Plant parasitic


Chapter One:

Research Proposal

1.1. Background or Justification of the Study

Plant parasitic nematodes are a major pest of many crop species. They form a very diverse group, of which the sedentary root endoparasites, comprising the cyst and root-knot nematodes, are the most evolved and interact intricately with their host (Tytgat et al., 2000). Upon infection, the second-stage juveniles of cyst nematodes penetrate into the root tissue and migrate intracellularly toward the vascular cylinder, where they induce a syncytium by injecting pharyngeal gland secretions into a selected initial feeding cell (Davis et al., 2000). In response to the nematode stimulus, the initial feeding cell rapidly enlarges, the central vacuole disintegrates into several small vacuoles, and organelles, such as the endoplasmic reticulum, ribosome, mitochondria, and dictyosomes, rapidly proliferate (Fenoll et al., 1997). In the process of syncytium formation, neighboring cells react in a similar way, and partial cell wall dissolution between these cells occurs a few hours later, followed by fusion of the protoplasts. When the feeding cell further enlarges, the normal vascular structure is destroyed, resulting in a severely disturbed solute transport (Gheysen and Fenoll, 2002).

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According to Gheysen and Fenoll (2002) feeding site formation involves drastic changes in gene expression in these root cells. The feeding site serves as nutrient source for the developing nematode, which alternates cycles of cytoplasm withdrawal and injection of pharyngeal gland secretions, both by means of a hollow protrusible stylet (Wyss and Zunke, 1986). The cause of the formation of the feeding cells and the reactivation of the cell cycle is still unknown. Three pharyngeal glands are recognized, two sub ventral and one dorsal. In pre parasitic juveniles, the two sub ventral glands are very active, but their activity and size drastically decrease during root penetration and migration prior to feeding cell induction (Tytgat et al., 2002; Wyss, 1992). In contrast, the dorsal pharyngeal gland increases enormously at the onset of the parasitic life stages. Until now, several genes encoding pharyngeal gland secretions have been identified (Davis et al., 2000; Gao et al., 2003).

However, most of those with a known function are sub ventral gland-specific and are involved in weakening of the cell wall during intracellular migration of the juvenile in the root tissue (De Meutter et al., 2001; Smant et al., 1998).

A novel type of secreted Ubiquitin extension protein is expressed in the dorsal pharyngeal gland of both H. glycines (Gao et al., 2003) and H. schachtii (Tytgat et al., 2004). Ubiquitin is a protein of 76 amino acids found in all eukaryotic cells, whose sequence is very well conserved. It is a globular protein, the last four C-terminal residues (Leu-Arg- Gly-Gly) extending from the compact structure to form a tail important for its function. Ubiquitin is known to play crucial roles in a large variety of biological processes (Ciechanover, 1994; Ciechanover and Schwartz, 1994). The C-terminal domains of the cyst nematode proteins are novel sequences of currently unknown function. The short C-terminal domain peptide may have a regulatory role in syncytial formation, with the Ubiquitin domain acting as its chaperone (Tytgat et al., 2004).

In eukaryotes, there are many genes coding for Ubiquitin (Hershko et al., 2000; Jentsch and Pyrowolakis, 2000). They are classified into three groups, polyubiquitin, ubiquitin-like, and Ubiquitin extension proteins. In the Ubiquitin extension proteins, a ubiquitin monomer is fused to a C-terminal extension protein. The initial translation products of these genes are accurately and rapidly cleaved in vivo by Ubiquitin C-terminal hydrolases (Wilkinson, 1997). The extension proteins are constituents of mature ribosomes (Finley et al., 1989). The actual function of the monoubiquitin domain in these fusion proteins is still unclear, although it could act as a chaperone. Many Ubiquitin extension proteins have been described with C-terminal domains of various lengths (Callis et al., 1990; Jones and Candido, 1993). However, all C-terminal domains of these proteins are highly basic, contain a 4-cysteine residue sequence in consensus with the zinc finger domain present in nucleic acid-binding proteins (Klug and Rhodes, 1987), have a consensus sequence for nuclear localization (Kalderon et al., 1984), and show a high degree of homology among different species (Callis et al., 1990).

In recent months, Vivian Blok and John Jones have identified a secreted Ubiquitin-like protein from Globodera pallida. In Glododera pallida there are two forms of the protein present.

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The two forms vary in the sequence of the C-terminal extension present in the protein.One form of the protein seems to be selected for if G.pallida grows on potato lines that have partial resistance. However, in parent lines, nematodes that have this selected version of the protein seem to produce fewer offspring than those with the "unselected" version - there seems to be some sort of penalty for having this "selected" version of the protein ( Despite these significant insights into the characterization of ubiqutin-like protein from Globodera pallida much remains to be elucidated about the features of it in nematode -plant interaction interface. Therefore, this research proposal is designed for the following objectives.


  • To explain the negative roles of the selected version of Ubiquitin like protein in reproductive ability of Globodera pallida.
  • To understand the positive roles of the selected version of Ubiquitin like protein in parent line R genes
  • To be able to identify the rationales behind the dependency of selection of the ‘selected Ubiquitin like proteins version' from partial resistance potato cultivars.

1.2. Questions and Hypotheses of the Study

The research investigates two questions:

  1. Does the presence of the selected version of Ubiquitin like protein in ‘Newton population' affect the reproduction rate?
  2. Does the selection of the selected version of Ubiquitin like protein potato cultivar dependant?

By comparing susceptible potato cultivar (Desirre) inoculated with and without this ‘selected version' of Ubiquitin-like protein the effect on the number of offspring produced can be investigated. By comparing partial resistant and strongly partial resistant potato cultivars (santé and vales Everest), respectively with and without the ‘selected Ubiquitin-like protein version', the effect can be determined (Santé/Newton, Vales Everest/Newton Vs Santé/Halton, Vales Everest/Halton).

The selected version of protein will only be selected from partially resistant cultivar (Santé) not from (Desiree & Vales Everest). The Newton population will have fewer offspring in parent lines. Therefore, the negative role of having this selected version of Ubiquitin like protein in reproduction for Globodera pallida and the positive role in R genes in parent lines can be inferred.

Null hypothesis (Ho) 1: G. pallida (Newton)population with selected version of Ubiquitin like protein will have no difference towards the number of offspring being produced from G. pallida (Halton) population with unselected version of proteins in parent lines.

