Pathogenic oomycetes have evolved RXLR effectors to thwart plant defense mechanisms and invade host tissues. We analysed the function of one of these effectors (Penetration-Specific Effector 1 (PSE1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete Phytophthora parasitica.
Expression of PSE1 protein in tobacco (Nicotiana tabacum and Nicotiana benthamiana) leaves and in Arabidopsis thaliana plants was used to assess the role of this effector in plant physiology and in interactions with pathogens. A pharmacological approach and marker lines were used to charcterize the A. thaliana phenotypes.
Expression of PSE1 in A. thaliana led to developmental perturbations associated with low concentrations of auxin at the root apex. This modification of auxin content was associated with an altered distribution of the PIN4 and PIN7 auxin efflux carriers. The PSE1 protein facilitated plant infection: it suppressed plant cell death activated by Pseudomonas syringae avirulence gene AvrPto and Phytophthora cryptogea elicitin cryptogein in tobacco and exacerbated disease symptoms upon inoculation of transgenic A. thaliana plantlets with P. parasitica in an auxin-dependant manner.
We propose that P. parasitica secretes the PSE1 protein during the penetration process to favour the infection by locally modulating the auxin content. These results support the hypothesis that effectors from plant pathogens may act on a limited set of targets, including hormones.
Plant–pathogen interactions are often viewed as a merciless battle. Pathogens strive to invade host tissues to obtain nutrients and complete their life cycle; plants attempt to restrict microbial colonization to ensure their survival. The outcome of this interaction depends on molecular events during penetration and contact with the first plant cells. Plants have evolved two layers of defence to protect themselves against pathogens (Dodds & Rathjen, 2010). The first layer of the defence system allows the recognition of conserved pathogen- or microbial-associated molecular patterns (PAMPs/MAMPs) and is thus referred to as PAMP-triggered immunity (PTI; Boller & Felix, 2009). PAMPs include various molecules, some of which have been extensively studied, including bacterial flagellin, chitin fragments from fungal cell walls and oomycete elicitins. They are recognized by pattern-recognition receptors (PRRs; Zipfel, 2009) which activate downstream signalling including mitogen-associated protein kinase (MAPK) cascades, leading to oxidative burst, ethylene synthesis, changes in gene expression and cell wall reinforcement (Boller & Felix, 2009). Pathogens have evolved a large weaponry to overcome plant defence mechanisms. They secrete proteins called effectors which are targeted to the apoplast or to the plant cytoplasm to block the PTI, leading to effector-triggered susceptibility (ETS; Ellis et al., 2009). As a counter measure, plants have evolved a second layer of defence, referred to as effector-triggered immunity (ETI). ETI is activated when some effectors or their activity is detected by the product of a resistance (R) gene (Dodds & Rathjen, 2010). Such effectors are referred to as avirulence genes. ETI and PTI activate similar signalling pathways but ETI responses are usually amplified and associated with localized cell death at the infection site; this phenomenon is called the hypersensitive response (HR; Dodds & Rathjen, 2010). This kind of local, induced cell death protects plants from further invasion. However, successful pathogens secrete another set of effectors to overcome ETI, leading to a zigzag evolutionary pattern involving compatible and incompatible interactions (Jones & Dangl, 2006).
Major advances in understanding the function of effectors have been made during the past decade. Effectors from bacteria, fungi, oomycetes and nematodes have been shown to modulate plant defence responses and to contribute to plant susceptibility (Alfano, 2009; Oliva et al., 2010; Wawra et al., 2012). However, only a few of these proteins have had their molecular function elucidated. They were shown to interfere with PTI pathways and general host metabolism including gene silencing, vesicular trafficking and hormone physiology (Gimenez-Ibanez et al., 2010; Hann & Rathjen, 2010; Dou & Zhou, 2012; Zvereva & Pooggin, 2012). A few reports concerned modulations of plant hormone physiology by pathogen effectors. For example, the Pseudomonas syringae jasmonate analogue coronatin induces the jasmonic acid (JA) signalling pathway and thereby interferes with salicylic acid (SA)-dependent defence responses in Arabidopsis thaliana (Brooks et al., 2005). The HopI1 effector from the same bacterium interacts with a host heat-shock protein (HSP70) and suppresses SA accumulation, thus contributing to virulence (Jelenska et al., 2007). The corn pathogen Ustilago maydis secretes a chorismate mutase, Cmu1, that impairs SA biosynthesis by depleting the plant cell of chorismate (Djamei et al., 2011). Similarly, pathogen effectors have been shown to target the phytohormone auxin. The P. syringae effector AvrRpt2 induces indole acetic acid (IAA) accumulation and increases plant susceptibility (Chen et al., 2007). Recently, the cyst nematode Heterodera schachtii was shown to secrete the effector Hs19C07 which interacts with the auxin influx carrier LAX3 to facilitate plant infection (Lee et al., 2011).
