The soybean Rhg1 locus for resistance to the soybean cyst nematode Heterodera glycines 1 regulates expression of a large number of stress- and defense-related genes in degenerating 2 feeding cells 3

of findings this ABSTRACT 2 To gain new insights into the mechanism of soybean resistance to the soybean cyst nematode 3 ( Heterodera glycines ), we compared gene expression profiles of developing syncytia in soybean 4 near-isogenic lines (NILs) differing at Rhg1 , a major QTL for resistance, by coupling laser 5 capture microdissection with microarray analysis. Gene expression profiling revealed that 1,447 6 genes were differentially expressed between the two lines. Of these, 241 (16.8%) were stress- 7 and defense-related genes. Several stress-related genes were up-regulated in the resistant line, 8 including those encoding homologs of enzymes that lead to increased levels of reactive oxygen 9 species and proteins associated with the unfolded protein response. These results indicate that 10 syncytia induced in the resistant line are undergoing severe oxidative stress and imbalanced 11 endoplasmic reticulum homeostasis, both of which likely contribute to the resistance reaction. 12 Defense-related genes up-regulated within syncytia of the resistant line included those 13 predominantly involved in apoptotic cell death, the plant hypersensitive response, and salicylic 14 acid-mediated defense signaling; many of these genes were either partially suppressed or not 15 induced to the same level by a virulent soybean cyst nematode population for successful 16 nematode reproduction and development on the resistant line. Our study demonstrates that a 17 network of molecular events takes place during Rhg1 -mediated resistance, leading to a highly 18 complex defense response against a root pathogen.


INTRODUCTION
with microarray analysis has been particularly useful in extending our understanding of the SCN-1 soybean interaction, as indicated by recently published studies (Klink et al., 2005;Ithal et al., 2 2007b). These studies have provided new insights into the underlying molecular events occurring 3 during syncytium development. More recently, the same technology has been applied to study 4 incompatible SCN-soybean interactions (Klink et al., 2007b;Klink et al., 2009;Klink et al., 5 2010). Two studies reported on a comparative microarray analysis of soybean genes induced in 6 response to either a virulent or an avirulent SCN population on Peking (Klink et al., 2007b;7 Klink et al., 2009), demonstrating that soybean can differentiate between nematode populations 8 prior to feeding cell establishment (Klink et al., 2007b). The same group also published a 9 microarray study that examined the transcriptional changes occurring in syncytia induced by an 10 avirulent SCN population on PI 88788 at three time points after infection (Klink et al., 2010). To 11 our knowledge, there are no reports of a direct comparative analysis of syncytia gene expression 12 profiles using near-isogenic lines (NILs) to identify transcripts regulated by specific soybean 13 resistance genes. NILs have several advantages over PIs for comparative analyses of plant gene 14 expression between resistant and susceptible soybean in response to SCN. Theoretically,NILs 15 can share up to 98% of their genome, differing only in a region encompassing a trait of interest 16 (Li et al., 2004); thus, NILs are powerful tools to study the effects of specific gene loci with 17 reduced genetic background effects. Consequently, the use of NILs for molecular studies is 18 becoming more prevalent. NILs have been used in a microarray analysis of iron efficient and 19 inefficient cultivars of soybean (O'Rourke et al., 2009) and a wheat leaf rust resistance gene Lr10 20 (Manickavelu et al., 2010). NILs also recently helped to identify the effects of the Arabidopsis 21 gene FLC on seed germination (Chiang et al., 2009). 