Endo, 1964Endo, , 1965Endo, , 1991Riggs et al. 1973;Kim et al. 1987;Mahalingam and Skorpska, 1996). Phase 1 and 2 span the periods including the initiation, development and maintenance of the syncytium (Figure 1). These observations are not unique to SCN since similar observations have also been made for the cyst nematode Rotylechulus reniformis (Robinson et al. 1997), indicating that a basic level of conservation may exist for the process of defense at the site of infection while genotype-specific gene activities also exist (Klink et al. 2011a;Matsye et al. 2011Matsye et al. , 2012. During phase 1, the cellular reactions leading to susceptibility or defense appear the same at the cytological level. The cellular events occurring during the earlier stages of syncytium development include hypertrophy, the dissolution of cell walls, the development of dense cytoplasm, an enlargement of nuclei and an increase in endoplasmic reticulum (ER) and ribosome content (Endo, 1964(Endo, , 1965Riggs et al. 1973;Kim et al. 1987;Kim and Riggs, 1992;Mahalingam and Skorpska, 1996). The enlargement of nuclei and increase in ER and ribosome content indicate an increase in gene expression and protein synthesis accompanies the activity of the nematode within the parasitized cells. Therefore, it is likely that the plant cell is being programmed to make specific materials to benefit the nutritional needs of the nematode. It is known that plant parasitic nematodes lack the ability to make materials such as sterols (Chitwood and Lusby, 1990). Therefore, altering the metabolism of the parasitized cell probably would involve the induction of metabolic activity that relates to these processes. Cell fate mapping experiments have demonstrated the metabolism that occurs during these stages of parasitism and some of it relates to enhanced plant sterol production (Klink et  After these earlier events, the cytology of susceptibility and defense become apparent and is referred to as phase 2. Phase 2 of the susceptible reaction is characterized by hypertrophy of nuclei and nucleoli. This process is accompanied by the reduction and dissolution of the vacuole. The reduction and dissolution of the vacuole suggests important events or structural features involved in membrane fusion and/or maintenance are perturbed. This topic will be described in a later section. Other cellular events that have been identified during the susceptible reaction include cell expansion as it incorporates and fuses with adjacent cells (Endo and Veech 1970; Gipson et al 1971; Jones and Northcote, 1972;Riggs et al. 1973;Jones, 1981). Additional activities include the proliferation of cytoplasmic organelles.
In contrast, the cellular aspects of the defense responses occurring during phase 2 depend on the soybean genotype being infected. Information that has been generated through a number of cytological studies have resulted in the development of a system that divides the PIs into cohorts having similar cytological reactions that is based on the cellular characteristics associated with how SCN responds during resistance (Colgrove and Niblack, 2008). Currently, the PIs have been categorized into those genotypes having the G. max [Peking] and G. max [PI 88788] -types of defense responses (Colgrove and Niblack, 2008). Much more work in this area of research is required for a comprehensive understanding of the different forms of the defense response. Such knowledge would allow the commonalities of the cytological features to be correlated with the molecular events that are occurring in the parasitized cell types. By doing so, it would allow for the identification of genes that always correlate to re-sistance, regardless of the cytology or genotype of soybean. It would be likely that these genes are central to all forms of the defense response (Klink et al. 2011a;Matsye et al. 2011Matsye et al. , 2012. Among the characteristics that define these cohorts, the G. max [Peking] -type of defense includes the development of a necrotic layer that surrounds the head of the nematode (Kim et al. 1987;Endo, 1991). This process is followed by necrosis of the initial cell that the nematode had parasitized. In contrast, in the G. max [PI 88788] -type of defense response, the necrotic layer that surrounds the head of the nematode is lacking and the initial cell that the nematode parasitized first experiences necrosis (Kim et al. 1987;Endo, 1991). In addition to these cytological characteristics found in the G. max [Peking] and G. max [ (Mahalingam and Skorpska, 1996). This makes the placement of G. max [PI 437654] in the G. max [Peking] cohort logical (Colgrove and Niblack et al. 2008). In contrast, CWAs are lacking in G. max [PI 88788] . More work is required in the understanding the role(s) that CWAs play, if any, during defense of soybean to SCN. However, the significance and role of CWAs during defense were first demonstrated by Collins et al. (2003), and followed by additional studies performed by Assaad et al. (2004) and Kalde et al. (2007). In those studies, it was shown at the molecular level that CWA formation involves the vesicular transport machinery protein component known as syntaxin. This was a striking discovery since the process of vesicular transport is a conserved cellular process, meaning it has been found in other organisms. The syntaxin gene was first identified in animal systems (Inoue et al. 1992 Kalde et al. 2007), among other proteins. Since these numerous studies have shown very specifically how the protein complex is assembled, it was then possible to determine how specific components of the CWA assembly process that are present during defense of soybean to SCN function (Matsye et al. 2012). However, even though CWAs are lacking in genotypes like G. max [PI 88788] , it does not mean that the proteins are not involved in defense through related activities. Membrane fusion has been shown to play a role in defense through a process known as autophagy. Autophagy is a process known in plants to play crucial roles in defense (

