The Novel Cyst Nematode Effector Protein 19C07 ... - BioMedSearch

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Chris Lee2, Demosthenis Chronis2,3, Charlotte Kenning4, Benjamin Peret, Tarek Hewezi, Eric L. Davis,. Thomas J. Baum, Richard Hussey, Malcolm Bennett, ...
The Novel Cyst Nematode Effector Protein 19C07 Interacts with the Arabidopsis Auxin Influx Transporter LAX3 to Control Feeding Site Development1[W][OA] Chris Lee2, Demosthenis Chronis2,3, Charlotte Kenning4, Benjamin Peret, Tarek Hewezi, Eric L. Davis, Thomas J. Baum, Richard Hussey, Malcolm Bennett, and Melissa G. Mitchum* Division of Plant Sciences, Interdisciplinary Plant Group and Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211 (C.L., D.C., C.K., M.G.M.); School of Biosciences and Center for Plant Integrative Biology, University of Nottingham, Nottingham LE12 5RD, United Kingdom (B.P., M.B.); Department of Plant Pathology, Iowa State University, Ames, Iowa 50011 (T.H., T.J.B.); Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27607 (E.L.D.); and Department of Plant Pathology, University of Georgia, Athens, Georgia 30602 (R.H.)

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.

Cyst nematodes are sedentary endoparasites of plant roots that cause substantive damage and yield loss to many agricultural crops (Chitwood, 2003). These microscopic worms hatch from eggs in the soil, locate and 1 This work was supported by the U.S. Department of Agriculture National Research Initiative (grant no. 2006–35607–16601 to E.L.D. and M.G.M. and grant no. 2008–35302–18824 to T.J.B., M.G.M., and E.L.D.) and by the United Soybean Board (project no. 7214 to T.J.B., M.G.M., and E.L.D.). 2 These authors contributed equally to the article. 3 Present address: U.S. Department of Agriculture-Agricultural Research Service, Robert W. Holley Center for Agriculture and Health and Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853. 4 Present address: Lisztstr. 13, 99423 Weimar, Germany. * Corresponding author; e-mail [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www. plantphysiol.org) is: Melissa G. Mitchum ([email protected]). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.110.167197

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penetrate a root, and then migrate through the root until they find and select a cell near the vasculature to initiate feeding (Hussey and Grundler, 1998). Using a hollow mouth spear, called a stylet, the nematode secretes esophageal gland-derived effector proteins into the host cell. These effector proteins developmentally reprogram the host cell into a specialized feeding site, called a syncytium (Davis et al., 2008). The nematode also induces an up-regulation of cell wall-modifying proteins (Goellner et al., 2001; Wieczorek et al., 2006, 2008; Karczmarek et al., 2008), causing the cell wall to dissolve. This allows the syncytium to expand in size by incorporating neighboring cells into the growing feeding site (Endo, 1964). The phytohormone indole-3-acetic acid (IAA), or auxin, has been implicated in syncytium development (Goverse et al., 2000; Grunewald et al., 2009b). Auxin is involved in a myriad of plant cellular and developmental processes, including vascular development (Mattsson et al., 1999), embryonic axis development (Friml et al., 2003), phyllotaxis patterning (Reinhardt et al., 2003), gravitropism (Friml et al., 2002), phototropism (Stone et al., 2008), and lateral root development (Bhalerao et al., 2002; Swarup et al., 2008). The

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Nematode Interactions with Auxin Influx Carriers

physiological responses associated with auxin are derived from a rapid transcriptional up-regulation of genes containing auxin-responsive elements (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). Polar transport of auxin, both into and out of the cell, controls the amount of intracellular auxin available, thus providing regulation of the physiological processes affected by auxin. Several classes of plasma membrane auxin efflux transporters have been discovered, including those belonging to the PIN (Chen et al., 1998; Luschnig et al., 1998; Utsuno et al., 1998; Petra´sek et al., 2006) and the MDR/PGP (Sidler et al., 1998) families. In Arabidopsis (Arabidopsis thaliana), the auxin influx transporter, AUX1 (Bennett et al., 1996), and three related family members, LAX1, LAX2, and LAX3, constitute the AUX/LAX family (Swarup et al., 2004), localize to the plasma membrane, and regulate auxin influx into the cell (Yang et al., 2006). LAX3 is required for lateral root emergence (Swarup et al., 2008) and is expressed specifically in cells overlaying lateral root primordia. By bringing auxin into these cells, LAX3 effectively up-regulates cell wall-modifying enzymes that loosen the cell wall and allow the newly formed lateral root to emerge through the existing root tissues (Swarup et al., 2008; Pe´ret et al., 2009). An increase in perceived auxin directly in the syncytium early during its development has been shown using auxin-responsive promoter-reporter constructs (Hutangura et al., 1999; Karczmarek et al., 2004; Wang et al., 2007; Grunewald et al., 2008). Cyst nematodes may manipulate auxin flow through the developing feeding site, as the Arabidopsis auxin influx transport protein, AUX1, is transcriptionally up-regulated in developing feeding sites (Mazarei et al., 2003) and the auxin efflux transporter, PIN3, relocalizes to the lateral plasma membrane of the developing syncytium (Grunewald et al., 2009a). Arabidopsis auxin-insensitive mutants with defects in auxin transport (pin) and signaling (axr2) support fewer cyst nematodes and produce smaller cysts compared with wild-type plants (Goverse et al., 2000; Grunewald et al., 2008). Furthermore, treatment with a polar auxin transport inhibitor, N-1-naphthylphthalamic acid, delays nematode development and inhibits nematode feeding site formation (Goverse et al., 2000). Taken together, these findings suggest a model in which an accumulation of auxin in the developing syncytium plays a substantive role in the formation of cyst nematode feeding sites from existing root cells. The mechanisms that cyst nematodes use to alter auxin transport and signaling pathways to promote feeding site development are not well understood. A leading hypothesis is that cyst nematodes secrete effector proteins into the host cell and that these proteins function, among other things, to manipulate auxin flow through the feeding site by modulating auxin transport proteins or auxin signaling pathway components. Microaspiration of esophageal gland cell contents, construction of gland-enriched cDNA libraries, and analyses that predict secretion signal peptides (Gao Plant Physiol. Vol. 155, 2011

