Arabidopsis spermidine synthase is targeted by an effector protein of the cyst nematode Heterodera schachtii.

Cyst nematodes are sedentary plant parasites that cause dramatic cellular changes in the plant root to form feeding cells, so-called syncytia. 10A06 is a cyst nematode secretory protein that is most likely secreted as an effector into the developing syncytia during early plant parasitism. A homolog of the uncharacterized soybean cyst nematode (Heterodera glycines), 10A06 gene was cloned from the sugar beet cyst nematode (Heterodera schachtii), which is able to infect Arabidopsis (Arabidopsis thaliana). Constitutive expression of 10A06 in Arabidopsis affected plant morphology and increased susceptibility to H. schachtii as well as to other plant pathogens. Using yeast two-hybrid assays, we identified Spermidine Synthase2 (SPDS2), a key enzyme involved in polyamine biosynthesis, as a specific 10A06 interactor. In support of this protein-protein interaction, transgenic plants expressing 10A06 exhibited elevated SPDS2 mRNA abundance, significantly higher spermidine content, and increased polyamine oxidase (PAO) activity. Furthermore, the SPDS2 promoter was strongly activated in the nematode-induced syncytia, and transgenic plants overexpressing SPDS2 showed enhanced plant susceptibility to H. schachtii. In addition, in planta expression of 10A06 or SPDS2 increased mRNA abundance of a set of antioxidant genes upon nematode infection. These data lend strong support to a model in which the cyst nematode effector 10A06 exerts its function through the interaction with SPDS2, thereby increasing spermidine content and subsequently PAO activity. Increasing PAO activity results in stimulating the induction of the cellular antioxidant machinery in syncytia. Furthermore, we observed an apparent disruption of salicylic acid defense signaling as a function of 10A06. Most likely, increased antioxidant protection and interruption of salicylic acid signaling are key aspects of 10A06 function in addition to other physiological and morphological changes caused by altered polyamines, which are potent plant signaling molecules.

Sedentary plant-parasitic nematodes are obligate biotrophs that meet their nutritional requirements solely from modified, but living, root cells of their host plants. The most economically important plantparasitic nematodes are the sedentary endoparasitic root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera and Globodera spp.). The soybean cyst nematode (Heterodera glycines) and the closely related sugar beet cyst nematode (Heterodera schachtii) are of particular importance in U.S. agriculture.
Cyst nematode infection involves the penetration of second-stage juveniles (J2) into host roots followed by intracellular migration to the vasculature, where the nematodes initiate specialized feeding structures called syncytia. Feeding site initiation and formation are characterized by complex morphological and physiological changes of the parasitized plant root cells. This process includes endoreduplication, cell wall modification and dissolution leading to cell fusion, disappearance of large vacuoles, increased numbers of organelles, and metabolic activity. In addition, these parasites must deploy countermeasures to defeat or evade plant defense responses that would otherwise jeopardize successful syncytium formation and function. The infection process is accompanied by an extensive alteration of gene expression in parasitized plant cells and roots. Host genes related to defense responses, primary metabolic pathways, cell wall modification, transport activities, signal transduction and transcription activity, the cell cycle, and hormone responses have been identified as differentially expressed in response to cyst nematode infection (Puthoff et al., 2003;Alkharouf et al., 2006;Ithal et al., 2007;Gheysen and Mitchum, 2009;Szakasits et al., 2009). Although these findings provide insights into the biological changes occurring during syncytium development, the molecular mechanisms safeguarding the redifferentiation of these plant cells into the complex feeding sites remain unclear.
A growing body of evidence shows that secreted proteins encoded by nematode parasitism genes act as effector molecules and play the central role in the initiation and formation of the feeding sites (Wang et al., 2005;Huang et al., 2006aHuang et al., , 2006bHewezi et al., 2008). These nematode effectors are the secreted protein products of parasitism genes expressed uniquely in the nematode esophageal gland and directed for secretion into parasitized plant cells and tissues through the stylet, a hollow mouth spear. The esophageal glands are composed of three large secretory cells (one dorsal and two subventral) that are connected through valves to the esophageal lumen, and thus the stylet. Changes in the content, morphology, and activity of the secretory gland cells are evident during parasitic stages (Davis et al., 2004). Identification of nematode effectors has been facilitated through the mining of cDNA libraries prepared from microaspirated esophageal gland cytoplasm of different parasitic stages (for review, see Davis et al., 2008). Candidate nematode effector proteins have been identified from the gland cDNA sequences through the application of different criteria, including the presence of an N-terminal signal peptide for secretion and the absence of transmembrane domain motifs in these protein sequences, along with the specific expression in the nematode esophageal gland cells. A subset of these nematode proteins has been shown to be secreted using immunolocalization technologies. These include, for example, cellulose-binding protein (Ding et al., 1998), pectate lyases (Doyle and Lambert, 2002), chorismate mutase (Doyle and Lambert, 2003), and calreticulin (Jaubert et al., 2005) from root-knot nematodes and cellulases (Wang et al., 1999) and SPRY domain-containing proteins (Rehman et al., 2009) from cyst nematodes. In addition, expression in the host plants of several effector genes identified by this approach affected plant phenotypes and nematode susceptibility (Doyle and Lambert, 2003;Wang et al., 2005;Huang et al., 2006b;Hewezi et al., 2008), validating esophageal gland effector protein involvement in plant parasitism.
More than 50 effector cDNAs have been identified from H. glycines (Gao et al., 2003). The majority of the corresponding proteins are novel, and their putative functions cannot be assigned due to the absence of significant sequence similarities to known proteins in sequence databases. Similarity to functionally characterized proteins in other organisms has been identified in only a few cases and is more often with proteins from bacteria, fungi, and plants than with proteins from the nonparasitic model nematode Caenorhabditis elegans . This again supports a function for these effectors in the parasitized plant cells and not in the nematode itself. Sequence similarity of a small set of effectors with proteins of known functions implicated these effector proteins in the softening of root cell walls, modification of metabolic pathways, regulation of gene expression via protein degradation, regulation of the cell cycle, inhibiting plant defense, and signaling pathway modification (Davis et al., 2004;. Subcellular localization prediction of cyst nematode effector proteins suggested nuclear or cytoplasmic localizations (Gao et al., 2003), and some of these subcellular localizations have been confirmed experimentally (Elling et al., 2007;Hewezi et al., 2008). A few of these proteins are targeted to the host nucleus and may act in regulation of the transcription machinery of the parasitized host cells, while others accumulate in the cytoplasm and most likely interact with specific cytoplasmic host proteins. In plant-nematode interactions, evidence for intermolecular interaction between nematode effector proteins and host proteins has been reported in only three cases (Huang et al., 2006b;Hewezi et al., 2008;Rehman et al., 2009). These direct interactions demonstrate that nematode effectors can modulate signal transduction pathways (Huang et al., 2006b), cell wall characteristics (Hewezi et al., 2008), and resistance protein signaling in their host plants (Rehman et al., 2009). These examples show that the identification and functional characterization of host target proteins is a powerful first step toward elucidating the mechanism by which nematode effectors mediate plant susceptibility.
Here, we report the functional characterization of a cyst nematode effector protein, during which we identified the triamine spermidine (Spd) as a novel and potent determinant of cyst nematode parasitism success. We uncovered that the 10A06 nematode protein acts as a cytoplasmic effector that directly targets Spermidine Synthase2 (SPDS2). We show that plant Spd levels are altered as a function of 10A06 and conclude that morphological and physiological polyamine effects likely are conducive to cyst nematode parasitism. For example, our data show that through the function of the cyst nematode effector in question, polyamine metabolism and signaling provide a protective antioxidant function to the syncytium and certain defense responses are inhibited. As a result of our study, we have uncovered, to our knowledge, a new plant pathogen effector mode of action and expanded the known functions of polyamines. and its host species, the 10A06 cDNA was identified from the sugar beet cyst nematode (H. schachtii), which can parasitize Arabidopsis. DNA gel-blot analysis showed that 10A06-type sequences in H. schachtii form a small gene family composed of at least three members (Fig. 1A). Using forward and reverse primers located in the untranslated regions of the H. glycines 10A06 cDNA, we amplified H. schachtii cDNA pools and cloned the amplification product. Sequence analysis of these 10A06 cDNA clones revealed the presence of three different isoforms (GQ373256, GQ373257, and GQ373258) that differed in only four nucleotides (Supplemental Fig. S1A). At the protein level, these three isoforms differed by only two amino acids (Supplemental Fig. S1B). Sequence alignment of H. glycines and H. schachtii 10A06 proteins revealed the H. schachtii sequence to be most similar to that of H. glycines, which showed 87% similarity and 86% identity (Supplemental Fig. S2). Given our current data, we consider this H. schachtii clone to be orthologous to the previously reported H. glycines 10A06 sequence and, therefore, conducted our characterization work using this clone (GQ373256). The H. schachtii 10A06 contained an open reading frame of 858 nucleotides encoding a 285-amino acid protein with an N-terminal signal peptide of 17 amino acids for secretion. The protein domain analysis program SMART (http://smart.embl-heidelberg.de) predicted a region of 40 amino acids located between amino acids 170 and 209 with weak similarity to the RING finger domain (SM00184; E value of 2.53e-06). However, this domain similarity per se is not high enough to deduce 10A06 function. Scanning the entire GenBank databases, including the recently published root-knot nematode Meloidogyne incognita and Meloidogyne hapla genomes (Abad et al., 2008;Opperman et al., 2008), revealed a strong conservation (98%) between the Figure 1. Characterization of H. schachtii 10A06. A, Genomic DNA of H. schachtii was digested with the EcoRI and SacI restriction enzymes and probed using the full-length radiolabeled 10A06 cDNA, which revealed that 10A06 belongs to a small gene family composed of at least three members. Molecular size markers are shown in kb. B, Developmental expression pattern of 10A06. The relative mRNA expression level of 10A06 was quantified using qPCR in six different H. schachtii life stages. The fold change values were calculated using the 2 2DDCT method and represent changes in mRNA level in preparasitic J2 (preJ2), parasitic J2 (parJ2), J3, J4, and females relative to that of eggs. Data are averages of three biologically independent experiments, each consisting of four technical replicates. H. schachtii Actin was used as an internal control to normalize gene expression level. C, In situ hybridization of a digoxigenin-labeled antisense 10A06 cDNA probe to transcripts exclusively expressed in the secretory dorsal gland cells of parasitic J2 of H. schachtii that was collected from host plant roots. When the control sense cDNA probe was used, no signal was detected. D, Subcellular localization of 10A06 in plant cells. 10A06 cDNA without signal peptide-coding sequence was fused to the joined GFP and GUS reporter genes and expressed in onion epidermal cells. GFP fluorescence is localized to the plant cell cytoplasm.
N-terminal amino acids (1-97) of 10A06 and those of another putative effector protein, 8H07 (accession no. AAP30763), from H. glycines but an absence of homologous sequences from any other organism, suggesting that 10A06 forms a cyst nematode-specific gene family.
Nematode effector genes are developmentally regulated with altering mRNA abundances throughout the parasitic stages. The developmental expression dynamics of 10A06 in six H. schachtii life stages was assessed using quantitative real-time reverse transcription-PCR (qPCR). 10A06 mRNA expression was highest in the parasitic J2 and third-stage juveniles (Fig. 1B), which suggests a function of 10A06 during the early stages of syncytium formation. Since glandspecific expression is one of the main characteristics of nematode effectors, in situ mRNA hybridization was used to localize 10A06 transcripts in different H. schachtii life stages. The digoxigenin-labeled antisense cDNA probes of 10A06 hybridized uniquely with transcripts accumulating in the dorsal esophageal gland cell of H. schachtii (Fig. 1C), the same localization pattern as reported for H. glycines (Gao et al., 2003).

