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Keywords:

  • cell death;
  • localization;
  • Phytophthora sojae;
  • plasma membrane;
  • RxLR effector;
  • virulence

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241.
  • To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role.
  • Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen’s full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection.
  • This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plants are attacked by many pathogens and defend themselves via multiple mechanisms of innate immunity (Jones & Takemoto, 2004; Nurnberger et al., 2004; Chisholm et al., 2006). Successful pathogens can suppress host defenses to invade and colonize plants. For example, the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) secretes > 30 effectors via the type III system (T3SS) to inhibit plant defense signaling events (Schechter et al., 2006). Fungi and oomycetes also secrete a wide diversity of effectors into host cells to manipulate plant immunity (De Wit et al., 2009; Stergiopoulos & de Wit, 2009; Tyler, 2009; Thines & Kamoun, 2010).

Microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs, respectively) trigger a set of plant defense responses referred to as PAMP-triggered immunity (PTI) (Medzhitov & Janeway, 1997). Elicitins are a class of highly conserved lipid transfer proteins secreted by Phytophthora species that can induce defense responses including hypersensitive cell death on most Nicotiana and a few other plant species (Huet et al., 1993; Kamoun et al., 1993; Keller et al., 1996). Plant responses to elicitins can block Phytophthora infection (Kamoun et al., 1998).

A second overlapping mode of innate immunity in plants is effector-triggered immunity (ETI) (Jones & Dangl, 2006), whereby plant disease resistance (R) proteins recognize pathogen effectors either directly or indirectly (Dangl & Jones, 2001; Jones & Dangl, 2006), thereby preventing the infection of host plants containing those R genes (Whisson et al., 1994; Tyler et al., 1995; Gijzen et al., 1996; May et al., 2002). Genes encoding pathogen effectors detected by plant R gene products are called avirulence (Avr) genes. Many Avr genes have been cloned from Phytophthora species, including P. sojae Avr1a (Qutob et al., 2009), Avr1b (Shan et al., 2004), Avr3a (Dong et al., 2011a; Qutob et al., 2009), Avr3b (Dong et al., 2011b), Avr3c (Dong et al., 2009), Avr4/6 (Dou et al., 2010), Avr5 (Dong et al., 2011a), P. infestans Avr2 (Gilroy et al., 2011), Avr3a (Armstrong et al., 2005), Avrblb1 (Vleeshouwers et al., 2008), Avrblb2 (Oh et al., 2009), and Avr4 (van Poppel et al., 2008).

All the Avr proteins from Phytophthora species contain N-terminal RxLR-dEER motifs following their signal peptide (Jiang et al., 2008). RxLR-dEER motifs are responsible for transporting effector proteins into plant cells (Whisson et al., 2007; Dou et al., 2008b; Jiang et al., 2008). The P. sojae, P. infestans, and H. arabidopsidis genomes contain > 390, 550, and 130 genes, respectively, encoding potential effector proteins carrying RxLR dEER motifs (Tyler et al., 2006; Jiang et al., 2008; Haas et al., 2009; Baxter et al., 2010).

Many effectors from oomycetes and bacteria can suppress cell death induced by elicitors (Bos et al., 2006; Dou et al., 2008a). For example, Avr1b from P. sojae can suppress programmed cell death (PCD) triggered in soybean, Nicotiana benthamiana, and Saccharomyces cerevisiae cells via the proapoptotic protein BAX (Dou et al., 2008a). Avr3a is required for full virulence of P. infestans and can suppress cell death induced by the MAMP INF1 (an elicitin) (Bos et al., 2006, 2010). Wang et al. (2011) conducted a broad survey of the transcription, variation, and functions of a large sample of candidate effectors from P. sojae. Of 169 effectors tested, most could suppress PCD triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves.

Increasing evidence indicates that effectors target different compartments of plant cells to execute their functions. For example, the effector ExoU of Pseudomonas aeruginosa contains a membrane localization domain (MLD) required for its activity (Rabin et al., 2006; Veesenmeyer et al., 2010). Similarly, the virulence effector HopM1 of P. syringae localizes to host endosomes where it targets the host protein AtMIN7 to suppress PTI and ETI (Nomura et al., 2011). The P. syringae effector HopL1 localizes to chloroplasts where it suppressing the synthesis of salicylic acid (Jelenska et al., 2007). The P. infestans RxLR effector AVRblb2 accumulates around haustoria and prevents the host papain-like cysteine protease C14 secreting into the apoplast and significantly enhances susceptibility of host plants to P. infestans (Bozkurt et al., 2011). Many pathogen effector proteins target the nucleus in order to modify host cell physiology, including transcription-activator-like (TAL) effectors from Xanthomonas spp. (Marois et al., 2002), the RxLR effector SNE1 from P. infestans (Kelley et al., 2010), and the crinkler effectors CRN8 from P. infestans (Schornack et al., 2010) and CRN63 from P. sojae (Liu et al., 2010).

In a survey of 49 RxLR effectors from Hyaloperonospora arabidopsidis (Caillaud et al., 2012), 16 localized in the nucleus exclusively, 16 localized in both the nucleus and the cytoplasm, three localized to the cytoplasm, nine localized to the endoplasmic reticulum, three localized to the plasma membrane and one each localized to the vacuole and tonoplast. One effector, HaRxL17, localized to the tonoplast in uninfected cells and to the membranes around haustoria, probably the extrahaustorial membrane, in infected cells. This effector enhanced plant susceptibility (Caillaud et al., 2012).