et al., 2001; Wang et al., 2001) have led to the discovery of many putative effector proteins from the soybean cyst nematode (Heterodera glycines), 38 of which are novel (Gao et al., 2003). Subsequent in situ analyses have verified that these effector protein transcripts are present in esophageal gland cells (Gao et al., 2003). One effector protein transcript, Hg19C07 (accession no. AF490250), codes for a 207-amino acid protein that contains a 21-amino acid secretion signal. The Hg19C07 transcript is not detectable in preparasitic juveniles but is expressed specifically in the secretory dorsal gland cell of early parasitic life stages during feeding site development (Gao et al., 2003; Elling et al., 2009). H. glycines and the beet cyst nematode (Heterodera schachtii) are closely related phylogenetically (Subbotin et al., 2001). Identification of putative effector proteins in H. schachtii has been accomplished, with many ortholog genes having a very high degree of sequence similarity and esophageal gland expression patterns (Patel et al., 2008). For example, H. schachtii ubiquitins (UBIs) have greater than 90% sequence identity with HgUBIs (Tytgat et al., 2004); Hs10A06 has 86% sequence identity with Hg10A06 (Hewezi et al., 2010); the H. schachtii cellulose-binding protein (CBP) has between 94% and 97% sequence homology to H. glycines CBP isoforms (Hewezi et al., 2008); the Hs4F01 annexin-like protein has 92% sequence identity to Hg4F01 (Patel et al., 2010); and H. schachtii CLAVATA3/ ESR-like (CLE) proteins have greater than 80% homology with HgCLEs (Wang et al., 2010b), with the H. schachtii transcripts localizing to the same esophageal gland cell(s) as the H. glycines orthologs. The H. schachtii-Arabidopsis pathosystem (Sijmons et al., 1991) is a useful tool for the study of cyst nematode parasitism on a molecular level. This infection system has led to the identification of several characterized protein-protein interactions between the H. schachtii effector proteins and Arabidopsis proteins expressed in tissues enriched in syncytial contents with yeast two-hybrid screens (Hewezi et al., 2008, 2010; Patel et al., 2010). In this paper, we report the results of a yeast two-hybrid approach that identified the Arabidopsis auxin influx transporter LAX3 as a potential interactor with the Hs19C07 effector protein. We demonstrate, with use of promoter-GUS lines, fluorescent protein fusions, and mutant lines, that the interaction is biologically relevant. Finally, we show that ectopic expression of Hs19C07 in Arabidopsis speeds up lateral root emergence, most likely by modulating the activity of LAX3. RESULTS Identification and Sequence Analysis of H. schachtii 19C07

To identify the H. glycines putative effector protein 19C07 ortholog in H. schachtii, primers were designed from the previously reported sequence (Gao et al., 2003) and used in RACE-PCR on parasitic H. schachtii 867

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cDNA. The Hs19C07 gene codes for a 207-amino acid protein with a predicted molecular mass of 21.9 kD (Fig. 1A; accession no. HM142892). The Hs19C07 protein shares 99% sequence identity with Hg19C07. Both proteins contain the same number of amino acids as well as a 21-amino acid predicted secretion signal (Fig. 1A). No significant database matches were identified, classifying 19C07 as a novel protein. An in situ analysis demonstrated that Hg19C07 mRNA is induced only upon the onset of parasitism and is present exclusively inside of the single dorsal esophageal gland cell in both early-stage (Fig. 1B; parasitic second-stage juveniles [pJ2]) and late-stage (Fig. 1C; parasitic fourth-stage juveniles [pJ4]) nematodes, indicating that Hs19C07 is also a secreted protein from the dorsal esophageal gland cell expressed specifically during parasitism. 19C07 Interacts with the Auxin Influx Transporter AtLAX3

A yeast two-hybrid analysis was used to identify potential proteins in Arabidopsis that interact with Hs19C07. The Hs19C07 bait construct pGBKT7-Hs19C07 was used to screen approximately 8.67 3 107 yeast colonies from an Arabidopsis prey library constructed from H. schachtii-infected root tissues at 7 d post infection (Hewezi et al., 2008). Seventeen independent Arabidopsis proteins were identified from 37 clones selected on quadruple dropout synthetic defined (SD) medium that were 5-bromo-4-chloro-3-indolyl-a-Dgalactopyranoside (x-a-gal) positive; seven were reconfirmed by cotransformation. One of these interactors (Y.C. 55) contained the complete 469-amino acid se-

quence coding for the auxin influx transporter, LAX3, as well as extra sequence upstream of LAX3 from another Arabidopsis gene, At3g18370, coding for 106 amino acids (amino acids 549–638). To further examine this interaction, both the fulllength LAX3 (At1g77690) and the 318 nucleotides from At3g18370 upstream of LAX3 in Y.C. 55 were cloned separately into the prey pGADT7 activation domain vector and then cotransformed with pGBKT7Hs19C07 into AH109 yeast cells along with appropriate controls (Fig. 2A). The full-length LAX3 cotransformed with Hs19C07 was x-a-gal positive, although the intensity was slightly weaker than that of the LAX3 Y.C. 55 and the positive control interaction between p53 and SV40 (Fig. 2A). A negative control cotransformation with pGBKT7-Hs19C07 and pGADT7 carrying SV40 did not turn as blue in the presence of x-a-gal, and neither did the cotransformation carrying the Y.C. 55 extra N-terminal (Y.C. 55 N-ter) sequence in pGADT7 cotransformed with pGBKT7-Hs19C07 when compared with full-length LAX3 coexpressed with Hs19C07. When plated on less stringent selective medium (SD-Leu-Trp-His), the full-length LAX3 did not grow as quickly as the original clone (Y.C. 55); however, no growth was observed with the Y.C. 55 N-ter or the negative control (Fig. 2A). Furthermore, no interaction was observed between the most closely related AUX/LAX family members, AUX1 or LAX2 and pGBKT7-Hs19C07 (Supplemental Fig. S1). On restrictive medium (SD-Leu-Trp- His-Ade), Y.C. 55 still interacted with Hs19C07, but the selection was too strong for the full-length LAX3 clone to overcome (Fig. 2A). On stringent selection plates (SD-/Leu-Trp-His-Ade), the full length LAX3 fusion may not drive enough of the activation

Figure 1. The cyst nematode effector protein 19C07. A, Sequence comparison of 19C07 orthologs from H. glycines and H. schachtii. 19C07 is a novel 207-amino acid protein containing a putative 21-amino acid secretion signal. 19C07 is highly conserved between these two cyst nematode species containing 99% homologous amino acid sequence. B and C, In situ analysis of 19C07 in parasitic H. glycines cyst nematodes. B, pJ2 nematode. C, pJ4 nematode. DG, Dorsal gland.

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Figure 2. H. schachtii 19C07 interacts with the Arabidopsis LAX3 auxin influx transporter. A, Yeast two-hybrid analysis. Left column, cotransformants grown on SD-Leu-Trp medium demonstrate that both bait and prey plasmids are present in yeast. Hs19C07 was cloned into the yeast twohybrid vector pGBKT7, while the original recovered clone (Y.C. 55), the extra N-terminal sequence from Y.C. 55 (Y.C. 55 N-ter), and full-length LAX3 were cloned into pGADT7. pGBKT7 with Hs19C07 was cotransformed with pGADT7 carrying SV40 for a negative control. pGBKT7 with p53 and pGADT7 with SV40 provided a positive control. Blue colonies represent positive interactions in the presence of x-a-gal. Middle and left columns, positive selection of yeast two-hybrid interactions with minimal selection (SD-Leu-Trp-His) and restrictive (SD-Leu-Trp-His-Ade) media. B to I, BiFC confirmation of Hs19C07 interaction with AtLAX3. Bright-field images (B, D, F, and H) and fluorescent images (C, E, G, and I) of onion epidermal cells bombarded with BiFC constructs. B and C, Cobombardment of LAX3 C-eYFPand 19C07 N-eYFP. D and E, Plasmolyzed onion epidermal cell cobombarded with 19C07 N-eYFP and LAX3 C-eYFP. F and G, Cobombardment of LAX3 C-eYFP and 4D09 N-eYFP. H and I, Cobombardment of 19C07 N-eYFP and NHL3 C-eYFP.