Subcellular Localization of 10A06 in Plant Cells
Nematode effector proteins are delivered into the host cell through the nematode stylet. The subcellular localization of 10A06 inside plant cells was analyzed by generating a construct that fused the mature 10A06 sequence (without signal peptide) to joined GFP and GUS reporter genes (10A06:GFP:GUS) under the control of a double cauliflower mosaic virus (CaMV) 35S promoter. This construct was delivered into onion (Allium cepa) epidermal cells by biolistic bombardment. As shown in Figure 1D, the 10A06:GFP:GUS fusion protein is localized inside the plant cell cytoplasm, suggesting that 10A06 acts as cytoplasmic effector.
Expression of 10A06 in Arabidopsis Affects Plant Phenotype and Increases Susceptibility to H. schachtii Expression of candidate nematode effector genes in plant tissues can provide a direct, albeit crude, indication of effector function. Therefore, we constitutively expressed the 10A06 cDNA without signal peptide in Arabidopsis driven by the CaMV 35S promoter. Three independent homozygous T3 lines (lines 2-7, 5-7, and 12-12) that showed elevated 10A06 mRNA levels (Supplemental Fig. S3) were phenotypically investigated. All transgenic lines developed higher numbers of leaves than wild-type plants ( Fig. 2A). At flowering, the average total leaf number per plant for the transgenic lines ranged from 24.0 6 0.82 to 29.0 6 1.69, whereas on wild-type plants the average leaf number was 16.6 6 0.58. Expression of 10A06 also produced plants with significantly increased root lengths, ranging from 47.15 6 1.21 mm to 50.20 6 1.35 mm compared with 39.26 6 1.34 mm of the wild-type control at 10 d after planting (Fig. 2B), equaling a 20% to 28% increase in root length. In addition, the transgenic lines were found to flower approximately 3 d earlier than the wild-type plants. These transgenic lines were subjected to cyst nematode susceptibility assays. Tenday-old plants were inoculated with J2 of H. schachtii, and the number of adult females was counted 3 weeks after inoculation for both the transgenic and wild-type lines. A clear effect of transgene expression on nematode susceptibility was observed. All three transgenic lines were dramatically more susceptible than the wild-type control, as evidenced by the statistically significantly higher number of adult females (Fig. 2C). This increased susceptibility of more than 80% cannot be explained by the approximately 20% to 28% longer roots and, therefore, suggests a key role of the 10A06 effector in increasing Arabidopsis susceptibility to the cyst nematode.

10A06 Enhances Susceptibility to Multiple Pathogens
In order to further explore the mechanism of this increased nematode susceptibility, we investigated whether 10A06 also modulated plant susceptibility to other pathogens. Two independent 10A06-expressing homozygous T3 lines (lines 2-7 and 12-12) were inoculated with Pseudomonas syringae pv tomato (Pst DC3000) or the yellow strain of Cucumber mosaic virus (CMVY). Three days following bacteria inoculation, the number of bacteria was determined. Growth of Pst DC3000 on the transgenic plants was significantly increased relative to that of the wild-type C24 (Fig.  2D). Likewise, 5 d post inoculation (dpi) of the transgenic plants with CMVY, RNA was isolated from leaves and qPCR was used to quantify the accumulation of viral RNA. A significant increase of viral RNA accumulation in the transgenic lines was detected relative to the wild type (Fig. 2E). Taken together, these data demonstrate that expression of 10A06 in Arabidopsis alters plant susceptibility to various pathogens.