Here, we characterize in depth the P. sojae RxLR effector Avh241. Avh241 triggers cell death in multiple plant species, and localization to the membrane is required to trigger cell death. Localization requires a sequence resembling a myristoylation motif. Avh241 is essential for the full virulence of P. sojae, and induces susceptibility when expressed in plant cells. The ability to trigger cell death is not required to promote susceptibility, suggesting that Avh241 interacts with the plant immune system in at least two difference ways.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plasmid construction and strains

The oligonucleotides used for plasmid construction and the constructs used in this study are documented in the Supporting Information, Tables S1 and S2. The Avh241 gene was cloned using cDNA from P. sojae. For the PVX assay, Avh241 without signal peptide and the Avh241 deletion mutants were amplified using combinations of primers (Table S1). The amplicons were cut using appropriate restriction enzymes (Table S2) and ligated into the PVX vector PVX::Flag (Lu et al., 2003; Wang et al., 2011). To make the particle bombardment construct pX-DG::Avh241::GFP and its mutants, we used Avh241 in the vector PVX::Flag as template to amplify the genes using Taq™ (code: DR001A; TaKaRa, Kyoto, Japan) with the chosen primers (Table S1). The PCR products were inserted into XcmI-digested pX-DG (Chen et al., 2009) creating a fusion with the green fluorescent protein (GFP) coding region. CHF3 is a pPZP211-based plant expression vector carrying a cauliflower mosaic virus 35S (CaMV35S) promoter. The oligonucleotides for the transient expression construct CHF3::GFP::Avh241 are shown in Table S1. The amplicons and the CHF3 vector were cut with restriction enzymes BamHI and PstI (Table S2). To make transient expression constructs pBinGFP2::Avh241 and pBinGFP2::Avh241AAAAAA, we used PVX-Flag::Avh241 or PVX-Flag::Avh241AAAAAA as templates to amplify the genes. The primers used are given in Table S1 and the restriction enzymes are shown in Table S2. Full-length Avh241 was cloned and cut with appropriate restriction enzymes (Table S2) to replace the RFP gene of the vector pTOR (Whisson et al., 2007). The other constructs are shown in Table S2. All plasmids were validated by sequencing by GenScript, Inc. (Shanghai, China).

Agrobacterium tumefaciens infiltration assays and the growth of plants

Constructs were introduced into A. tumefaciens strain GV3101 by electroporation (Hellens et al., 2000). The PVX::Flag transformants were selected using tetracycline (12.5 μg ml−1) and kanamycin (50 μg ml−1), and the CHF3 transformants were selected using tetracycline (12.5 μg ml−1) and spectinomycin (50 μg ml−1). Individual colonies were verified by PCR using vector primers. For infiltration of PVX::Flag into leaves, recombinant strains of A. tumefaciens were grown in Luria–Bertani medium plus 50 μg ml−1 kanamycin for 48 h, harvested, and washed with 10 mM MgCl2 three times, then resuspended in 10 mM MgCl2 to achieve a final OD600 of 0.4. For infiltration of CHF3 or pBinGFP2 into leaves, recombinant strains of A. tumefaciens were grown in Luria–Bertani medium with 50 μg ml−1 spectinomycin or kanamycin, respectively, for 48 h, harvested, suspended in MgCl2 (10 mM), 2-(N-morpholino)ethanesulfonic acid (MES, 1 mM, pH 5.7), acetosyringone (100 μM) for 3 h in the dark at room temperature (RT). N. benthamiana Domin and Solanum lycopersicum L. plants were grown in a glasshouse for 4–6 wk at 25 : 20°C, 16 : 8 h, day : night. To promote infiltration into N. benthamiana and S. lycopersicum leaves (Bos et al., 2006), a small nick was placed in each leaf with a needle and then 100 μl of A. tumefaciens cell suspension was infiltrated through the nick using a syringe without a needle. Symptom development was monitored visually 3–8 d after infiltration for N. benthamiana and 15–20 d for S. lycopersicum leaves. The photographs were taken at 8 d for N. benthamiana and 20 d for S. lycopersicum. Every experiment was repeated at least three times and each assay consisted of at least three plants inoculated on three leaves. Col-0 Arabidopsis thaliana (L.) Heynh plants for protoplast transformation were grown in the glasshouse for 5 wk at 25 : 20°C, 8 : 16 h, day : night. Humidity was kept between 75 and 80%. Arabidopsis cell suspension cultures were maintained as described in Mathur & Koncz (1998). Protoplast isolation and polyethylene glycol-mediated transfection were performed as described in Asai et al. (2002).

Charge-coupled device (CCD) imaging and luciferase activity measurement

The day after Arabidopsis protoplast transformation, 1 mM luciferin was added to the transformed protoplasts. To quench the fluorescence, the materials were kept in the dark for a few min, then a low-light cooled CCD imaging apparatus (CHEMIPROHT 1300B/LND, 16 bits; Roper Scientific, Trenton, NJ, USA) was used to capture the luciferase image. After cooling the camera to −110°C, we measured the relative luciferase (LUC) activity as described by He et al. (2004). An exposure time of 2 min with 3 × 3 binning was used for taking images. Relative LUC activity is equivalent to luminescence intensity/200 protoplasts. Each data point consisted of at least three replicates.

Fluorescence

Green fluorescent protein fluorescence in Arabidopsis protoplasts, onion (Allium cepa L.) epidermal cells, and N. benthamiana leaves was captured with an excitation wavelength of 488 nm and an emission wavelength of 505–530 nm using an LSM 710 laser scanning microscope with a 40× objective lens (Carl Zeiss). GFP fluorescence in onion epidermal cells in Fig. 3 was detected using a fluorescence microscope with a GFP filter and a 20× objective lens (IX71; Olympus, Tokyo, Japan).