domain expressed in the pGADT7 fusion to the nucleus, abolishing growth, while the N-terminal addition to LAX3 in Y.C. 55 may disrupt membrane localization, thus allowing the fusion to enter the nucleus, where it would be available for interaction. In contrast, overexpression of the full-length LAX3 on Plant Physiol. Vol. 155, 2011

less stringent selection plates (SD-Leu-Trp-His) may have allowed enough of LAX3 to traffic to the nucleus for interaction with Hs19C07, allowing yeast growth (Fig. 2A, second column). Furthermore, the lack of interaction between Y.C. 55 N-ter and Hs19C07 on the less restrictive medium (SD-Leu-Trp-His) suggests that LAX3 is responsible for the interaction observed in Y.C. 55 (Fig. 2A). Because of the discrepancy of this yeast twohybrid system to detect interactions with plasma membrane proteins, we used another approach to verify the interaction. To confirm a potential interaction between Hs19C07 and LAX3 in vivo, both proteins were transiently expressed using the pSAT4 bimolecular fluorescence complementation (BiFC) vector system (Citovsky et al., 2006) in onion (Allium cepa) peel epidermal cells. Hs19C07DSP fused to the N terminus of enhanced yellow fluorescent protein (eYFP), cobombarded into onion peels with LAX3 fused to the C terminus of eYFP, reconstituted the YFP signal specifically on the periphery of the cell (Fig. 2, B and C). After plasmolysis, the signal remained specifically with the plasma membrane (Fig. 2, D and E). To demonstrate the specificity of the interaction between Hs19C07 and LAX3, we coexpressed LAX3 C-eYFP with Hs4D09 N-eYFP, another H. schachtii esophageal gland protein (P. Howe and T.J. Baum, unpublished data). A slight YFP signal was detected, but at a much lower level than between LAX3 C-eYFP and Hs19C07 N-eYFP (Fig. 2, F and G). The YFP signal also was present within the cytoplasm of the cell surrounding the nucleus, unlike cells coexpressing LAX3 C-eYFP and Hs19C07 N-eYFP. We also tested Hs19C07 N-eYFP for interaction with another plasma membrane-localized protein, NHL3 (Varet et al., 2003), by fusing NHL3 to the C terminus of eYFP. Although a slight signal was detected, the signal was weak in comparison with the LAX3 and Hs19C07 interaction and was localized within the cytoplasm of the cell surrounding the nucleus (Fig. 2, H and I). LAX3 and a LAX3-Inducible Gene Are Up-Regulated in Developing Syncytia

To examine the biological relevance of the interaction between Hs19C07 and LAX3, LAX3pro:GUS Arabidopsis seedlings were infected with H. schachtii juveniles and then stained at various stages of nematode development. Feeding cells stained blue as early as 2 d post inoculation (dpi) and before pJ2 nematodes had undergone their first in planta molt (Fig. 3A). LAX3 gene expression was constant in developing syncytia after the first in planta molt to pJ3 nematodes (Fig. 3B) and then subsided after feeding sites had finished developing. Syncytia development requires the dissolution of neighboring cell walls for fusion of adjacent cells into the growing feeding site (Endo, 1964). During lateral root emergence, normal expression of LAX3 in cells overlaying lateral root primordia results in an influx of auxin into these cells, inducing a plethora of cell wall869

Lee et al. Figure 3. LAX3 and a LAX3-inducible gene are expressed in and surrounding developing syncytia. A and B, LAX3pro:GUS Arabidopsis roots infected with H. schachtii pJ2 (A) and pJ3 (B). C to E, PG expression demonstrated with PGpro:GUS Arabidopsis in uninfected roots. PG is expressed in cell layers overlaying developing lateral root primordia (C), emerging lateral roots (D), and in the cells shortly after the lateral root has emerged (E). F and G, PGpro:GUS Arabidopsis roots infected with H. schachtii. F, pJ2. G, Syncytium (Syn) at 8 dpi. Bars = 100 mm.

modifying enzymes (Swarup et al., 2008). Therefore, we examined the expression of a polygalacturonase (PG) gene that is regulated by LAX3 (Swarup et al., 2008) in response to nematode infection. PGpro:GUS transgenic plants infected with H. schachtii showed PG expression in cells overlaying the lateral root emergence site (Fig. 3C); expression was maintained after lateral root emergence (Fig. 3, D and E). PG was upregulated in feeding sites several days after inoculation (Fig. 3F). As feeding sites developed and enlarged, PG expression shifted from the syncytium to surrounding cells (Fig. 3, F and G). LAX3 Protein Is Expressed in the Syncytium and in Cells to Be Incorporated into the Syncytium

To examine LAX3 localization in roots following infection with H. schachtii, LAX3-YFP plants were grown on vertical plates and LAX3-YFP localization was imaged (Swarup et al., 2008). For comparative purposes, LAX3-YFP expression was also observed in noninfected plants. In noninfected plants, LAX3 protein expression was restricted to the vasculature and to the cells directly overlaying lateral root primordia, as described previously (Swarup et al., 2008; Fig. 4, A–D; Supplemental Fig. S2). LAX3-YFP expression was monitored and imaged at the same infection site every 24 h for 7 dpi. LAX3-YFP was specifically up-regulated in root cells associated with nematode infection sites. LAX3-YFP expression was first observed in the feeding cell and then in the developing feeding site within 2 dpi (Fig. 4, E and F, red arrows). Nematode-induced LAX3-YFP expression was easy to distinguish from the normal lateral root-induced expression pattern (Supplemental Fig. S2, A, B, E, and F). LAX3-YFP expression expanded substantially between 2 and 3 dpi and was localized in 870

the plasma membrane of cells to be incorporated into the developing syncytia (Fig. 4, G and H). The red arrows in Figure 4, E to H, point to the same location on the infected root at 2 and 3 dpi. At another feeding site at 3 dpi, the central structure of the syncytium was observable, and the remnants of cells merging into the feeding site through cell wall dissolution were also visible in the center of the developing feeding site (Fig. 4, I and J). LAX3-YFP expression was detectable in cells directly contacting the expanding feeding site (Fig. 4, I and J). At 4 dpi, the cell remnants observed at 3 dpi were completely incorporated into the growing feeding site, as were the perimeter syncytium cells that had LAX3-YFP in their plasma membrane. At 4 dpi, LAX3YFP expression was detectable in the plasma membrane of the new layer of cells surrounding the developing syncytium (Fig. 4, K and L). Cells expressing LAX3-YFP at 4 dpi were incorporated into the syncytium by 7 dpi, with LAX3-YFP continually being expressed in the layer of cells proximal to the feeding site (Fig. 4, M–P). Both rapid expansion of the LAX3-YFP expression pattern and incorporation of cells into a developing feeding site are evident in a single root section between 5 and 6 dpi (Supplemental Fig. S2, A–D, N*). LAX1 Is Up-Regulated in Developing Syncytia

Up-regulation of LAX3 and AUX1 (Mazarei et al., 2003) in developing syncytia led us to examine the expression of the other Arabidopsis AUX/LAX family members during cyst nematode infection. Both LAX1pro:GUS and LAX2pro:GUS Arabidopsis seedlings were infected with H. schachtii and stained at various stages of nematode development. Uninfected roots were examined from each line to determine basal expression patterns of LAX1 and LAX2 in Arabidopsis roots. LAX1 is expressed in vasculature cells surrounding the site of Plant Physiol. Vol. 155, 2011