Increased Susceptibility of 10A06-Expressing Plants Is Associated with Repression of Salicylic Acid Signaling
We evaluated the contribution of salicylic acid (SA), jasmonic acid (JA), and ethylene toward the increased susceptibility of 10A06-expressing plants by assaying the expression of a set of pathogenesis-related (PR) genes. The mRNA abundances of PR-1, PR-2, and PR-5, whose induction patterns are used as molecular markers for the activation of the SA signaling pathway, were analyzed in transgenic plants expressing 10A06 and in wild-type control plants under both infected and noninfected conditions. Furthermore, we assayed the mRNA expression of the JA-induced PR-3 and PR-4 genes as well as the PDF1.2 gene whose activation occurs via JA/ethylene-mediated signaling. While under noninfected conditions only PR-1 showed significant down-regulation in transgenic plants expressing 10A06 relative to the wild type, under infected conditions all SA-responsive genes (PR-1, PR-2, and PR-5) showed clear down-regulation as a function of 10A06, Figure 2. Constitutive expression of 10A06 in Arabidopsis alters plant morphology and enhances plant susceptibility to H. schachtii and distinct pathogens. A and B, Homozygous T3 lines expressing 10A06 displayed a higher number of leaves (A) and longer roots (B) than the wild type (WT) C24. C, Transgenic Arabidopsis plants expressing 10A06 showed enhanced susceptibility to H. schachtii. Homozygous T3 lines expressing 10A06 without the native signal peptide (lines 2-7, 5-7, and 12-12) were planted on modified Knop's medium, and 2-week-old seedlings were inoculated with approximately 250 surface-sterilized J2 H. schachtii nematodes. Two weeks after inoculation, the number of J4 female nematodes per root system was determined. Data are presented as means 6 SE. Mean values significantly different from the wild type are denoted by asterisks, as determined by unadjusted paired t tests (P , 0.05). Identical results were obtained from at least two independent experiments. D, Transgenic Arabidopsis plants expressing 10A06 exhibit enhanced susceptibility to Pst DC3000. The wild type (C24) and two independent transgenic lines (2-7 and 12-12) were inoculated with Pst DC3000 at an initial density of 4 310 5 colony-forming units (cfu) cm 22 , and bacteria growth was quantified 3 dpi. Data represent means of eight independent experiments 6 SE. Mean values significantly different from the wild type, as determined by paired t tests (P , 0.05), are denoted by asterisks. E, Expression of 10A06 in Arabidopsis enhanced the accumulation of CMVY. The wild type (C24) and two independent transgenic lines (2-7 and 12-12) were inoculated with CMVY, and leaf samples were collected 5 dpi for RNA extraction and qPCR analysis to quantify the accumulation of viral RNA using primer pairs specific to the RNA1 replicase gene. Shown are the expression levels in the transgenic lines relative to the wild type after normalization using Arabidopsis Actin8 as an internal control. Data are means of four biologically independent experiments, each consisting of four technical replicates. Mean values significantly different from 1.0 (no change) are indicated by asterisks, as determined by paired t tests (P , 0.01).
with PR-1 being the most responsive gene (Fig. 3). In contrast, 10A06 expression had only slight or no effect on the expression of PR-3, PR-4, and PDF1.2 under both infected and noninfected conditions (Fig. 3). These results suggest that the increased susceptibility of 10A06-expressing plants to H. schachtii and other pathogens is associated with a suppression of SA signaling.

10A06 Specifically Interacts with SPDS2 in a Yeast Two-Hybrid Assay
Hypothesizing that 10A06 acts in concert with host plant proteins, we screened three yeast two-hybrid libraries prepared from Arabidopsis roots harvested at 3, 7, and 10 d after H. schachtii infection (Hewezi et al., 2008) using a full-length 10A06 (minus signal peptide)-GAL4 DNA-binding domain fusion as bait. More than 15 million yeast transformants were screened, and only one clone was found to interact specifically and consistently with 10A06. This clone encoded the C-terminal 243 amino acids of SPDS2 (At1g70310). SPDS2, a 340-amino acid protein, is a key enzyme of polyamine biosynthesis involved in the conversion of the diamine putrescine (Put) to the triamine Spd. This interaction was further confirmed by cotransformation and a-galactosidase (a-Gal) quantitative assays, in which yeast AH109 cells were cotransformed with the SPDS2-GAL4 transcriptional activation domain fusion together with either the empty GAL4 DNAbinding domain vector or the human Lamin C-GAL4 DNA-binding domain vector. The transformed yeast cells were unable to grow on SD/2Ade2His2Leu2Trp medium or to activate the MEL1 reporter gene, which encodes a-Gal. In contrast, when yeast cells were cotransformed with the SPDS2-GAL4 transcriptional activation domain fusion together with the 10A06-GAL4 DNA-binding domain fusion, they were able to grow on SD/2Ade2His2Leu2Trp and activate the MEL1 reporter gene to high levels in a-Gal quantitative assays (Fig. 4, A and B). These experiments clearly showed that the interaction between 10A06 and Arabidopsis SPDS2 is due to a specific binding between the two proteins. To confirm the 10A06-SPDS2 interaction in planta, bimolecular fluorescence complementation (BiFC) assays (Citovsky et al., 2006) were carried out. Coding sequences of 10A06 without a signal peptide and full-length SPDS2 were fused N terminally to those of nonfluorescent halves of yellow fluorescent protein (YFP) and coexpressed in onion epidermal cells. The interaction between 10A06 and SPDS2 brought the two halves of YFP into proximity of each other and reconstituted the fluorescent YFP in the cytoplasm of transformed cells (Fig. 4C). Onion epidermal cells bombarded with single plasmids or in combination with empty vectors or in combination with an unrelated nematode gene yielded no YFP fluorescence.
10A06 was further characterized in the yeast twohybrid system by deletion analysis to identify the region(s) important for binding to SPDS2. Because the RING finger domain is known to be involved in protein-protein interaction, we first tested whether the 40-amino acid region with weak similarity to the RING finger domain in 10A06 itself is sufficient to interact with SPDS2. A bait construct of the full-length 10A06 lacking this region (Fig. 4D, Del1) and a construct of the 40-amino acid region itself (Fig. 4D, Del2) were generated, and their ability to interact with SPDS2 was assayed using a-Gal quantitative assay to measure the strength of the protein interactions. In both cases, the a-Gal activity was significantly reduced relative to that observed with the full-length 10A06, indicating that the 40-amino acid region is essential but not sufficient to confer a high interaction with SPDS2. Bait constructs containing parts of this region along with flanking regions (Fig. 4D, Del3 and Del4) or the whole 40-amino acid region with the flanking regions ( Fig. 4D, Del5) failed to give rise to positive interactions. Two additional bait constructs containing the whole 40-amino acid sequences with the flanking N-terminal region (Fig. 4D, Del6) or C-terminal region (Fig. 4D, Del7) were tested. a-Gal quantitative assays revealed that the C-terminal region can bind to SPDS2, but not as strongly as the complete 10A06 (Fig. 4D). This finding suggests that the C-terminal region may have a role in mediating the interaction.
Because the Arabidopsis genome contains two additional genes coding for SPDS proteins (Hanzawa et al., 2002), we assayed whether SPDS1 (AT1G23820), which shares the strongest sequence homology with SPDS2, also interacts with 10A06 in the yeast twohybrid assay. The full-length SPDS1 coding sequence was inserted into the prey vector and transformed into . Increased susceptibility of 10A06-expressing plants is associated with repression of SA signaling. qPCR was used to quantify the expression levels of PR genes in wild-type and transgenic plants expressing 10A06 (line 12-12) under noninfected and infected conditions (7 dpi). The fold change values were calculated using the 2 2DDCT method and represent changes of mRNA abundance in transgenic plants relative to the wild-type control. Data are averages of three independent biological experiments, each consisting of four technical replicates. Arabidopsis Actin8 was used as an internal control to normalize gene expression level. yeast cells in combination with the bait vector containing 10A06 or human Lamin C or the empty bait vector. The interactions in all cases were assayed by plating the transformed cells onto the appropriate medium and quantified by a-Gal activity produced by at least 10 colonies. a-Gal quantitative assays revealed no activity of the MEL1 reporter gene beyond the background level, indicating that the physical interaction between 10A06 and SPDS2 but not SPDS1 is highly specific (Supplemental Fig. S4). . 10A06 specifically interacts with SPDS2. A, The yeast two-hybrid interaction between 10A06 and SPDS2 visualized by differential growth on nonselective medium (left) and on selective medium (right). Yeast cells containing the SPDS2 prey plasmid and the 10A06 bait vector grow on the selective medium. In contrast, yeast cells containing the SPDS2 prey plasmid along with either the empty bait vector or bait vector containing the human Lamin C gene fail to grow on the selective medium. B, a-Gal quantitative assays of the 10A06-SPDS2 interaction. Yeast strain AH109 was cotransformed with the prey plasmid in combination with the 10A06 bait vector, or bait vector containing the human Lamin C gene, or the empty pGBKT7 bait vector and plated on SD/2Leu/2Trp. Three days after culture, 10 independent colonies per combination were picked to quantify the interaction using a-Gal activity. Activity was seen only in yeast cells containing the SPDS2 prey plasmid and the 10A06 bait vector. The experiment was repeated three times with identical results. C, BiFC visualization of the 10A06-SPDS2 interaction. Onion epidermal cells were cobombarded with constructs expressing nEYFP-10A06 and cEYFP-SPDS2. Bright-field, YFP, and overlay of bright-field and YFP images were taken 24 h after bombardment. D, Schematic representation of intact and truncated 10A06 sequences used as bait in a-Gal quantitative assay demonstrating that the full-length 10A06 is required for strong interaction with SPDS2. Yeast strain AH109 cotransformed with prey plasmid and the different bait constructs indicated in the scheme were streaked on SD/2Leu/2Trp, and 3 d after culture, 10 separate colonies per construct were picked to quantify a-Gal activity. The assays were repeated three times with identical results. 2, No interaction detected; +, weak interaction; + + +, strong interaction.