Genomics-based studies of SCN
A number of "omics" studies in the soybean-SCN pathosystem have been performed to understand both plant and nematode gene expression at the organismal level. Many of the gene expression studies that relied on the microarray technology were modeled after earlier experiments that were performed in the model plant Arabidopsis thaliana that were infected with the beet cyst nematode, Heterodera schactii (Puthoff et al. 2003 The parasitism of soybean by SCN begins with and is sustained through the injection of materials that are synthesized in subventral and esophageal glands into the root cell. It is a costly life strategy since typically only about 10% of the nematodes ever make it to maturity in a fully susceptible genotype like G. max [Williams 82/PI 518671] . Identifying the genes involved in parasitism would likely occur through collecting the cytoplasm of the cells composing the subventral and esophageal glands. It was hypothesized that these genes would be important for the events of parasitism and would be involved in altering the metabolic processes of the soybean to benefit the nematode. The experiments were performed by microaspirating the cytoplasm of the gland cells, constructing cDNA libraries and sequencing the genes, allowing for downstream bioinformatics analyses to help elucidate what the genes could actually be (Smant et al. 1998). The experiments were then repeated for the SCN, identifying a number of putative parasitism genes Gao et al. 2001Gao et al. , 2003. With the development of the Affymetrix® Soybean GeneChip, it was possible to examine the expression of thousands of SCN genes simultaneously. This was made possible because 7,539 H. glycines probe sets representing 7,431 transcripts (genes) were printed onto the array. One analysis examined the expression of SCN genes that were expressed specifically during infection of the G. max [Williams 82/PI 518671] genotype that lacks a functional defense response (Ithal et al. 2007). This means that gene expression occurring during a susceptible reaction was monitored. The work examined the expression of previously identified Gao et al. 2001Gao et al. , 2003 and analyzed (Bakhetia et al. 2007) putative parasitism genes (Ithal et al. 2007). The remaining genes that were fabricated onto the array were not a focus of the analysis. The experiments confirmed the expression pattern of dozens of putative parasitism genes (Ithal et al. 2007). A gap in the knowledge was that the experiments were not designed to determine what genes were expressed as the nematode experienced a resistant reaction in a soybean genotype that was capable of a defense reaction. This information would be important because it would provide knowledge on the metabolic pathways that may be sensitive to genetically-based control measures. That gap in knowledge was filled in experiments that performed population-specific analyses of gene expression, comparing the susceptible and resistant reactions experienced by SCN as they infected the G. max ) that was available for essentially every gene in the genome housed in the C. elegans database at http://www.wormbase.org. Since the genomic sequence of C. elegans is known, it is possible to find highly conserved and related genes in SCN. The working hypothesis was that if the genes in C. elegans and SCN are nearly identical in primary sequence, it would be likely that they have similar function. If the genes have similar function, for example an essential function for survival in C. elegans, knocking out that gene in SCN would probably result in lethality for those nematodes if the gene could be knocked out. The annotation of the SCN genes was driven by a homology criterion whereby the SCN genes were pooled into six bins referred to as Groups 1-6 ( Alkharouf et al. 2007). The six bins were based on the level of homology the sequence had to C. elegans genes. Group 1 had the highest level of homology and Group 6 had the lowest level. For example, Group 1 had E-values between 0 and 1E-100; Group 2 had E-values between 1E-100 and 1E-80; Group 3 had E-values between 1E-80 and 1E-60; Group 4 had E-values between 1E-60 and 1E-40; Group 5 has E-values between 1E-40 and 1E-20 while Group 6 has E-values > 1E-20 (Alkharouf et al. 2007). The gene annotation process resulted in taking the nearly 8,334 conserved genes between H. glycines and C. elegans and identifying 1,508 that have been shown to have lethal phenotypes/phenocopies in C. elegans (Alkharouf et al. 2007). The research then was poised to test the function of the 1,508 genes, but it was an unmanageable number of genes. To narrow down the 1,508 genes to a manageable number for functional studies, the genes underwent further annotation procedures (Alkharouf et al. 2007). To do this annotation procedure, firstly, a pool of 150 highly conserved, Group 1, H. glycines homologs of genes having lethal mutant phenotypes or phenocopies from the free living nematode C. elegans were identified from the pool of 1,508 genes that were fabricated onto the Affymetrix® microarray. Secondly, it was determined that of those 150 genes on the Affymetrix® soybean GeneChip, a subset of 131 genes could have their expression monitored during the parasitic phase of their life cycle. Thirdly, a microarray analyses identified a core set of 32 genes with induced expression occurring during the parasitic stages of infection. The identification of 32 genes that had known expression during the parasitic stages of infection provided a small, but feasible, core set of genes that could be targeted in RNAi-based, reverse genetic screens (Table 1).