Null hypothesis (Ho) 2: The selection of the ‘selected version of Ubiquitin like protein' will not be cultivar dependant. It will be isolated from cysts that will come out of the susceptible, partially resistant and strongly resistant potato cultivars

Chapter Two: Literature Review

2.1. Plant Parasitic Nematodes

Plant-parasitic nematodes are microscopic roundworms tapering towards the head and tail. Females of a few species lose their worm shape as they mature, becoming pear-, lemon- or kidney- shaped. Plant parasitic nematodes possess all of the major organ systems of higher animals except respiratory and circulatory systems. The body is covered by a transparent cuticle, which bears surface marks helpful for identifying nematode species. Plant-parasitic nematodes have rigid, pointed mouth-spears or stylets, usually hollow that are used to penetrate plant cells and feed. The presence of a stylet differentiates plant parasites from free-living nematodes in soil (although some fungal-feeding nematodes also possess stylets).

Most plant parasitic species feed on roots, but some feed on plants' foliar tissues. Among those nematodes infecting plant roots, some are ectoparasitic (the stubby-root nematode, the sting nematode, and the ring nematode) and others are endoparasitic (Root knot nematodes and cyst nematodes). They differ in that ectoparasitic nematodes feed on plant tissues from outside of the plant, whereas endoparasitic nematodes feed on plant tissues from within the plant. Nematodes can be further broken down into migratory (e.g. lesion nematode)and sedentary types. If the adult female moves freely through the soil or plant tissues, the nematode is said to be migratory. An adult female that is immobile and remains at one area of the root is termed sedentary.

The life cycle of plant-parasitic nematode begins as an egg. The embryo develops into a juvenile. Juveniles grow and molt to the next juvenile stage. Usually, the first molt occurs inside the egg so that it is the second-stage juvenile that hatches from the egg. After four molts and four juvenile stages, nematodes mature into adult males and females. Since plant-parasitic nematodes are obligate parasites, they must feed on living plant tissue at some point in their life to complete their life cycle.

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Plant-parasitic nematodes damage plants in a number of ways. Feeding causes wounding, this creates openings through which other pathogens may enter the plant.

They also disrupt the vascular tissue, which reduces the transport of water and minerals from the root system up to the leaves and stems of the plant. Evidence of nematodes feeding on roots includes the following symptoms: lesions, stubby, curled, or galled roots, and a reduced root system.

Although the majority of plant-parasitic nematodes feed on the roots of plants, there are some that feed on above ground plant parts. Two important types are the foliar nematodes (Aphelenchoides) and stem and bulb nematodes (Ditylenchus dipsaci). The nematodes swim up from the soil in water films when plants are wet. Feeding nematodes use water, minerals, and nutrients made by the plant during photosynthesis, which would otherwise be available to support plant growth. Nematodes produce damaging enzymes and other disease-inducing compounds. Such damage causes stunting of plant growth, reduced crop yields, and may lead eventually to plant death.

Plant-parasitic nematodes may attack plants together with other plant-pathogenic microorganisms, such as fungi. The result is often synergistic, in that the incidence and severity of the disease caused when both organisms are present are much greater than that caused by either organism alone. Some plant-parasitic nematodes transmit viruses; they acquire the virus when feeding on a virus-infected plant and later pass the virus on when they feed on other plants.

Determining if plant parasitic nematodes are involved in a plant growth problem is difficult because few nematodes cause distinctive diagnostic symptoms. A sound diagnosis should be based on as many as possible of symptoms above and below ground, field history, and laboratory assay of soil and/or plant samples.

2.2. Potato Cyst Nematodes (PCN)

Potato Cyst Nematode (PCN) is a serious pest of potatoes, tomatoes and eggplants world-wide and is subject to stringent quarantine and/or regulatory procedures wherever it occurs. PCN can reduce crop yield significantly, increase production costs, and result in trade restrictions for potatoes. PCN lives in the soil and can be easily spread by the movement of host plants or soil attached to plants, bulbs, advanced trees and agricultural equipment. The female forms cysts on the roots of host plants which can detach and survive in the soil for up to 20 years.

There are two species of PCN, Globodera rostochiensisand Globodera pallid.PCN can be a devastating pest of potatoes in temperate regions if not controlled. Current approaches to combat agricultural losses are the use of nematicides, cultural techniques and resistant varieties that may be used in an integrated manner. Nematicides include some of the most hazardous compounds used in agriculture and alternative control is required urgently, because of health and environmental concerns over their use.

2.2.1. Life Cycle and Biology

The complex cyst nematode life cycle consists of eggs and a distinct free living pre- parasitic stage in the soil and parasitic stages inside the root tissue. Once hatched from eggs, the second-stage juvenile (J2) of cyst nematodes does not feed in soil and thus must have a behavioral strategy that makes efficient use of its lipid reserves to find a host plant; if these reserves are depleted by more than 65% of the original level the juvenile is unable to invade plants and establish a feeding site (Robinson et al., 1987). Therefore, to establish a successful parasitic relationship with plants, nematodes rely on behavioral strategies based on their well developed nervous system, including specific sense cells and also on special structures (such as the stylet and large pharyngeal glands) for efficient root-cell penetration and modification, and food withdrawal and digestion.

The J2s of cyst forming nematodes are attracted to and penetrate plant roots to migrate intracellularly towards the vascular cylinder, where they establish an intimate nutritional relationship with their host through the development of syncytial feeding sites. During nematode development, the syncytium continuously increases in volume by incorporation of neighboring cells through cell wall breakdown. The syncytium becomes a large multinucleated hypertrophied cell generated by the fusion of as many as 200 neighboring protoplasts after partial cell wall dissolution (Jones, 1981). The juveniles undergo three additional molts before reaching the adult stage.

When feeding commences, the juvenile body grows and becomes saccate and immobile. The vermiform males regain their mobility and leave the root to migrate in the soil, where they are attracted to the females by a pheromone and fertilization occurs. The fertilized females produce eggs, most of which remain inside their bodies.


They become a protective cyst when they die eventually the cyst becomes detached from the roots at the end of the growing season (Evira, 2006) and remains dormant in soil until presence of a suitable host crop is detected. The dormant period allows the nematode to survive until favorable conditions return and nematodes can persist in soil in cyst form for more than 20 years (Karssen and Moens, 2006). When favorable conditions commence the J2 will hatch which is driven by or under stimulus from host root exudates (hatching factors) and migrate towards a new host root (Jones, 1981).

2.2.2. The Hatching Process

In plant-nematode interrelationships the hatching process is critical (Perry, 1987). There are three major factors that have an effect on cyst nematode hatching, namely host root diffusate, physical conditions before and during juvenile emergence, and hereditary preconditions of the cysts (Manduric, 2004). The hatching mechanism of PCN has been shown to be stimulated by PRD and by different hatching factors such as temperature, oxygen availability, soil type and water content of the soil.

