Ethylene signalling and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis between the endophytic fungus Piriformospora indica and Arabidopsis thaliana

Authors


Author for correspondence:
Ralf Oelmüller
Tel: +49 3641 949231
Email: b7oera@uni-jena.de

Summary

  • The endophytic fungus Piriformospora indica colonizes the roots of the model plant Arabidopsis thaliana and promotes its growth and seed production. The fungus can be cultivated in axenic culture without a host, and therefore this is an excellent system to investigate plant–fungus symbiosis.
  • The growth of etr1, ein2 and ein3/eil1 mutant plants was not promoted or even inhibited by the fungus; the plants produced less seeds and the roots were more colonized compared with the wild-type. This correlates with a mild activation of defence responses. The overexpression of ETHYLENE RESPONSE FACTOR1 constitutively activated defence responses, strongly reduced root colonization and abolished the benefits for the plants.
  • Piriformospora indica-mediated stimulation of growth and seed yield was not affected by jasmonic acid, and jasmonic acid-responsive promoter β-glucuronidase gene constructs did not respond to the fungus in Arabidopsis roots.
  • We propose that ethylene signalling components and ethylene-targeted transcription factors are required to balance beneficial and nonbeneficial traits in the symbiosis. The results show that the restriction of fungal growth by ethylene signalling components is required for the beneficial interaction between the two symbionts.

Introduction

Like other members of the Sebacinaceae, the endophyte Piriformospora indica colonizes the roots of many plant species, thereby promoting their growth, development and seed production. The fungus confers resistance to various biotic and abiotic stresses (cf. Sahay & Varma, 1999; Peškan-Berghöfer et al., 2004; Pham et al., 2004; Shahollari et al., 2005; Sherameti et al., 2005, 2008a,b; Waller et al., 2005; Johnson & Oelmüller, 2009; Oelmüller et al., 2009). Piriformospora indica is a cultivable fungus and can grow on synthetic media without a host (Varma et al., 1999, 2001; Peškan-Berghöfer et al., 2004). As the fungus can colonize the roots of many plant species, including agricultural and horticultural as well as medicinal plants and mosses (Varma et al., 2001; Glen et al., 2002; Peškan-Berghöfer et al., 2004; Weiss et al., 2004; Barazani et al., 2005; Shahollari et al., 2005, 2007; Sherameti et al., 2005; Waller et al., 2005), the interaction between the symbiotic partners should be based on general recognition and signalling processes. We studied this beneficial interaction using the model plant Arabidopsis to identify the plant genes and signalling processes targeted by the fungus.

Ethylene (ET) affects many aspects of plant–microbe interactions, and thus might also be involved in the beneficial interaction between P. indica and Arabidopsis. The hormone controls plant development, fitness, germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responsiveness to stress and pathogen attack (Johnson & Ecker, 1998; Bleecker & Kende, 2000; Broekaert et al., 2006). Many ET-response mutants have been isolated, which can be divided into four groups: constitutive triple-response mutants [i.e. ET overproduction (eto) 1, 2 and 3, constitutive triple response (ctr) 1 and responsive to antagonist (ran) 1/ctr2)]; ET-insensitive mutants [i.e. ET receptor (etr) 1 and 2, ET insensitive (ein) 2–6)]; tissue-specific ET-insensitive mutants [i.e. hookless 1, ET insensitive root (eir) 1]; and several auxin-resistant mutants (Bleecker et al., 1998; Johnson & Ecker, 1998; Bleecker & Kende, 2000; Stepanova & Ecker, 2000). ET is perceived by a family of membrane-associated, two-component systems at the endoplasmic reticulum, including ETR1/ETR2, ET-response sensor (ERS) 1 and 2, and EIN4 in Arabidopsis (Chang et al., 1993; Hua et al., 1995, 1998; Sakai et al., 1998). The hormone binds to its receptors via a copper cofactor, which is probably delivered by the copper transporter RAN1, which results in the inactivation of receptor function (Hua & Meyerowitz, 1998), indicating that, in the absence of ET, the receptors are active. In the absence of ET, the receptors activate a Raf-like serine/threonine (Ser/Thr) kinase, CTR1, which is a negative regulator of the pathway (Kieber et al., 1993; Chang & Stadler, 2001). EIN2, EIN3, EIN5 and EIN6 are positive regulators of ET responses, acting downstream of CTR1. CTR1 derepresses EIN2, leading to the activation of EIN3 and EIN3-like (EIL) transcription factors. EIN2 is an integral membrane protein of unknown function with similarities to Nramp metal transporters (Alonso et al., 1999). EIN5 is an exoribonuclease that targets specific F-box proteins (Olmedo et al., 2006; Potuschak et al., 2006). EIN6 has not yet been characterized, and EIN3 is a transcription factor which, in concert with other EIL transcription factors, activates a cascade that results in the regulation of the expression of target genes, such as ET-response factor (ERF) 1 (Chao et al., 1997; Solano et al., 1998; Tieman et al., 2001), in the nucleus. ERF1 belongs to a large family of APETALA2 domain-containing transcription factors that bind to a GCC-box present in the promoters of many ET-inducible defence genes (Hao et al., 1998; Brown et al., 2003). ERF1, 2 and 5 activate and ERF3, 4, 7, 10, 11 and 12 repress GCC-box-containing genes (Fujimoto et al., 2000; Ohta et al., 2000). Overexpression of ERF1 reduces resistance against several fungal pathogens (Berrocal-Lobo et al., 2002; Berrocal-Lobo & Molina, 2004; McGrath et al., 2005).