Protein extraction and western blots

Agroinfiltrated N. benthamiana leaves were harvested at 60 h postinoculation (hpi). Total protein extracts were prepared by grinding 400 mg of leaf tissue in 1 ml radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (no. P0013B; Beyotime, Jiangsu, China) in the presence of 0.1 mM protease inhibitor PMSF (no. ST506; Beyotime). The membrane and cytosol fractions were extracted using a membrane and cytosol protein extraction kit (P0033; Beyotime). The treated leaves were ground in liquid nitrogen and added to an Eppendorf tube with 1 ml of buffer A, shaken on a vortex intensely for 30 s, and then centrifuged at 700 g for 10 min at 4°C. The resulting supernatant was transferred to a new Eppendorf tube without any precipitation and was centrifuged at 14 000 g for 10 s at 4°C. The supernatant was transferred to a new Eppendorf tube (cytosol fraction), and 200 μl of buffer B was added to the precipitate. The sample was then shaken on a vortex intensely for 10 s and kept on ice for 5–10 min. This shaking and ice step was repeated twice and then centrifuged at 14 000 g for 5 min at 4°C to recover the supernatant’s membrane fraction. Proteins from the sample lysate were fractionated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred from the gel to an Immobilon-PSQ polyvinylidene difluoride membrane (pretreated with methanol for 15 s; Millipore) using a transfer buffer (20 mM Tris, 150 mM glycine). The membrane was then blocked using phosphate-buffered saline (PBS; pH 7.4) plus 3% nonfat dry milk (PBSM) for 30 min at RT with 50 rpm shaking, followed by one wash with PBST (PBS with 0.1% Tween 20). Mouse anti-Flag monoclonal antibodies (Sigma-Aldrich) were added to the PBSTM (PBS with 0.1% Tween 20 and 3% nonfat dry milk) at a ratio of 1 : 5000 and incubated at RT for 90 min, followed by three washes (5 min each) with PBST. The membrane was then incubated with a goat anti-mouse IRDye 800CW (Odyssey, no. 926-32210; Li-Cor, Lincoln, NE, USA) at a ratio of 1 : 10 000 in PBSTM at RT for 30 min with 50 rpm shaking. The membrane was washed three times (5 min each) with PBST and one time for 5 min with PBS. The membrane was then visualized using Odyssey with excitation 780 and 800 nm.

Particle bombardment assays

Particle bombardment assays were performed using a conventional barrel for onion epidermis and a double-barreled extension for soybean (Glycine max Merrill) leaves, and the He/1000 particle delivery system (Bio-Rad) (Scott et al., 1999; Dou et al., 2008a,b; Kale et al., 2010). Avh241 and its mutants were cloned into the CaMV 35S promoter-driven transient expression vector pX-DG (Chen et al., 2009), creating a fusion with the GFP coding region. The plasmid of Avh241 and its mutants fused with GFP were mixed with tungsten powder and then transiently expressed into onion epidermal cells using particle bombardment. Twenty-four hours later, the onion epidermal cells were detected using a fluorescence microscope. The cell death-inducing activities of Avh241 or Avh241AAAAAA constructs were measured as the difference in the number of blue spots comparing the Avh241 (Avh241AAAAAA)+GUS bombardment with a GFP+GUS control bombardment. The following differential soybean cvs were used: Williams (rps), Harlon (Rps1a), L77-1863 (Rps1b), PI103091 (Rps1d), Williams 82 (Rps1k), PRX146-36 (Rps3b), PRX145-48 (Rps3c), L85-2352 (Rps4), L85-3059 (Rps5), Harosoy62xx (Rps6, Rps7), and Harosoy (Rps7). Seedlings of each soybean cv were grown in a glasshouse for 14 d.

P. sojae isolates, transformation, and characterization

Phytophthora sojae strain P6497 (race 2) (Forster et al., 1994) was routinely grown and maintained on V8 agar (Erwin & Ribeiro, 1996). For stable transformation, Avh241 was ligated into vector pTOR (Whisson et al., 2007). Stable transformation was performed as previously described (Dou et al., 2008a). For DNA or RNA extraction, mycelia of P. sojae transformants were cultured in V8 liquid then harvested, frozen in liquid nitrogen, and ground into a powder. Genomic DNA was isolated from mycelia using Multisource Genomic DNA Miniprep Kit (Axygen, Union City, CA, USA) following the manufacturer’s protocol. The DNA was screened for Avh241 transgenes by PCR using primers pTOR-F and Avh241-1-SmaI-F (Table S1). To screen transformants for Avh241 silencing, total RNA was extracted from the mycelia. Silenced transformants were identified by quantitative real-time PCR (qPCR) assays using cDNA reverse transcripted from mycelia RNA.

The pathogenicity phenotypes of selected transformants were determined by inoculation of etiolated soybean seedlings (Dong et al., 2009; Wang et al., 2011). For inoculation of etiolated soybean seedlings, the Chinese susceptible cv HF47 was used. To measure the virulence of the pathogen, the ratio of P. sojae DNA to soybean DNA in the infected tissue was determined using qPCR. Etiolated soybean tissue infected by P. sojae transformants was harvested from 1 cm above the inoculation site to 1 cm below. Tissue samples from five seedlings were pooled, and total genomic DNA was extracted using the Multisource Genomic DNA Miniprep Kit (Axygen). The P. sojae Actin gene (VMD GeneID: 108986) was used as the pathogen target gene, and the soybean housekeeping gene CYP2 (TC224926) was used as the host target gene. Reactions were performed on an ABI Prism 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The degree of infestation of the wildtype (WT) strain P6497 was assigned the value 1.0, and the transformant amounts were related to P6497 using ABI SDS Software V1.4. All qPCR assays for infection were performed in replicates of three.

SYBR Green quantitative reverse transcription polymerase chain reaction (RT-PCR) assay

Total RNA was isolated from the hyphae, sporangia, zoospores, cysts, and germinating cysts of the infected susceptible soybean cv Williams, 0.5, 1, 3, 6, 12, and 24 hpi, using the NucleoSpin RNA II kit (Invitrogen) following the manufacturer’s protocol. The integrity of total RNA was confirmed by agarose gel electrophoresis. The RNA was quantified using a spectrophotometer (ND-1000; NanoDrop, Wilmington, DE, USA). To remove contaminating genomic DNA in RNA preparations, 3 μg of total RNA was treated with 2 units of RNase-free DNase I (TaKaRa) at 37°C for 30 min. First-strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (RNase-free) and an oligo(dT)18 primer (Invitrogen). qPCR was performed in 20 μl reactions including 20 ng of cDNA, 0.2 mM gene-specific primer or reference actin gene (Table S1), 10 μl of SYBR Premix ExTaq (TaKaRa), and 6.8 μl of deionized water. PCR was performed on an ABI Prism 7500 Fast Real-Time PCR System (Applied Biosystems) under the following conditions: 95°C for 30 s and 40 cycles at 95°C for 5 s, 60°C for 34 s, followed by a dissociation step of 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.