  1. Potato root diffusate: Peak production of PRD is limited to a short period in the plant's life and in this short period the juveniles are stimulated to hatch. Maximum activity of PRD (with hatch inhibitors and stimulants) is reached two weeks after planting (Perry, 1998).
  2. Temperature: It is an essential factor in the hatching of PCN. PCN requires a relative stable temperature regime to hatch (Perry, 1998). Franco et al.(1998) showed that the optimum hatching temperature in the field is 13.4 but substantial emergence can be observed at 10. G. pallida is adapted to lower temperatures (16) (Robinson et al., 1987). At the optimum temperature the fastest growth, reproduction, high metabolic activities etc. occur. At temperatures below and above the optimum, the rates are slower or death occurs (Wharton, 2002).
  3. Soil and Water Status: Soil structure and soil water status are vital factors for maximum hatching of PCN and all other soil borne nematodes. Coarse- textured soils favor hatching and when the water content is at field capacity the maximum hatching occurs. Drought and water-logging inhibit the hatching of plant parasitic nematodes (Perry, 1998).
  4. Potato cultivar: Potato cultivars can be divided into fully susceptible, partially susceptible and resistant cultivars. A fully susceptible plant allows the nematode to multiply freely on the roots while the partially susceptible plant allows less multiplication. The resistant cultivars multiplication does not occur, but the juvenile can still enter the plant root (Whitehead and Turner, 1998).
  5. Signals between plants and organisms: Nematodes and other organisms are influenced by host signals as regards hatching, host location and selection, feeding-site location, sex determination and intra-host migration. Signals contain information, which react with a receptor and elicit a response. Most signals between organisms and their host plant/s are based on phyto- chemicals from the host plant, but the organism can also release chemicals to the host plant. The host plant then starts to defend itself (Hirsch et al., 2003).
  6. Biotic factors: Studies have demonstrated that soil micro-organisms living in the rhizosphere play a significant role in the hatching of cyst nematodes (Ryan and Jones, 2004).

To sum up, at least three requirements must be fulfilled for efficient hatch: the physiological mechanism of the nematode must be operating efficiently; the nematode must be highly mobile; and it has to possess the anatomical requirements for locomotion and penetration out of the egg (Wallace, 1973). Movements inside the egg can be considered an adaptation that enables the nematode to reach a pitch of physiological efficiency at the time it hatches. The stylet, which is necessary for the escape out of the cyst, is produced. The juvenile of both PCN species makes a line of overlapping punctures with its stylet, forming a slit through which the larva escapes (Wallace, 1973).

2.2.3. Pathotypes and Races

Variations in the ability of nematodes to reproduce on a given plant species or cultivars are of great agricultural significance and are of two principal types. Nematode populations distinguished by their ability or inability to reproduce on designated plant species are known as host races. Pathotypes are variants of a host race or species, which are distinguished by their

ability to reproduce on a designated host plant genotype (e.g. cultivar, line, etc.).

As resistant varieties were developed as a means of controlling cyst nematodes in several major crops, it became apparent that genetic variation existed within populations able to overcome such resistance (Cook and Rivoal, 1998); these are called virulent populations. This led to the growing realization that within cyst nematode species, populations that were morphologically identical had distinct differences in their virulence. Based on the ability or(inability) of populations within each species to reproduce on a range of ‘differential' host plants various pathotype schemes for the major cyst nematodes were proposed, with ‘pathotype' being regarded as a group of individual nematodes with common gene(s) for (a)virulence and differing from gene or gene combinations found in other groups. The function of pathotype classifications is to provide evidence as to the potential effectiveness of resistance against a range of populations of particular crop nematode pests. The information from these schemes has to be useful in plant breeding in directing the choice of resistance sources. Such a practical function means that the classifications do not always provide precise evidence on gene-for-gene interactions. Thus, many of the differential potatoes have ‘cryptic' resistance genes whose existence was only revealed as the differentials were tested against more populations from wider areas. However, since the pathotype scheme was published (Kort et al., 1977), various attempts have been made to use biochemical and molecular techniques to find markers to identify pathotypes rapidly. All these studies have shown that it is not possible to link these groupings with the present pathotype designation of populations.

Despite the various limitations of the various Pathotypes/race schemes for cyst nematodes, they continue to give a useful indication of the virulence characteristics of particular nematode gene pools. As such, they can provide critical information necessary for effective management and the emergence of new virulent strains.

2.2.4. Management and Control Strategies

Management of PCN is difficult. Various control strategies have been developed, including chemical control, crop rotation (Evans & Haydock, 2000) and nematode resistance in different potato cultivars.

From an economic point of view the resistant cultivars are the best option in managing PCN, but are not always available. Some control methods such as chemicals and hot water treatment are recommended to eradicate nematodes in the soil. To prevent nematode spread by machinery, it is important to have compulsory cleaning of all machinery after use (Evira, 2006). To reduce the risk of heavier infestations, use of an appropriate crop rotation is recommended. The crop rotation must include resistant cultivars so that adult nematodes die of starvation (Karssen and Moens, 2006).

  1. Prevention: Fundamentals to the prevention of cyst nematodes spreading into uninfested regions is the use of certified planting material, and strict legislation for those commodities being traded both internationally and locally. Efficient management and containment of an infestation may be compromised by the ease with which cysts can be dispersed by, wind, in small aggregates of soil, on small roots attached to other parts of plants, by flood water runoff or by adhering to machinery or animals passing through infested land. General hygiene practices should be adopted in higher risk situations when the pest is known to be present in the locality. Such measures would include cleaning machinery both before and after use, restricting movement of soil outside the field boundary and construction of natural wind breaks.
  2. Crop rotation: Where host range is limited, crop rotation has proved an important component in managing cyst nematode levels. Alternative non-host crops can safely be cultivated, during which time a combination of spontaneous hatch and natural mortality will reduce the field population to below threshold levels.
  3. Resistance: Cultivar resistance remains the most economical practice for managing cyst nematodes, although these are not always available. Resistance of major crop hosts to Globodera spp has been found and attempts made to incorporate it into commercial cultivars. In many cases resistance is found only in wild species, with the accompanying inherent difficulties of transferring this trait into commercial cultivars (Riggs and Schuster, 1998). The inappropriate continuous growing of resistant cultivars is now known to increase selection pressure for virulent populations, limiting the durability of resistant in some cultivars, or resulting in the increase of other nematode problems.
  4. Biological control: Cyst nematodes would appear to be the perfect target for the use of biological agents in their management. Eggs of cyst nematodes are contained either inside the female's body/cyst or in gelatinous sac, so they should be very susceptible to parasitism by fungi or bacteria in the rhizosphere (Riggs and Schuster, 1998).
  5. Chemical control: Nematicides have been very effective for controlling cyst nematodes (Whitehead, 1998) but several of the most effective have now been withdrawn due to health and safety concerns and others are under threat. Another factor in the effectiveness of nematicides is biological degradation by soil organisms, which may be increased by the multiple use of nematicides .nematicides have been used extensively as management strategy for cyst nematodes that produce only one or two generations a year, such as PCN on potatoes.
  6. Integrated control: The repeated use of a single control measure is likely to fail, sooner or later, from selection of virulent biotypes, accelerated microbial degradation of nematicide or possible selection of more persistent populations of the nematode; in general, selection of individuals unaffected by any control measure that may be applied. The potential in managing the cyst nematodes by combining two or more control strategies in an integrated program has been widely demonstrated. Usually a crop rotation is practiced, alongside additional measures. The advantage of this approaches include the use of partially effective strategies and protection of highly effective ones that are vulnerable to nematode adaptation or environmental risk; examples include integrated control of G. rostochiensis and G.pallida in Europe (Roberts, 1993).