22 To gain new insights into the cause of the aberrant syncytia development that occurs in 1 resistant soybean in response to SCN infection, we analyzed gene expression in syncytia induced 2 in soybean NILs differing at the Rhg1 locus (Mudge, 1999). LCM coupled with comparative 3 microarray profiling of syncytia isolated from the resistant NIL (NIL-R) and susceptible NIL 4 (NIL-S) resulted in the identification of 1,447 differentially expressed genes using a false 5 discovery rate set at 10%. Many of the genes induced in the NIL-R are soybean homologs of 6 genes known to play important roles in disease resistance responses of other plant species to 7 various pathogens, including canonical resistance genes (e.g., CC-NB-LRR class of receptors), 8 genes associated with the hypersensitive-like response (HR), apoptotic cell death, the salicylic 9 acid (SA)-mediated resistance pathway, and several transcription factors with defense-related 10 roles. These results were validated by syncytia-specific qPCR, time course qPCR on infected 11 whole root pieces, and promoter-GUS reporter experiments. Our study reveals that Rhg1 12 mediates a complex defense response within syncytia formed in resistant soybean plants, 13 ultimately limiting the growth and development of the nematode. 14 15 RESULTS 16

Response of NILs to SCN 17
Soybean NILs, derived from a cross between the susceptible cultivar Evans and the resistant PI 18 209332, were chosen for these studies. These NILs are predicted to share 98% of their genome, 19 differing at the major SCN resistance locus, Rhg1 (Mudge, 1999). NIL-S is susceptible and NIL-20 R is resistant to SCN inbred line PA3 (HG type 0). The Rhg1 allele in PI 209332 is likely similar 21 to the Rhg1 allele in PI 88788, the source of resistance found in greater than 90% of 22 commercially available SCN-resistant soybean cultivars. Field populations of SCN that can 23 break PI 88788 resistance typically can break PI 209332 resistance, suggesting that these PIs 1 1 3 microarray analysis. This clearly illustrates the dilution effect attributed to using infected whole 1 root pieces for gene expression analyses in this pathosystem. For Gma.7623.1.A1_at, regulation in the NIL-R peaks at 6 dpi ( Figure 3a). The pattern is similar for 3 GmaAffx.46603.1.S1_at (Figure 3c). The expression pattern is slightly different for the gene 4 represented by probe set GmaAffx.68498.1.S1_at in that the maximum up-regulation is observed 5 at 4 dpi. These peaks in expression levels are consistent with the timing of the resistance 6 response observed in syncytia of the NIL-R (Figure 1). The observed trend in expression for each 7 gene over the time course of infection was reproducible in three independent infection 8 experiments; however, the level of up-regulation varied among experiments. This difference can 9 be attributed to the biological variation inherent to nematode infection experiments, where it is 10 impossible to achieve identical rates of infection. Similar to what was observed in qPCR 11 analyses of RNA isolated from syncytia, infection by the virulent and avirulent SCN populations 12 results in differential up-regulation of these genes. For all three genes tested, the level of up-13 regulation is lower in response to infection by the virulent nematode population (Figure 3a-c). 14 Additionally, in contrast to infection of the NIL-R, infection of the NIL-S with the avirulent 15 population shows only a slight up-regulation for all three genes tested. 16 17

Promoter-GUS expression analysis 18
Promoter-GUS fusions were generated to provide further validation of the spatial expression 19 pattern of the differentially expressed genes identified by microarray analysis and to isolate 20 nematode-responsive soybean promoter sequences with high levels of expression within 21 syncytia. For these experiments, primers corresponding to the 5' upstream sequences of 10 genes 22 (Figure 4a), chosen from the differentially expressed microarray data set (Table S1), were 23 1 4 designed by using the recently released Williams 82 soybean genome sequence (Schmutz et al., 1 2010). Genes selected for promoter analysis were those whose expression was highly 2 upregulated based