Reverse genetic screens to identify essential SCN genes
Unlike C. elegans, SCN is not an ideal system for genetic studies because of its obligate endoparasitic life cycle. However, from information learned in C. elegans, gene function can be studied by an mRNA nuclease process called RNA interference  . 2007, 2008). The problem with these experiments, from a nematode biocontrol perspective, is that it would be virtually impossible to synthesize, apply and deliver enough dsRNA to nematodes that are living in the environment to obtain a positive effect even though crude dsRNA extracts can be used (Tenllado et al. 2003). Other problems would be whether the dsRNA remained residually in the soil. Therefore, a second method would be needed that could express the genes as dsRNA in soybean, allowing greater control over the delivery of the dsRNA to SCN.  The problem with the transgenic approach is that soybean is a difficult to genetically engineer. However, strategies whereby composite plants (Collier et al. 2005) that are chimeras having nontransformed aerial stocks having transgenic root stocks can be readily made in soybean (Klink et al. 2008(Klink et al. , 2009a. . These observations demonstrated that broad spectrum resistance for PPNs in soybean was probable.

Proteomic studies of SCN
The prior experiments have discussed gene expression in SCN at the RNA level. These experiments are technologically simplistic to perform, because of major advances in sequencing and detection technologies. However, in these experiments using hybridization to study gene expression, little to no information is obtained as to how much protein is actually synthesized from the RNA or modifications that are known to exist on the protein molecules.

Soybean gene expression
To understand how soybean was reacting to infection, it was going to be imperative to develop ways to monitor gene expression during infection. Unlike the model system, A. thali-  (Klink et al. 2007b). All of these proteins were suppressed in their activity during the resistant reaction. However, the problem with the vast amounts of data that was being generated at the time was in obtaining a meaningful annotation that would provide an understanding of the global events occurring in the sample types.