2.3. Establishment and Maintenance of Parasitism

2.3.1. Penetration and Migration

Endo-parasitic nematode species must penetrate host tissues directly, using mechanical and/or biochemical methods. For plant-parasitic nematodes, a cell wall composed primarily of cellulose poses a formidable barrier to penetration. Thrusts of the nematode stylet combined with esophageal gland secretions mediate penetration and migration through plant tissues (Sijmons et al., 1994; Hussey and Grundler, 1998). Plant-parasitic nematodes possess a magazine of hydrolytic enzymes for digesting cell wall polymers. Genes encoding secreted cell-wall-modifying enzymes have been localized to nematode esophageal gland cells including enzymes

that degrade the pectic polysaccharides (pectate lyases and polygalacturonases) comprising the middle lamella between plant cells (Popeijus et al., 2000; Jaubert, 2002) and enzymes that degrade the cellulose (endoglucanases) and hemicelluloses (xylanase) structural components of the cell wall (Davis et al., 2000; Hussey et al., 2002; Jasmer et al., 2003). Fascinatingly, the cell-wall-modifying enzymes appear to be active only in the sub ventral gland cells and, in the case of the cyst nematode endoglucanases, they are only active during nematode migration within roots (Wang et al., 1999; Davis et al., 2000; Goellner et al., 2001; Jasmer et al., 2003), whereas plant endoglucanases up regulated in feeding sites probably modify the walls comprising these specialized cells (Goellner et al., 2001). The discovery of a gene encoding a secreted homolog of plant expansins (Cosgrove et al., 2002) is expressed in the sub ventral glands of the potato cyst nematode is novel, and the observed plant cell wall expansion activity of the encoded protein suggests a unique mechanism for nematodes to ‘loosen' cell walls to accommodate intracellular migration of infective juveniles through plant tissues (Qin, 2000).

2.3.2. Feeding Cell Formation and Feeding Tubes

Nematodes arrange complex root cell modifications leading to the development of specialized feeding cells (Sijmons et al., 1994; Hussey and Grundler, 1998; Gheysen and Fenoll, 2002; Jasmer et al., 2003). Upon hatching, pre-parasitic J2 have a series of parasitism proteins already packaged in secretory granules within their sub ventral gland cells (Hussey, 1989). Although many sub ventral gland secretory proteins consist of cell wall- degrading enzymes used by J2 to facilitate the invasion of roots (Jasmer et al., 2003), other parasitism proteins synthesized in the sub ventral gland cells (Ding et al., 1998; Lambert et al., 1999; Ding et al., 2000; Gao et al., 2001; Gao et al., 2003; Huang et al., 2003) might have other roles in the parasitic processes, for example, the induction of feeding cells. The growth and predominant activity of the dorsal gland cell during and after feeding cell formation suggests both a role in feeding site induction and regulation of feeding cells (Hussey, 1989). To complete their life cycle, sedentary nematodes depend entirely on the successful induction and maintenance of specialized feeding cells (Davis et al., 2000)

Secretions of cyst, root-knot and a few other sedentary endoparasitic nematodes produce a ‘feeding tube' that appears to serve as a molecular sieve for host cell contents that enter the stylet orifice during ingestion (Hussey and Mims, 1991; Hussey and Grundler, 1998). The crystalline feeding tube of sedentary phyto-nematodes is a unique structure with no known counterpart in other parasitic interactions. A new feeding tube is produced through the stylet orifice directly into host cell cytoplasm at the end of each secretion cycle before the ingestion phase, and the cycles are repeated throughout the duration of parasitism. The timing of feeding tube production would allow secretion of relatively large molecules from the stylet orifice before its formation but could limit ingested molecules to < 40 kDa after its formation (Hussey and Grundler, 1998). Since the stylet does not pierce the plasma lemma of the feeding cell, the destination of different secreted molecules from the nematode might be directed to the extracellular space or directly into host cell cytoplasm through the opening in the plasma lemma at the stylet orifice.

2.3.3. Peptide Signaling Mechanisms

One depiction that is emerging is the potential role of secreted nematode peptide signaling molecules in feeding cell formation. Phytohormones are well known to regulate many processes in the growth and development of plant cells, and have been implicated as having roles (direct or indirect) in the parasitic interaction between nematodes and plants (Goverse et al., 2000). By contrast, small peptides represent a relatively newly recognized group of signal molecules in plants (Lindsey, et al., 2002). Plant peptide signal molecules regulate a variety of physiological processes, and a protein domain has identified the predicted product of the parasitism gene encoding H. glycines SYV46 (Wang et al., 2001) to be similar to the CLAVATA3/ESR-related (CLE) class of plant signal peptides (Olsen and Skriver, 2003). Plant CLAVATA3 exists in several isoforms, and if bound to extracellular CLAVATA1, which is a receptor-like kinase, CLAVATA3 promotes differentiation of stem cells in shoots meristems (Clark, 2001). The potential for nematodes to have evolved a mechanism to mimic this type of peptide signaling and thereby regulate feeding cell differentiation in plants is astonishing, yet is consistent with the hypothesis of de-differentiation of plant cells to become nematode feeding sites (Sijmons et al., 1994; Hussey and Grundler, 1998; Gheysen and Fenoll, 2002).

Support for a role of peptide signals in nematode-host interactions comes from a study that showed a low-molecular-weight peptide(s) secreted by G. rostochiensis enhanced cell proliferation (Goverse et al., 1999).