Effectors from filamentous pathogens were first identified as an arsenal of glucanase and protease inhibitors that counteract plant defence-related enzymes (Kamoun, 2007). Recently, filamentous pathogens were also shown to secrete proteins that enter the plant cytoplasm to modulate plant immunity (Oliva et al., 2010). Most of the findings regarding cytoplasmic effectors from filamentous pathogens were obtained with oomycete pathogens and concerned a family of proteins characterized by the presence of a signal peptide and an RXLR motif (Schornack et al., 2009). The RXLR motif is required for the internalization of effectors into plant cells (Whisson et al., 2007), after which they are addressed to various plant cell compartments (Caillaud et al., 2012). Originally identified as avirulence genes, RXLR effectors have now been shown to contribute to virulence and to modulate plant defence responses. This was demonstrated in the model plant Nicotiana benthamiana by monitoring the ability of these proteins to block cell death caused by various microbial and animal proteins. For example, Phytophthora sojae Avr1b suppresses cell death caused by the mouse pro-apoptotic protein BAX (Dou et al., 2008). Similarly, Phytophthora infestans AVR3a suppresses cell death caused by the elicitin INF1 in tobacco plants (Bos et al., 2006). It was recently shown that AVR3a also interferes with several avirulence/resistance protein pairs by stabilizing the CMPG1 ubiquitin ligase (Gilroy et al., 2011). Phytophthora sojae Avr3b encodes an ADP-ribose/NADH pyrophosphorylase that modulates plant immunity (Dong et al., 2011) and P. infestans AVRblb2 prevents secretion of the C14 cysteine protease to interfere with the activation of plant defences (Bozkurt et al., 2011). Evidence that the RXLR effectors AVR3a and AVRblb2 are secreted by specialized feeding structures called haustoria was obtained, and it is now commonly accepted that RXLR effectors are secreted by these structures during the biotrophic phase of the interaction (Whisson et al., 2007; Bozkurt et al., 2011). However, two reports suggest that transcripts encoding RXLR effectors may accumulate earlier during the infection process. Judelson et al. (2008) showed that some RXLR-encoding transcripts accumulate during the differentiation of appressorium-like structures. We also reported accumulation of three RXLR effectors during appressorium-mediated penetration of plant roots by Phytophthora parasitica (Kebdani et al., 2010). Furthermore, Wang et al. (2011) recently suggested that effectors with early or late expression patterns may target different plant defence signalling pathways. The function of effectors expressed early deserves further invstigation.
We conducted a functional analysis of Penetration-Specific Effector 1 (PSE1), an RXLR effector produced by the broad-host-range pathogen P. parasitica. The transcripts encoding this effector were accumulated in appressoria differentiated on a simplified penetration system and in appressoria during the penetration of tomato (Lycopersicon esculentum) roots (Kebdani et al., 2010). We investigated the role of this effector during the development of disease in the pathosystem involving P. parasitica and A. thaliana. We report that the PSE1 transcript was specifically expressed during the first hours of infection of the model plant. The PSE1 protein suppressed plant cell death as a part of the plant defence responses and enhanced plant susceptibility to P. parasitica. Expressing the effector in A. thaliana resulted in a series of developmental defects associated with perturbations of auxin physiology. In particular, the PSE1 protein modulated the auxin content of the plant root, thereby facilitating infection.
Materials and Methods
Strains and plant lines
Phytophthora parasitica Dastur isolate 310 was grown and sporulation was induced as described previously (Galiana et al., 2005).
Arabidopsis thaliana (L.) Heynh. ecotype Columbia (Col-0) was used. Seeds were surface-sterilized for 3 min (70% ethanol and 0.05% SDS). Seeds were sown on 1X Murashige and Skoog (MS) medium (Sigma) supplemented with 20 g l−1 sucrose and 10 g l−1 agar. Seedlings were grown under short-day conditions (8-h light : 16-h dark photoperiod) at 20°C for 2 wk and were used for subsequent experiments as described below.
The pDR5::GFP (Ottenschläger et al., 2003) reporter line was kindly provided by Mathilde Clément (CEA, Cadarache, France). The pPIN1::PIN1-GFP (Benková et al., 2003), pPIN2::PIN2-GFP (Xu et al., 2006), pPIN4::PIN4-GFP (Vieten et al., 2007) and pPIN7::PIN7-GFP (Vieten et al., 2007) reporter lines were kindly provided by Yvon Jaillais (ENS, Lyon, France). The HS::AXR3NT-GUS (Gray et al., 2001) reporter line was kindly provided by Ottoline Leyser (The Sainsbury Laboratory, Cambridge, UK).
Nicotiana benthamiana (Domin) and Nicotiana tabacum (L.) var Xanthi were grown on soil for 3–4 wk at 24°C with a 16-h light : 8-h dark photoperiod.
Arabidopsis thaliana plantlets, grown as previously described by Attard et al. (2010), were inoculated with 500 P. parasitica zoospores. Experiments were carried out in duplicate. At least 20 plantlets were analysed for each experiment. The disease progression was ranked according to Attard et al. (2010). Healthy plants were assigned rank 1. Rank 2 was assigned when one or two old leaves were invaded, rank 3 when three to four old leaves were invaded, rank 4 when only one or two old leaves remained healthy, rank 5 when the first stage of the rosette was completely invaded and only young leaves remained healthy, rank 6 when two to three young leaves remained healthy and rank 7 when the plant was dead.
Total RNA extraction and qRT-PCR analyses
RNA was extracted from four biological replicates as previously described (Laroche-Raynal et al., 1984). After DNAse I treatment (Ambion, Austin, TX, USA), RNA (1 μg) was reverse-transcribed using IScript cDNA synthesis kits (BioRad, Hercules, CA, USA). Real-time PCR experiments, in triplicate, were performed using 5 μl of a 1 : 50 dilution of first-strand cDNA and SYBRGreen (Eurogentec SA, Seraing, Belgium) and Opticon 3 (BioRad). Gene-specific oligonucleotides were designed using primer3 software (http://frodo.wi.mit.edu) and their specificity was validated by the analysis of dissociation curves. The following internal controls were used: ubiquitin-conjugating enzyme (UBC) and 40S ribosomal protein S3A (WS21 ) genes from (Yan & Liou, 2006) from P. parasitica; mitochondrial inner membrane protein (OXA1) and NADH genes for A. thaliana (Quentin et al., 2009; Attard et al., 2009) and NAPDH (GQ256517.1) and β-actin (Archana et al., 2009) actin genes for N. benthamiana. Normalization was performed according to Vandesompele et al. (2002).