Measurement of ion leakage from leaf discs

Cell death was assayed by measuring ion leakage from leaf discs (Mittler et al., 1999). For each sample, five leaf discs (9 mm diameter) were floated on 5 ml distilled water for 3 h at RT. Then the conductivity of the bathing solution was measured with a conductivity meter (Con 700; Consort, Tutnhout, Belgium) to give ‘value A’. The leaf discs were then returned to the bathing solution and boiled in sealed tubes for 25 min. After cooling the solution to RT, the conductivity was measured again to obtain ‘value B’. For each measurement, ion leakage was expressed as percent leakage, that is (value A/value B) × 100. All assays were repeated three times.

Assay of Phytophthora capsici virulence on N. benthamiana

Agrobacterium-infiltrated N. benthamiana plants were grown in a glasshouse for 48 h. Symptomatic leaves were detached and maintained on half-strength MS medium in a Petri dish. Next, 2.5% V8 juice agar plugs (0.5 × 0.5 cm) infested with fresh P. capsici mycelia were inoculated onto the infiltrated regions. Diseased plant tissues at 16 hpi were stained by Trypan blue as described by Dong et al. (2008. The diameter of the disease lesion was photographed and measured at 36 hpi. Total DNA extracted from P. capsici-infected regions (2 × 2 cm) was isolated at 36 hpi. Real-time PCR was used to quantify the ratio of host-to-pathogen DNA sequences, employing primers specific for the N. benthamiana and P. capsici housekeeping Actin genes (Table S1). Three independent biological replicates were conducted.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

RxLR effector Avh241 induces cell death

Previous data have shown that some RxLR effectors of P. sojae can trigger cell death in N. benthamiana following Agrobacterium-mediated transient expression (Wang et al., 2011). Among these effectors, Avh241 triggered cell death in N. benthamiana and S. lycopersicum the most quickly and intensely when transiently expressed without its signal peptide (Fig. 1a,b). To determine whether Avh241-triggered cell death was affected by any R genes against P. sojae (Rps, resistant to Phytophthora sojae), we performed soybean bombardment assays on the soybean cv Williams (containing no known Rps genes). The ratio of blue spots between Avh241 + GUS (chimeric p-glucuronidase) and empty vector + GUS was calculated to assess cell death induced by Avh241 expression. The ratio of blue spots was significantly lower in the presence of Avh241 than on empty vector soybean leaves (Fig. 1c). This experiment demonstrated that Avh241 expression can trigger cell death in soybean independent of known Rps genes. We further confirmed it by transiently expressing Avh241 on different soybean cvs (with different Rps genes). Transient expression of Avh241 together with the LUC gene in Arabidopsis protoplasts completely abolished LUC activity when compared with the empty vector genotype (Fig. 1d), indicating that Avh241 expression can trigger cell death in Arabidopsis protoplasts. Taken together, these results demonstrate that Avh241 can trigger cell death in a variety of plant species regardless of the Rps genes present.

image

Figure 1. Avh241-induced cell death in a variety of plant species. (a) Leaves of Nicotiana benthamiana were infiltrated with Agrobacterium tumefaciens carrying PVX-Flag::Avh241. Photographs were taken 8 d postinfiltration. The experiment was repeated three times. (b) Leaves of Solanum lycopersicum were infiltrated with A. tumefaciens carrying PVX-Flag::Avh241. Photographs were taken 20 d postinfiltration. The experiment was repeated three times. (c) Avh241 induced cell death in soybean irrespective of R (Rps1a, Rps1b, Rps1d, Rps1k, Rps3b, Rps3c, Rps4, Rps5, Rps6, or Rps7) genes. The Williams cultivar contains no Rps genes. Soybean leaves were bombarded using a double-barreled device that delivered Avh241 DNA-bearing particles (encoding mature protein) to one side of the leaf and control (empty vector) DNA to the other side; both sides received GUS DNA. CK represents both leaf sides being delivered the empty vector and GUS DNA, and the spot ratio is nearly 1. The ratio of GUS-positive spots with Avh241 as a percentage of the control is shown. Data are the means of three independent experiments and the error bars represent SE. (d) Avh241 suppressed expression of luciferase (LUC) in Arabidopsis protoplasts. Protoplasts were transfected with a 35S-LUC reporter construct along with 35S-pUC19 or 35S-pUC19::Avh241. The means and SEs of luciferase activity from three replicates are shown. The statistical analyses were performed with PASW statistics 18 (SPSS Inc., Chicago, IL, USA). **, P < 0.01.

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Avh241 protein localization in the plasma membrane

To determine the cellular localization of Avh241, we conducted localization experiments with Arabidopsis protoplasts, onion epidermis cells, and Nbenthamiana leaf cells. When a Avh241–GFP fusion protein was transiently expressed in Arabidopsis protoplasts using the CHF3::GFP::Avh241 construct, the GFP fluorescence almost completely accumulated on the plasma membrane (PM), while control GFP fluorescence was detected in the cytoplasm as well as the nucleus (Fig. 2a). When Avh241–GFP was transiently expressed in onion epidermal cells using particle bombardment with pX-DG::Avh241::GFP, the Avh241–GFP fluorescence accumulated on the PM of the onion cells (Fig. 2b). When we transiently expressed the Avh241–GFP fusion protein with the pBinGFP2 vector using agroinfiltration in Nbenthamiana leaves, we also found that the Avh241 protein localized to plant PMs within 2 d (Fig. S1). Three days after transiently expressing Avh241 in Nbenthamiana leaves via agroinfiltration with PVX-Flag::Avh241, we extracted the membrane and cytosolic proteins. Using western blotting, we found that Avh241 could only be detected in the membrane fraction, while the control GFP (PVX-Flag::GFP) protein was detected in both cytosol and membrane fractions (Fig. 2c). Pm-rk-CD3-1007 is a plasma membrane localization protein from Arabidopsis fused with RFP(Nelson et al., 2007), and it can only detected in membrane fractions by western blotting (Fig. 2c).These results are consistent with the localization of Avh241 to the plant PM.