2.3.4. Altered Cellular Metabolism and Cell-Cycle Augmentation

A number of nematode parasitism proteins that induce potential direct modifications of recipient host cells for parasitic benefit have been identified that correlate with observed augmentation of host metabolism, cell cycle , cellular development, and defense response (Gheysen and Fenoll, 2002; Ithal et al., 2007). Some report demonstrating that some nematode parasitism proteins have nuclear localization signals that are functional in plant cells (Elling et al., 2007) supports the exciting scenario that some host cell regulation by nematodes may occur at the transcriptional level (Huang et al., 2006). Secreted Chorismate mutase (CM) from root-knot nematode and cyst nematodes (Mitchum et al., 2007) can complement a CM-defective bacterial mutant and may modify the cellular shikimic acid pathway to affect a number of outcomes. Over expression of nematode CM in plant roots leads to altered plant tissue development and potential modification of cellular partitioning of indole-3-acetic acid (IAA) (Doyle and Lambert, 2003). Secreted nematode RanBPM like, calreticulin, SXP-RAL-2, and 14-3-3 family proteins may play roles in alteration of host cell cycle, calcium binding, defense modulation, and as cellular chaperones, respectively, although functional data are lacking at present (Baum et al., 2006; Mitchum et al., 2007). Amusingly, both nematode CM and RanBPM have also been associated with response to plant R genes (Lambert et al., 2005; Moffett and Sacco, 2006), suggesting that variant alleles encoding nematode parasitism proteins may function as a-virulence genes. The venom allergen-like proteins (formerly designated Ancylostoma secreted proteins) secreted by animal-parasitic nematodes that invoke host immune responses (Jasmer et al., 2003) are conserved among parasitism genes in several species of plant-parasitic nematodes (Baum et al., 2006) although their roles in compatible/incompatible plant-nematode interactions remain unclear.

2.3.5. Nuclear Localization and Protein Degradation

Previously, Gao et al (2003) found that a group of H. glycines parasitism proteins contain putative nuclear localization signal (NLS) motifs and proposed that these proteins are targeted to the plant cell nucleus after secretion by the cyst nematode into the host cell cytoplasm.

Cyst nematode parasitism proteins that contain a functional NLS could be hypothesized to play regulatory roles for processes in the host nucleus that are required for successful parasitism, like modifying host gene expression and/or cell cycle regulation.

Selective protein degradation by the ubiquitin-proteasome pathway plays a key position in cell signaling and cellular regulation (Estelle, 2001), and the potential secretion of putative members [S-phase kinase-associated protein 1 (Skp-1), RING-H2 and Ubiquitin (Gao et al., 2003) of this complex into host cells by nematodes might represent a mechanism of cellular regulation and mitigation of host defense to promote parasitism by nematodes. The Skp-1-cullin-Fbox (SCF)-type E3 complex modulates a variety of cellular processes including cell signaling and cell cycle by selective protein degradation and interaction with phytohormone proteins (Estelle, 2001). The RING-H2 class contains monomeric E3 proteins that facilitate the transfer of Ubiquitin to target proteins for subsequent degradation within the cell (Estelle, 2001). The potential for nematodes to target specific host cell proteins for degradation by Ubiquitin secreted directly into feeding cells would be a truly unique mechanism evolved for parasitism. According to Gao et al (2003) the H. glycines ubiquitins have two novel features: they contain a signal peptide and a novel 19 amino acid extension peptide at the C-terminus. By contrast, plant Ubiquitin extension proteins are not secretory proteins and their extension proteins are ribosomal proteins (Callies et al., 1990). Because Ubiquitin is an abundant protein in the plant cell, the secretion of Ubiquitin as such by H. glycines might not affect the parasitized cell. An alternative role could be that the novel secretory Ubiquitin extension proteins of H. glycines serve as a chaperone for the unique extension peptide that might function as a peptide signal within the host cell (Gao et al., 2003).

2.4. Approaches to Parasitism gene Analysis

Though a few genetic loci that form parasitic ability have been identified in plant nematodes, the identification of nematode parasitism genes by forward genetics has proven difficult (Davis et al., 2000). So far, the successes in identifying nematode parasitism genes have resulted from efforts focused on isolation of nematode factors (secretions) that promote responses in host cells and, more recently, on the isolation of expressed genes from parasitic nematodes (Davis et al., 2000; de Meutter et al., 2001; Hussey et al., 2002; Jaubert et al., 2002; Jasmer et al., 2003).

2.4.1. Isolation of Parasitism Proteins

Peptide sequence from an antigen that was affinity purified with an esophageal-gland-specific monoclonal antibody (MGR 48) was used to identify the first parasitism gene from a phytonematode (Smant et al., 1998), encoding a -1,4-endoglucanase (cellulase). Subsequent studies established the expression of cyst nematode endoglucanase genes and their products specifically within the sub ventral gland cells, cellulolytic activity of the enzymes, and secretion of cellulases from the stylet in planta (Wang et al., 1999; Goellner et al., 2001) during migration of infective J2 through host plant roots.

2.4.2. Differential Gene Expression and Function Analysis

Profiles used to identify genes differentially expressed in several nematode life stages and different nematode tissues led to the discovery of gland-expressed parasitism genes that had their highest similarity to prokaryote genes (Ding et al., 1998; Lambert et al., 1999). Global analyses of gene expression using expressed sequence tags (ESTs) of hatched J2 of root-knot and cyst nematodes combined with cluster analyses have also been used to identify and group nematode genes based upon predicted function, including potential parasitism gene candidates (Popeijus et al., 2000; Dautova et al., 2001; Grenier et al., 2002; McCarter et al., 2003). A targeted approach to identify expressed parasitism gene candidates from cyst nematodes focused upon regulated activity in the esophageal gland secretory cells (Qin et al., 2000).

Identifying the functions of different nematode parasitism gene members remains an essential but intimidating task. Experimental limitations imposed by obligate parasites, including the use of mutants for forward genetics and the few technologies available for genetic manipulation of parasitic nematodes, are slowly being avoided. The potential to clone and express nematode parasitism genes and/or their regulatory elements within host cells and tissues to assess effects on host gene expression and phenotype (Doyle and Lambert, 2003) provides a measure of gene function in the absence of associated foreign signals. This could be accomplished in plant and animal hosts, including C.elegans (Kampkotter et al., 2003), but the utility of C.elegans for this purpose might be limited by genes that have evolved or have been acquired to promote parasitism directly (Davis et al., 2000; Geary and Thompson, 2001).16

The potential to knockout a target parasitism gene or itsproduct (Davis et al., 2000) provides a mechanism to assess host changes in the absence of one signal, providing a more-comprehensive functional analysis. Target gene knockout by RNA interference (RNAi) using introduced complimentary double stranded RNA (dsRNA), as developed (Fire et al., 1998) and utilized extensively in C.elegans, represents a powerful functional approach for nematode parasitism genes. The potential to direct the expression of dsRNA within host cells and tissues for ingestion, and RNAi of target nematode genes, raises the possibility for in vivo functional analyses of nematode parasitism genes, with the exciting dual potential of nematode management applications (Hussein et al., 2002; Urwin et al., 2002; Aboobaker et al., 2003).