Improvements in annotation
The described experiments resulted in the generation of a massive amount of gene expression data and gene lists for the 38,099 genes fabricated on the Affymetrix® soybean Gene-Chip. Annotated gene lists for soybean genes are very useful because no information is lost from the analysis (Table 1). However, the gene lists do not provide higher order knowledge of how the many genes are functioning during a process under study. It is possible that various metabolic pathways that pertain to a specific process could be identified if the data could be organized into a higher order structure. Since the aforementioned work was done in soybean, often considered a non-model organism, it was difficult to translate the information into gene pathway analyses applications in a manner that would reveal how the gene expression is orchestrated during the process under study. However, an investigation that had been done in A. thaliana infected with Pseudomonas syringae pv. tomato did show how useful the higher order gene expression knowledge could be in allowing for a visualization of the switch in metabolism from housekeeping to pathogen defense during infection (Scheideler et al. 2001

Genomics of the syncytium
While strides were being made in obtaining a deep analysis of the physiological processes occurring in whole infected roots, the greater challenge would be to identify gene expression that occurred within the syncytium because it would require either drawing the cytoplasm out of the syncytium or a way to physically isolate the cells. The original studies that attempted to determine gene expression in nematode nurse cells was done by Bird et al. (1994) and Wilson et al. (1994). The hypothesis was that by extracting the cytoplasm of the cells that are specifically undergoing the parasitism, it would be possible to determine the gene expression that pertains specifically to parasitism. However, it is noted that gene expression in the cells surrounding the syncytium probably plays some role in the maintenance and development of the susceptible and resistant reactions. This approach to isolate the cytoplasm Wilson et al. 1994) would be more challenging for syncytia because it is virtually impossible to determine what cells are infected by SCN. Therefore, instead of collecting the cytoplasm, the collection of the cells would have to occur and it would have to be done through their physical isolation.
The physical isolation of syncytia undergoing a susceptible reaction to the SCN was first described by Klink et al. (2005). The study collected syncytia by a procedure called laser microdissection (  In these studies, a number of genes were identified. However, to understand gene expression as it specifically pertains to defense, it would be required to study the cells undergoing the defense response. The main obstacle in performing studies with this goal in mind was determining whether the cells undergoing the defense response were already dead at the time of cell collection. The prediction is that cells that were dead would have already halted their physiological processes that pertained to defense and also may not provide RNA of suitable quality for microarray studies. However, it was unlikely that the cells progressing through the earlier stages of defense were dead (Figure 1) since the EM studies revealed very specific progression of cellular architecture during the defense response, suggesting that the cells had to be alive to progress through this developmental process (Endo, 1965;Kim et al. 1987;Endo, 1991). The initial collection of syncytia undergoing the developmental process that leads to their eventual collapse and death were then performed (Klink et al. 2007a). These experiments demonstrated that the cells would be a suitable source for RNA collection and genomics-based analyses. The first set of experiments to use laser microdissected cells undergoing an incompatible reaction for genomics studies determined that the expression of lipoxygenase (LOX), arabinogalactan-protein (AGP18), annexin, a thioesterase family protein heat shock protein (HSP) 70 and superoxidase dismutase (SOD) (Klink et al. 2007a). Many of the genes have very well known roles in plant defense. Subsequent experiments examined more time points occurring during the defense response, spanning phase 1 and phase 2 (Klink et al. 2009b(Klink et al. , 2010b. The experiments identified a number a genes that were very highly expressed during the resistant reaction, specifically within the syncytium. In contrast, a number of genes were very highly suppressed (>1,000 fold) during the resistant reaction (Klink et al. 2009b(Klink et al. , 2010b. The experiments were repeated later by Kandoth et al. (2011) in the G. max [PI 209322] genetic background that either has or lacks the rhg1 resistance locus. During this time, studies were also performed that examined and compared multiple forms of the resistant reaction that were found in the G. max [ and G. max [PI 88788] genotypes are well known to undergo different forms of the resistant reaction at the cellular level (Kim et al. 1987;Endo, 1991;Mahalingham and Skorupska, 1996). The G. max [Peking/PI 548402] and G. max [PI 88788] PIs are also important genotypes to obtain knowledge from because they are the source of >97% of the resistance germplasm used in commercial breeding programs (Concibido et al. 2004). In some of the earlier studies (Klink et al. 2009b), a number of genes were identified that were induced preferentially in their expression during the resistant reaction. The genes included lipoxygenase, S-adenosylmethionine synthetase, a dnaK domain-containing protein, GRF2 GENERAL REGULATORY FACTOR 2, ACT7 (actin 7), major latex protein-related protein, xyloglucan endotransglucosylase/ hydrolase protein 26, cytochrome P450 monooxygenase CYP93D1, pyruvate dehydrogenase E1 beta subunit isoform 2, nitrate transporter (NTP2), endo-1,4-beta-glucancase that were all expressed preferentially between 100 to 383-fold higher in syncytia undergoing the defense response as compared to syncytia undergoing the early stages of a susceptible reaction (Klink et al. 2009b). Additional experiments aided by Illumina® deep sequencing technology which is a sequence by synthesis procedure much like quantitative PCR, but for every gene in the genome simultaneously, identified genes that were expressed only in syncytia undergoing the defense response (Matsye et al. 2011). Some of the genes were expressed at all times during the defense response. Importantly, the Illumina® deep sequencing technology revealed that some of the transcripts that are genes known to be important in defense responses represented between 1 and 17% of the sequenced tags from RNA isolated from the syncytia undergoing the defense response (