SPDS2 Accumulates in the Plant Cytoplasm, and Its Transcript Abundance Is Influenced by H. schachtii and 10A06
Because 10A06 is localized to the cytoplasm, we needed to determine if SPDS2 also accumulates in the cytoplasm in order to enable an interaction with the nematode effector. These assays clearly showed a cytoplasmic accumulation of SPDS2 (Supplemental Fig. S5). To test whether the SPDS2 mRNA level is modulated in response to H. schachtii infection, we used qPCR to examine SPDS2 gene expression responses in nematode-infected wild-type plants. Root tissues were collected 3, 7, and 14 dpi from infected and control plants and assayed for SPDS2 mRNA accumulation. Data obtained from three biological experiments revealed up-regulation of SPDS2 at the 3-and 7-dpi time points (Fig. 5A) and no difference in mRNA level between inoculated and control plants at the 14-dpi time point (data not shown). These expression profiles suggest a role of SPDS2 during the initiation and development of syncytia, which coincides with the developmental expression profile of 10A06 in the nematode. To investigate whether the expression of SPDS2 is directly influenced by 10A06, as our yeast two-hybrid and expression analyses suggest, we assayed SPDS2 mRNA abundance in one of the transgenic plant lines expressing 10A06 (line 12-12). In three independent experiments, the mRNA abundance of SPDS2 was higher in 10A06-expressing plants than in wild-type plants, and this effect was evident with both infected (3-and 7-dpi time points) and noninfected plants (Fig. 5A). These data strongly suggest that a mechanism triggered by the 10A06-SPDS2 protein-protein interaction, possibly a feedback loop, causes the observed SPDS2 mRNA steady-state level changes.
Transgenic Plants Expressing 10A06 Exhibited Elevated S-Adenosyl-L-Met Decarboxylase Expression, Spd Content, and Polyamine Oxidase Activity As shown above, 10A06 in Arabidopsis stimulated the expression of SPDS2 under both infected and noninfected conditions. Because S-adenosyl-L-Met decarboxylase (SAMDC) is another key enzyme involved in Spd biosynthesis, we tested whether its mRNA expression level is affected in the transgenic plants expressing 10A06 as well. Under both noninfected and infected conditions, the expression of SAMDC was elevated as a function of 10A06 expression (Supplemental Fig. S6A). These data confirmed a profound perturbation of the plant polyamine biosynthesis as a result of the nematode 10A06 gene expression.
Our findings prompted us to determine whether in planta Spd content is affected by 10A06. We quantified free Spd levels in transgenic plants expressing 10A06 (line 12-12) and wild-type plants under both noninfected and infected (7-dpi) conditions. Without nematode infection, Spd content was increased by 25% in the transgenic 10A06 plants relative to the wild type, although this increase was statistically nonsignificant (Fig. 5B). However, under infected conditions, a statistically highly significant Spd increase of 62% was detected in 10A06 plants when compared with the infected wild-type control (Fig. 5B).
Because Spd is the main substrate for polyamine oxidase (PAO), we further tested whether the increased Spd content of 10A06 plants was associated with increased PAO expression and activity. While the Arabidopsis genome contains five putative PAOs (Tavladoraki et al., 2006), we focused on AtPAO2, which was previously shown to be the most responsive to H. schachtii infection (Szakasits et al., 2009). We tested AtPAO2 mRNA abundance in 10A06-expressing plants both under infected and noninfected conditions relative to wild-type plants using qPCR. AtPAO2 showed higher mRNA abundance in the transgenic plants when compared with the wild-type under both infected and noninfected conditions (Supplemental Fig. S6B). Similarly, we measured the activity of PAO. While we could not detect significant differences of PAO activity under noninfected conditions (Fig.  5C), a statistically significant increase of PAO activity (73%) in 10A06 plants was evident following nematode infection (Fig. 5C).

SPDS2 Is Highly Expressed in the H. schachtii-Induced Feeding Sites
If in fact 10A06 and SPDS2 bind in planta when present in the same cell and if this interaction is the causal trigger for the observed SPDS2 mRNA increase, SPDS2 mRNA abundance should be increased in H. schachtii-induced syncytia. Because our qPCR analysis of SPDS2 gene expression reflects whole root responses rather than specific spatial activity, we generated multiple transgenic lines expressing a SPDS2 promoter:GUS construct, and the expression of the reporter gene was histochemically assayed in both noninfected and infected plants. In noninfected plants, GUS staining was infrequently detected in vascular leaf bundles (Fig. 6A). In roots, GUS expression also was confined to vascular cells, with strong expression only in young roots but not in the root tip regions (Fig.  6B). As hypothesized, the SPDS2 promoter-GUS reporter gene showed a strong increase in GUS expression in the H. schachtii-induced feeding sites as early as 3 dpi (Fig. 6C). The observed expression increase prevailed through the 7-and 14-dpi sample times, when GUS activity was very strongly elevated in the feeding sites (Fig. 6, D and E). These data provide additional support for a 10A06-SPDS2 interactive role in cyst nematode parasitism.
Polyamine Biosynthetic Genes Are Differentially Expressed in Response to H. schachtii Infection SPDS2 is a key enzyme of polyamine biosynthesis, and its modulation through the nematode 10A06 effector should have an effect on polyamine metabolism in general. Furthermore, because expression of polyamine biosynthetic genes is believed to be under feedback regulation by polyamines (Moschou et al., 2008b), it was of interest to test the expression profile of other key genes involved in polyamine biosynthesis in response to H. schachtii infection. The mRNA abundance of the Arg decarboxylase genes ADC1 and ADC2, which are involved in the generation of the diamine Put from Arg, the Spd synthase gene SPDS1, which is involved in the conversion of Put to the triamine Spd, SAMDC, which acts as the propylamine group donor in the synthesis of the polyamines Spd and spermine (Spm) from Put, and the ACAULLS5 (ACL5) and spermine synthase (SPMS) genes, which are involved in the synthesis of the tetraamine Spm from Spd, were quantified by qPCR in root tissues of wild-type Arabidopsis seedlings at 3, 7, and 14 dpi with H. schachtii. Data obtained from three independent experiments showed that ADC1, ADC2, SPDS1, and SAMDC all were up-regulated at all time points relative to noninfected roots. In contrast, ACL5 and SPMS were down-regulated, with the exception that SPMS was highly up-regulated at 14 dpi (Fig. 7). These data strongly support that H. schachtii infection has profound effects on polyamine biosynthetic gene expression and thus support our discovery of 10A06 functioning as a regulator of polyamine synthesis.