on our microarray analysis or genes presumed to either not be expressed or 3 expressed weakly in roots based on expression data of Arabidopsis orthologs, because at the time 4 we performed this experiment we did not have reliable expression data for these genes in the 5 roots of soybean. Promoter fragments were amplified by PCR using Williams 82 genomic DNA 6 as a template and cloned upstream of a β -glucuronidase (GUS) reporter gene in the gateway 7 binary vector pYXT1 (Xiao et al., 2005;Figure 4b). Transgenic soybean hairy roots were 8 generated in the NIL-R soybean background for each reporter construct. As a positive control, 9 the Arabidopsis WRKY23 promoter (At2g47260) was tested in soybean; At2g47260 is induced 10 within syncytia in Arabidopsis upon infection with the beet cyst nematode, Heterodera schachtii 11 (Grunewald et al., 2008). The transgenic hairy roots were infected with the avirulent (PA3) SCN 12 population. The positive control and all 10 promoter-GUS lines show induced GUS gene 13 expression at the nematode feeding sites at 5 dpi ( Figure 5). For several promoter-GUS lines, 14 GUS expression is observed throughout the root but further induced at nematode feeding sites 15 (Figure 4c,d,i,and j;Figure S1b,c). Several promoters have an expression pattern that is very 16 low or more restricted to specific cell types within roots and an up-regulation of GUS expression 17 is clearly distinguishable at the nematode feeding sites (Figure 4a, b, e, f, g, h, and k; Figure S1a, 18 d). RNA-seq expression data for each soybean gene model in uninfected roots according to 19 Severin et al. (2010) andLibault et al. (2010) is provided in Figure 4a for comparison. Although 20 there are some discrepancies (Fig 4d,

Differential expression of stress-and defense-related genes 1
We specifically looked at stress-and defense-related genes to gain a better understanding of the 2 Rhg1-associated syncytium collapse that occurs in the NIL-R in response to SCN. The 3 differential expression of this class of genes varies from 87-fold up-regulated to 17-fold down-4 regulated (Tables 2, 3, S1, and S2). Soybean orthologs of many known plant defense genes have 5 not yet been identified; therefore, we relied on their similarity to Arabidopsis homologs. A total 6 of 241 probe sets representing 16.8% of the total number of differentially expressed genes 7 identified are classified in this group. These included genes involved in apoptosis and disease 8 resistance. Several soybean genes showing high similarity to defense genes of Arabidopsis that 9 play a role in incompatible responses to other plant pathogens were found to be differentially 10 expressed in syncytia of the NIL-R in response to SCN (Tables 2, 3, S1, and S2). These include a 11 highly upregulated AtBag6 homolog, a CC-NB-LRR gene, several heat shock protein genes and 12 heat shock transcription factors, defense-related WRKY transcription factors, pathogenesis-13 related (PR) genes, and genes that modulate salicylic acid (SA) and jasmonic acid (JA) levels. A 14 large number of genes involved in oxidative, drought, cold, osmotic, and salt stress responses are 15 also differentially regulated (Tables 2, 3, S1, and S2). 16

DISCUSSION 18
The planting of resistant soybean cultivars is the primary strategy used to manage SCN 19 population levels in the field. Despite the widespread use of SCN-resistant soybean, this 20 pathogen still causes an estimated $1.286 billion annually in yield losses. A lack of 21 understanding of the molecular basis of resistance to this pathogen continues to hinder progress 22 to enhance the effectiveness and durability of natural plant resistance and enable the design of 23 novel strategies for resistance through biotechnological approaches. Rhg1, a major resistance 1 locus in almost all SCN-resistant germplasm, is required for resistance against multiple SCN Hg 2 types (Concibido et al., 2004); however, the molecular nature of the resistance gene underlying 3 Rhg1 (Melito et al., 2010) and the downstream signaling and response genes