Transient expression on tobacco
The PSE1 coding sequence (GenBank accession no. FK937603.1) with its signal peptide deleted was cloned into the vectors pK2GW7, pK7WGF2 and pK7FWG2 (Plant Systems Biology, Ghent University, Belgium). The Phytophthora cryptogea cryptogein coding sequence (GenBank accession no. Z34459.1) was cloned into the pK7FWG2 vector (Plant Systems Biology). The coding sequence of mouse BAX was amplified from a PVX vector (kindly supplied by Dr Brett Tyler; Dou et al., 2008) and cloned into pH2GW7 (Plant Systems Biology).
Agrobacterium tumefaciens strain GV3301 was used for transient expression in N. tabacum and N. benthamiana. Agrobacterium tumefaciens cultures were grown overnight at 28°C in Luria–Bertani (LB) medium supplemented with 50 μg ml−1 rifampicin, 20 μg ml−1 gentamicin and 100 μg ml−1 spectinomycin. Bacteria were resuspended in infiltration buffer (10 mM MES, pH 5.7, 10 mM MgCl2 and 100 μM acetosyringone) and incubated for 3 h at 28°C before infiltration. Infiltrations were performed at a final optical density (OD600) of 0.4. Results were observed after 48 h for PSE1 in planta localization and after 8 d for cell death suppression assays.
Production of recombinant PSE1 protein and of anti-PSE1 polyclonal antibodies
The coding sequence of PSE1 with its signal peptide deleted was cloned into the pET300/NT-DEST expression vector (Invitrogen Corp., Carlsbad, CA, USA) and expressed in Escherichia coli Rosetta strain (Novagen, Madison, WI, USA). Recombinant 6xHis-PSE1 protein was purified using immobilized metal ion affinity chromatography (IMAC). Polyclonal antibodies were obtained in rabbit by Covalab (Villeurbane, France).
Total protein extraction and SDS-PAGE
Protein extracts were prepared from 40 mg (fresh weight) of the infiltrated areas collected 36 h after agroinfiltration. Tissues were ground in liquid nitrogen and total proteins were extracted in boiling Laemmli buffer containing 63 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS and 0.0025% Bromophenol Blue for 5 min. Proteins were seperated on a 12% polyacrylamide gel, transferred to a nitrocellulose membrane (GE Healthcare, UK) and stained with Ponceau Red.
Western blot analysis
Nitrocellulose membranes were blocked with 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20 and 2% milk powder. Primary antibodies were incubated for 3 h at 1 : 5000 (mouse anti-GFP antibodies; Roche) or 1 : 20 000 (rabbit anti-PSE1 antibodies; Covalab, Lyon, France). Horseradish peroxidase (HRP) -conjugated secondary antibodies were incubated for 1 h at 1 : 20 000. Detection was performed by chemiluminescence using the ECL kit according to the manufacturer's instructions (Pierce, Rockford, IL, USA).
Stable transformation of Arabidopsis thaliana
Transformation was performed as previously described (Clough & Bent, 1998) using A. tumefaciens strain GV3101 carrying the pK2GW7::PSE1 construct.
Root growth analyses
For root length assays, seedlings were transferred to vertically oriented 1% agar plates. For coiled root assays, seedlings were transferred to 2% agar plates. Coiled root phenotype complementation assays were performed on MS medium supplemented with 50 nM 2,4-dichlorophenoxyacetic acid (2,4-D), 100 nM α-naphthaleneacetic acid (NAA), 100 nM indole-3-acetic acid (IAA), 100 nM indole-3-butyric (IBA), 10 μM 2,3,5-triiodobenzoic acid (TIBA) or 10 μM 1-N-naphthylphthalamic acid (NPA; Sigma). Plantlets were analysed after 10 d of growth in short-day conditions.
Lugol staining of starch granules
Starch granules from 8-d-old seedlings were stained for 30 min with 1% Lugol solution (Sigma) and mounted in chloral hydrate/lactophenol.
A Zeiss LSM 510 META confocal microscope was used (Carl Zeiss GmbH, Jena, Germany). Green fluorescent protein (GFP) and propidium iodide excitation was obtained at 488 and 543 nm, respectively. For pDR5::GFP assays, 1-wk-old plantlets were analysed. Acquisition parameters were calibrated on the pDR5::GFP reporter in the wild-type background and unchanged for the mutant background. For pPIN::PIN-GFP assays, 3-wk-old plantlets were grown in liquid 0.1X MS medium as previously described (Attard et al., 2010). Propidium iodide was used at 10 mg ml−1. GFP fluorescence was quantified with ImageJ software (Schneider et al., 2012).
Histochemical staining for GUS activity
Plantlets were grown for 2 wk in 0.1X liquid MS medium, inoculated with 105 zoospores, incubated in darkness at 24°C for 6 h and then immediately heat-shocked at 37°C for 2 h to induce the transgene expression. Roots were fixed overnight in 80% acetone (−20°C), rinsed with distilled water and stained for 2 h in 100 mM sodium phosphate, pH 7.0, 0.1% Triton X-100, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 and 2 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc).
PSE1 is specifically expressed during plant penetration
Previous investigations of P. parasitica appressorium-mediated penetration led to the identification of transcripts encoding RXLR effectors accumulating in penetrating appressoria differentiated both on onion epidermis, used as a simplified penetration assay, and on roots of the natural P. parasitica host tomato (Kebdani et al., 2010). We used the recently published P. parasitica–A. thaliana pathosystem (Attard et al., 2010) to study the expression profile of the gene for one of these effectors, PSE1, previously referred to as seq350.