The rate-limiting enzymes in ET biosynthesis are the highly regulated 1-aminocyclopropane-1-carboxylate synthases (ACSs; Chae et al., 2003; Wang et al., 2004; Chae & Kieber, 2005). For instance, ETO1 interacts with ACS5 and functions as a negative regulator by the direct inactivation of ACS5 activity via the targeting of the protein for degradation through the 26S proteasome. Eto1 insertion lines contain approximately 10-fold higher ET levels in Arabidopsis (Guzmán & Ecker, 1990; Wang et al., 2004).

ET and jasmonic acid (JA) often function synergistically in the plant defence response. Defence genes, such as PLANT DEFENSIN 1.2 (PDF1.2) and pathogenesis-related protein (PR)-3, encoding the basic chitinase, are activated against necrotrophic fungi primarily by the ET/JA pathway. Both hormones are also required for the induced systemic resistance triggered by beneficial rhizobacteria and fungi (Pieterse et al., 1998; Van Wees et al., 2008). By contrast, biotrophic pathogens are more efficiently countered by salicylic acid (SA)-controlled defence mechanisms (Thomma et al., 1998, 1999), resulting in the activation of PR-1, PR-2 and PR-5.

Here, we demonstrate that the ET signalling components ETR1, EIN2 and EIN3/EIL1 are required for P. indica-induced growth promotion in Arabidopsis. Growth and seed production of etr1, ein2 and ein3/eil1 mutants are inhibited rather than promoted by P. indica, indicating that the ET signalling components might be required to balance beneficial and nonbeneficial traits in the symbiosis. We demonstrate that ETR1, EIN2 and EIN3/EIL1 participate in the restriction of root colonization in adult plants and the repression of defence responses, whereas the overexpression of ERF1 reduces the P. indica-induced benefits for the plants, presumably because the fungal performance is restricted by the activated defence responses.

Materials and Methods

Growth conditions of plant and fungus

Wild-type (WT) and transgenic [ein2-1, ein3-1, ein3-1/eil1-1; eto1-1; 35S::eto1 (#481, Wang et al., 2004), 35S::ERF1 (Berrocal-Lobo et al., 2002), jar1, aoc3/4 (Delker et al., 2006), opr3 (Stintzi & Browse, 2000)] Arabidopsis seeds (Arabidopsis thaliana ecotype Columbia) and seeds with promoter::uidA constructs (oac3, At3-1-6 and At3-2-27; aoc4, At4-4-1 and At4-9-1; cf. Delker et al., 2006) were surface sterilized and placed on Petri dishes containing Murashige and Skoog nutrient medium (Murashige & Skoog, 1962). The mutants used in this study are listed in Table S1 (see Supporting Information). After cold treatment at 4°C for 48 h, the plates were incubated for 7 d at 22°C under continuous illumination (100 μmol m−2 s−1). Piriformospora indica was cultured as described previously (Verma et al., 1998; Peškan-Berghöfer et al., 2004) on Kaefer medium (Hill & Kaefer, 2001). For solid medium, 1% (w/v) agar was included.