Manipulation of SPDS2 Expression
To obtain a comprehensive view of the functional role of SPDS2 in mediating Arabidopsis susceptibility to H. schachtii, we manipulated the expression of SPDS2 using gene knockout and overexpression plants. First, we identified two independent T-DNA insertional null alleles (spds2-1 and spds2-2) in the Columbia-0 (Col-0) background from the randomly mutagenized T-DNA lines (the SALK collection) at The Arabidopsis Information Resource (Alonso et al., 2003). No phenotype changes were observed in these two mutants when they were compared with Col-0 wild-type plants under standard growth conditions, confirming the previously reported results of Imai et al. (2004) that spds2 mutants are morphologically indistinguishable from wild-type plants. Furthermore, Figure 5. Transgenic plants expressing 10A06 stimulated the expression of SPDS2 and exhibited significantly higher Spd content and PAO activity than the wild type (WT). A, Up-regulation of SPDS2 in response to H. schachtii infection. qPCR was used to quantify SPDS2 expression levels in wild-type and transgenic plants expressing 10A06 (line 12-12) under noninfected and infected conditions (3 and 7 dpi). Data obtained from three biologically independent experiments show higher SPDS2 expression levels in the transgenic plants than in the wild type under both infected (3-and 7-dpi time points) and noninfected conditions. Arabidopsis Actin8 was used an internal control to normalize gene expression level. B, Transgenic plants expressing 10A06 contained significantly higher Spd contents than wild-type plants. Free Spd levels were quantified in tissue samples collected from wild-type and trans-genic plants expressing 10A06 (line 12-12) under noninfected and infected conditions (7 dpi) using HPLC. The values are reported in nmol g 21 fresh weight (FW). Data are presented as means of four biologically independent experiments 6 SE. The asterisk indicates a statistically significance difference from wild-type plants at P , 0.01. C, Transgenic plants expressing 10A06 exhibited higher PAO activity than wild-type plants. PAO activity was measured in tissue samples collected from wild-type and transgenic plants expressing 10A06 (line 12-12) under noninfected and infected conditions (7 dpi) using a spectrophotometer. PAO activity levels are expressed on a fresh weight basis. Data are presented as means of three biologically independent experiments 6 SE. The asterisk indicates a statistically significance difference from wild-type plants at P , 0.01. no significant differences in susceptibility to H. schachtii between spds2 mutants and wild-type plants were detected (Fig. 8A). These negative results most likely are due to redundant SPDS functions in Arabidopsis, which have been reported by Imai et al. (2004), as well as by redundant functions in the nematode effector arsenal. We also overexpressed the full-length SPDS2 cDNA in Col-0 under the control of the 35S promoter. Multiple, independent homozygous lines were identified and subjected to qPCR to quantify the expression of SPDS2. Four lines expressing between 2-and 10fold higher SPDS2 mRNA relative to the wild type were chosen and used in nematode infection assays. Three out of the four lines showed statistically significant increases in susceptibility to H. schachtii relative to the wild-type control (Fig. 8B), indicating that the susceptibility-increasing effect of 10A06 most likely is accomplished through an elevated SPDS2 activity.

Expression of Antioxidant Machinery Genes Is Activated in Transgenic Plants Overexpressing 10A06 or SPDS2
Degradation of Spd and other polyamines through PAO produces hydrogen peroxide, which at low concentration has been shown to function as a signaling molecule stimulating the induction of antioxidant machinery genes in plants (Papadakis and Roubelakis-Angelakis, 2005;Moschou et al., 2008a). In order to test whether the SPDS2-and nematode-induced alterations of polyamines have such an antioxidant effect, we used qPCR to quantify the expression of the Arabidopsis antioxidant genes Catalase1 (CAT1) and CAT2, Glutathione Peroxidase2 (GPX2) and GPX6, and Ascorbate Peroxidase1 (APX1) and APX3 in transgenic plants overexpressing SPDS2 in response to H. schachtii infection. Data obtained from three independent experiments showed a significant up-regulation of all assayed genes relative to the infected wild-type control (Fig. 9A). If 10A06 functions through its interaction with SPDS2 to activate the antioxidant machinery, one would expect that transgenic plants expressing 10A06 would activate the expression of the antioxidant genes as well. Thus, we quantified the expression of the above-mentioned antioxidant genes in transgenic plants expressing the nematode effector 10A06 under infected conditions relative to the infected wild-type plants. Interestingly, these genes also exhibited a statistically significant up-regulation in these transgenic plants compared with the wild type (Fig. 9B). Thus, these data further confirm that 10A06 in fact functions through SPDS2 and that the parasitism-enhancing effect of 10A06 and SPDS2 could at least partially be grounded in an induction of the antioxidant machinery.
Because accumulation of hydrogen peroxide associated with high levels of PAO activity participates in the induction of programmed cell death (PCD) rather than an activation of the antioxidant machinery, we analyzed DNA fragmentation in the transgenic plants expressing 10A06 as a tell-tale sign of PCD. Genomic DNA was extracted from root tissues of transgenic 10A06 plants along with the wild type at 4 dpi with H. schachtii, and DNA fragmentation was examined with gel electrophoresis. As shown in Supplemental Figure  S7, the extracted DNA was intact in both wild-type and transgenic plants. This lack of any DNA laddering indicates that the elevated PAO activity detected by us is sufficiently moderate in amplitude to cause the activation of the antioxidant machinery and does not trigger PCD.