2.4.3. cDNA Libraries from Nematode Secretary Cells

A cDNA library contains the cDNA molecules synthesized from mRNA molecules in a cell. The complete cDNA library of an organism gives the total of the genes it can possibly express, also called its transcriptome. The advantage of a cDNA library is that only the expressed mRNA sequences are templates for creation of cDNA and are being cloned.

The direct approach of micro-aspirating the contents of esophageal gland cells of parasitic phytonematode stages to generate cDNA libraries of gland-cell-expressed genes has provided a range of parasitism gene candidates that profile the entire nematode parasitic cycle (Gao et al., 2001; Wang et al., 2001; de Boer et al., 2002; Gao et al., 2003; Huang et al., 2003). Initial attempts to select for candidates from gland-cell cDNA libraries using a secretion signal peptide selection system (Wang et al., 2001), or microarrays (De Boer et al., 2002), or suppression subtractive hybridization (Gao et al., 2001) yielded several parasitism gene candidates. Very few of the putative parasitism genes identified thus far had homologs in C. elegans, reinforcing the hypothesis that some genes present in parasitic nematodes that are relatively divergent or absent from the C. elegans genome could be considered as potential adaptations for parasitism (Davis et al., 2000; Geary and Thompson, 2001).

2.5. Nematode Secreted Proteins involved in Parasitism

The majority of candidate plant nematode parasitism genes identified to date encode polypeptides that are predicted to be secreted from the nematode esophageal gland cells into plant tissues via the stylet (Baum et al., 2006; Davis et al., 2004; Mitchum et al., 2007; Vanholme et al., 2004). As genomic, bioinformatics, and functional analyses progress, the roles of the parasitism genes encoding novel proteins (that now include the majority of candidates) will be elucidated. Based on current knowledge of the putative parasitism genes that have database homologues or identifiable functional domains is summarized below.

2.5.1. Cell wall Degrading Enzymes

1, 4 Endoglucanases: Early evidence of the production and secretion of cell wall-degrading enzymes by plant-parasitic nematodes (Deubert and Rohde, 1971) was confirmed by the first report of expressed beta-1,4 endoglucanase genes in cyst nematodes (Smant et al., 1998). The transcripts and translated products of the cyst endoglucanase genes were produced exclusively within the sub ventral glands as early as the developing J2 stage within the eggshell, and expression persisted until the early J3 stage of cyst nematodes within host roots.

Endoglucanase expression was absent in subsequent stages of developing sedentary cyst nematode females, but interestingly, endoglucanase expression resumed during the development of the motile cyst nematode males that exit root tissues (De Boer et al., 1999 ; Goellner et al., 2000). Secretion of cyst nematode endoglucanase was detected during intracellular migration within host roots (Wang et al. 1999) but not in developing host feeding cells. However, up-regulation of plant endoglucanases was detected subsequently in nematode feeding sites as one (endogenous) component of the extensive cell wall modifications of these cells (Goellner et al. 2001). It is hypothesized that a combination of mechanical force of stylet thrusts and nematode cell wall-digesting enzymes promotes the breach of plant cell walls. Support for this hypothesis was provided from experiments in which genes for cell wall digesting enzymes were silenced by RNAi-soaking techniques and host infectivity of the treated nematodes was reduced (Chen et al., 2005) .

Other Hydrolytic Glucanases: In addition to beta-1, 4 endoglucanases, other parasitism genes encoding cell wall modifying proteins produced in the sub ventral esophageal gland cells of J2 plant parasitic nematodes have been identified. Expressed pectinase genes encoding pectate lyase and polygalacturonase have been isolated from several nematode species (Vanholme et al., 2004), and the ability to digest pectolytic substrates was related to the ability to migrate through host root tissues. The expression of an active chitinase predicted to be secreted from the sub ventral esophageal glands of the J2 soybean cyst nematode hatched from eggshells is also curious (Gao et al., 2002) and may reflect an adaptation to mitigate concurrent infection by organisms with chitin-containing walls. The first expressed xylanase (Mitreva-Dautova et al., 2006) and beta-galactosidase (Vanholme et al., 2006) genes of animal origin have been reported from root-knot and cyst nematodes, respectively, providing the first evidence of digestion of cell wall hemi-cellulose as a component of nematode migration through root tissues.

Expansin: The occurrence of several nematode genes encoding an expressed carbohydrate binding domains (CBD) joined with a peptide of non endoglucnanase origin (Ding et al., 1998; Gao et al., 2004b) is inquiring since over-expression of a bacterial CBD gene has been reported to increase elongation of plant cells (Shpigel et al. 1998). One gene (GR-exp1) expressed in the sub ventral gland cells of the potato cyst nematode (PCN) that encoded a CBD domain was an expansin-like protein that represented the first confirmed report of such a protein outside the plant kingdom (Qin et al., 2004). Structural analyses derived from the predicted domains of one PCN expansin suggested a best fit with the three-dimensional structure of extracellular proteins from soil Actinobacteria (Kudla et al., 2005). Different than the cell wall-digesting enzymes above, expansins soften cell walls by breaking non covalent bonds between cell wall fibrils, thereby allowing a sliding of fibrils past each other (Cosgrove, 2000). Expansin activity in plant cells was confirmed in proteins derived directly from PCN as well as from the expressed GR-exp1 product (Qin et al., 2004).

2.5.2. Chorismate mutase

The esophageal gland cells of both root-knot (Huang et al., 2005; Lambert et al., 1999) and cyst nematodes (Gao et al., 2003 ; Jones et al., 2003) express parasitism genes encoding Chorismate mutase, a pivotal enzyme in the shikimate pathway that converts Chorismate to prephenate

(Romero et al., 1995). The activity of Chorismate mutase is a key regulatory mechanism that determines the cellular balance of the aromatic amino acids phenylalanine, tyrosine, and tryptophan (Romero et al., 1995). The metabolites that have these amino acids as precursors, among which auxin, salicylic acid, and phenylpropanoid derivatives are of particular interest in plant-parasite interactions, would in theory be influenced by introduction of Chorismate mutase into host cells by nematodes. Cytoplasmic expression of a root-knot nematode Chorismate mutase gene in soybean hairy root cells produced tissues with an auxin-deficient phenotype that could be reversed by the application of exogenous auxin (Doyle and Lambert, 2003).