mediated by Rhg1 4 are not known. The Rhg1 gene has been mapped to chromosome 18 and is within 0.4 cm of SSR 5 marker satt_309 (Cregan et al., 1999), enabling the generation of NILs differing only at this 6 locus (Mudge, 1999). These NILs are useful for molecular studies to dissect the SCN-soybean 7 incompatible interaction because of the multigenic nature of resistance. Therefore, to develop a 8 better understanding of the molecular events associated with Rhg1-mediated resistance against 9 SCN, we employed the use of NILs for a comparative analysis of syncytial gene expression 10 using LCM and microarrays. These NILs have been used previously to study the effects of Rhg1 11 on root penetration and development of SCN (Li et al., 2004). Although root penetration by SCN 12 juveniles is similar between NIL-R and NIL-S, the growth, development, and fecundity of 13 nematode females is suppressed on NIL-R (Li et al., 2004), suggesting that Rhg1 may have a 14 negative impact on syncytium development and maintenance. The histological characteristics of 15 syncytia in resistant soybean cultivars to SCN infection are well documented (Ross, 1958;Endo, 16 1965;Riggs et al., 1973;Acedo et al., 1984). Second-stage SCN juveniles (J2s) induce the 17 formation of syncytia in all resistant cultivars, but syncytial collapse occurs several days later. In 18 one type of resistance (PI 437654; Peking type), syncytial collapse is very rapid and begins to 19 occur within 48 hours of induction of the syncytium (Mahalingam and Skorupska, 1996). The 20 NILs used in our study, however, show a delayed type of resistance (Acedo et al., 1984;Li et al., 21 2004) with notable histological changes to syncytia occurring by 8-10 dpi ( Figure 1). Thus, 5 and 22 onset of syncytium collapse. The comparison of syncytia gene expression between NIL-R and 1 NIL-S by microarray analysis identified 1,447 differentially expressed probe sets. We found a 2 high representation of stress-and defense-related genes (241 probe sets representing 16.8% of 3 the total; Figure 2; Tables S1 and S2), including genes involved in oxidative, heat, cold, salt, and 4 drought stress. Due to space constraints, we limited our analysis to stress-and defense-related 5 genes. 6 A gene coding for a BAG (BCL2-associated athanogene) domain protein with highest 7 homology to the Arabidopsis BAG6 protein (AtBAG6) is the most highly up-regulated gene in 8 syncytia of the resistant line (87-fold, Table 2). Bag6 encodes a stress-induced calmodulin-9 binding BAG domain protein that is homologous to mammalian BAG proteins, which are 10 regulators of BCL2 involved in apoptosis (Kang et al., 2006). BAG proteins are anti-apoptotic in 11 animals; however, overexpression of AtBag6 in yeast and Arabidopsis causes cell death (Kang et 12 al., 2006). The increased expression of this gene in the resistant line suggests that the syncytia 13 may be undergoing an apoptotic-like cell death response. 14 Several heat shock proteins (HSPs) of the small HSP superfamily are up-regulated in 15 syncytia of the resistant line (Table 2). HSPs are stress responsive proteins that have a protective 16 function in promoting cellular stress tolerance (Wang et al., 2004). Small HSPs bind and 17 stabilize denatured proteins to which other high molecular weight HSPs act as chaperones under 18 stress conditions. Several other HSPs, including an HSP70 homolog (Gma.11115.2.S1_at), 19 HSP70B homolog (GmaAffx.30428.1.S1_at), HSP90.1 homolog (GmaAffx.80951.1.S1_at), and 20 two heat shock transcription factors (HSFs), Hsf-A2 homolog (GmaAffx.71308.2.A1_at, 4.0 21 fold) and Hsf-A3 homolog (GmaAffx.19934.1.S1_at, 2.6 fold), are up-regulated in syncytia of 22 the NIL-R. HSP90 is a highly conserved molecular chaperone rapidly induced during pathogen 23 challenge and a variety of environmental stresses. It interacts with the R protein, RPM1 (Hubert 1 et al., 2003), and is required for RPS2-mediated resistance against Pseudomonas syringae pv. 2 tomato DC 3000 (avrRpt2) (Takahashi et al., 2003). HSFs are involved in a variety of 3 environmental stresses; Hsf-A2, for example, is a key inducer of defense responses and is up-4 regulated during environmental stress and H 2 O 2 treatment (Nishizawa et al., 2006). AtBAG6 is 5 up-regulated by heat stress, and the Hsf-A2 is involved in its regulation (Nishizawa et al., 2006). 