Cocultivation experiments and estimation of plant growth

Nine days after plating A. thaliana seeds on Murashige and Skoog medium, the seedlings were transferred to nylon discs (mesh size, 70 μm) and placed on top of a modified plant nutrient culture medium (5 mm KNO3, 2 mm MgSO4, 2 mm Ca(NO3)2, 0.01 μm FeSO4, 70 μm H3BO3, 14 μm MnCl2, 0.5 μm CuSO4, 1 μm ZnSO4, 0,2 μm Na2MoO4, 0.01 μm CoCl2, 10.5 g l−1 agar, pH 5.6) in Petri dishes. One seedling was used per Petri dish and one fungal plug of 5 mm in diameter was placed at a distance of 1 cm from the roots. The plates were incubated at 22°C under continuous illumination from the side (80 μmol m−2 s−1). Fresh weights were determined directly after seedlings were removed from the plates.

Experiments on soil

For the experiments on soil, Arabidopsis seedlings were first cultivated on plates with or without the fungus as described above. Eighteen days after cocultivation (or mock treatment), the seedlings were transferred to heat-sterilized soil. For experiments with the fungus, the soil was mixed carefully with the mycelium (1%, w/v), which was obtained from liquid cultures after the medium had been removed and the mycelium had been washed with an excess of distilled water. Cultivation was performed in small plastic pots with Aracon tubes in a temperature-controlled growth chamber at 22°C under long-day conditions (light intensity, 80 μmol m−2 s−1). The sizes of the plants were monitored continuously. Seed production (gram seeds per plant) was monitored by collecting seeds from individual plants grown under the standardized conditions described above. RNA was isolated from the roots 7 wk after transfer to soil. The roots were carefully washed (× 20) with an excess of sterile water to remove soil and loosely attached fungal hyphae.

Staining fungal hyphae and spores

To monitor root colonization, small parts of the roots from seedlings cocultivated with P. indica were transferred to 10% KOH and boiled for 10 min. After washing with water for 1 min, the roots were placed into a 0.01% acid fuchsin–lactic acid solution and boiled again for 10 min. Excess dye was removed with water before microscopy.

Fluorescence measurements

Autofluorescence in the developing root hairs as a result of coculture with P. indica was detected with an LSM 510 META microscope (Carl-Zeiss Jena GmbH, Jena, Germany). Relative values (595 nm) were obtained from the emission spectra (cf. Peškan-Berghöfer et al., 2004).

Microarrays

Arabidopsis seedlings, grown as described above, were cocultivated (or mock treated) with P. indica for 2, 6 or 10 d. RNA was extracted from 70 mg of root material with an RNeasy Plant Mini Kit, followed by an On-Column DNAse treatment (Qiagen, Hilden, Germany). Microarray hybridization was performed with the Arabidopsis Genome Array ATH1 from Affymetrix, and the data were analysed with GCOS1.4 software (Affymetrix, Charleroi, Belgium).

The expression levels of the following ET-related genes have been analysed in the microarray data: ETO1 (At3g51770); ETO2/ACS5 (At5g65800); ETO3/ACS9 (At3g49700); CTR1 (At5g03730); CTR2/RAN1 (At5g44790); ETR1 (At1g66340); ETR2 (At3g23150); ERS1 (At2g40940); ERS2 (At1g04310); EIN4 (At3g04580); EIN5 (At1g54490); EIN3 (At3g20770); EIL1 (At2g27050); EIL2 (At5g21120); EIL3 (At1g73730); EIL4 (At5g10120); EIL5 (At5g65100); HLS1 (At4g37580); EIR1 (At5g57090); ERF1 (At3g23240); ERF2 (At5g47220); ERF3 (At1g50640); ERF4 (At3g15210); ERF5 (At5g47230); ERF6 (At4g17490); ERF7 (At3g20310); ERF8 (At1g53170); ERF9 (At5g44210); ERF10 (At1g03800); ERF11 (At1g28370); ERF12 (At1g28360); ERF13 (At2g44840); ERF14 (At1g04370); ERF15 (At2g31230); EBF1 (At2g25490); EBF2 (At5g25350); ACS1 (At3g61510); ACS2 (At1g01480); ACS3 (At5g28360); ACS4 (At2g22810); ACS6 (At4g11280); ACS7 (At4g26200); ACS8 (At4g37770).