Nematode Interactions with Auxin Influx Carriers Figure 4. LAX3 is induced early in developing feeding sites and expressed in cells to be incorporated into syncytia. Arabidopsis lax3 and LAX3-YFP lines were observed before and after infection. A, C, E, G, I, K, M, and O, Bright-field images. B, D, F, H, J, L, N, and P, Fluorescent images. A and B, LAX3-YFP expression in the cells directly above an emerging lateral root primordium. C and D, LAX3-YFP expression persists in the cells above the emerged lateral root. E and F, H. schachtiiinfected root 2 dpi with LAX3-YFP expression initiated in the feeding site as well as in several cells surrounding the developing feeding site. Blue N is on the infecting nematode. G and H, The same root infection site from E and F at 3 dpi. The LAX3-YFP signal has spread about 1 mm from the original infection site. Red arrows point to the nematode head. I to P, LAX3-YFP expression in the same syncytium at 3 dpi. Blue arrows point to the nematode (N), and red arrows point to the syncytium. Times are as follows: 3 dpi (I and J), 4 dpi (K and L), 5 dpi (M and N), and 7 dpi (O and P). Bars = 100 mm.

emergence of lateral roots (Supplemental Fig. S3, A–C). Expression starts coincident with the expansion of the emerging lateral root tip (Supplemental Fig. S3A) and increases in cells directly adjacent to the site of emergence shortly after lateral root tip emergence (Supplemental Fig. S3B). LAX1 expression continues for several days past the emergence of the lateral root (Supplemental Fig. S3C), then expression subsides. LAX1 is induced shortly after feeding site cell selection (Supplemental Fig. S3D) and stays up-regulated until after the J3 molt. LAX2 is expressed in lateral root primordia and emerging lateral roots (Supplemental Fig. S3, E and F) and in the root tips of both lateral roots (Supplemental Fig. S3G) and the primary root (Supplemental Fig. S3H). LAX2 is not expressed in nematode feeding sites at any time during infection (Supplemental Fig. S3I). Auxin Influx Transport Mutants Decrease Infectivity

A role for auxin in developing syncytia is well documented (Hutangura et al., 1999; Goverse et al., 2000; Mazarei et al., 2003; Karczmarek et al., 2004; Grunewald et al., 2008, 2009a). However, infection assays on auxin influx transporter mutants are limited to a single allele Plant Physiol. Vol. 155, 2011

of the aux1 mutant, aux1-7 (Goverse et al., 2000). Therefore, we examined H. schachtii infection on Arabidopsis auxin influx transporter single mutant lines, aux1-22 and lax3, as well as on the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant to determine if the AUX/LAX family plays a critical role in syncytia development. We first tested whether H. schachtii nematodes could perceive and penetrate into the roots of these mutant lines. We found no statistical difference between penetration in wild-type and lax3 lines (data not shown) or between the wild type and the aux1lax3 double mutant or the aux1lax1lax2lax3 quadruple mutant (Fig. 5A). Infection was assayed by measuring the development of parasitic J4 females at 14 dpi and adult females at 30 dpi. We found no significant reduction in nematode development in lax3 (Fig. 5B) or aux1 (Fig. 5C) single mutants. The aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant are agravitropic and fail to produce lateral roots up until 14 d post germination (Swarup et al., 2008; B. Peret and R. Swarup, unpublished data). Hence, an infection assay was performed on both the double and quadruple mutant seedlings at 7 d post germination prior to emergence of the first lateral root in order to start with the same 871

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amount of root tissue for infection; females were counted per plate rather than by individual plants, leading to larger infection numbers. Both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant had significant decreases in nematode numbers at both 14 and 30 dpi (Fig. 5D). Nematode Infection Induces Lateral Root Formation in AUX/LAX Double and Quadruple Mutants

The severe lateral root phenotype of both the aux1lax3 double and aux1lax1lax2lax3 quadruple mutants, in which lateral root formation is blocked until at least 14 d after germination, has been documented (Swarup et al., 2008; B. Peret and R. Swarup, unpublished data). During infection assays with both the double and quadruple mutants, we observed hyper lateral root formation at the site of infection; this was examined further. Seven-day-old seedlings were infected with H. schachtii and then monitored for lateral root formation specifically from developing feeding sites. Infection of all lines resulted in decreased root growth as compared with uninfected roots (Fig. 6A). Although lateral root formation from syncytia occurred in all lines examined (Fig. 6), it was very apparent on the aux1lax3 double mutant and aux1lax1lax2lax3 quadruple mutant (Fig. 6A, insets, red arrowheads) because of the lack of lateral root formation in the uninfected plants at 5 dpi. By 11 dpi, lateral roots had grown substantially from feeding sites (Fig. 6B). Infection induced about one lateral root per infection site in both the wild type and the lax3 single mutant and around 2.5 lateral roots in both the aux1lax3 double and aux1lax1lax2lax3 quadruple mutants (Fig. 6C). Ectopic Expression of 19C07 in Arabidopsis Reduces the Infectivity of H. schachtii

Because Hs19C07 interacted with AtLAX3 in the yeast two-hybrid screen, we generated Arabidopsis lines overexpressing Hs19C07 without the signal peptide to examine the effect of the nematode protein. Multiple independent transformants expressing Hs19C07DSP driven by the 35S promoter were selfed to homozygosity, and then expression levels were measured by reverse transcription (RT)-PCR and quantitative RT-PCR (qPCR). Four independent Hs19C07 overexpression lines were chosen with varying levels of transcript

Figure 5. Auxin influx transport mutants decrease nematode infectivity. A, At least 30 roots from Arabidopsis Col-0, aux1lax3, and aux1lax1lax2lax3 infected with H. schachtii were compared for penetration 48 h after infection. Means were not significantly different from the wild type using a two-tailed t test assuming unequal variances. Error bars indicate SE. For aux1lax3, P = 0.901; for aux1lax1lax2lax3, P = 0.414 B, Three replicates of 36 individuals from Col-0 and lax3 lines were infected on 12-well plates with 250 infective J2 of H. schachtii. pJ4 female nematodes were counted per plant at 14 dpi, and adult female 872

nematodes were counted at 30 dpi. Error bars indicate SE. C, Three replicates of 36 individuals from Col-0 and aux1 lines were infected on 12-well plates with 250 infective J2 of H. schachtii. pJ4 female nematodes were counted per plant at 14 dpi, and adult female nematodes were counted at 30 dpi. Error bars indicate SE. D, Three replicates of at least four vertical plates with eight individuals of Col-0, aux1lax3, and aux1lax1lax2lax3 genotypes were infected with H. schachtii. pJ4 female nematodes were counted per plate at 14 dpi, and adult female nematodes were counted at 30 dpi. Error bars indicate SE. * P , 0.05 using a two-tailed t test. Plant Physiol. Vol. 155, 2011

Nematode Interactions with Auxin Influx Carriers Figure 6. Nematode infection induces lateral root formation in the aux1lax3 double and aux1lax1lax2lax3 quadruple mutants. A, Col-0, lax3, aux1lax3, and aux1lax1lax2lax3 seedlings either uninfected or infected (5 dpi) grown on vertical plates. Insets show infected aux1lax3 and aux1lax1lax2lax3 mutant lines. Arrowheads point to infection sites. Edge of squares = 1.3 cm. B, Lateral roots emerging from syncytia in infected aux1lax3 and aux1lax1lax2lax3 roots at 11 dpi. C, Lateral roots from all feeding sites were counted on three replicates of at least two vertical plates sown with six individuals from each line. Error bars indicate SE. Letter grouping above bars indicates significance groups (P , 0.05). Plant lines that share a letter are not significantly different from each other using a two-tailed t test.

abundance (i.e. from relatively low expression [7-10-5] to high expression levels [6-13-3; Fig. 7A]). No obvious phenotypes were observed in the root systems of the Hs19C07 overexpression lines when grown on vertical plates. When examined for increased susceptibility to H. schachtii infection, we found a significant decrease in nematode number correlated to Hs19C07 expression level compared with the wild type at both 14 and 30 dpi, with greater amounts of Hs19C07 leading to fewer nematodes (Fig. 7B).