6 Several genes related to ER stress were also found to be up-regulated (e.g., BZIP60 7 homolog (GmaAffx.3568.1.S1_at), BIP2 homolog (Gma.17631.1.S1_at), Calnexin 8 ( Gma.6427.2.S1_a_at), Bax inhibitor genes (GmaAffx.1991.1.S1_at; GmaAffx.34450.1.S1_at; 9 GmaAffx.92919.1.S1_at), and several protein disulphide isomerases) (Tables 2, S1). ER stress is 10 a cellular condition in which unfolded proteins accumulate in the ER. Misfolding of proteins is a 11 result of mutations, disturbances in calcium homeostasis, and the heightened need for protein 12 folding. In order to maintain ER homeostasis under such conditions, signaling pathways are 13 activated that are collectively known as the unfolded protein response (UPR). When ER stress is 14 not relieved by different measures, an apoptotic cell death occurs (Urade, 2009). Recently, it was 15 reported that water deficit or drought leads to programmed cell death mediated by the ER stress 16 response pathway in Arabidopsis roots (Duan et al., 2010). Several drought and ABA-induced 17 genes were found to be up-regulated in syncytia of the resistant NIL. Taken together, these data 18 suggest that multiple stress response pathways are induced by an upstream signaling event in the 19 resistant plants during nematode infection that may ultimately lead to the activation of an HR-20 like programmed cell death (PCD) causing the pathogen to starve and die. It is also possible that 21 pathogen death occurs before the syncytial HR-PCD. The HR may represent the final stages of 22 the resistance response where a certain threshold of defense-related responses has been reached 23 (Morel and Dangl, 1997). For example, the Arabidopsis dnd1 mutant expresses resistance to 1 pathogens that otherwise induce HR on wild-type plants (Clough et al., 2000). 2 The production of reactive oxygen species (ROS) is a key aspect of the HR during R-3 mediated resistance to other pathogens (Lamb and Dixon, 1997). An NADPH thioredoxin 4 reductase, similar to Arabidopsis NTRC, is up-regulated (5.8 fold, GmaAffx.92590.1.S1_at). 5 Alternative oxidase (Gma.1439.1.S1_at, 4.2 fold; Gma.8204.1.A1_at, 3.46 fold), glutathione S-6 transferase (Gma.620.1.S1_at, 3.9 fold), and a gene similar to RCD1-5 involved in ROS 7 regulation (Gma.7922.1.A1_a_at, 2.9 fold) are up-regulated. Several genes related to oxidative 8 stress and regulation of ROS are also down-regulated (Table 3) as are many peroxidases 9 (Gma.5629.2.S1_a_at, Gma.5629.1.S1_at, Gma.5971.1.S1_at, GmaAffx.74124.1.S1_at, 10 Gma.1539.1.S1_at, Gma.4919.1.S1_at, Gma.338.1.S1_at, Gma.4189.1.S1_at, fold changes 11 ranging from -5.4 to -1.9). Peroxidases are involved in H 2 O 2 catabolism, and their down-12 regulation may suggest a positive impact on ROS generation; although, they can also generate 13 ROS species (Passardi et al., 2004). Other down-regulated oxidative stress genes include two 14 NADPH quinone oxidoreductases (GmaAffx.65280.1.A1_at, -2.57 fold; 15 GmaAffx.90444.1.S1_s_at, -1.3 fold), glutathione peroxidase 2 and 3 homologs, and a protein 16 disulphide isomerase-like 4 (PDI like-4) that belongs to the thioredoxin family. 17 A gene coding for CC-NB-LRR protein is upregulated (Gma.1622.1.A1_s_at, 5.2 fold).. 18 The up-regulation of a MAP3K homolog (GmaAffx.48022.2.A1_at) implicates MAPK signaling 19 in the regulation of resistance to SCN. A soybean gene encoding a protein with homology to 20 Arabidopsis syntaxin 121 (SYP121), a secretory pathway protein with known roles in defense 21 responses, is also induced (GmaAffx.1338.1.S1_at, 2.6-fold GmaAffx.20155.1.S1_at 2-fold) as 22 are several genes involved in cold, drought, dehydration, and ABA responses 23 www.plantphysiol.org on August 20, 2017 -Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved.