Real-time quantitative PCR and reverse transcriptase-PCR (RT-PCR)

Real-time quantitative PCR was performed using the iCycler iQ real-time PCR detection system and iCycler software version 2.2 (Bio-Rad). Total RNA was isolated from 4–10 independent replicates of Arabidopsis roots. For the amplification of the PCR products, iQ SYBR Supermix (Bio-Rad) was used according to the manufacturer’s instructions in a final volume of 20 μl. The iCycler was programmed to 95°C for 2 min, × 35 (95°C for 30 s, 55°C for 40 s, 72°C for 45 s), 72°C for 10 min, followed by a melting curve programme (55–95°C in increasing steps of 0.5°C). All reactions were repeated at least twice. The mRNA levels for each cDNA probe were normalized with respect to the actin message level. Fold induction values were calculated with the ΔΔCP equation of Pfaffl (2001), and related to the mRNA levels in the target genes in WT roots, which were defined as 1.0. The primer pairs used for the analyses are given in Table S2 (see Supporting Information).

RT-PCR was performed after reverse transcription of total RNA with gene-specific primers. After PCR, the products were analysed on 1.5% agarose gels and stained with ethidium bromide, and the visualized bands were quantified with the Image Master Video System (Amersham, GE Life Science).

Statistics

Samples were evaluated with a two-sample t-test (± P. indica) and ANOVAs (comparison of all datasets).

Results

Response of the seedlings of ET mutants to P. indica

Growth regulation  As reported previously (Peškan-Berghöfer et al., 2004; Sherameti et al., 2005, 2008a,b; Shahollari et al., 2007; Vadassery et al., 2009), we have established cultivation conditions in which the growth of Arabidopsis seedlings is stimulated when cocultivated with P. indica. Growth of ein2 seedlings was not promoted and that of etr1 and ein3/eil1 seedlings was inhibited by the fungus (Fig. 1a,c). After transfer to soil for 7 wk, growth of the etr1 and ein3/eil1 plants was still inhibited in the presence of the fungus (reduction of fresh weight compared with the uncolonized control: etr1, 22 ± 4.6%; ein3/eil1, 36.2 ±5.8%), whereas that of the ein2 mutant was comparable with the uncolonized control (Fig. 1b,c). Also, flowering of colonized etr1 and ein3/ein1 was retarded by ca. 2–3 wk and started only after the uncolonized controls had set seeds. The flowering time of ein2 plants was not affected by the fungus, but the flowering of P. indica-colonized WT plants occurred 2–3 wk earlier than in uncolonized controls (cf. Shahollari et al., 2007). In addition, the number of seeds per colonized etr1 and ein3/eil1 mutant was reduced by ∼30%, which is probably a secondary effect because of the retarded development. Again, no significant difference was observed for colonized and uncolonized ein2 plants, but colonized WT plants produced > 30% more seeds (Shahollari et al., 2007). Thus, ET perception and signalling, as well as ET-targeted transcription factors, are crucial for P. indica-induced growth promotion in Arabidopsis seedlings.

Figure 1.

