Cyst nematode species of the genus Heterodera are well known for their distinct host ranges. In susceptible hosts, these nematodes use a stylet to penetrate the cell wall of a selected root cell and secrete effector proteins to induce the formation of a syncytium. While the mechanisms underlying and driving the differences in host range among species are unknown, a role for nematode effectors in this process is anticipated. To date, effector discovery in Heterodera has focused on two species, Heterodera schachtii and Heterodera glycines. Unlike other cyst nematodes, the sugar beet (Beta vulgaris) cyst nematode H. schachtii infects Arabidopsis thaliana, and was adopted as a model system for studies of plant–nematode interactions nearly 20 yr ago (Sijmons et al., 1991; Gheysen & Fenoll, 2011). Heterodera glycines, the soybean (Glycine max) cyst nematode, has demonstrated utility as a genetic model system with which to study parasitism (Opperman & Bird, 1998; Niblack et al., 2006), but functional genomic approaches targeting identification of stylet-secreted effector proteins have accelerated the process. The earliest approaches focused on affinity purification of proteins using monoclonal antibodies to antigens in nematode stylet secretions (De Boer et al., 1996). This approach was successful in identifying the first nematode parasitism genes, encoding β-1-4-endoglucanases, in H. glycines and Globodera rostochiensis (Smant et al., 1998). The search for a higher throughput approach resulted in the identification of over 50 candidate parasitism genes encoding putative secreted effectors of H. glycines from cDNA libraries constructed from RNA isolated from cytoplasm microaspirated from esophageal gland cells of parasitic life stages (Gao et al., 2001a, 2003; Wang et al., 2001). A microarray-based expression atlas of the parasitism genes across H. glycines life stages followed (Elling et al., 2007b, 2009). The identification of orthologous H. glycines parasitism genes in the closely related H. schachtii has facilitated studies to acquire insight into the biological function of several nematode effectors using A. thaliana as a model system (Wang et al., 2005, 2010a; Hewezi et al., 2008a, 2010; Patel et al., 2010; Lee et al., 2011; Hamamouch et al., 2012). Additionally, approaches such as direct sequencing of in vitro-produced esophageal gland secretions and mining of expressed sequence tags (ESTs) generated from infective juveniles have identified a number of putative secreted effector proteins from H. schachtii (De Meutter et al., 2001; Vanholme et al., 2006).
To date, 70% of all effectors identified from Heterodera are novel proteins, which presents a considerable challenge in ascribing function. Among those identified from H. glycines and H. schachtii with sequence similarity to known proteins is an elaborate suite of cell wall-modifying proteins (CWMPs), including ENGs (Smant et al., 1998; De Meutter et al., 2001; Gao et al., 2002a, 2004a; Vanholme et al., 2006), CBPs (Gao et al., 2004b; Vanholme et al., 2006), pectate lyases (PELs; Vanholme et al., 2007), and an arabinogalactan endo-1,4-β-galactosidase (Vanholme et al., 2009b). These nematodes also produce secreted effector proteins with homology to chitinase (CHI; Gao et al., 2002b), chorismate mutase (CM; Bekal et al., 2003; Gao et al., 2003), ubiquitin (Gao et al., 2003; Tytgat et al., 2004), and venom allergen proteins (VAPs; Gao et al., 2001b, 2003), although their functions in nematode parasitism remain unknown. The first nematode-secreted peptide hormones sharing homology with plant CLE peptides were identified from H. glycines (Wang et al., 2001; Gao et al., 2003; Olsen & Shriver, 2003; Mitchum et al., 2012), and more recently from H. schachtii (Patel et al., 2008; Wang et al., 2011). These peptides were shown to functionally mimic the effects of plant CLE peptides in exogenous peptide assays and through complementation of the A. thaliana clavata3 mutant (Wang et al., 2005, 2010a, 2011). Although the biological function of these peptide mimics in feeding cell formation is unclear at present, they were recently shown to signal through A. thaliana LRR-RLPs CLV1, CLV2, and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2; Replogle et al., 2011, 2013), probably triggering developmental changes necessary for syncytium formation. Another nematode-secreted effector identified from H. glycines and H. schachtii that may mimic endogenous host proteins is annexin. Annexins are calcium- and membrane-binding proteins with wide-ranging cellular functions. Nematode annexin was able to complement the reduced germination phenotype of a plant annexin mutant under high salt stress and was found to interact with an A. thaliana oxidoreductase, possibly promoting parasitism by modulating host stress and defense responses (Patel et al., 2010). Despite the challenges, tremendous progress has been made to elucidate the function of several novel effector proteins, including 10A06, 19C07, and 30C02, identified from both H. glycines (Gao et al., 2003) and H. schachtii (Hewezi et al., 2010; Lee et al., 2011; Hamamouch et al., 2012). The 10A06 and 30C02 effector proteins were found to interact with spermidine synthase (Hewezi et al., 2010) and β-1,3-endoglucanase (Hamamouch et al., 2012), respectively, and functional studies have suggested a role for these proteins in modulating host defense responses. The 19C07 effector protein was found to interact with an auxin influx transporter, LAX3, presumably to regulate auxin balance during the early stages of feeding cell formation (Lee et al., 2011).