A model derived from these results suggests that nematode-secreted Chorismate mutase will deplete the cytoplasmic Chorismate pool leading to an increased export of Chorismate from host cell plastids into the cytoplasm, effectively decreasing synthesis of plastid-produced Chorismate-dependent metabolites like auxin or salicylic acid (Doyle and Lambert, 2003). Reduction in cellular salicylic acid or phenylpropanoid production in response to the introduction of nematode Chorismate mutase in host cell cytoplasm could result in a down-regulation of plant defense against the invading nematode. Consistent with a putative function in defense inactivation, Chorismate mutase genes are polymorphic and apparently selected among soybean cyst nematode isolates that differ in capacity to infect soybean genotypes with different sources of resistance (Bekal et al., 2003; Lambert et al., 2005). Alternatively, it is possible that the nematode Chorismate mutase may lead to changes in local levels of flavonoids. Flavonoides are derived from prephenate and can act as inhibitors of auxin transport in plants. An increase in prephenate levels caused by the nematode Chorismate mutase may, therefore, lead to the presence of compounds that can interfere with auxin transport, changing levels of this important plant hormone in or around the feeding site precursor.

2.5.3. CLAVATA3-Like Peptides

Perhaps the most interesting group of parasitism genes are those that encode signaling peptides. Secretions collected and fractionated from hatched juveniles of the potato cyst nematode contained a peptide or peptides of less than 3 kDa that induced mitogenic activity in tobacco leaf protoplasts and human peripheral blood mononuclear cells (Goverse et al., 1999).

The nature and origins of this bioactive peptide or peptides from PCN are unknown. Two parasitism genes that encode secreted bioactive peptides produced in the esophageal gland cells of plant nematodes have been the subject of considerable characterization and functional analyses. HG-SYV46, the parasitism gene expressed most strongly in the dorsal gland cell of H. glycines during parasitism (Gao et al., 2003), was first isolated from a screen of an expressed H. glycines gland cell-specific cDNA library for signal peptides that function in secretion (Wang et al., 2001) . Database searches of the complete predicted protein provided no significant homology, but the C-terminus of the SYV46 protein contained the consensus domain of known CLAVATA3/ESR-like (CLE) plant signaling peptides (Olsen and Skriver, 2003). Plant CLV3 peptide regulates the balance of stem cell proliferation and differentiation in the shoot meristems through interactions with a CLAVATA1/ CLAVATA2 receptor complex to negatively regulate expression of WUSCHEL (Fletcher, 2002).

2.5.4. Cytokinins

Cytokinins are plant hormones that have a variety of roles in plant development, often acting in concert with auxins. Secretions of root-knot nematodes contain Cytokinins at biologically significant levels. Thus, it is feasible that root-knot nematodes directly introduce Cytokinins into plant cells and that these molecules influence plant developmental pathways. The major Cytokinins present in root knot nematode secretions are Zeatins. These molecules are present at far lower levels in cyst nematodes. Zeatins are known to influence the G2 to M phase transition of the cell cycle. Formation of cyst nematode feeding site (NFS) does not involve mitosis, whereas root knot nematode giant cells show mitosis uncoupled from Cytokinins. It is possible that zeatins are secreted by root knot nematodes in order to drive the cell cycle to another phase and that the absence of these molecules from cyst nematodes is correlated with the different developmental patterns observed in syncytia.

2.5.5 Ubiquitin and Ubiquitin-Like Proteins

Ub is a 76 amino acid globular protein found in all eukaryotes; its sequence is highly conserved and only two residues differ between yeast and human species (Callis et al., 1995). It is the prototypical member of the ubiquitin-like (Ubl) protein family which covalently modify target

proteins to alter various aspects of their regulation (Jentsch and Pyrowolakis, 2000). Several parasitism genes expressed in cyst nematode esophageal gland cells encode secreted isotypes of cytoplasmic proteins involved in the ubiquitination pathway, namely ubiquitin itself, along with proteins (i.e. RING-Zn-Finger-like and Skp1- like proteins) similar to those found in the host E3 ubiquitin protein ligase complex (Gao et al., 2003).

2.5.6. Function of Ubiquitin-Like Proteins

Targeted and timed protein degradation (Estelle, 2001) may provide a powerful and unique means for regulation of host cell phenotype by nematodes, including potential effects within the host cell nucleus and cytoplasm. Secreted proteasome proteins from cyst nematodes could be involved in polyubiquitination of target host cell proteins for degradation to modulate cellular defense and cell signaling, to influence host cell cycle, or simply to provide a substrate for nutrient uptake by the nematode. Precedent for a potential role in modulating host defense is demonstrated by the activity of a domain of Pseudomonas syringae AvrPtoB that functions as a mimic of host plant E3 ubiquitin ligase (Janjusevic et al., 2006).

The ubiquitin extension proteins that are predicted to be secreted from cyst nematodes contain a unique non ribosomal extension peptide that is distinct from those of plants (Gao et al., 2003; Tytgat et al., 2004). The potential exists that Ubiquitin functions as a chaperone of this unique extension peptide signal of nematode origin within host cells. Though no functional data on secreted proteasome members from nematodes exists, these putative parasitism gene products are prime candidates for protein interaction studies with host cell components.

2.6. Plant Defense Mechanisms

2.6.1. Pre-existing Defenses

The first line of defense in plants is present on its surface. Several characters of plant surface function as barrier to penetration which pathogen must breach to enter the host. The pathogens enter the plant host through the epidermis (mechanical enzymatic) or through wounds or natural openings. The structure of epidermis along with cuticle and cuticular wax and number of natural openings existing before the onset of pathogenesis can obstruct penetration.

If pathogen succeeds in penetration it encounters pre-existing internal structural barriers. The external and internal structural barriers existing before pathogen attack are called pre-existing defense structures or passive /static or anti infection structures (Singh, 2002). Starting from periphery pathogens face barriers like wax and cuticle, epidermal layer, natural openings (stomata, hydathodes, lenticels, nectarines, etc), internal structures (the thickness and toughness of cell walls of internal tissues). In addition to the structural barriers plants liberate different chemicals such as release of anti-microbial compounds, inhibitors present in plant cells, lack of essential factors (recognition factors, host receptors and sites for toxins, essential nutrients and growth factors) (Singh, 2002).

2.6.2. Basal Defense and Hypersensitive Response

All plants have basal defense, the general immune response to pathogens and other environmental stresses. Against pathogens that can overcome constitutive barriers as mentioned in the previous section, plants evolved a so-called non host immune system that has many similarities to the innate immune system of animals (Zipfel and Felix, 2005). This type of resistance is based on the recognition of nonspecific factors, which can be wound- and injury-related structures indirectly derived from pathogens upon infection (Matzinger, 2002) or pathogen-associated molecular patterns (PAMPs) directly derived from pathogens themselves (Janeway Jr and Medzhitov, 2002).