19C07 Expression Speeds up Lateral Root Emergence

To examine the effects of Hs19C07 on LAX3 directly, the LAX3-YFP line was crossed with the 6-13-3 Hs19C07DSP overexpression line (Fig. 8). F1 progeny from the cross were examined. The first lateral root emergence site was monitored daily as soon as LAX3YFP was detectable. LAX3-YFP 3 Hs19C07 was comPlant Physiol. Vol. 155, 2011

pared with the LAX3-YFP line. Lateral root emergence was about 24 h sooner in the Hs19C07 background, with lateral roots emerging within 48 h of the onset of LAX3-YFP expression (Fig. 8B) compared with 72 h in the LAX3-YFP line (Fig. 8A). However, lateral roots were quantified at 7 and 11 d after planting, and no difference in the number of lateral roots was observed between the LAX3-YFP 3 19C07 overexpression line and the LAX3-YFP line (Fig. 8C). From these data, we can conclude that 19C07 does not block LAX3 function, leading to a decrease in lateral root emergence, as reported in the lax3 mutant line (Swarup et al., 2008), but rather may stimulate its activity.

DISCUSSION

Auxin is most likely involved in several aspects of syncytium development. Auxin-responsive genes, including the transcription factor AtWRKY23, are up873

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Auxin Influx Transporters Play a Role in Syncytium Development

LAX3 plays a role in lateral root emergence. Its upregulation in cells overlaying lateral root primordia results in an influx of auxin to promote the emergence of lateral roots. This auxin influx is followed by the upregulation of cell wall-modifying enzymes, such as PG (Wen et al., 2006; Swarup et al., 2008), that loosen the cell wall and allow the lateral root to emerge. Likewise, auxin influx into the developing feeding site from LAX3 and, presumably, AUX1 (Mazarei et al., 2003) and LAX1 (Supplemental Fig. S3), induces PG expression directly in the feeding site early (Fig. 3F) and most likely the other cell wall-modifying enzymes that are induced by LAX3-associated auxin influx in the roots (Swarup et al., 2008). Furthermore, in early syncytium development, PIN1, an auxin efflux protein, is downregulated, presumably increasing the influx transporter-

Figure 7. Ectopic expression of 19C07 in Arabidopsis reduces infectivity of H. schachtii. A, Relative expression assayed by qRT-PCR of Hs19C07 in four independent homozygous Arabidopsis overexpression lines. Transcript levels were compared with Col-0 and are expressed in a logarithmic scale. Error bars indicate 95% confidence intervals. The bottom shows RT-PCR analysis of the coding region of the 19C07 transgene in the lines tested. B, Three replicates of 36 individuals from Col-0 and 19C07 overexpression lines were infected on 12well plates with 250 infective J2 of H. schachtii. pJ4 female nematodes were counted on individuals at 14 dpi, and adult female nematodes were counted at 30 dpi. Error bars indicate SE. * = P , 0.05.

regulated in H. schachtii-derived syncytia (Grunewald et al., 2008; Szakasits et al., 2009). Thus, auxin most likely triggers a developmental cascade of events similar to the developmental cues triggered by auxin in developing organs, such as lateral roots (Swarup et al., 2008), or in the development of the root meristem early in embryogenesis (Friml et al., 2003). Although auxin has been shown to be required for cyst nematode infection, nothing is known about the role that nematode effector proteins may play in controlling the auxin flow into and out of developing feeding sites. Our results suggest that the cyst nematode effector protein 19C07 may modulate the activity of the auxin influx transporter LAX3 directly in the host cell destined to become the syncytium as well as in surrounding cells subsequently incorporated into the growing feeding site. 874

Figure 8. Ectopic expression of 19C07 in Arabidopsis leads to earlier lateral root emergence. A and B, Initial lateral root emergence of lax3 and LAX3-YFP (A) and lax3 and LAX3-YFP 3 19C07 6-13-3 overexpression line 5 d after seeding (T = 0) through 8 d after seeding (T = 72 h). Bars = 100 mm. C, The number of LAX3-YFP sites of expression on 7- and 11-d-old seedlings. Lateral root initiation sites were counted on two replicates of two vertical plates seeded with six individuals for each line. No significant difference was found using a two-tailed t test. Error bars indicate SE. Plant Physiol. Vol. 155, 2011

Nematode Interactions with Auxin Influx Carriers

mediated auxin concentration in the developing feeding site that triggers the syncytium-specific auxininduced physiological changes (Grunewald et al., 2009a). Within several days of syncytium initiation, LAX3 (Figs. 3B and 4, G to P; Supplemental Fig. S2, A–D) and PG (Fig. 3G) expression is shifted to cells neighboring the developing feeding site. This may be accomplished by a similar mechanism to LAX3 expression in cells overlaying emerging lateral roots (i.e. auxin flow from the lateral root primordia to the overlaying cells leads to the degradation of SLR/IAA14 repressor protein). Degradation of the repressor allows the auxininducible LAX3 to influx auxin into the cells, leading to a loosening of the cell wall by up-regulating a suite of cell wall-modifying proteins (Swarup et al., 2008; Pe´ret et al., 2009). Thus, cyst nematodes may use existing auxin transport mechanisms to accomplish the same auxin-induced LAX3 expression in surrounding cells as the auxin efflux transporter PIN3 localizes to the periphery of developing syncytium (Grunewald et al., 2009a). LAX3 induction in cells surrounding the syncytium leads to an auxin influx into these cells, amplifying the auxin signal and, most likely, priming them for incorporation into the feeding site (Grunewald et al., 2009b). After several weeks, when adult females are present and feeding, LAX3-YFP expression is relegated to the locale farthest from the nematode or dissipated altogether (data not shown), suggesting that auxin is necessary for early events in syncytium setup and development. When the syncytium is large enough to maintain feeding, it stops expanding and incorporating cells, thus relieving the necessity for auxin-induced cell wall-modifying enzymes. Prior experimental evidence and the work described herein now demonstrate that AUX1 (Mazarei et al., 2003), LAX1 (Supplemental Fig. S3), and LAX3 (Figs. 3, A and B, and 4, E–P) may be involved in auxin influx in the developing syncytium. Nevertheless, nematode infection and development observed on the aux1lax1lax2lax3 quadruple mutant (Fig. 5D) suggest that auxin may not be required for, but may enhance, syncytium development. In our studies, H. schachtii juveniles were able to sense, locate, and penetrate auxin transporter mutants (Fig. 5A), which suggests that auxin transporter mutants only affect syncytia initiation and development. The lack of a significant decrease in nematode infection on both aux1 and lax3 single mutants compared with the wild type (Fig. 5, B and C) suggests that removing one of four potential transporters does not affect infectivity, likely due to functional redundancy among these transporter proteins, as is observed in phyllotactic patterning (Bainbridge et al., 2008) and embryonic root cell organization (UgartecheaChirino et al., 2010). When more than one of the four potential AUX/LAX influx transporters were removed, overall auxin flow into the developing syncytia most likely decreased, leading to a delay in nematode development (Fig. 5D; 14 d). Although feeding sites matured and nematode development proceeded, a significant decrease in female numbers was observed 30 d Plant Physiol. Vol. 155, 2011