DISCUSSION
Sedentary plant-parasitic nematodes secrete effector proteins that act synergistically on the parasite's host plant (Davis et al., 2004. These effectors are thought to be critical for host invasion, formation of feeding sites, feeding, and negating host defenses. There are only a few examples of in vivo functional characterization of nematode effectors due to the inability to transform plant-parasitic nematodes and the complexity of the infection process. In this study, we have characterized the H. schachtii 10A06 effector protein, which is specifically synthesized in the dorsal esophageal gland during early stages of parasitism, when the syncytium is developing. 10A06 belongs to a small gene family without similarity to proteins in databases, suggesting a unique functional role in cyst nematode parasitism. Previously, we showed that expression of nematode effectors in plants provides a direct way to investigate their potential involvement in host parasitism (Huang et al., 2006b;Hewezi et al., 2008). A critical role of 10A06 in cyst nematode parasitism of Arabidopsis plants was shown in this study by the substantial increase in nematode susceptibility of the transgenic lines expressing 10A06 relative to wild-type control plants. Increasing nematode susceptibility by expressing 10A06 lacking the predicted signal peptide suggests that this secretory protein acts within the plant cell as a cytoplasmic effector, which is consistent with the subcellular localization results for this protein. Also, constitutive expression of 10A06 resulted in increased susceptibility to CMVY and Pst DC3000, demonstrating that 10A06 expression alters plant susceptibility to different pathogens. The enhanced susceptibility of 10A06-expressing plants Figure 7. Expression profile of key genes involved in polyamine biosynthesis in response to H. schachtii infection. The mRNA expression levels of ADC1, ADC2, SPDS1, SAMDC, ACL5, and SPMS were measured by qPCR in wild-type (Col-0) root tissues. Infected and noninfected tissues were collected at 3, 7, and 14 dpi. The fold change values were calculated using the 2 2DDCT method and represent changes of mRNA abundance in infected tissues relative to noninfected controls. Data are averages of three independent biological experiments, each consisting of four technical replicates. Arabidopsis Actin8 was used as an internal control to normalize gene expression level. The spds2 knockout mutant alleles (spds2-1 and spds2-2) and wild-type Col-0 (WT) plants were planted on modified Knop's medium, and 2-week-old seedlings were inoculated with approximately 250 surfacesterilized J2 H. schachtii. Three weeks after inoculation, the number of J4 female nematodes per root system was counted. Data are presented as means 6 SE. Identical results were obtained from at least three independent experiments. B, Transgenic Arabidopsis plants overexpressing SPDS2 showed increased susceptibility to H. schachtii. Homozygous T3 lines overexpressing SPDS2 (lines 5-6, 6-5, 10-6, and 4-6) were assayed for nematode susceptibility as indicated in A. Data are presented as means 6 SE. Mean values significantly different from the wild type (Col-0) as determined by unadjusted paired t tests (P , 0.05) are denoted by asterisks. Identical results were obtained from at least two independent experiments. seems to be associated with a repression of SA signaling. Recently, it has been reported that successful cyst nematode parasitism involves a local suppression of SA signaling in roots (Wubben et al., 2008). Several secreted effector proteins have been shown to negatively impact basal defense pathways, leading to increased susceptibility to other pathogens. For example, the expression of Cladosporium fulvum Avr2 in both Arabidopsis and tomato (Solanum lycopersicum) showed enhanced susceptibility toward various pathogens (van Esse et al., 2008).
The functionality of certain nematode effector proteins may require them to interact with host proteins when secreted into plant cells. Using yeast two-hybrid analyses, we identified Arabidopsis SPDS2 as a 10A06 interactor. SPDS2 (EC 2.5.1.16) is a key enzyme of polyamine biosynthesis and catalyzes the synthesis of Spd. These data, therefore, identify polyamines as a target of cyst nematode manipulation in the syncytium during the infection process. Our findings are of particular interest also because the SPDS2 promoter was activated to very high levels in the developing syncytium, which supports a 10A06 and SPDS2 binding and implicates a likely feedback regulation of SPDS2 transcription. The fact that SPDS2 overexpression lines, just like 10A06 overexpression lines, also exhibited elevated cyst nematode susceptibility relative to wild-type plants suggests that the 10A06-SPDS2 interaction results in a net increase of SPDS2 activity. However, no significant differences in cyst nematode susceptibility between the spds2 mutant lines and the wild-type control were seen. Possible explanations are that the nematode has a complex and robust system of effectors that can overcome limited loss of effector function and the fact that the Arabidopsis genome contains two additional genes coding for Spd synthases, SPDS1 and SPDS3, which share 81% to 85% similarity with SPDS2 and could be involved in cyst nematode infection, possibly through other so far uncharacterized effectors, as well. These proteins likely have redundant functions, and the spds2 single mutant could possibly be functionally replaced by SPDS1 or SPDS3. In accord with this interpretation, it has been demonstrated that SPDS1 and SPDS2 are enzymatically active SPDS isologs (Panicot et al., 2002). The spds1/spds2 double mutant plants exhibit an embryo-lethal phenotype (Imai et al., 2004), precluding us from testing this hypothesis.
We also detected increased Spd content and PAO activity as a function of 10A06 and SPDS2. How do these 10A06-SPDS2-triggered changes increase the parasitic success of H. schachtii? One possible role centers on a protective function against reactive oxygen species (ROS). Generation of ROS is a common characteristic of plant responses to plant-parasitic nematode infection in both compatible and incompatible interactions (Waetzig et al., 1999). In support, plant-parasitic nematodes themselves are believed to possess an antioxidant machinery to protect their bodies against ROS produced by the host. Putatively secreted enzymes to scavenge ROS, such as superoxide dismutase, CAT, peroxidase, and GPX, have been identified in root-knot and cyst nematodes (Molinari and Miacola, 1997;Robertson et al., 2000;Jones et al., 2004;Dubreuil et al., 2007). It could be that the developing syncytium, required for successful cyst nematode parasitism, needs ROS protection as a safeguard for nematode survival. This function could be fulfilled by Spd and other polyamines, which have ROS-scavenging properties due to their anion-and cation-binding capacity (Løvaas, 1991;Groppa et al., 2001;Groppa and Benavides, 2008). Therefore, nematode activation of SPDS2 via secreting 10A06 during feeding site formation may increase the antioxidant potential of syncytial cells by a mere increase in polyamines. In addition, the SPDS product Spd is the main substrate for PAO (Yoda schachtii. Plant tissues were collected 7 dpi. The fold change values were calculated using the 2 2DDCT method and represent changes of mRNA abundance in infected tissues relative to infected wild-type controls. Data are averages of three independent biological experiments, each consisting of four technical replicates. Arabidopsis Actin8 as was used as an internal control to normalize gene expression level.  , 2003), and Spd catabolism by PAO results in hydrogen peroxide production, which at low concentration functions as a signaling molecule stimulating the induction of the antioxidant machinery (Papadakis and Roubelakis-Angelakis, 2005;Moschou et al., 2008a). Our documentation of the activation of antioxidant genes in 10A06-overexpressing as well as in SPDS2-overexpressing Arabidopsis plants infected with H. schachtii provides support for ROS scavenging as a likely mechanism of 10A06 effector function.
On the other hand, 10A06-mediated polyamine changes may have other functions. Phenotypic changes in transgenic plants expressing 10A06 included early flowering, accelerated root growth, and increased leaf numbers, indicating that 10A06 can influence various morphological and physiological processes in plants.
Morphological and physiological alterations in transgenic plants caused by the expression of other nematode effectors also have been reported (Wang et al., 2005;Huang et al., 2006b;Hewezi et al., 2008). Based on the documented 10A06-SPDS2 interaction and the resultant changes in polyamine biosynthetic gene expression and Spd content, the morphological alterations associated with 10A06 expression in transgenic plants most likely can be attributed to polyamine metabolism and signaling in these plants. A connection between polyamines and the physiological events leading to flowering and plant growth has been reported (Kakkar and Rai, 1993;Kakkar et al., 2000;Bais and Ravishankar, 2002;Zieliń ska et al., 2006). It appears likely that increased Spd content as a result of cyst nematode infection will influence the morphological and physiological changes during syncytium formation in addition to ROS protection. Identification of the regulatory components that connect the plant morphological and physiological changes and nematode susceptibility on the one hand and SPDS-mediated polyamine changes on the other hand requires further analyses.
Finally, a third role may center on the extensive experimental evidence that has been reported on the essential role of polyamines in plant defense responses to a wide range of biotic and abiotic stresses (Bouchereau et al., 1999;Shen et al., 2000;Kasukabe et al., 2004;Moschou et al., 2008bMoschou et al., , 2009. Supporting evidence for the involvement of polyamines in plantmicrobe interactions comes from the fact that polyamines rapidly accumulate in plant tissues upon pathogen infection (Marini et al., 2001;Cowley and Walters, 2002;Yoda et al., 2003Yoda et al., , 2006Yoda et al., , 2009Moschou et al., 2009). Moreover, Spd was identified as an effective inducer of nitric oxide (Tun et al., 2006), which in turn plays significant signaling roles in plant-pathogen interactions (Romero-Puertas et al., 2004). In animal cells, polyamines have contradictory roles in inducing apoptosis and in its prevention through two different functional roles (Schipper et al., 2000;Thomas and Thomas, 2001). Similar dual functional roles of polyamines in plants would not be unexpected. In other words, a multitude of polyamine effects could be re-sponsible in addition to the scenarios detailed above. In this context, it is important to mention that the enhanced susceptibility of 10A06-expressing plants to CMVY and Pst DC3000 sheds light onto a possible connection between polyamine signaling and the basal defense response. The down-regulation of PR-1, PR-2, and PR-5 in 10A06-expressing plants reinforces this possibility. In accordance with this interpretation, the expression of PR genes was found to be affected in transgenic tobacco (Nicotiana tabacum) plants overexpressing or downregulating PAO as well as in response to exogenous application of the polyamines Put, Spd, and Spm (Moschou et al., 2009).
Collectively, the data reported here provide strong support for 10A06 and SPDS2 functioning together in modulating the outcome of the cyst nematode-plant interaction. All our data can be reconciled in the following working model of how 10A06 secretion increases cyst nematode parasitic success. The binding of the secreted 10A06 to SPDS2 posttranslationally increases this enzyme's activity, ultimately leading to an increase in Spd content and subsequently PAO activity. Thus, elevated degradation of Spd by PAO results in hydrogen peroxide production, which at low concentration functions as a signaling molecule stimulating the induction of antioxidant genes in the syncytium. Because the expression of polyamine biosynthetic genes, including SPDS2, is under tight feedback regulation by polyamines, PAO degradation of Spd in turn activates the SPDS2 promoter in the syncytium. Transcriptional activation of SPDS2 further amplifies Spd synthesis and results in an overall change of polyamine metabolism in the syncytium.