Transcript accumulation was tested by quantitative RT-PCR (Fig. 1) in A. thaliana plantlets inoculated with motile zoospores from appressorium differentiation at the host surface (2.5 h after inoculation (hai)) to established necrotrophy (4 d after inoculation (dai)) (four independent samples). RNA enriched for appressorium-derived sequences prepared from onion epidermis was similarly analysed (Kebdani et al., 2010). PSE1 transcripts were not detected in mycelium (Myc) or motile zoospores before infection (Fig. 1). Transcripts accumulated during the appressorium-mediated penetration of host roots (2.5 hai) to a peak during the penetration process (6 hai); the amount of PSE1 transcript then declined during early biotrophy (10 hai), and became barely detectable during the invasive growth of the oomycete along the stele (30 hai), and later during the necrotrophic phase of the infection (4 dai). Transcript accumulation was observed in samples enriched for penetrating appressoria (PA), 3 h after inoculation of onion epidermis (Fig. 1). Therefore, the PSE1 transcript is transiently accumulated during the early stages of infection.
PSE1 suppresses cell death triggered by P. syringae AvrPto and P. cryptogea cryptogein and is rapidly degraded in planta
We used A. tumefaciens-mediated transformation for transient expression of the PSE1 protein in the plant tissues. In tobacco leaves expressing PSE1 protein alone, there was no cell death in the infiltrated areas, whereas in leaves expressing the mouse pro-apototic factor mBAX there was rapid necrosis (Fig. 2a; Dou et al., 2008). Thus, the PSE1 effector had no toxic effect and did not trigger a hypersensitive response in the two tobacco species.
Agroinfiltrations were also used to assess the ability of the PSE1 protein to suppress plant defence responses. The PSE1 protein was co-produced with a subset of proteins that trigger necrosis on tobacco leaves, including P. cryptogea cryptogein elicitin (Bonnet et al., 1996; Ponchet et al., 1999), the P. syringae effector AvrPto (Yeam et al., 2010) and mBAX. The PSE1 protein efficiently suppressed cryptogein-triggered cell death in N. tabacum leaves (Fig. 2b) and AvrPto-triggered cell death in N. benthamiana (Fig. 2c), but did not inhibit BAX-triggered cell death (Fig. 2d). Co-infiltration with AvrPto led to a yellow chlorosis of the infiltrated area (Fig. 2c). It has been suggested that this is a consequence of suppression of PTI by AvrPto, favouring growth of the bacterium (Hann & Rathjen, 2010; Gilroy et al., 2011). These results show that PSE1 is able to suppress plant cell death triggered by proteins from two different pathogens.
Fusions with the GFP reporter were obtained to determine the in planta localization of the PSE1 protein. The functionality of these PSE1 alleles was determined. Neither PSE1-GFP nor GFP-PSE1 fusions were able to block the plant cell death caused by cryptogein in N. tabacum (Fig. 2b) or by AvrPto in N. benthamiana (Fig. 2c). These chimeric proteins may be misfolded as a result of steric hindrance caused by the GFP, or incorrectly addressed in plant cells. Fusion with fluorescent proteins are routinely used for investigating the subcellular localization of effectors into plant cells. Our example showed that the results obtained using this strategy should be interpreted with caution, especially when the functionality of the fusion proteins is not known. We obtained a Myc-tagged PSE1 protein (Myc-PSE1), differing from PSE1 by only 10 extra amino acids. The Myc-PSE1 protein suppressed both cryptogein-triggered cell death (Fig. 2b) and AvrPto-triggered cell death (Fig. 2c) as efficiently as the native PSE1 protein, suggesting that the Myc-tag had little or no effect on PSE1 functionality. We were unable to detect the Myc-PSE1 protein in N. benthamiana leaves by immunolocalization. Abundance of the fusion protein in the infiltrated areas was assessed by immunoblotting on extracts from leaves infiltrated with A. tumefaciens strains carrying a GFP-PSE1 construct or both Myc-PSE1 and endoplasmic reticulum (ER)-targeted GFP constructs (Fig. 2e). Co-expression of the ER-targeted GFP with the Myc-PSE1 protein was used as a control for transformation efficiency. Nonfunctional GFP-PSE1 fusion was detected using both anti-GFP and anti-PSE1 antibodies (Fig. 2e). In contrast, the Myc-PSE1 protein was not detected using anti-PSE1 antibodies while the ER-targeted GFP was detected by anti-GFP antibodies in the same extracts. Quantitative RT-PCR analyses on the same samples confirmed that the transcripts encoding the Myc-PSE1 fusion protein were properly accumulated. Moreover, these transcripts were eight times more abundant than the transcripts encoding the ER-GFP protein, suggesting a high expression level (Fig. 2f). These results showed that the Myc-PSE1 protein is not accumulated in plant cells. As nonfunctional GFP fusions strongly accumulated in crude protein extracts, these results suggested that functional Myc-PSE1 may be rapidly degraded in plant cells. Thus, the in planta localization of the PSE1 protein appears challenging.
PSE1 perturbs A. thaliana development
Three independent A. thaliana transgenic lines (referred to hereafter as 1.8, 3.2 and 4.2) expressing the coding sequence of the PSE1 gene under control of the cauliflower mosaic virus (CaMV) 35S promoter were analysed. They strongly accumulated PSE1 transcripts (Fig. 3a). When grown in soil, the three lines showed growth rate, rosette morphology, inflorescence length, flowering time and fertility similar to those of the wild-type Col-0 ecotype. Nevertheless, a detailed phenotypic analysis of these lines revealed several subtle developmental abnormalities which co-segregated with the PSE1 transgene. Dark-grown PSE1 transgenic lines showed a significantly higher number of lateral roots than the control (Fig. 3b). Eight-day-old PSE1 transgenic plantlets developed more than two lateral roots per seedling (means of 2.64, 2.70 and 3.29 for lines 1.8, 3.2 and 4.2, respectively), whereas wild-type plantlets developed less than one (mean of 0.60).