after infection (Fig. 5D). A similar delay in nematode development was also observed previously in plants treated with an auxin transport inhibitor (Goverse et al., 2000). Decreased auxin transport in the double and quadruple mutants results in a severe lateral root defect (i.e. no lateral roots form for 14 d) and a complete loss of gravitropism (Swarup et al., 2008). Interestingly, however, we observed a hyperinduction of lateral roots from aux1lax3 and aux1lax1lax2lax3 mutant feeding sites (Fig. 6). The fact that auxin promotes lateral root emergence (Pe´ret et al., 2009) suggests that other classes of auxin influx transporters may be induced during syncytium development to fulfill the auxin requirement for developing feeding sites in the double and quadruple mutants. For example, the NRT1.1 nitrate transporter has also been shown to influx auxin in Xenopus oocytes, yeast, and in planta (Krouk et al., 2010). While auxin may presumably still accumulate in the feeding sites from other influx transporters on the aux1lax3 and aux1lax1lax2lax3 lines, the auxin accumulation that promotes lateral root growth may trigger the signaling events that precede lateral root formation, or alternatively, the nematodes may provide these lateral root induction signals. Following infection, the nematodes may potentially bypass the auxin requirement and up-regulate auxin-inducible genes with an unidentified parasitism factor (Grunewald et al., 2008). This might be the case during infection of the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant that still support nematode growth and development, although the numbers are substantively decreased from wild-type plants (Fig. 5D). Potential nematode-induced auxin-independent up-regulation of auxin-inducible genes (Grunewald et al., 2008) could come from either uncharacterized nematode effector proteins or other nematode-derived signals, such as nematode-synthesized auxin (De Meutter et al., 2005), as other classes of plant pathogens have been shown to produce auxin or related compounds during parasitism (Glickmann et al., 1998; Chung et al., 2003). The upregulation of auxin-inducible genes might also be explained by auxin biosynthesis occurring directly within developing syncytia, as auxin synthesis genes have been shown to be up-regulated in rapidly dividing root cells (Petersson et al., 2009), and from meristematic cells originating in both the primary and lateral roots (Ljung et al., 2005). The regulation of auxin biosynthesis by cytokinin has also been demonstrated (Jones et al., 2010). Therefore, alterations to cytokinin metabolism within developing syncytia might also explain the up-regulation of auxin-inducible genes in response to nematode infection. Hs19C07 May Modulate AtLAX3 Activity in Developing Syncytia

Here, we describe the identification of the H. schachtii effector protein ortholog of H. glycines 19C07. As with Hg19C07 and Hs19C07, many H. glycines effector 875

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proteins have high sequence identity (80%–97%) to orthologs in H. schachtii (Tytgat et al., 2004; Hewezi et al., 2008, 2010; Patel et al., 2008, 2010; Wang et al., 2010b). The first transcript synthesis can be detected specifically within the dorsal gland cell after the nematode has penetrated the root (Gao et al., 2003). The increased expression of 19C07 at the onset of parasitism is similar to other known effector proteins (Ithal et al., 2007; Elling et al., 2009). Furthermore, the presence of an N-terminal secretion signal implies that this protein can enter the secretory pathway to the gland’s ampulla, where it is released by exocytosis and delivered through the feeding stylet into the host plant either intracellularly or extracellularly, as has been demonstrated with the cyst nematode HgCLEs (Wang et al., 2010a), the cyst nematode b,1-4-endoglucanases (Wang et al., 1999; Goellner et al., 2001), and the rootknot nematode Meloidogyne incognita calreticulin (Jaubert et al., 2005). We demonstrate that Hs19C07 interacts directly with the syncytia-expressed auxin influx transporter AtLAX3 in situ. Using BiFC, a robust reconstitution of the YFP signal occurred specifically on the plasma membrane of onion peel epidermal cells when both Hs19C07 and AtLAX3 were coexpressed (Fig. 2, B–E) compared with the negative controls. When AtNHL3, an unrelated plasma membrane protein (Varet et al., 2003), and Hs19C07 were coexpressed, a faint signal was produced in the cytoplasm and surrounding the nucleus (Fig. 2, H and I), as is often observed in cytoplasmically expressed proteins, which may suggest that this signal is not associated with real interaction at the plasma membrane. Furthermore, substituting Hs19C07 with Hs4D09 (P. Howe and T.J. Baum, unpublished data), another effector protein expressed in the dorsal esophageal gland cell with a similar expression profile (Elling et al., 2009), and coexpressing with LAX3 produced a weak YFP signal in the cytoplasm surrounding the nucleus (Fig. 2, F and G), suggesting that the observed fluorescence was not comparable to the specific interaction observed between 19C07 and LAX3 at the plasma membrane. We observed that overexpression of Hs19C07 in a LAX3-YFP background led to the faster emergence of lateral roots (24 h earlier; Fig. 8, A and B) compared with the LAX3-YFP line, suggesting that Hs19C07 increased the auxin influx rate from LAX3 to increase the rate of lateral root emergence. Thus, we propose that the nematode-secreted Hs19C07 most likely functions to increase the activity of LAX3 in such a way that the increased auxin accumulation in the developing feeding site stimulates cell wall hydrolysis to accelerate syncytia expansion and development. However, the results from our infection assays of Hs19C07 overexpression lines are counterintuitive to this idea. In previous studies, expression of H. schachtii effector proteins in Arabidopsis led to greater susceptibility to nematode infection (Hewezi et al., 2008, 2010; Patel et al., 2010). In contrast, we found that overexpression of Hs19C07 using the constitutive 35S promoter led to 876

a slightly decreased cyst number inversely correlated to the amount of transcript expression. We speculate that this may have been an effect of Hs19C07 activity on normal auxin flux throughout the root, thereby dissipating the auxin concentrations in the root and decreasing the amount of auxin available for import into the developing feeding sites. To more carefully examine the effects of 19C07 overexpression on nematode infectivity, future studies will need to be performed using promoters more specific to developing feeding sites. In summary, we present data that suggest that a nematode effector protein, Hs19C07, most likely modulates the activity of a plant auxin influx transporter protein, LAX3, during feeding site formation. This work adds to the growing body of evidence that auxin is an integral part of syncytia formation (for review, see Grunewald et al., 2009b). Like other symbiotic and parasitic interactions with plants (Mathesius et al., 1998; Boot et al., 1999; Huo et al., 2006; Chen et al., 2007; van Noorden et al., 2007; Felten et al., 2009), cyst nematodes also appear to use and manipulate auxin transport and sensing mechanisms in their host in order to dedifferentiate and up-regulate many genes leading to the development of transcriptionally unique syncytia (Szakasits et al., 2009). Further studies will be performed to examine the effects of Hs19C07 coexpression with LAX3 on auxin influx in cultured cells. MATERIALS AND METHODS Plant Materials The LAX3pro::GUS, PGpro::GUS, and aux1-22lax3 lines were as described (Swarup et al., 2008). The LAX3pro::LAX3-YFP line was as described (Swarup et al., 2004, 2008). The aux1-22 and lax3 lines were as described (Tissier et al., 1999). The LAX1pro::GUS, LAX2pro::GUS, and aux1lax1lax2lax3 quadruple mutant were as described (Bainbridge et al., 2008). Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 (Col-0) was used for wild-type controls. The 19C07 overexpression lines were generated by the floral dip method (Clough and Bent, 1998) of Arabidopsis Col-0 using Agrobacterium tumefaciens strain GV3101 carrying pMHs19C07DSP. Lines were self-pollinated and carried to the T3 generation; homozygous lines were used in this study. To create the LAX3YFP 3 19C07 6-13-3 line, the LAX3pro::LAX3-YFP line was crossed to the T3 19C07 6-13-3 overexpression line as a female. Heterozygous F1 individuals were analyzed from this cross.