Plant Materials and Growth Conditions
Arabidopsis (Arabidopsis thaliana) ecotype C24 was used as the wild type for expression of the 10A06 gene, while ecotype Col-0 was used for overexpression of SPDS2 and expression of SPDS2 promoter:GUS construct. Arabidopsis plants were grown under sterile conditions on Murashige and Skoog (MS) solidified medium containing 2% Suc, or in potting soil in a growth chamber under long-day conditions (16 h of light/8 h of dark) at 23°C.

DNA Gel-Blot Analysis
Total genomic DNA was isolated from Heterodera schachtii according to Bline and Stafford (1976). Ten micrograms of DNA was digested overnight at 37°C with the EcoRI and SacI restriction enzymes (Invitrogen). DNA transfer, probe hybridization, and signal detection were performed as described by Hewezi et al. (2006).

Plasmid Construction and Generation of Transgenic Arabidopsis Plants
The coding sequences of 10A06 without the nematode signal peptide sequences were amplified from the full-length cDNA clones. The forward and reverse primers that contain BamHI and SacI restriction sites, respectively, were used (Supplemental Table S1). For the SPDS2 overexpression construct, the coding region of SPDS2 was amplified from Arabidopsis cDNA using gene-specific primers designed to create the XbaI and SalI restriction sites in the forward and reverse primers, respectively (Supplemental Table S1). PCR amplification was performed using the Expand High Fidelity plus PCR System (Roche) according to the manufacturer's instructions. The PCR products were digested, gel purified, ligated into the binary vector pBI121, and verified by sequencing. For the SPDS2 promoter construct, a 1.383-kb fragment upstream of the start codon of the SPDS2 gene was amplified from Arabidopsis genomic DNA using the forward and reverse primers containing SalI and BamHI restriction sites, respectively. The purified PCR product was digested by SalI and BamHI, gel purified, cloned into SalI-BamHI restriction sites of binary vector pBI101, and confirmed by sequencing. Agrobacterium tumefaciens strain C58 was transformed with the binary plasmids by the freeze-thaw method and used to transform Arabidopsis wild-type C24 or Col-0 as described previously by Clough and Bent (1998). Transformed T1 plants were screened on MS medium containing 50 mg L 21 kanamycin, and transgenic plants were identified. Homozygous T3 seeds were collected from T2 lines after segregation analysis on kanamycin-containing medium and used in this study. The histochemical detection of GUS activity was performed according to Jefferson et al. (1987).

Morphological Change Measurements
Seeds were surface sterilized and transferred to Nunc four-well Polystyrene Rectangular Dishes (Thermo Fisher Scientific) containing MS medium. Plates were incubated in a growth chamber at 23°C under 16-h-light/8-h-dark conditions. Ten days after planting, the root length (distance between the crown and the tip of the main root) of at least 10 plants was measured in four independent experiments. Flowering time was determined when the petals of the first flower were entirely expanded. The total leaf number was counted after complete flowering. Statistically significant differences between lines were determined by unadjusted paired t test (P , 0.01).

Nematode Infection Assay
Transgenic Arabidopsis seeds (T3 generation), homozygous knockout spds2 mutants, as well as wild-type controls (Col-0 and C24) were surface sterilized and planted, in a random block design, on 12-well Falcon tissue culture plates (BD Biosciences) containing modified Knop's medium (Sijmons et al., 1991) solidified with 0.8% Daishin agar (Brunschwig Chemie). Plants were grown at 24°C under 16-h-light/8-h-dark conditions. Ten-day-old seedlings were inoculated with approximately 250 surface-sterilized J2 H. schachtii nematodes per plant, as described previously by Baum et al. (2000). The inoculated plants were maintained under the same conditions described above for an additional 3 weeks before counting the J4 adult females. Mean values significantly different from that of the wild type were determined in a modified t test using the statistical software package SAS (P , 0.05).

Bacterial Growth Assay
Bacterial strain Pseudomonas syringae pv tomato DC3000 was grown overnight at 30°C in King's B medium with appropriate antibiotics. Final cell densities were adjusted to A 600 = 0.05 with 10 mM MgCl 2 for plant inoculation. Fully expanded leaves of 4-to 5-week-old plants were hand infiltrated with the bacterial suspensions. Bacterial growth was determined at 0 and 3 dpi. Leaf tissues (four no. 4 cork-borer leaf discs) were collected, ground in 1 mM MgCl 2 , diluted, and then plated on King's B agar medium containing appropriate antibiotics. Bacterial populations were determined using four replicate plants per line, and the data are represented as means of eight independent experiments 6 SE of log (colony-forming units cm 22 ).

Viral Infection Assay
Fully expanded leaves of 4-to 5-week-old plants were mechanically inoculated with 10 mL of CMVY at a concentration of 100 mg mL 21 in phosphate buffer. Leaf samples were collected 5 dpi, from five to six plants per line, for RNA extraction and qPCR analysis to quantify the accumulation of viral RNA using primer pairs specific to the RNA1 replicase gene (Supplemental Table S1). The data are represented as means of three independent experiments 6 SE.

Yeast Two-Hybrid Assays
A yeast two-hybrid screening was performed as described in the BD Matchmaker Library Construction and Screening Kits user manual (Clontech). The coding sequence of 10A06 was amplified using forward and reverse primers containing EcoRI and PstI restriction sites, respectively (Supplemental Table S1), and fused to the GAL4 DNA-binding domain of pGBKT7 vector to generate pGBKT7-10A06 and then introduced into Saccharomyces cerevisiae strain Y187 to generate the bait strains. Three Arabidopsis cDNA libraries from roots of ecotype C24 at 3, 7, and 10 d after H. schachtii infection were generated in S. cerevisiae strain AH109 as a fusion to the GAL4 activation domain of pGADT7-Rec2 vector (Hewezi et al., 2008). Screening for interacting proteins and subsequent analyses were performed as described by Clontech protocols. To test the potential interaction between 10A06 and SPDS1, the coding sequence of SPDS1 was amplified using forward and reverse primers containing EcoRI and ClaI restriction sites, respectively (Supplemental Table  S1), and fused to the GAL4 DNA activation domain of pGADT7 prey vector to generate pGADT7-SPDS1, then introduced into S. cerevisiae strain AH109 in combination with the bait vector containing 10A06 or human Lamin C or the empty bait vector, and the interaction was tested following Clontech protocols.