Root growth was slower for PSE1 transgenic lines (mean root length after 8 d growth in light: 13.9, 14.2 and 14.8 mm for lines 1.8, 3.2 and 4.2, respectively) than for the wild-type (mean of 21.2 mm; Fig. 3c).
The PSE1 transgenic lines showed severely impaired root hair development. Root hairs were absent from the hypocotyle/root junction (Fig. 3d, left) and very scarce on the roots. However, in some rare cases, small areas showed aberrant root hair development with swollen and depolarized root hairs (Fig. 3d, right). PSE1 transgenic root hairs were 10-fold shorter than those of the wild-type (Fig. 3e).
Dark-grown PSE1 transgenic plantlets displayed bigger cotyledons than the wild-type, with premature formation of primary leaves (Fig. 3f, left). They also showed disorganized root growth (Fig. 3f, right). Grown on vertically oriented hard (2%) agar plates, they showed right-handed (clockwise) coiled roots (Fig. 3g, left); such coiled roots have previously been described for wild-type A. thaliana grown horizontally on hard surfaces, but not on vertically oriented plates (Migliaccio et al., 2009). Indeed, no coil was observed in wild-type plantlets after 10 d of growth on vertically oriented, hard agar plates. By contrast, in the same growth conditions the lines expressing the PSE1 transgene showed at least two coils per plantlet (n =20 for each line), and most of the coils were right-handed (80% for line 1.8 (n =20), 79% for line 3.2 (n =19) and 77% for line 4.2 (n =26)). This phenotype was not observed on soft (1%) agar plates, suggesting that it may be an exaggerated response to thigmotropism. This coiled-root phenotype was not associated with abnormal root morphology (Supporting Information Fig. S1). Modification of the statoliths can lead to abnormal perception of gravity, so we assessed the integrity of these organelles. Starch granules at the root apex of the transgenic and control plants were stained with Lugol. Starch granules were observed in both the transgenic and wild-type plants. There was no evidence that the coiled-root phenotype could be explained by modified statolith content (Fig. 3g, right; Fig. S2).
PSE1 interferes with auxin accumulation in A. thaliana
Most of the developmental phenotypes observed for PSE1 transgenic lines are consistent with those associated with perturbations of auxin physiology (Leyser et al., 1996; Sabatini et al., 1999; Tian & Reed, 1999). We used a root inhibition assay to study the response of PSE1 transgenic lines to exogenous auxin. The response of PSE1 transgenic lines to IAA and 2,4-D was similar to that of wild-type plantlets (Fig. S3). This suggests that neither auxin import into plant cells nor auxin signalling pathways are altered by the expression of the PSE1 transgene.
Addition of exogenous auxin (IAA, IBA, NAA and 2,4-D) did not restore normal root hair shape or number, cotyledon size or lateral root number. However, following exogenous application of 50 nM 2,4-D to PSE1 transgenic lines, no coil was observed after 10 d of growth on hard agar MS medium (Figs 4a, S4). Neither IAA nor IBA significantly reverted the coiled-root phenotype, even at high concentrations (100 nM; Fig. 4a). By contrast, NAA, an auxin that diffuses across plant cell membranes with high efficiency, significantly reduced the number of coiled roots in PSE1 transgenic lines. Nevertheless, NAA did not fully revert the phenotype. The synthetic auxin 2,4-D is not exported from plant cells (Delbarre et al., 1996), so these observations suggested that the observed phenotype may be the consequence of an altered auxin efflux from plant cells. Therefore, wild-type and PSE1 transgenic plants were treated with the auxin efflux inhibitors TIBA and NPA. Treatment with both TIBA and NPA fully impaired coil formation by PSE1 transgenic lines (Fig. 4b). Consequently, increasing auxin content by inhibiting auxin efflux can revert the coiled-root phenotype.
A transgenic auxin sensor pDR5::GFP line (Col-0 background) was crossed to the PSE1 transgenic line 4.2 to study perturbations of auxin physiology. The GFP fluorescence signal at the root apex of the progeny of this cross (n =20) was compared with that for progeny of the pDR5::GFP line crossed with wild-type plants (n =20). GFP fluorescence, and thus expression of the pDR5::GFP transgene, was detected in the same cell types in both cell lines: the quiescent centre and columella cells (Fig. 5a). Nevertheless, GFP signal intensity was more than four times weaker in the p35S::PSE1 than the wild-type background (Figs 5b, S5). As our root growth inhibition assays showed that the signalling pathways downstream of auxin were functional in the PSE1 transgenic plants, one could interpret this result as a reduction of the auxin content at the root apex. Next, we studied the accumulation of PIN auxin efflux carriers at the root apex. PSE1 transgenic line 4.2 and wild-type Col-0 plants were crossed to GFP-tagged PIN1, PIN2, PIN4 and PIN7 reporter lines in a Col-0 background. No significant difference in PIN1 and PIN2 accumulation was observed (Fig. S6). Conversely, both PIN4 and PIN7 showed abnormal accumulation in PSE1 transgenic lines. Both the PIN4 (Figs 5c, S7) and PIN7 (Figs 5c, S7) proteins were detected in more cell layers in PSE1 transgenic than wild-type plants. Moreover, when considering the PIN7 reporter, the GFP signal ratio between the columella and the stele was 2.8 times higher in the PSE1 background than in the wild-type background (Figs 5c,d, S7), consistent with abnormally high accumulation of the PIN7 protein in the columella cells in PSE1 transgenic plants. The analysis of the PIN4 and PIN7 reporter lines also revealed significant perturbations of columella organization at the root apex (Figs 5b,c, S7).