Nematode Materials The beet cyst nematode (Heterodera schachtii) was propagated on greenhouse-grown Beta vulgaris ‘Monohi’. The soybean cyst nematode (Heterodera glycines) was propagated on Glycine max roots. Preparation and hatching of nematode eggs were performed as described (de Boer et al., 1999; Goellner et al., 2001; Mitchum et al., 2004). After 48 h of hatching, second-stage preparasitic juveniles were collected by low-speed centrifugation (8,000 rpm) in a tabletop centrifuge and surface sterilized for 10 min in a 0.004% mercuric chloride, 0.002% sodium azide, and 0.001% Triton X-100 solution, followed by five washes in sterile distilled water. Sterilized nematodes were resuspended in sterile 0.1% agarose. Nematodes were resuspended at a concentration of 200 nematodes 25 mL21 with 25 mL applied to each root for GUS staining experiments. Nematodes were resuspended at a concentration of 50 nematodes 25 mL21 with 25 mL applied to each root for LAX3-YFP line infections. Nematodes were resuspended at a concentration of 250 nematodes 25 mL21 with 25 mL added to each individual plant for the 12-well plate, vertical plate, and penetration infection assays as well as the lateral root emergence from the feeding site assay.

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In Situ Analysis

Yeast Two-Hybrid Screening

Parasitic H. glycines was isolated from G. max, fixed with paraformaldehyde, prepared, and labeled with a Hg19C07 digoxigenin probe as described (de Boer et al., 1998; Gao et al., 2001).

Yeast two-hybrid screening was carried out as described in the BD Matchmaker Library Construction and Screening Kits according to the manufacturer’s instructions (Clontech). Three Arabidopsis cDNA libraries from roots of ecotype C24 at 3 d (pJ2), 7 d (pJ3), and 10 d (pJ4) post H. schachtii infection were generated in Saccharomyces cerevisiae strain AH109 as a fusion to the GAL4 activation domain of pGADT7-Rec2 (Hewezi et al., 2008). Screens were conducted using the mating method according to the manufacturer’s instructions. pGBKT7-Hs19C07 was used to screen approximately 8.67 3 107 yeast colonies from the pJ2, pJ3, and pJ4 prey libraries.

H. schachtii 19C07 Sequence Identification Hs19C07 sequence was obtained using RACE with the BD SMART cDNA amplification kit (Clontech) according to the manufacturer’s directions. To obtain Hs19C07 flanking sequences, both 5# and 3# RACE were performed utilizing primers (5# RACE primer, 5#-GTTCTCTCCGCCCCGTTTCTCTTCGT-3#; 3# RACE primer, 5#-ACGAAGAGAAACGGGGCGGAGAGAAC-3#) designed from the H. glycines 19C07 sequence (Gao et al., 2003). The full-length Hs19C07 clone was obtained by using the primers Hs19C07 long 5#UTR (5#-CTCTCTCCCATTGGAATTATTCATTG-3#) and Hs19C07 long 3#U (5#-CCGATCAGTCGTTCCCTTTCG-3#) and then cloned into the PCR4TOPO vector to create PCR4TOPO-easy-Hs19C07MC1. Hs19C07 was independently cloned multiple times and sequence verified. The Hs19C07 GenBank accession number is HM142892.

Constructs Overexpression Construct Hs19C07 without the signal peptide was PCR amplified using the primers Hs19C07BamHIDSPF (5#-CCAAGGATCCATGGAAGAAAATGGGGCGACAGA-3#) and Hs19C07XhoIR (5#-GGTTCTCGAGTCAGTTCATCGGCCCCTCC-3#). The resulting PCR product was cloned into pGEM-T-Easy (Promega) to make pGHs19C07DSPw. Hs19C07DSP was cut from pGHs19C07DSPw as a BamHI and SalI fragment and ligated into pMD1, a derivative of pBI121 (Clontech), under the control of the 35S promoter to make pMHs19C07DSP.

BiFC Onion (Allium cepa) peel epidermal cells were bombarded with 0.5 mg of each pSAT4 plasmid (nEYFP or cEYFP) coated onto 1.6-mm-diameter gold particles (Bio-Rad) at 1,100 p.s.i. and 9-cm distance with a Biolistic Particle Delivery System (PDS-1000/He; Bio-Rad). Onion peels were incubated at room temperature in the dark for 24 h on half-strength Murashige and Skoog medium, pH 5.7, prior to imaging for YFP signal. At least 20 onion peel cells were examined from each of three replicates for each cobombardment.

GUS Staining Seedlings were sown on modified Knops medium plates (Sijmons et al., 1991), grown vertically for 8 d, and then subjected to infection with H. schachtii. Infected seedlings were collected at various time points and vacuum infiltrated with a GUS substrate buffer (1 mM 5-bromo-4-chloro-3-indolyl glucuronide, 100 mM Tris, pH 7.0, 50 mM NaCl, 0.06% Triton X-100, and 0.5 mM potassium ferrocyanide) and then incubated in GUS substrate buffer overnight (Jefferson et al., 1987; Wang et al., 2007) at 37°C.

RNA Isolation, Quantitative Real-Time PCR, and RT-PCR Yeast Two-Hybrid Constructs Hs19C07 without the signal peptide was PCR amplified using the primers Hs19C07NdeIY2HDSPF (5#-CCAACATATGGAAGAAAATGGGGCGACAGA-3#) and Hs19C07Y2HBamHIR (5#-GGTTGGATCCTCAGTTCATCGGCCCCTCCTTTTC-3#) and then cloned into pGEM-T-Easy (Promega) to make pGHs19C07NB. Hs19C07DSP was digested from pGHs19C07NB as an NdeI-BamHI fragment and cloned into the GAL4 DNA-binding domain of pGBKT7 (Clontech) to generate the bait construct pGBKT7-Hs19C07. Fulllength LAX3 was amplified from Arabidopsis root cDNA using the primer set LAX3EcoRIY2HF (5#-CCAAGAATTCATGGCGGCAGAGAAAATAGA-3#) and LAX3Y2HBamHIR (5#-GGTTGGATCCTCATGGCTTGTGAGGAGGGC-3#) and then cloned into pGADT7 as an EcoRI-BamHI fragment to make pGADT7-Lax3. The extra N-terminal sequence recovered in Y.C. 55 was amplified from the recovered pGADT7-Rec2 Y.C. 55 plasmid using the primers Y.C. 55 extra F (EcoRI) (5#-GCAAGAATTCCACCCAAGCAGTGGTATCAACG-3#) and Y.C. 55 extra R (XhoI) (5#-GCAACTCGAGCTATTTTCTCTTCTTCTCTCCATATTG-3#) and cloned into pGADT7 as an EcoRI-XhoI fragment to make pGADT7-Y.C. 55 N-ter.