Perception of conserved microbe structural components termed PAMPs leads to the prompt activation of plant defenses through PTI. PAMPs have to be functionally important components because they are shared between all members of a certain pathogen group (Chisholm et al., 2006). To date, a number of PAMPs have been identified including flagellin from bacteria, and xylanase and chitin from fungi (Nürnberger and Lipka, 2005). Currently, two plant receptors have been identified that are involved in the recognition of nonspecific elicitors, i.e. FLS2 from A. thaliana, which recognizes flagellin (Gomez-Gomez and Boller; 2000) and LeEIX from tomato, which recognizes xylanase (Ron and Avni, 2004). Non host immunity in plants manifests itself by local changes and enforcement of the plant's basal defense system due to the enhanced production of various defense compounds like waxes, cutin, suberin, lignin, and callose, or the production of oxidative reaction elements like reactive oxygen species (ROS), free radicals, and

peroxidases (Kawalleck et al., 1995). In response to nematode infection, plants were shown to reinforce the cell wall by depositing callose at the site of stylet penetration (Grundler et al. 1997). There are numerous examples of plants that have an innate immune response to invasion by specific nematode species (Starr et al., 2000b). This resistance response is characterized by two features. First, it is dependent on the specific plant resistance genes that detect the invading nematodes, and second, the plant responds by a localized hypersensitive response (HR) that can include cell death, atrophy, or abnormal development of the feeding sites. Nematodes remaining at these feeding sites are either dead or greatly diminished in their size or fertility (Williamson and Hussey, 1996; Williamson and Kumar, 2006). The HR is thought to act to suppress biotroph infection by restricting pathogen access to water and nutrients (Nimchuk et al., 2003).

2.6.3. Gene for gene and guard hypotheses

Specific recognition in plant-pathogen interactions requires the presence of cognate resistance (R) and avirulence (Avr) genes in the host and pathogen, respectively. The requirement for paired genetic components is referred to as the ‘gene-for-gene' effect. The absence of either determinant leads to the breakdown of resistance. This simple genetic relationship embodies the intellectual fascinations of the field: first, that the requirement for specific R-Avr gene-pairs implies an elaborate, co-evolved molecular recognition of the pathogen by the host; and second, the apparent evolutionary contradiction that a pathogen should retain Avr genes that specify its demise. Recent publications are beginning to shed light on both of these areas, with an emerging theme that the phenomena might be fundamentally linked. Ellingboe (1981) first proposed that direct interaction between cognate R and Avr gene products might underlie gene-for-gene resistance. Effectors triggered immunity (ETI) activation causes elevated salicylic acid (SA) accumulation which induces transcription of various pathogenesis-related (PR) genes and the activation of systemic acquired resistance (SAR) (Durrant and Dong, 2004).

The activation of common disease resistance signaling pathways results from the perception of bacterial, viral, fungal, oomycete, and nematode pathogen effectors by their associated R proteins (Dangle and Jones, 2001). Despite the broad taxonomic origins of known plant pathogens and the presumed diversity in their effector molecules, only five structural classes of R protein have been

reported, with the presence of LRR domains being a recurring theme in the majority of cases (Dangle and Jones, 2001). By far the largest class encodes a loosely predicted amino-terminal coiled-coil (CC) or TIR (Toll, interleukin receptor homology) domain, fused to a central NB-ARC (i.e. a nucleotide-binding site typical of Apaf-1, R proteins and CED4) domain and a carboxy terminal region that contains leucine-rich repeats (LRRs). These genes are referred to as CNL (CC-NB-LRR) or TNL (TIR-NB-LRR).

The ‘guard hypothesis' proposes that the R proteins associate with the cellular targets of the Avr proteins (Van der Biezen and Jones,1998; Dangle and Jones,2001).These cellular targets could be proteins involved in plant defense, for example, or be required to provide nourishment for the pathogen. The guard hypothesis proposes that when a pathogen Avr gene product binds to a target in a resistant plant cell this complex is recognized by the R protein, which initiates the plant's defence. Therefore, in a susceptible host, no R protein is present and the plant target is unguarded from the pathogen virulence elicitor, resulting in disease.

2.7. Resistance to Plant Parasitic Nematodes

2.7.1. Virulence and a-virulence in Plant Pathogens

Numerous genes that confer resistance against plant parasitic nematodes have been described, and several of these have now been cloned (Williamson and Hussey, 1996; Williamson, 1999). The best-studied of these genes is the tomato gene Mi, which confers resistance against three species of root-knot nematode. Mi also confers resistance to some isolates of the potato aphid Macrosiphum euphorbiae and to the white fly Bemisia tabaci (Goggin et al., 2001; Nombela et al., 2003). The encoded protein contains a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region, protein motifs that are found in numerous plant resistance genes (R genes) against a variety of pathogens (Dangle and Jones, 2001). Other recently cloned nematode R genes, Gpa2 and Hero, also belong to the NBS-LRR family (Van Der Vossen et al., 2000; Ernst et al., 2002). However, nematode resistance gene sequences do not cluster together. The sequence of Gpa2, for example, is much more similar to that of the virus resistance gene Rx than to those of other nematode resistance genes (Van Der Vossen et al., 2000).

Avirulence genes — that is, single pathogen genes that are required for R-gene-mediated resistance—have been identified in bacteria, viruses and fungi (Dangle and Jones, 2001). To date, no avirulence genes have been conclusively isolated from nematodes, although there has been progress in this area. There is genetic evidence for avirulence genes in Globodera rostochiensis that correspond to the resistance gene H1 (Janseen et al., 1991).

To establish a parasitic relationship with their host plants, cyst and root-knot nematodes have to overcome multiple layers of defense responses to modify host root cells into beneficial feeding structures. Several cyst and root-knot nematode genes encoding secreted proteins produced in the esophageal glands were shown to have the features of pathogenicity factors (Vanholme et al., 2004; Davis et al., 2004). Avr proteins are generally considered to be virulence factors required for successful infection of the host plant and as such, these nematode parasitism genes are good candidates to encode avirulence proteins. However, proof has not been obtained yet for any of them to act as a resistance gene-dependent plant defense elicitor. The amount and variability of putative nematode pathogenicity factors and limited knowledge about nematode resistance mechanisms make it very difficult to predict which proteins secreted by nematodes can play a role in avirulence. Besides secreting a mixture of cell-wall degrading enzymes that act on the plant cell walls and facilitate migration, nematodes also release secretions into the feeding site initiation cell (Williamson and Hussey, 1996). Most of the proteins have predicted signal peptides and are proven to be produced exclusively in esophageal glands, although some proteins lack such a signal and have been shown to be secr