PSE1 increases auxin-dependent A. thaliana susceptibility to P. parasitica
The effect of the constitutive expression of PSE1 on the interaction with P. parasitica was assessed by monitoring disease development on PSE1 transgenic plantlets inoculated with zoospores. PSE1-accumulating lines were more susceptible to P. parasitica than wild-type (P-values of 3.6 × 10−27, 3.5 × 10−45 and 1.5 × 10−25 for lines 1.8, 3.2 and 4.2, respectively; Fig. 6a). The first wilting symptoms appeared 1 d earlier on PSE1 transgenic lines than on wild-type plants; 90% of the transgenic plantlets were totally invaded (ranks 6 and 7 of the disease index) after 10 d, whereas this stage was not reached after 16 d for wild-type plantlets (Fig. 6a,b). Therefore, PSE1 expression increases susceptibility of A. thaliana to P. parasitica.
We tested whether the perturbation of auxin content in PSE1 transgenic roots was responsible for the increased susceptibility to P. parasitica infection. Wild-type and PSE1 transgenic lines were treated with 50 nM 2,4-D, 100 nM IAA or 100 nM NAA and inoculated with motile zoospores. Disease symptoms on the wild-type plantlets treated with 2,4-D and NAA were slightly more severe when compared with untreated wild-type plantlets (P-values of 0.027 and 0.012, respectively; Figs 6c, S8). This is consistent with a slight toxicity of auxins, even at a low concentration, on A. thaliana. By contrast, 2,4-D treatment significantly decreased the susceptibility of PSE1 transgenic lines to P. parasitica (P-value of 9.1 × 10−5; Fig. 6c). No reduction of disease susceptibility was observed following IAA and NAA treatments (Fig. S8). As PSE1-induced increased A. thaliana susceptibility was complemented by treatment with an auxin, the perturbation of auxin physiology in PSE1 transgenic plant roots may account, at least in part, for their increased susceptibility to P. parasitica infection. Moreover, only the poorly exported auxin 2,4-D could partially complement the increased susceptibility of PSE1 transgenic line. An enhanced auxin efflux might be involved in this phenotype, as also suggested by modification of PIN efflux carrier accumulation at the root apex.
Searching for perturbations of host auxin physiology by P. parasitica during the penetration process, we inoculated wild-type A. thaliana plantlets expressing the pDR5::GFP reporter and monitored GFP fluorescence 6 h after inoculation, when one to three host cells are invaded (Fig. 6d). We found that massively inoculated roots displayed a significantly reduced GFP signal when compared with uninfected roots from the same plants (Figs 6d,e, S9).
The possibility that the reduction of GFP fluorescence could be caused by the destruction of plant cells in massively invaded roots could not be ruled out. To investigate this possibility, we inoculated A. thaliana plantlets carrying a second auxin sensor reporter, HS::AXR3NT-GUS. This construct encodes a fusion between the coding sequences of the amino terminus (NT) of the auxin-response repressor AXR3 and the GUS-encoding uidA gene driven by a heat-shock-inducible promoter (pHSP70). The AXR3NT-GUS fusion protein is rapidly destabilized by auxin and thus GUS staining is only obtained in plant cells having a reduced auxin response (Gray et al., 2001). Consistent with the result obtained with the pDR5::GFP reporter line, we observed significantly increased GUS staining at the apex of massively infected roots (Fig. 6e, middle) when compared with uninfected roots (Fig. 6f). Moreover, GUS signal was locally detected in single cells at the penetration site of P. parasitica while adjacent uninfected cells remain unstained (Fig. 6g). Taken together, these results suggest that P. parasitica locally modulates auxin physiology during the penetration process. However, in our experimental conditions, we did not observe any modulation of the PIN4 and PIN7 efflux carriers following infection by P. parasitica zoospores (Fig. S10).
We previously identified P. parasitica effectors which accumulate during appressorium-mediated penetration of tomato roots (Kebdani et al., 2010). We analysed the expression pattern of the PSE1 gene, encoding a member of the RXLR family of effectors, during A. thaliana infection by P. parasitica. PSE1 transcripts were not detected in P. parasitica mycelium or motile zoospores before infection. The transcripts accumulated during appressorium-mediated penetration of host roots, and their abundance dropped rapidly following penetration. Most RXLR effectors studied previously accumulate during the biotrophic phase of the interaction and some are secreted by haustoria (Whisson et al., 2007; Bozkurt et al., 2011). Nevertheless, Judelson et al. (2008) described transcripts encoding RXLR effectors that accumulate in P. infestans appressorium-like structures differentiated on artificial surfaces, and Wang et al. (2011) showed that a series of transcripts encoding RXLR effectors accumulated during the first hours of soybean (Glycine max) infection by P. sojae. However, these studies did not determine whether the corresponding transcripts were down-regulated during the later stages of the interaction. Ours is the first report of an oomycete RXLR effector that is transiently accumulated during the penetration process.