BiFC Constructs Hs19C07DSP was amplified using the primers 19C07 nC (cC) Sense (XhoI) (5#-GCAACTCGAGCTGAAGAAAATGGGGCGACAGA-3#) and 19C07 nC (cC) AS (BamHI) (5#-GCAAGGATCCTCATCAGTTCATCGGCCCCTCC-3#) and then cloned into pSAT4-nEYFP-C1 (Citovsky et al., 2006) to make pSAT4-nEYFP-Hs19C07DSP. AtLAX3 was amplified from root cDNA using the primers Lax3 nC (cC) Sense (XhoI) (5#-GCAACTCGAGCTATGGCGGCAGAGAAAATAGAGAC-3#) and Lax3 nC (cC) AS (BamHI) (5#-GCAAGGATCCTCATCATGGCTTGTGAGGAGGGC-3#) and then cloned into pSAT4cEYFP-C1-B (Citovsky et al., 2006) to make pSAT4-cEYFP-Lax3. AtNHL3 (Varet et al., 2002) was amplified from seedling cDNA using the primers NHL3 cC Sense (XhoI) (5#-GCAACTCGAGCTATGGCGGACTTAAACGGTGCGTATTAC-3#) and NHL3 cC AS (BamHI) (5#-GCAAGGATCCTCATCAAAAGTCAACGTCACACTTGGTC-3#) and then cloned into pSAT4-cEYFPC1-B to make pSAT4-cEYFP-NHL3.

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Total RNA was extracted from fresh root tissue isolated from the Hs19C07 overexpression Arabidopsis lines using the RNeasy Plant Mini Kit (Qiagen) and treated with RNase-free DNaseI (Qiagen). cDNA was synthesized from 750 ng of RNA using the First Strand cDNA Synthesis Kit for RT-PCR (Roche Diagnostics) according to the manufacturer’s instructions. qPCR was performed using the Applied Biosystems 7500 Real-Time PCR system. qPCR primers were designed from the Hs19C07 coding sequence and an endogenous control gene (GAPDH; accession no. AAG51517.1) by using Primer Express (Applied Biosystems). The following primers were designed to analyze Hs19C07: 19C07 qRT F (5#-CTGTATGTTGTTCGGCTTTTTCC-3#) and 19C07 qRT R (5#-CCCGCTTTTATATCGTTTTTGC-3#). The following primers were used for the GAPDH internal control: ATGAPDHF (5#-TCCCGTGTGGTCGACTTGA-3#) and ATGAPDHR (5#-CCCTATCATTCGAGATCTGCTTCT-3#). Triplicate qPCRs were set up and analyzed as described (Wang et al., 2007), except that the primer dimer amplification of the Hs19C07 primers in the wildtype background enabled us to set the expression level of 19C07 in the ecotype Col-0 background to zero for a relative comparison with the overexpression lines. Confidence intervals were calculated as described (Wang et al., 2007). RT-PCR was performed on root cDNA from the Arabidopsis Hs19C07 overexpression lines to visually examine the expression of the Hs19C07 transcript. PCR was performed using the primer set 19C07 SSdel (HindIII) (5#-GCAAAAGCTTATGGAAGAAAATGGGGCGACAGA-3#) and 19C07 AS (SalI) (5#-GCAAGTCGACGGGTTCATCGGCCCCTCCTTTTC-3#) with ExTaq DNA polymerase (Takara) with the following conditions: 95°C for 90 s followed by 30 cycles of 95°C for 30 s, 63°C for 45 s, and 72°C for 45 s.

Microscopy Images for the onion peels for BiFC cobombardment were acquired with an LSM 510 META confocal system mounted on an Axiovert 200M inverted microscope (Carl Zeiss) equipped with a 203 Plan Apochromat objective lens. YFP was excited with a 514-nm laser, and emitted fluorescence was detected through a 535- to 590-nm long-pass filter. Several onion peels were plasmolyzed with a plasmolysis buffer (50 mM Tris, pH 7.1, 2 M NaCl) before imaging. GUS-stained roots were mounted on glass microscope slides, and images were acquired with an Olympus Vanox AHBT3 AH-3 equipped with 103 and 43 objectives fitted with a Leica DFC295 digital camera.

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LAX3-YFP plant lines were grown on vertical plates with a thin layer of medium, keeping the root system in the dark until imaging. Images were acquired through the plate and medium with an Olympus IX 70 inverted microscope equipped with either a 203/0.7 Plan Apochromat or a 103/0.3 UPlanFL objective lens and a Hamamatsu Orca AG cooled CCD camera. For fluorescent images, a filter set with 490- to 510-nm excitation, a 515-nm longpass dichroic mirror, and 520- to 550-nm emission was used (Chroma). Infected root systems for the lateral root counts were imaged on an Epson Perfection V200 photo scanner and processed with Photoshop.

Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Hs19C07 yeast two-hybrid screen of AUX/LAX family members. Supplemental Figure S2. LAX3 induction in feeding sites and in cells immediately overlaying emerging lateral roots. Supplemental Figure S3. LAX1 and LAX2 expression in response to nematode infection.

Infection Assays All Arabidopsis seeds used in infection assays were surface sterilized with Cl2 gas for 6 h, incubated at 4°C for 48 h, and then seeded on modified Knops medium (Sijmons et al., 1991).

ACKNOWLEDGMENTS

Penetration Assay

We thank Amy Replogle and Dr. Jianying Wang for critical discussions of the work; Melody Kroll for editing the manuscript; Robert Heinz for maintenance of the nematode lines; Peter Howe for the Hs4D09 BiFC control; and Esteban Fernandez and Stephanie Boyle for help with imaging.

Two replicates of at least 30 Arabidopsis seeds from each genotype were grown on vertical plates for 8 d prior to infection with H. schachtii. Seedlings were collected 48 h after infection, and nematodes were stained with acid fuchsin fixation/staining solution (Hewezi et al., 2008) and counted on an inverted microscope. Nematodes penetrated into each root were scored.

Received October 8, 2010; accepted December 8, 2010; published December 14, 2010.

LITERATURE CITED Twelve-Well Plate Infection Assays Three replicates of 36 seeds from each Arabidopsis genotype under investigation were individually and randomly distributed on 12-well plates and grown for 14 d prior to infection. At 14 and 30 d post infection, pJ4 females or adult females were counted on each individual.

Vertical Plate Infection Assay Three replicates of at least four vertical plates with eight seeds from each genotype tested were grown vertically for 8 d prior to infection. pJ4 females or adult females were counted at 14 and 30 d post infection, respectively. Nematode counts were scored for each plate.

Lateral Root Scoring from Feeding Sites of Auxin Transport Mutant Lines Three replicates of at least two vertical plates with six seeds from each genotype tested were grown vertically for 7 d in 16-h-light/8-h-dark conditions such that there was similar root length with almost no lateral root development when infection occurred. Lateral roots that emerged from all feeding sites at 11 dpi were counted and scored for each syncytium.

Analysis of Hs19C07 in the LAX3-YFP Background LAX3-YFP 3 19C07 6-13-3 seeds were surface sterilized for 6 h with Cl2 gas and then planted on vertical plates. Seedlings were grown on vertical plates on half-strength Murashige and Skoog medium, pH 5.7, with 2% Suc for 5 d. Seedlings were monitored and visualized for the next 4 d at 24-h intervals at the site of the first lateral root emergence on all seedlings. The total number of lateral roots per individual was counted from two replicates of two vertical plates for each genotype sown with six seeds each at 7 and 11 d after planting.

Statistical Analysis Data analysis was performed with SAS version 9.2. Penetration assays, all infection assays, and lateral root counts were all compared with the wild type using unpaired two-tailed t test and assuming equal variance. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AF490250 (H. glycines 19C07), HM142892 (H. schachtii 19C07), AF469061 (H. schachtii 4D09), At5g01240 (LAX1), At2g21050 (LAX2), At1g77690 (LAX3), At5g06320 (NHL3), At5g14650 (PG), and AAG51517.1 (GAPDH).

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