image

Figure 2. Avh241 protein localized in the plasma membrane fraction. (a) Green fluorescent protein (GFP)–Avh241 fusion protein or GFP were expressed in Arabidopsis protoplasts following polyethylene glycol (PEG)-mediated transformation of the protoplasts. The nuclei of the plant cells were stained by DAPI and imaged for comparison with the GFP fluorescence. Photographs were taken 16 h after transformation. Bar, 20 μm. (b) GFP–Avh241 fusion protein or GFP were expressed in onion epidermal cells following bombardment. The nuclei of the plant cells were stained by 4’,6-diamidino-2-phenylindole (DAPI) and imaged for comparison with the GFP fluorescence. Photographs were taken 16 h after particle bombardment. Bar, 50 μm. (c) Western blot analysis of proteins from Nicotiana benthamiana leaves transiently expressing Avh241, GFP fused with 3 × Flag tags or an Arabidopsis plasma membrane protein Pm-rk-CD3-1007 (a plasma membrane protein fused with RFP). Equal amounts of protein (20 μg) were loaded in each lane. C, cytosolic protein fraction; M, microsomal membrane protein fraction; T, total protein.

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Functional domains of Avh241

To identify the domains of Avh241 required for cell death induction and membrane localization, we analyzed Avh241 deletion mutants. Sequence analysis showed that Avh241 encodes a 188-amino-acid (aa) protein with a predicted signal peptide (SP) (aa 1–18). Avh241 has two W and two Y motifs (Dou et al., 2008a; Jiang et al., 2008) (Fig. 3a). W and Y motifs form a structural scaffold upon which a diversity of amino acids is displayed to mediate the interactions of the effectors with components of the plant immune system (Boutemy et al., 2011; Win et al., 2012). To delineate the functional domains of Avh241, we assayed N-terminal, C-terminal deletion mutants and W/Y motif deletion mutants for the ability to trigger cell death using agroinfiltration in N. benthamiana, and for localization by transformation of onion epidermal cells using particle bombardment or of N. benthamiana using agroinfiltration (Fig. 3a, S1). The N-terminal deletion mutant Avh241-3 triggered cell death and was localized to the PM, while the N terminal deletion mutant Avh241-4 could not trigger cell death and was localized in the cytoplasm. All the C-terminal deletion mutants exclusively localized to the PM regardless of triggering cell death (e.g. Avh241-1) or not (e.g. Avh241-2) (Fig. 3a). The deletion mutants Avh241-5 (W1 domain deleted), Avh241-6 (Y1 domain deleted), and Avh241-7 (W2 domain deleted) could not induce cell death (Fig. 3a) and still localize to the PM (Fig. S1). This demonstrated that these domains are required for triggering cell death but are not essential for localization. A western blot assay showed that all mutant Avh241 proteins accumulated to comparable degrees in N. benthamiana leaves (Fig. 3b). Our results therefore demonstrated that a 110 aa region (59–168 aa) of Avh241 is necessary and sufficient for triggering cell death. As expected, the RxLR motif is not required for triggering cell death or localization. The N terminal region from aa 59–80 determines subcellular localization and is also required for triggering cell death.

image

Figure 3. Deletion analysis of PsAvh241. (a) Left column, schematic view of deletion mutants for PsAvh241. Middle column, deletion mutants were expressed by agroinfiltration in Nicotiana benthamiana to assay triggering of cell death. Typical symptoms were photographed 8 d after infiltration. Right column, deletion mutants were transiently expressed as green fluorescent protein (GFP) fusions in onion epidermal cells by particle bombardment; photographs of typical symptoms were taken 24 h after transformation. Bar, 100 μm. (b) Western blot analysis of proteins from N. benthamiana leaves transiently expressing Avh241 and its deletion mutants from a PVX-Flag vector.

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Function of motif located at the Avh241 N-terminus

The N-terminal region of 59–80 aa of Avh241 is required for localization and cell death induction. This region contains a motif, GAAKAK, that resembles the myristoylation motif that normally occurs at the very N-terminus of proteins. We mutated the motif to AAAAAA to generate the mutant Avh241AAAAAA (Fig. 4a). After the Avh241AAAAAA–GFP fused protein was transiently expressed in onion epidermal cells by particle bombardment, the protein did not exclusively localize to the PM, and the GFP fluorescence showed marked relocalization toward the cytosol and nucleus (Fig. 4b). We observed the same result when the fused protein Avh241AAAAAA–GFP was transiently expressed in N. benthamiana leaves using agroinfiltration (Fig. S2). Avh241AAAAAA-Flag was detected in the cytosolic fraction similar to the GFP-Flag control by western blotting, whereas no WT (Avh241) or Pm-rk-CD3-1007 was detected in the cytosolic fraction (Fig. 4c). When transiently expressed in N. benthamiana leaves using the PVX-Flag vector, Avh241AAAAAA triggered significantly less cell death than the WT (PVX-Flag::Avh241) (Fig. 4d). Measurement of ion leakage (Mittler et al., 1999) was used to quantify cell death triggered by Avh241 and Avh241AAAAAA, since ion leakage is positively correlated with cell death. Ion leakage from leaves transiently expressing Avh241AAAAAA was significantly less than in Avh241-expressing plants (Fig. 4e). We also transiently expressed the mutant Avh241AAAAAA and GUS in soybean leaves using particle bombardment and additionally found that the mutant triggered much less cell death than the WT (Fig. 4f). These experiments demonstrate that this N-terminal motif is required for Avh241 localization and that localization is required for the triggering of cell death by Avh241.