PSE1 transgenic lines showed growth, morphology, flowering time and fertility similar to those of the wild-type. This shows that the constitutive accumulation of PSE1 protein had no substantial toxic effect on plant cells. The transgenic lines were more susceptible to P. parasitica infection than wild-type plants, suggesting that the PSE1 protein favours disease development. Similarly, Caillaud et al. (2012) showed that expression of the effectors ATR13, HaRxL17 and HaRxL77 from Hyloperonospora arabidopsidis increased A. thaliana susceptibility to H. arabidopsidis. Transgenic A. thaliana plants accumulating the P. infestans ipiO protein became susceptible to infection by Phytophtora brassicae (Bouwmeester et al., 2011). All these effectors have been reported to be expressed once the interaction is underway and not during the penetration process (van West et al., 1998; Caillaud et al., 2012). Our results show that an RXLR effector from the ‘early expressed’ expression pattern group can also increase susceptibility to infection when expressed in A. thaliana.
The PSE1 protein abolished cell death in tobacco plants triggered by the P. cryptogea elicitin cryptogein and the P. syringae AvrPto avirulence protein. This property may contribute to the enhancement of plant susceptibility. The P. infestans RXLR effector AVR3a interferes with the response to the P. infestans elicitin INF1 and to the AvrPto protein (Gilroy et al., 2011). This RXLR effector is secreted by haustoria once the biotrophic process is underway (Whisson et al., 2007). Thus, our results suggest that effectors transiently accumulated during the penetration process may target similar defence mechanisms as effectors accumulated later during the interaction. This raises the issue of the function of this particular expression pattern for an effector that targets functions similar to those targeted by other effectors expressed later during the interaction. Wang et al. (2011) suggested that effectors with different expression patterns may target different plant responses: effectors expressed before infection may impair plant responses activated by effectors expressed after penetration that in turn target responses activated by PAMP, such as INF1. Our observations are not consistent with this model, because PSE1 impairs both ETI (AvrPto) and PTI (cryptogein). Wang et al. (2011) analysed effector expression during the first 12 h after inoculation; additional information about effector expression during the necrotrophic phase of the P. sojae/soya interaction would be necessary to determine the transient nature of the effectors described as immediate early and early. The role of additional transiently accumulated effectors in plant infection will have to be addressed to determine whether they have specific functions, or are part of a relay between penetration-specific and biotrophy-specific effectors. The PSE1 protein had no effect on pro-apoptotic mouse BAX-induced cell death. This is consistent with data obtained by Wang and co-workers demonstrating that only 53% of the analysed effectors effective against BAX-induced cell death could also impair PTI. That some effectors could impair all three pathways (PTI-, ETI- and BAX-induced cell death) while others (such as PSE1) could impair only a subset of these plant cell death responses suggests that RXLR effectors target various steps of the signalling pathways involved in cell death activation. Gilroy et al. (2011) suggested that AVR3a could impair signalling pathways activated in response to the recognition of pathogen molecules at the plasma membrane. Since both elicitins (cryptogein) and AvrPto are recognized at the plasma membrane while BAX is effective on intracellular components, the results obtained for PSE1 suggest that this effector could also target signalling pathways triggered by recognition events occurring at the plasma membrane.
Arabidopsis thaliana lines transgenic for PSE1 showed supernumerary lateral roots, reduced root length, altered root hair formation, disorganized root growth and enlarged cotyledons when etiolated. The phenotypes were not caused by a perturbation of the signal transduction downstream of auxin, as shown by our root growth inhibition assays. Similar phenotypes have been described for mutants affected in auxin physiology, such as agr1 (agravitropic), axr3 (auxin resistant), and shy2 (short hypocotyl) (Leyser et al., 1996; Tian & Reed, 1999; Chen et al., 2007). Mutants of the SHY2 gene, encoding a member of the AUX/IAA family of transcription factors, showed perturbed root gravitropism, reduced root growth, enhanced lateral root number and enlarged cotyledons when grown in the dark (Tian & Reed, 1999). Unlike PSE1 transgenic plants, shy2 mutants have longer root hairs than the wild-type (Tian & Reed, 1999). Nevertheless, Knox et al. (2003) showed that weak expression of a mutant axr3 allele in a shy2 mutant background altered root hair formation, leading to small apolar root hair development similar to that observed for our PSE1 transgenic plants. Thus, shy2 mutants appear to be the most similar to the PSE1 transgenic plants. Most of the mutations described for the AXR3 and SHY2 genes lead to forms of the proteins that are resistant to degradation and thus mimic reduced auxin content. Their similar phenotypes are in agreement with PSE1 perturbing auxin accumulation.
The coiled-root phenotype of PSE1 transgenic lines was fully reverted after elevation of the cellular auxin content by treatment with either the exogenous auxin 2,4-D or the auxin efflux inhibitors TIBA and NPA. These results suggested a modulation of the auxin content in PSE1 transgenic roots. In agreement with this hypothesis, the pDR5::GFP auxin reporter system showed a reduced activity at the root apex. As our root growth inhibition assays confirmed that auxin signalling pathways are still functional, this result was interpreted as PSE1 altering auxin accumulation at the root apex. To link this defect to the enhanced plant susceptibility, we assessed the effect of auxin supplementation on the interaction between PSE1 transgenic plants and P. parasitica. Exogenous treatment with low amounts of 2,4-D slightly increased wild-type susceptibility to P. parasitica infection and, conversely, significantly reduced the susceptibility of the transgenic plants. This shows that the PSE1-mediated enhanced susceptibility is caused, at least in part, by the perturbation of auxin accumulation in plant roots. As we were aware that overexpression of PSE1 in A. thaliana was an artificial system, we looked for modulations of the auxin content in wild-type A. thaliana roots during early infection by P. parasitica. Using two independent reporter systems, we were able to demonstrate that P. parasitica modulates auxin physiology during the first hours of infection. In particular, we showed a down-regulation of auxin signalling in the first infected cell as soon as 6 h after inoculation. These findings demonstrate that a modulation of auxin signalling does occur during the first hours of plant infection by P. parasitica. Even if PSE1 is not the only effector expressed during the penetration process of P. parasitica (Kebdani et al., 2010), these results confirm those obtained through overexpression of PSE1 in A. thaliana. Taken together, our results demonstrate that P. parasitica modulates auxin physiology at the penetration site. The PSE1 effector is at least in part responsible for this modulation and facilitates P. parasitica infection of the host.
