Soybean cyst nematode (SCN, Heterodera glycines) is an obligate, sedentary endoparasite that is consistently the most damaging pest of soybean in the U.S. . Once SCN is present in a field it cannot feasibly be eradicated.
The SCN life cycle consists of five stages. After the first molt within the egg, SCN second stage juveniles (J2) hatch, move through the soil, penetrate roots and move toward the vascular cylinder [2, 3]. Migratory juveniles select a host cell in the cortex, endodermis, or pericycle and induce host cell fusion as part of the formation of a permanent feeding site called a syncytium. At this point the nematode becomes sedentary and differentiates to the third (J3) and fourth (J4) juvenile stages and then matures to an adult female or male. Males undergo a metamorphosis to resume a vermiform shape at the J4 stage and migrate back out of the root to fertilize adult females. Following fertilization, the female produces eggs, most of which remain inside the body. After dying, the female body develops into a hardened cyst that encases the eggs. At 25°C, some nematodes reach the adult stage 12 days after entering roots, and most become adults by 30 days post-infection .
Soybean cyst nematodes infect and grow in the roots of both resistant and susceptible cultivars [2, 5]. Nematode growth and development depends on the successful establishment and maintenance of a syncytium, and impairment of the syncytium can give outcomes that range from reduced growth and reproduction to death. The available SCN resistance in soybean is partial, and can be observed as a reduced number of females that develop compared to the number that develop on similarly inoculated susceptible cultivar controls . In intact soybean plants, resistance is often expressed as the female index (FI): the number of fully developed females (cysts) on the tested soybean genotype divided by the number of females on a susceptible standard [2, 7]. Soybean cultivars are generally classified as strongly resistant to SCN if the FI is less than 10%; partial levels of resistance can also be useful .
The SCN resistance in current commercially grown soybeans is derived from a very small number of sources . These sources include 'Peking' (PI 548402), PI 88788 and PI 437654, which have each been shown to carry resistance loci effective against multiple nematode races [10, 11]. Inheritance of resistance to SCN was first reported in the 'Peking' plant introduction, and three genes for resistance (rhg1-rhg3) were assigned and initially classified as recessive . Of the resistance sources, PI 88788 has been the most widely used in breeding programs. More than 95% of the SCN resistant cultivars available for planting in Illinois during 2009 received their resistance from this PI .
The Rhg1 locus has been shown to have the greatest impact on SCN development in several resistance sources including Peking, PI 88788, PI 437654, PI 209332 and PI 90763. This locus provides resistance to many common SCN populations such as Hg type 0 (race 3) . Multiple research groups have mapped the Rhg1 locus to a sub-telomeric region on chromosome 18 [11, 14, 15], to a location approximately 0.4 centimorgans (cM) from the simple sequence repeat (SSR) marker Satt309  (chromosome 18 was formerly known as linkage group G; http://www.phytozome.net/). Although originally reported as a recessive locus, "rhg1" has more recently been characterized as exhibiting incomplete dominance. Soybean lines heterozygous for resistant and susceptible alleles at the Rhg1 locus often allow SCN cyst formation at a rate intermediate between that of plants genotyped with Satt309 as homozygous resistant or homozygous susceptible at the Rhg1 locus [18–20](Kim et al. submitted; incomplete dominance also has been observed in unpublished work with the Ina × E98076 material used in this study). A second QTL (Rhg4) has been identified as being necessary for full resistance to some SCN populations in Peking and in PI 437654, but not PI 88788 or PI 209332 [7, 9]. Rhg4 exhibits dominant gene action, and in Peking-derived material the relevant alleles of both Rhg1 and Rhg4 are necessary to exhibit the full resistance phenotype. Other loci that make smaller and/or more race-specific contributions to SCN resistance have also been identified throughout the soybean genome, but often a given locus was identified in only one study [10, 21–25].