(Gma.14272.1.S1_at, 11.7 fold; Gma.2044.1.S1_at, 10.8 fold; GmaAffx.68621.1.A1_at, 9.4 fold; 1 Gma.7526.1.A1_at, 7.6 fold) and two transcription factors of the AP2/ERF family involved in 2 drought responses (GmaAffx.29929.1.S1_at, 11.2 fold; Gma.9553.1.A1_at, 8.1 fold). The high 3 up-regulation of these genes may suggest new roles in HR against a biotrophic pathogen, or they 4 are secondary physiological responses that potentiate HR. 5 The most highly down-regulated probe set (Gma.5283.1.S1_at, -17.6 fold) corresponds to 6 a gene encoding a predicted natriuretic peptide with an expansin-like domain sharing homology 7 to AtPNP-A (Table 3), which is involved in plant growth and homeostasis (Morse et al., 2004). 8 AtPNP-A is induced by SA and is expressed at higher levels in Arabidopsis mutants with 9 increased SA levels (Meier et al., 2008). Another highly down-regulated gene is a cyclic 10 nucleotide-gated channel (CNGC) (GmaAffx.84317.1.S1_at, -5.5 fold, Table 4), which shares 11 homology with Arabidopsis CNGC4/HLM1. Arabidopsis mutants of CNGC4/HLM1 produce a 12 lesion mimic phenotype and altered HR (Balague et al., 2003). 13 Several PR genes are also up-regulated. A soybean osmotin (Gma.2821.1.S1_at, Table  14 2), which is described as a salt stress-induced acidic isoform of PR-5 (Onishi et al., 2006) and 15 has similarity to Arabidopsis osmotin 34, is up-regulated 6.4 fold. Osmotins are components of 16 incompatible reactions against bacterial pathogens (Jia and Martin, 1999). Another up-regulated 17 PR-protein is a hevein-like protein belonging to the PR-4 family, which is up-regulated during 18 salt stress, in response to viral infection, and in systemic acquired resistance (SAR) (Potter et al., 19 1993). We found a 3.5-fold up-regulation of a defensin homologous to Arabidopsis defensin 20 PDF2.1 (GmaAffx.36259.1.S1_s_at), but a 3.5-fold down-regulation of another member of the 21 same defensin family, PDF2.5 (Gma.4126.1.S1_at, Table 3 WRKY transcription factors are known to take part in defense responses to viral, 1 bacterial, and fungal pathogens (Eulgem and Somssich, 2007). Several WRKY transcription 2 factor homologs are up-regulated in syncytia of the NIL-R (Table 2) including a homolog of  3 AtWRKY33, a known regulator of defense pathways mediating resistance to P. syringae and 4 fungal necrotrophic pathogens (Zheng et al., 2006) (GmaAffx.6438.1.S1_at, 5.9 fold); an 5 AtWRKY48 homolog (GmaAffx.93596.1.S1_at, 2.67 fold); and a homolog of AtWRKY18, 6 which is involved in SA-mediated defenses against viruses, bacteria, and fungi 7 (GmaAffx.19777.1.A1_at, 1.4 fold). Interestingly, a soybean homolog of AtWRKY23 8 (Gma.8336.1.S1_at, 2.4 fold), which is involved in nematode feeding site establishment 9 (Grunewald et al., 2008), is also up-regulated. Down-regulated WRKYs (Table 3) include a 10 homolog of AtWRKY11 (GmaAffx.6478.1.S1_s_at, -2.05 fold; Gma.3504.1.S1_at, -2 fold; 11 Gma.3504.2.S1_a_at, -1.9 fold), a negative regulator of basal defense responses against bacterial 12 pathogens (Journot-Catalino et al., 2006). Down-regulation of a negative regulator would lead to 13 an enhanced defense response. 14 In general, the SA pathway has been shown to be activated in resistance against biotrophs 15 and the JA pathway in resistance to necrotrophs and insects, although exceptions exist 16 (Glazebrook, 2005;Bari and Jones, 2009). The SA pathway also has been implicated in 17 resistance to the root-knot nematode in tomato (Branch et al., 2004). Here, we identified several 18 homologs of genes belonging to the SA-mediated defense signaling pathway to be up-regulated 19 in SCN-induced syncytia of the NIL-R. A soybean MYB protein homologous to AtMYB30, an 20 SA-dependent R2-R3 MYB that acts as a positive regulator of HR cell death and is a modulator 21 of SA levels (Vailleau et al., 2002;Raffaele et al., 2006)  are key signal transducers in SA-mediated signaling. NDR1 is a plasma membrane localized 1 protein required for disease resistance to P. syringae pv. tomato DC3000 carrying avirulence 2 genes avrRpm1, avrRpt2, avrPph3, and avrB. It is also required for resistance against avirulent 3 isolates of the fungal pathogen Peronospora parasitica (Century et al., 1995;Century et al., 4 1997). The