Auxin antagonizes SA signalling and represses Pathogenesis Related (PR) gene expression, thereby contributing to increased susceptibility to some pathogens (Kazan & Manners, 2009). Mutations of the PIN auxin efflux carriers were shown to affect interaction with cyst nematodes (Grunewald et al., 2009). Conversely, auxin signalling is required for resistance to the fungus Botrytis cinerea and to oomycetes including Pythium irregulare and Hyaloperonospora arabidopsidis (Tiryaki & Staswick, 2002; Tör et al., 2002; Wang et al., 2007). These reports are in agreement with auxin playing an important role in the regulation of plant responses to pathogens. To date, PSE1 is the only effector from a filamentous pathogen that has been shown to modulate the physiology of a hormone. The effector AvrRpt2 from the bacterium P. syringae increases plant auxin content and enhances A. thaliana susceptibility (Chen et al., 2007). Similarly, the effector Hs19C07 from the cyst nematode H. schachtii interacts with the auxin influx protein lax3; Hs19C07 may trigger lax3-mediated auxin influx to increase plant susceptibility (Lee et al., 2011). When delivered in planta by P. parasitica appressoria, PSE1 may locally target auxin efflux components to modulate auxin content at the penetration point and increase plant susceptibility.
Our results also provide the first clues as to how PSE1 could modulate auxin accumulation. The analysis of the PIN efflux carriers at the root apex showed that PIN4-GFP and PIN7-GFP accumulated at the columella of PSE1 transgenic plants. Also, the coiled-root phenotype was complemented by the auxin efflux inhibitors TIBA and NPA. Thus, the low auxin content of the root apex of PSE1 transgenic plants may be the consequence of enhanced auxin efflux resulting from the overexpression of PIN4 and PIN7 carriers at the columella. Accordingly, only 2,4-D, an auxin that is poorly exported from plant cells through PIN efflux carriers, complemented the increased susceptibility of the PSE1 transgenic line. We showed that only the nonfunctional forms (GFP fused versions) of PSE1 could be detected in plant cells, while the active versions of the protein (native or Myc-tagged versions) were not detected. This result suggests that functional forms of PSE1 are rapidly degraded in plant cells. The P. syringae effector HopM1 was shown to perturb auxin physiology by targeting AtMIN7 (a guanine nucleotide-exchange factor involved in trafficking of the PIN1 auxin efflux carrier) to the proteasome (Nomura et al., 2006; Kleine-Vehn et al., 2008). Similarly, P. infestans AVR3a stabilizes the ubiquitin ligase CMPG1 and inhibits plant immunity by modulating proteasome activity (Bos et al., 2010), and P. infestans AVRblb2 prevents secretion of the C14 papain-like cysteine proteases to modulate plant immunity (Bozkurt et al., 2011). PSE1 could modulate auxin content at the penetration points of P. parasitica by altering similar functions. PSE1 may reduce, for example, the turnover of auxin efflux carriers or their recycling from the plasma membrane. According to the hypothesis formulated earlier relating to PSE1 targeting recognition events occurring at the plasma membrane, one could hypothesize that PSE1 may modulate general mechanisms involved in the control of vesicular trafficking. In this case, modulation of auxin physiology would be a side effect. Nevertheless, the complementation of increased disease susceptibility by 2,4-D treatment is in agreement with the auxin signalling pathway being the direct target. Unfortunately, we were unable to detect any modulation of the PIN4-GFP and PIN7-GFP reporters at the penetration point of P. parasitica. This is not surprising as these reporters are expressed at very low levels, mainly in the columella. Faint modulation occurring at the penetration point of the epidermis could probably not be detected using these reporters. The identification of PSE1 targets in the plant is thus required to understand the molecular mechanisms involved in PSE1-mediated auxin-dependent enhancement of plant susceptibility to P. parasitica.
Our study provides an additional example of a pathogen effector targeting auxin signalling pathways. This work with PSE1 supports the suggestion by Mukhtar et al. (2011) that effectors from diverse pathogens converge onto a limited set of plant targets. Indeed, using a large yeast two-hybrid screen, 18 targets were identified that are common to effectors from the bacterium P. syringae and the oomycete H. arabidopsidis (Mukhtar et al., 2011). A significant enrichment in GO annotations related to hormones was observed for the effector targets identified in this project. The results obtained for PSE1, an oomycete effector, together those for the bacterium P. syringae and the nematode H. schachtii, are all consistent with auxin accumulation and signalling being one of these common targets.
We thank Dr Harald Keller (INRA, Sophia Antipolis, France), Dr Mathilde Clément (CEA, Cadarache, France) and Dr Marie-Noëlle Rosso (INRA, Marseille, France) for helpful discussions. We thank Dr Janice De Almeida-Engler (INRA, Sophia Antipolis, France) and Dr Gilbert Engler (INRA, Sophia Antipolis, France) for their help with microscopic analysis. We thank Dr Yvon Jaillais (ENS, Lyon, France), Dr Brett Tyler (Virginia Bioinformatics Institute, Blacksburg, USA), Dr Paul Birch (University of Dundee, Dundee, UK), Dr Ottoline Leyser (Cambridge, UK) and Dr Joanna Jelenska (University of Chicago, Chicago, IL, USA) for providing constructs and biological material. We thank Catherine Mura (INRA, Sophia Antipolis, France) for technical assistance.