Soybean resistance clusters
The major SCN resistance trait, rhg1, was first identified by Caldwell et al. (1960). In and around the same time, four other major loci, the recessive rhg2, rhg3 (Caldwell et al. 1960) and the dominant Rhg4 (Matson and Williams, 1965) and Rhg5 (Rao Arelli 1994) have been identified. In all, there are approximately 61 QTLs that associate with resistance to SCN (Kim et al. 2010). Many of the details of the numerous mapping studies can be found in a review by Concibido et al. (2004). Of all of the loci that associate with resistance to SCN, the best studied is rhg1. It is a major resistance locus and has been fine mapped to a region defined in a span of approximately 611,794 nucleotides between the molecular markers ss107914244 and Satt038 on chromosome 18 (Concibido et al. 1994 (Matsye et al. 2011). What was notable about the analysis was that AAT and α-SNAP were shown to be expressed throughout the defense response in experiments that sampled time points at 3, 6 and 9 days post infection (dpi), spanning phase 1 and 2 (Matsye et al. 2011). The AAT and α-SNAP genes did not appear to be expressed in syncytia undergoing the susceptible reaction. This difference in expression that was occurring between the resistant and susceptible reaction made it possible that the genes could be involved in the defense response. However, this would only be determined in functional studies that tested how the gene acted during infection (Matsye et al. 2012).

Genetic engineering as a solution for SCN
A number of approaches like conventional breeding programs have been shown for decades to generate resistance to SCN (Brim and Ross, 1966). The resistant cultivars have been shown to result in savings of hundreds of millions of dollars (Bradley and Duffy, 1982). One drawback of conventional breeding programs is that along with the resistance genes that are bred in, a number of genes are also introgressed that could have undesirable characteristics. This is especially a problem when is desirable traits are tightly linked to the undesirable traits. To circumvent this problem, it is possible to genetically engineer in genes of interest. A number of strategies that have been described in this chapter have shown promise in disrupting the soybean-SCN interaction. As noted earlier, RNAi of nematode parasitism genes has been shown in the Arabidopsis thaliana-Meloidogyne sp. system to perturb giant cell formation (Huang et al. 2006). This was also shown to work in the soybean-SCN pathosystem (Steeves et al. 2006

Conclusion
The soybean-SCN pathosystem has been under study for over 60 years. Through a massive amount of basic studies involving agricultural production practices, genomics and genetic engineering, solutions to the chronic and global SCN problem are emerging. The difficulty of studying the system has been met with many improvements in technology that are allowing for basic features of the pathosystem to be exploited so that agricultural practices and economic returns are improved. The basic knowledge gained in this system can now be ap-