requirement for resistance against a diverse group of pathogens suggests that this is a 5 common downstream element in R-gene-mediated resistance in plants. Arabidopsis ndr1 mutants 6 have reduced ROS production and SA accumulation in response to avirulent bacteria (Shapiro 7 and Zhang, 2001). Conversely, overexpression of NDR1 in Arabidopsis leads to enhanced 8 resistance to virulent P. syringae pv. tomato (Coppinger et al., 2004). NHL proteins have 9 sequence homology to NDR1 of Arabidopsis and HIN1 of tobacco and are pathogen-induced in 10 Arabidopsis (Varet et al., 2002). NHL3 overexpression in Arabidopsis is associated with 11 enhanced resistance to virulent strains of P. syringae (Varet et al., 2003). We also identified 12 homologs of Arabidopsis ACD11 (Gma.6474.1.A1_s_at, 1.7 fold) and PBS3 13 (Gma.3755.1.S1_at, 1.5 fold), which are involved in SA-mediated defense. PBS3 (WIN3), which 14 interacts with the P. syringae effector protein HopW1-1, is important for responses induced by 15 several effectors in Arabidopsis. PBS3 is an important component of NDR1-dependent RPS2-16 mediated resistance against P. syringae pv. tomato carrying avrRpt2 and also plays a role in 17 basal resistance (Lee et al., 2007). PBS3 is an acyl adenylase, and the Arabidopsis pbs3 mutant 18 exhibits enhanced susceptibility to P. syringae pv. tomato carrying avrPphB (Nobuta et al., 19 2007 including HopW1-1 (Lee et al., 2008), which indicates WIN1 is a negative regulator. Thus, 1 down-regulation of this gene would be predicted to have a positive impact on SA levels. 2 Previously, we found a majority of JA pathway components to be suppressed during a 3 compatible soybean-SCN interaction (Ithal et al., 2007b). In this study, we found a homolog of 4 Arabidopsis lipoxygenase 1 (AtLOX1) ( Table 2)  Here, we present evidence for the potential involvement of a complex stress and defense-19 related response, including increased expression of genes involved in the production of ROS, the 20 unfolded protein response, SA-mediated signaling, and plant PCD in Rhg1-mediated resistance 21 to SCN. Involvement of almost all hormones shows an intricate network of cross-talk associated 22 with this defense response. Our study also highlights the importance of conducting a direct 23 comparison between syncytia transcriptomes in the resistant versus susceptible NILs using the 1 same nematode population to identify genes potentially involved in resistance. Inadvertently, a 2 large number of genes would be overlooked in a direct comparison of syncytia transcriptomes 3 induced in the resistant line by an avirulent versus a virulent SCN population. Here, we 4 demonstrate that the plant still mounts a defense response against the virulent nematode 5 population, albeit somewhat attenuated compared to the avirulent nematode population. In 6 contrast, the response of the susceptible line to the avirulent population is minimal. 7 Additionally, our study led to the identification of nematode-inducible soybean 8 promoters, several of which have restricted expression in roots but are highly up-regulated in 9 syncytia, which can be employed as a tool for more targeted RNAi silencing experiments of 10 soybean genes in the resistant background. Our study, together with the newly developed 11 functional analysis tools in soybean such as VIGS (Zhang et al., 2009;Zhang et al., 2010)  The promoter sequences for the genes used in the GUS reporter assays were identified and 12 downloaded from the soybean genome database (Phytozome, www.phytozome.net). Primers 13 (Table S3)  Hairy roots transgenic for each promoter-GUS construct were generated using the method 1 described by Wang et al. (2007) with the following modifications. The cotyledons were excised 2 from 9-day-old aseptically grown soybean seedlings (NIL-R or cv. Williams 82) and vacuum 3 infiltrated for 20 min with A. rhizogenes culture resuspended in ¼ Gamborg's salt solution 4 (Phytotechnology Lab, Shawnee Mission, KS, USA) carrying various reporter constructs. 5 Cotyledons were co-cultivated with A. rhizogenes for 3 days. The cotyledons were later placed 6 on MXB medium [1x MS basal nutrient salts (Gibco BRL), 1x Gamborg's vitamins, 3% w/v 7 sucrose, and 0.8% w/v Daishin agar, pH 5.7] supplemented with kanamycin (200 µg ml -1 ) and 8 timentin (238 µg ml -1 ) and incubated in a growth chamber at 26°C set to a long-day photoperiod 9 (16h light/8h dark). Hairy roots that emerged after 14 days were root-tip propagated twice on 10 MXB medium with kanamycin (200µg ml -1 ) and timentin (238µg ml -1 ), after which the roots 11 were transferred to MXB medium with timentin (237µg ml -1 ). Hairy roots at this stage were 12 either used immediately for nematode inoculation experiments or maintained by subculturing for 13 later use. 14 15