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Figure 4. Functional characterization of a localization decision motif for Avh241. (a) Overview of the mutant construct. The amino acid residues 73–78 GAAKAK are mutated to AAAAAA. (b) Green fluorescent protein (GFP)-Avh241AAAAAA, GFP-Avh241, and control GFP were transiently expressed in onion epidermal cells using particle bombardment. Confocal microscopy was used to investigate fusion protein distributions. The epidermal cell transformed by Avh241AAAAAA showed the same amount of cytosolic fluorescence as the control GFP, whereas the GFP fluorescence of Avh241 existed only in the plasma membrane. Values on the y-axis indicate the relative intensity of GFP fluorescence signal when normalized using nuclear GFP fluorescence. White lines depict the transects used for the intensity plots. M, membrane; N, nuclei. Photographs of typical symptoms were taken 16 h after transformation. Bar, 50 μm. (c) Western blot analysis of proteins from Nicotiana benthamiana leaves transiently expressing Avh241, Avh241AAAAAA, GFP fused with 3 × Flag tags or Pm-rk-CD3-1007. Equal amounts of protein (20 μg) were loaded in each lane. C, cytosolic protein fraction; M, microsomal membrane protein fraction; T, total protein. (d) Expression of Avh241 and mutants in N. benthamiana leaves by agroinfiltration. The OD600 of the Agrobacterium culture for infiltration was 0.02. Photographs of symptoms were taken 5 d after infiltration. (e) Quantification of cell death by measuring electrolyte leakage. N. benthamiana leaves were agroinfiltrated with constructs encoding Avh241, Avh241AAAAAA, and GFP. After 4 d, electrolyte leakage from the infiltrated leaf discs was measured as a percentage of leakage from boiled discs. Data are the means of three independent experiments and the error bars represent SE. (f) The cell death induced by Avh241AAAAAA was decreased in soybean compared with the cell death induced by the wildtype construct (Avh241). The Williams soybean cultivar leaves were bombarded using a double-barreled device that delivered Avh241AAAAAA DNA-bearing particles (encoding mature protein) to one side of the leaf and control (empty vector) DNA to the other side, while both sides received GUS DNA. The ratio of GUS-positive spots with Avh241AAAAAA is > 60% and much higher than the WT. The control (CK) represents both sides receiving the empty vector and GUS DNA. The spot ratio was nearly 1. Values are the means of three independent experiments and the error bars represent SEs. The statistical analyses were performed with PASW statistics 18 (SPSS Inc). *, < 0.05; **, < 0.01.

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Involvement of mitogen-activated protein kinase (MAPK) cascades

Mitogen-activated protein kinase cascades play an important role in PTI and ETI (Pedley & Martin, 2005). The kinases MAPKKKα, MEK1, MEK2, WIPK, SIPK, and NTF6 and the transcription factors WRKY1 and WRKY2 perform essential functions in plant defense and cell death induction during interactions between plants and bacterial pathogens (Eulgem et al., 2000; Maleck et al., 2000; del Pozo et al., 2004; Naoumkina et al., 2008; Oh & Martin, 2010). We silenced these eight genes in N. benthamiana using virus-induced gene silencing (VIGS) (Ruiz et al., 1998; Baulcombe, 1999) to determine their role in cell death triggered by Avh241. Avh241 was transiently expressed in these silenced plants using agroinfiltration with CHF3::Avh241 4 wk after silencing was initiated. Silencing of MAPKKKα, MEK1, SIPK, NTF6, WRKY1, or WRKY2 had no effect on the triggering of cell death by Avh241 (Fig. S3). The silencing of MEK2 or WIPK, however, significantly depressed Avh241-triggered cell death (Fig. 5a). As a positive control, we used INF1, which triggers cell death independently of MEK2 and WIPK (Takahashi et al., 2007). Measurement of ion leakage was used to quantify the cell death triggered by Avh241 or INF1 in these experiments. Ion leakage from leaves transiently expressing Avh241 following MEK2 or WIPK silencing was significantly less than that from control plants (Fig. 5b). Fig. 5(c) shows the transcript abundances of MEK2 and WIPK in the silenced plants, validating successful silencing.

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Figure 5. Avh241-induced cell death requiring NbMEK2 and NbWIPK. (a) Avh241 was transiently expressed in Nicotiana benthamiana leaves in which NbMEK2 or NbWIPK had been silenced. INF1 is a control protein that induces cell death independently of NbMEK2 and NbWIPK. The left two sites were treated with Avh241, the upper site on the right was treated with INF1, and the lower site was treated with GFP control. Three replicate leaves are shown, photographed 5 d after agroinfiltration. (b) Quantification of cell death by measuring electrolyte leakage. N. benthamiana leaves were silenced using NbMEK2, NbWIPK, or control (CK) constructs, then agroinfiltrated with constructs encoding Avh241 (dark gray), INF1 (black), or green fluorescent protein (GFP) (light gray). After 4 d, electrolyte leakage from the infiltrated leaf discs was measured as a percentage of leakage from boiled discs. Data are the means of three independent experiments and the error bars represent SEs. (c) Transcript abundance of NbMEK2 or NbWIPK in silenced N. benthamiana tissues, measured by quantitative real-time PCR. The statistical analyses were performed with PASW statistics 18 (SPSS Inc). *, < 0.05; **, < 0.01.

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Impact of Avh241 silencing on P. sojae virulence

To investigate the role of Avh241 during infection, we determined the expression patterns of Avh241 in different stages of development, including mycelium, sporulating hyphae, zoospores, cysts, germinated cysts and infection 0.5, 1, 3, 6, 12, and 24 hpi. qPCR showed that Avh241 is highly expressed during both cyst and early infection stages (Fig. 6a).