Cytological studies suggest that Peking-type resistance displays host cell necrosis and cell wall appositions not seen in PI 88788 type resistances in response to SCN Hg type 0 [26, 27]. The Peking and PI 88788 Rhg1 sources also exhibit distinct differential behaviors in their strength of resistance against particular SCN test populations, suggesting at least partially different mechanisms in the SCN resistance controlled by different Rhg1 alleles [28, 29].
Two groups first filed applications with the U.S. Patent Office in 2000 identifying apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) genes currently annotated as Glyma18g02680.1 and Glyma08g11350.1 http://www.phytozome.net/ as the likely SCN resistance genes at both rhg1 and Rhg4 [18, 30], see also [19, 31]. Sequences supporting the claims were released to Genbank between 2000 and 2005. The basis for these claims was the presence of these genes at the Rhg1 and Rhg4 loci, their similarity to the known rice bacterial blight resistance gene Xa21, and the presence of derived amino acid sequence differences between the alleles from resistant and susceptible plant genotypes. However, a decade later, no functional evidence for a role of these LRR-kinase genes in SCN resistance has been reported in a peer-reviewed forum. There are numerous reasons why identification of the Rhg1 gene that confers SCN resistance is a high priority, including the extreme economic significance of SCN for yield loss, the major reliance on the Rhg1 locus in commercial soybean breeding, the detection of SCN populations that overcome currently available Rhg1-mediated resistance, the potential to respond to these challenges with engineered improvements in Rhg1, and basic scientific interest in the nature of plant resistance to SCN.
An experimentally tractable transgenic assay system is a crucial component for the identification, study and manipulation of genes controlling SCN resistance as well as many other soybean traits. Generation of transgenic fertile soybean lines is still a difficult and expensive process that requires close to a year to obtain transgenic seed lines. Agrobacterium rhizogenes has been used by many researchers as a transgene delivery system to study legume root biology, both in soybean and Medicago truncatula [32–36]. Transgenic roots generated using A. rhizogenes retain the SCN resistance phenotypes of the parental soybean genotypes, and have been used to test genes that may impart resistance [37, 38].
Function is often attributed to specific genes by the methods of gene mutation/positional cloning, phenotypic complementation via transformation with a cloned full-length gene, and/or by gene silencing. Experimental silencing of plant genes has generally been elicited using hairpin RNA-forming inverted repeat DNA constructs or by virus-induced gene silencing [39–42]. All silencing approaches are prone to incomplete penetrance (partial silencing, often for unpredictable reasons), but have nevertheless been useful for assigning function to specific genes, and for engineering useful traits. These gene silencing techniques have been used effectively in soybean and other legumes [43–49]. With the more recent discovery of endogenous microRNAs as a major mode of gene regulation in many eukaryotes, artificial microRNA (amiRNA) methods have been developed for investigator-initiated silencing of target genes [50–52]. Potential advantages of amiRNAs may include better penetrance, absence of undesired phenotypes associated with VIGS virus infections, and the capacity to limit off-target silencing of related sequences by elicitation with constructs specific for very short (19-24 bp) target sequences. However, use of amiRNA technology with soybean, M. truncatula, Phaseolus or other legumes has not been reported.
For the present study we refined assays that test SCN resistance in transgenic roots generated with A. rhizogenes. A nematode demographic assay was developed that discriminates resistant and susceptible responses by monitoring the infecting population for progression through nematode life stages. We also developed an amiRNA vector system for induction of gene silencing in legume roots using A. rhizogenes assay systems. We used these tools to investigate the impact on SCN resistance of the LRR-kinase gene from the Rhg1 genomic region (the candidate SCN resistance gene). Our experiments expressing the LRR-kinase from a resistant (Peking/PI 437654-source) Rhg1 locus in susceptible test lines, with or without the resistant allele at Rhg4, and silencing the LRR-kinase (PI 88788 source) in resistant lines, provided no evidence for a contribution of this gene to SCN resistance.