Nematode infection of transgenic hairy roots and GUS staining 16
Infective second-stage juveniles (J2) were hatched from eggs as described in Wang et al. (2007). 17 Nematodes were surface-sterilized with sterilizing solution (0.004% w/v mercuric chloride, 18 0.004% w/v sodium azide and 0.002 % v/v Triton X-100) for 8 min followed by 5 washes with 19 sterile water and resuspended in 0.1% w/v agarose. Hairy roots (3-4 cm) grown on MXB 20 medium were inoculated ~1 cm above the root tip with 200 ± 25 J2s per root in a 25-µl volume. 21 The roots were cut and stained for GUS expression at 5 dpi. GUS staining was done according to 22 Jefferson et al. (1987). Briefly, hairy roots were cut 1-2 cm above the infection zone and placed 23 in GUS staining solution (100 mM Tris pH 7.0, 50 mM NaCl, 1 mM X-Gluc, 1.5 mM potassium 1 ferricyanide pH 7.0, 0.06% v/v Triton X-100). The root tissues were vacuum-infiltrated twice for 2 10 min each and incubated at 37°C overnight. The GUS staining reaction was stopped by 3 replacing staining solution with 70% v/v ethanol. GUS stained roots were photographed under a 4 Leica MZFLIII stereoscope (Leica Microsystems, Bannockburn, IL) fitted with an Optronics 5 MagnaFire, version 2.0, camera (Optronics, Goleta, CA). Sectioning was done according to 6 Wang et al. (2007). 7 8

Sample preparation for time-course qPCR analysis 9
Infected root tissues for time course qPCR analysis were prepared as described in Ithal et al. 10 (2007a), except that samples were collected at 2, 4, 6, and 8 dpi. Excised root pieces from 12-15 11 different plants were pooled for each genotype/inoculum combination. Samples were quick 12 frozen in liquid nitrogen and stored at -80 o C until RNA isolation. Nematode penetration was 13 verified by staining the nematodes in at least five sample roots for each treatment at 24 hours 14 post-inoculation as described by Ithal et al. (2007a). Infected root tissues from three independent 15 biological replicates were prepared. 16

RNA isolation and qPCR 18
Total RNA was isolated from root tissues using the RNeasy plant miniprep kit (Qiagen,19 Valencia, CA, USA), according to the manufacturer's instructions. First strand cDNA synthesis 20 was carried out using a Superscript III first strand synthesis kit (Invitrogen, USA), according to 21 the manufacturer's instructions. Real-time qPCR was carried out using an Applied Biosystems 22 7500 real-time PCR system. Gene-specific primers (Table S4) were designed using the primer 23 express software (Applied Biosystems, CA, USA). All qRT-PCR reactions were carried out in 1 triplicate. PCR was performed using the following cycling parameters: 50°C for 2 min, 95°C for 2 10 min, and 40 cycles of 95°C 15 s and 60°C for 1 min. A soybean ubiquitin gene (Acc. No 3 D28123) was used as an endogenous control. We determined by qRT-PCR that expression of 4 this gene is stable across the treatment groups in our experiment. Expression was quantified 5 using the Δ Δ C T method in comparison to the endogenous control. Fold-changes were determined 6 relative to the NIL-R mock-inoculated sample for each time point. There were no significant 7 expression differences between mock-treated NIL-S and NIL-R roots. 8 9

Supplemental data 10
The following materials are available in the online version of this article. 11 Supplemental Table S1. Classification of up-regulated genes at FDR < 0.1. 12           The qPCR results are normalized to a soybean ubiquitin (Acc No D28123) endogenous control. The graph is representative of 3 independent experiments, and the bars represent the difference between min and maximum relative quantification values, which are calculated from the standard deviation of Ct values by the ABI software.