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Figure 6. Role of Avh241 in Phytophthora sojae infection. (a) Expression of Avh241 before and during the early stages of soybean infection measured by quantitative real-time PCR (qPCR). My, nonsporulating mycelium grown in V8 broth; Sp, sporangia; Zo, zoospores; Cy, cysts; GC, germinating cyst; and infected susceptible Williams soybean 0.5, 1, 3, 6, 12, and 24 h postinoculation (hpi). Transcript abundances are expressed relative to mycelium. Means and SEs are from three replicates. (b) Silencing of Avh241. Avh241 mRNA concentrations in three stably silenced P. sojae transformants were determined by qPCR 2 d after mycelial growth in V8 medium relative to actin mRNA concentrations and then normalized to a nontransformed control (WT). CK is a line subjected to the transformation procedure with empty vector. Standard errors from three technical replicates are shown. (c) Virulence of silenced lines. Virulence was determined using qPCR to measure the ratios of P. sojae to soybean DNA in etiolated seedlings 16 h after inoculation with 100 zoospores each. Error bars represent SEs from three technical replicates. WT indicates the nontransformed parent strain P6497; CK is a line subjected to the transformation procedure with empty vector. The statistical analyses were performed with PASW statistics 18 (SPSS Inc). **, P < 0.01.

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To determine the contribution of Avh241 to P. sojae infection, we silenced this gene by polyethylene glycol (PEG)-mediated transformation with the antisense construct pTOR::Avh241 (Whisson et al., 2007; Dou et al., 2008a). Three stable transformants (T72, T111, T157) were recovered in which the constitutive level of Avh241 mRNA was reduced to < 40% of the parent strain (P6497) according to qPCR (Fig. 6b). All three transformants showed significantly reduced virulence on etiolated soybean seedlings as quantified by qPCR measurements of host and pathogen DNA 12 h after inoculation (Dong et al., 2009; Wang et al., 2011) (Fig. 6c). This result indicates that Avh241 plays a role in virulence during infection.

Suppression of plant immunity by Avh241

Avh241 contributes to virulence during infection. Although Avh241 can trigger cell death when overexpressed in plants, many P. sojae RxLR effectors can suppress the cell death induced by Avh241 (Wang et al., 2011). We hypothesized that Avh241 might suppress plant immunity when the cell death induced by it is blocked. To test this hypothesis, we used Avh172 to suppress Avh241-triggered cell death (Wang et al., 2011). We expressed Avh172 in N. benthamiana leaves using agroinfiltration with PVX-HA (PVX vector with HA tag) 12 h before expressing Avh241 using agroinfiltration with a PVX-Flag vector. Following agroinfiltration with Avh241 (48 h later), we inoculated the infiltrated regions with an agar plug (5 × 5 mm) containing freshly grown mycelia of P. capsici (a Phytophthora pathogen of N. benthamiana), and evaluated disease development at different times following inoculation. At 16 hpi, abundant P. capsici hyphae were observed by Trypan blue staining in Avh172- and Avh241-expressing tissues, but few hyphae were present in the region expressing Avh172 and GFP (PVX-Flag::GFP) (Fig. 7a). At 36 hpi, the disease lesions on the leaves infiltrated with strains carrying control genes (Avh172 and GFP) were c. 1.0 cm in diameter, but on leaves infiltrated with strains carrying Avh172 and Avh241, the lesion diameter expanded to c. 1.5 cm, as shown in Fig. 7(b,c). To more precisely measure P. capsici infection, we measured the ratio of P. capsici DNA to N. benthamiana DNA using real-time PCR to determine the Phytophthora biomass in the infected plant tissues (Fig. 7d). The P. capsici biomass in the leaves expressing both Avh172 and Avh241 was significantly greater than in the control leaves expressing GFP and Avh172. Western blot assays showed that the Avh172, Avh241 and GFP proteins could be readily detected in the infiltrated leaves (Fig. 7e,f). Both the lesion size data and the biomass data suggest that Avh241 expression in plants in the presence of Avh172 enhanced the susceptibility of N. benthamiana to P. capsici, while the expression of GFP or Avh172 alone did not (Fig. 7, S4). These observations support the hypothesis that Avh241 can suppress immunity in N. benthamiana, when the cell death it triggers is suppressed.

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Figure 7. Avh241 promotes the infection of Nicotiana benthamiana by Phytophthora capsici. N. benthamiana leaves were first separately infiltrated with Agrobacterium tumefaciens cells carrying T-DNAs encoding GFP-Flag, Avh241-Flag, GFP-Flag, or Avh241-4-Flag. The former two treatments were preceded 12 h earlier by infiltration with A. tumefaciens cells carrying T-DNA encoding Avh172-HA. After 48 h, the four transformed leaves were inoculated with P. capsici mycelia on the infiltrated regions. (a) The treated leaves were stained with Trypan blue 16 h postinoculation (hpi). Photographs of typical symptoms were taken after decolorizing with chloral hydrate (the arrows denote hyphae). Bar, 50 μm. (b, c) Leaves were photographed 36 hpi (b) and diameters of the lesions were measured (c). Error bars represent SEs from three technical replicates. (d) Infection was quantified using quantitative real-time PCR to measure the ratios of P. capsici and N. benthamiana DNA 36 hpi. Error bars represent SE from three technical replicates. (e, f) Western blot analysis of proteins from N. benthamiana leaves 60 h after agroinfiltration in the absence of P. capsici. GFP, Avh241, and Avh241-4 were fused with Flag tag. Avh172 was fused with HA tag. The total proteins were stained with Ponceau S. These experiments were repeated three times with similar results. Four leaves were used for each treatment in each experiment. The statistical analyses were performed with PASW statistics 18 (SPSS Inc). *, < 0.05; **, < 0.01.