RNA Isolation and qPCR
Total RNA was extracted from 100 mg of frozen ground plant tissues using the RNeasy Plant Mini Kit (Qiagen) or from 50 mg of nematode tissues using the Versagene RNA Tissue Kit (Gentra Systems) following the manufacturer's instructions. DNase treatment of total RNA was performed using deoxyribonuclease I (Invitrogen). Ten nanograms of DNase-treated RNA was used for cDNA synthesis and PCR amplification using the One-Step RT-PCR Kit (Bio-Rad) according to the manufacturer's protocol. Gene-specific primers for 10A06, SPDS2, SPDS1, ADC1, ADC2, ACLS, SPMS, H. schachtii Actin (AY443352), and Arabidopsis Actin (AT1G49240) were designed (Supplemental Table S1). The PCRs were run in an iCycler (Bio-Rad) using the following program: 50°C for 10 min, 95°C for 5 min, and 40 cycles of 95°C for 30 s and 60°C for 30 s. Following PCR amplification, the reactions were subjected to a temperature ramp to create the dissociation curve, determined as changes in fluorescence measurements as a function of temperature, by which the nonspecific products can be detected. The dissociation program was 95°C for 1 min, 55°C for 10 s, followed by a slow ramp from 55°C to 95°C.
In all cases, at least three independent experiments each with four technical replicates of each reaction were performed. Arabidopsis and nematode Actin, as constitutively expressed genes, were used as internal controls to normalize gene expression levels. Quantification of the relative changes in gene expression was performed using the 2 2DDCT method (Livak and Schmittgen, 2001).

BiFC Analysis of 10A06 and SPDS2
The 10A06 cDNA without signal peptide was PCR amplified using forward and reverse primers containing EcoRI and XbaI restriction sites, respectively (Supplemental Table S1), and cloned into EcoRI-XbaI sites of pSAT4-nEYFP-C1 to generate pSAT4-nEYFP-10A06. Meanwhile, the full-length SPDS2 cDNA was PCR amplified using forward and reverse primers containing EcoRI and XbaI restriction sites, respectively, and cloned into EcoRI-XbaI sites of pSAT4-cEYFP-C1(B) to generate pSAT4-cEYFP-SPDS2. Both plasmids were confirmed by sequencing. For coexpression, particle bombardment was performed using onion (Allium cepa) epidermal cells. Gold particles (1.6 mm diameter; Bio-Rad) were washed with 100% ethanol and coated with 1.5 mg of each DNA using standard procedures. cDNA-coated gold particles were bombarded at 1,100 p.s.i. and 9 cm distance using a Biolistic Particle Delivery System PDS-1000/He (Bio-Rad). Bombarded tissues were incubated at 25°C in darkness for approximately 16 h before being assayed for YFP activity. The bright-field and fluorescent images were taken using the Zeiss Axiovert 100 microscope with appropriate YFP filters.

In Situ Hybridization
Specific forward and reverse primers for the 10A06 cDNA clone (Supplemental Table S1) were used to synthesize digoxigenin-labeled sense and antisense cDNA probes (Roche) by PCR. In situ hybridizations were performed using mixed parasitic stages of H. schachtii as described by de Boer et al. (1998). Hybridization signals within the nematodes were detected with alkaline phosphatase-conjugated anti-digoxigenin antibody and substrate, and specimens were observed with a Zeiss Axiovert 100 inverted light microscope.

Subcellular Localization
The 10A06 without signal peptide-encoding regions and SPDS2 coding sequences were amplified using gene-specific primer pairs containing ApaI and SalI restriction enzyme sites in the forward and reverse primers, respectively (Supplemental Table S1). The resulting amplified fragments were cloned into the respective sites in the modified pRJG23 vector (Grebenok et al., 1997) before the start codon of GFP fused into the GUS reporter gene and under the control of double CaMV 35S promoter. Both constructs were confirmed by DNA sequencing. These constructs were delivered into onion epidermal cells by biolistic bombardment using standard procedures. After bombardment, epidermal peels were incubated at 26°C for 24 h in the dark. The subcellular localization of the fused proteins was visualized using a Zeiss Axiovert 100 microscope. The transient transformation experiments were repeated at least three times independently.

Identification of spds2 Mutants
Two independent T-DNA insertional null alleles (spds2-1 and spds2-2) in the Col-0 background were obtained from the randomly mutagenized T-DNA lines (the SALK collection) at The Arabidopsis Information Resource. Homozygous plants were identified from segregating T3 populations. Sequence analysis revealed that the T-DNA is inserted in exon 7 of the SPDS2 gene in spds2-1 (SALK_139824) and in intron 7 in spds2-2 (SALK_096270), 1,673 and 1,708 bp, respectively, downstream of the translation initiation codon (Supplemental Fig. S8A). The mRNA expression level of SPDS2 was quantified in wild-type plants and spds2 knockout lines using qPCR. Using forward and reverse primers designed to amplify a 130-bp product from the center region of the SPDS2 coding region, the PCR product was only detected in wild-type plants and not in spds2-1 and spds2-2 homozygous plants (Supplemental Fig.  S8B), indicating that spds2-1 and spds2-2 lines are null alleles.

Determination of Free Spd Level
Plant material (100-150 mg) was ground in liquid nitrogen, extracted in 1 mL of 5% cold perchloric acid containing 1 mmol of 1,6-hexanediamine (Acros Organic) as an internal standard, and incubated on ice for 4 h. The samples were then centrifuged for 45 min at 14,000 rpm at 4°C. The supernatant phase, containing the free Spd fraction, was derivatized with dansyl chloride (Fluka) according to the method described by Smith and Davies (1985). Two hundred microliters of supernatant was mixed with 200 mL of saturated sodium carbonate (13%, w/v) and 400 mL of dansyl chloride in acetone (7.5 mg mL 21 , w/v). After vortexing, the mixture was incubated overnight at room temperature in the dark. Excess dansyl chloride was removed by adding 100 mL of L-Pro (100 mg mL 21 , w/v; Acros Organic) followed by an incubation for 30 min at room temperature. The dansylated Spd was further extracted with 500 mL of toluene. The organic phase containing Spd was vacuum evaporated, and the residue was dissolved in 600 mL of methanol. Free Spd was separated and quantified by HPLC with a reverse-phase (C18) column and a UV detector (254 nm) at room temperature. A standard curve was generated by measuring known amounts of Spd and used for the estimation of Spd content.

Measurement of PAO Activity
The level of PAO activity in plant tissues was determined using the spectrophotometric method described by Angelini et al. (2008) to measure the formation of a pink adduct (« = 515 nm = 2.6 3 104 m -1 cm -1 ), as a result of the hydrogen peroxide-dependent oxidation of 4-aminoantipyrine (4-AAP) catalyzed by horseradish peroxidase and the subsequent condensation of the oxidized 4-AAP with 3,5-dichloro-2-hydroxybenzenzenesulfonic acid. Plant materials were homogenized in 0.2 M sodium phosphate buffer (tissue:buffer ratio of 1:3, w/v). The homogenate was centrifuged at 14,000g for 30 min. A 26-mL aliquot of the supernatant was used for enzyme activity assay. Assays were performed in 0.2 M sodium phosphate buffer, pH 6.5, containing 80 mg of horseradish peroxidase (Sigma), 0.1 mM 4-AAP (Sigma), 1 mM 3,5-dichloro-2hydroxybenzenzenesulfonic acid (Sigma), and 2 mM Spd as a substrate in a 1-mL total volume. Enzyme activity was expressed as units g 21 fresh weight.

DNA Fragmentation Analysis
DNA was isolated from root tissues of transgenic plants expressing 10A06 (line 12-12) and wild-type plants 4 d following infection with H. schachtii as described by Fulton et al. (1995). Five micrograms was separated on a 2% agarose gel and visualized by staining with SYBR safe stain.

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Sequence alignment of 10A06 isoforms identified in beet cyst nematode H. schachtii.
Supplemental Figure S2. Protein alignment of 10A06 identified in beet cyst nematode H. schachtii and soybean cyst nematode H. glycines.
Supplemental Figure S3. Quantification of 10A06 expression levels in transgenic Arabidopsis lines using qPCR.
Supplemental Figure S6. Quantification of SAMDC and PAO2 expression levels in transgenic plants expressing 10A06 using qPCR.
Supplemental Figure S7. Lack of DNA laddering in transgenic plants expressing 10A06.
Supplemental Table S1. Primer sequences used in this study.