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To test the importance of the localization of Avh241 in the plant PM in suppressing immunity, these experiments were repeated using the N-terminal deletion mutant Avh241-4 (PVX-Flag::Avh241-4). This mutant does not trigger cell death either. The results showed that Avh241-4 could suppress immunity equally as well as the WT Avh241 with Avh172 present (Figs 7, S4). This indicates that neither localization nor the ability to trigger cell death is required for the suppression of immunity in N. benthamiana when Avh241 is overexpressed via agroinfiltration.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Hundreds of predicted RxLR effector-encoding genes exist in the genomes of sequenced oomycete plant pathogens (Whisson et al., 2007; Win et al., 2007; Birch et al., 2008; Jiang et al., 2008). The encoded proteins potentially play an important role in the establishment of infection and conversely a role in plant defense. Surveys of Phytophthora RxLR effectors identified many that could suppress cell death and a few that could induce cell death (Oh et al., 2009; Wang et al., 2011). Many of the suppressors, such as SNE1 of P. infestans and Avr1b and Avh331 of P. sojae, can suppress both PTI and ETI (Bos et al., 2006; Dou et al., 2008b; Wang et al., 2011). Avh241 is a typical RxLR effector identified as one that can trigger cell death in both N. benthamiana and soybean from a survey of P. sojae effectors. Here we showed that it also triggered cell death in Arabidopsis protoplasts and in tomato. Previous work showed that Avh241 induces cell death in soybean cv Williams, which does not contain known Rps genes, suggesting that no known Rps gene mediates this cell death (Wang et al., 2011). We confirmed this finding.

The wide host range of Avh241-triggered cell death suggests either that Avh241 targets a conserved and critical host protein guarded by R genes in many plants, or that R genes are not involved in Avh241-triggered cell death. Silencing of MAPK and transcription factor genes revealed that Avh241-triggered cell death requires the MEK2/WIPK MAP kinase signal transduction pathway. MEK2 and WIPK are orthologs of the Arabidopsis proteins AtMEK4, 5 and AtMPK3, respectively, which are required for transducing signals from the PAMP receptor FLS2. The broad range of species’ responses to Avh241, and the dependence on the MEK2/WIPK pathway, suggest that plants respond to this effector via a PAMP-signaling pathway. Avh241, however, has no close homologs in other sequenced oomycetes and so does not resemble a widespread PAMP. We speculate therefore that Avh241 targets a conserved upstream component of plant PAMP-signaling pathways, possibly a pattern recognition receptor. The localization of Avh241 in the plant plasma membrane, mediated by an N-terminal motif, would be consistent with this mechanism. The membrane localization motif of Avh241 resembles a myristoylation motif; however, its localization is atypical for a myristoylation motif. In future studies we plan to examine whether Avh241 is myristoylated at this site. AvrPto of P. syringae pv. tomato localizes in the PM of plant cells via myristoylation (Shan et al., 2000) and targets the kinase domains of the PAMP receptors FLS2 and EFR2 (Xiang et al., 2008). The PM localization of P. syringae pv. tomato DC3000 effector HopF2 is required for suppressing a plant’s innate immunity triggered by PAMPs or MAMPs (Robert-Seilaniantz et al., 2006; Wu et al., 2011). AvrXccC of Xanthomonas campestris pv. campestris is anchored to the plant PM, and therefore membrane localization is considered essential for host recognition (Wang et al., 2007).

Sequencing of the genomes of several physiological races of P. sojae revealed no polymorphisms in Avh241 (Wang et al., 2011), which we confirmed by cloning and sequencing the genes (data not shown). Unexpectedly, Avh241 does not appear to be under diversifying selection despite the fact that it triggers defense-related cell death in its host, soybean. Wang et al. (2011) found that nearly all P. sojae effectors tested could suppress the cell death triggered by Avh241. Whether Avh241 expression under natural conditions would trigger cell death, or whether cell death is an artifact of elevated expression in the agroinfiltration assay is currently unknown. Thus, under conditions of physiological infection, the plant responses triggered by Avh241 may not actually result in cell death, alleviating selection pressure for changes in Avh241. However, physiological expression levels of Avh241 may trigger responses such as ion leakage that could benefit the pathogen but would cause cell death at high levels.

Our data show that the Avh241 transcript abundance is enhanced during early infection. Silencing Avh241 impaired P. sojae virulence during infection, demonstrating that this effector plays an essential role in virulence during infection that is not replaced by any other effector. Avh241 also promotes Phytophthora infection of N. benthamiana when cell death is suppressed by the coexpression of Avh172 or by the removal of the membrane localization motif of Avh241. These results concur with previous findings (Bos et al., 2010; Wang et al., 2011) that several individual oomycete RxLR effectors are indispensable for virulence, despite the presence of many hundreds of RxLR effector genes in oomycete genomes. Furthermore, our results suggest that Avh241 interacts with plant immune systems via at least two different mechanisms, one that involves MEK2/WIPK-dependent cell death and requires the plasma membrane localization of Avh241, and one that suppresses immunity against Phytophthora but does not require the plasma localization of Avh241. Future studies will help to further identify and characterize signaling components targeted by Avh241 and provide more insight into the mechanisms that regulate host cell death, pathogenesis, and virulence.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the National Basic Research Program of China (973 Program; number 2009CB119200) to Y.W. and X.Z., and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and National Natural Science Foundation of China (30900932) to S.D. We acknowledge Professor Jianmin Zhou (CAS) for providing vector pUC19-35S::LUC and CHF3 and for help with Arabidopsis protoplast transformation; Daolong Dou (NJAU) for the pX-DG and pBinGFP2 vectors; Xiaorong Tao (NJAU) for the Pm-rk-CD3-1007 vector; and Dr Stephen Whisson (James Hutton Institute) for the pTOR vector. We also thank Brett Tyler (Oregon State) for critical reading of the manuscript and for suggestions.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

Fig. S1 Localization of Avh241-5, Avh241-6 and Avh241-7.

Fig. S2 Localization of Avh241 and Avh241AAAAAA.

Fig. S3 Nicotiana benthamiana genes that are not required for cell death triggered by Avh241.

Fig. S4 Avh241 promoted infection of Nicotiana benthamiana by Phytophthora capsici.

Table S1 Primers used in this study

Table S2 Plasmid constructs used in the study

FilenameFormatSizeDescription
nph4241_sm_FigsS1-S4.docx5209KSupporting info item
nph4241_sm_TableS1-S2.xls36KSupporting info item