 Arabidopsis seedlings (a) and adult plants (b) grown in the absence (left) or presence (right) of Piriformospora indica. (a) Wild-type (WT), etr1, ein2 and ein3/eil1 seedlings cocultivated with the fungus for 8 d (right seedling) or mock treated (left seedling). Each panel shows pairs of seedlings which were identical in size at the beginning of the cocultivation experiments (time point, 0 d). (b) WT, etr1, ein2 and ein3/eil1 plants after 7 wk on soil. Before transfer to soil, the seedlings were either cocultivated with P. indica for 18 d (right plant) on Petri dishes or mock treated (left plant). (c) Changes in the fresh weights of the seedlings (filled bars) and adult plants (open bars) cocultivated with P. indica relative to the uncolonized controls. Data are based on eight independent experiments with 20 seedlings per treatment (bars, + SE). (d–f) Colonization of the roots of WT and mutant seedlings and adult plants by P. indica shown by RT-PCR. (d) Colonization of the root by P. indica is not altered in etr1, ein2 and ein3/eil1 seedlings relative to the WT control. PCR analysis for the translation elongation factor 1α cDNA (Pitef1) from P. indica and the Arabidopsis actin cDNA (actin), either mock treated (−P. indica) or inoculated with P. indica (+P. indica) for 0, 6 or 12 d. (e) Quantified data for the colonization of the roots from seedlings cocultivated with P. indica for 12 d. The Pitef1 cDNA to actin cDNA ratio (generated from RNA) for WT seedlings was set as 100% and all other values were expressed relative to it. After RT-PCR, the intensities of the bands on an agarose gel were quantified as described in Materials and Methods. Based on three independent experiments (bars, + SE). (f) As (e), except that cDNA was obtained from the roots of 7-wk-old plants grown on soil.

To test whether ET production in the plant itself is important, we analysed the response of eto1 insertion and overexpressor (35S::eto1) lines to P. indica. Although 35S::eto1 seedlings showed slower growth (Wang et al., 2004), seedlings of both lines responded to P. indica, similar to WT (Fig. 2). This suggests that severe alterations in ET levels (cf. Wang et al., 2004) do not affect P. indica-induced growth promotion in Arabidopsis seedlings per se, and that ET perception and signalling are more important for the response to the fungus. Because of the quite different growth rates of eto1, 35S::eto1 and WT adult plants on soil, we could not obtain meaningful results for P. indica-mediated effects under these conditions.

Figure 2.

 Fresh weight of wild-type (WT), 35S::eto1 and eto1 Arabidopsis seedlings cocultivated with Piriformospora indica for 10 d (grey bars) or mock treated (white bars). Based on four independent experiments with 60 seedlings per treatment (bars, ± SE). The weight of wild-type seedlings without P. indica was 29.8 mg.

ET biosynthesis or signalling genes do not respond to the fungus in WT seedlings, but expression of a subset of P. indica-induced genes requires ETR1, EIN2 and/or EIN3/EIL1  Whole-genome expression profiles with RNA from the roots of Arabidopsis seedlings cocultivated with and without P. indica for 2 or 6 d revealed that only nine of the 226 genes annotated to be regulated by ET in Arabidopsis (genes taken from de Paepe et al., 2004 and Zhong & Burns, 2003; and Arabidopsis Databases) are also regulated more than two-fold by P. indica in Arabidopsis roots (Table 1). In addition, none of the tested 43 genes encoding components involved in ET biosynthesis or signal transduction were regulated in Arabidopsis roots in response to the fungus (data not shown, but see below). This indicates that P. indica-mediated growth promotion of Arabidopsis seedlings does not require the regulation of ET-responsive genes, but depends on ET perception and signalling.

Table 1.   Ethylene (ET)-responsive genes regulated by Piriformospora indica in Arabidopsis roots
ATG#Gene descriptionFold induction
  1. The expression levels of ET-responsive genes, taken from de Paepe et al. (2004) and Zhong & Burns (2003), were analysed in microarrays with RNA from Arabidopsis roots co-cultivated with P. indica for 2 d or mock treated. Nine of the analysed 226 genes showed an expression level significantly above background and were regulated more than two-fold in response to P. indica in two independent microarray experiments. These results were confirmed by real-time PCR, and the fold induction values of three independent real-time PCRs are given.

At2g20870Putative cell wall protein, precursor−2.91 ± 0.33
At2g25510Unknown3.61 ± 0.41
At2g43510Trypsin inhibitor protein 14.72 ± 0.44
At3g27650LOB domain-containing protein 25−2.31 ± 0.33
At3g46230Heat shock 172.21 ± 0.20
At4g19690Fe(II) transporter2.65 ± 0.37
At5g15950S-Adenosylmethionine decarboxylase 2−3.18 ± 0.35
At5g45950GDFL-motif lipase7.70 ± 0.53
At5g64110Peroxidase, ATP3a homologue3.88 ± 0.29

The beneficial interaction between P. indica and Arabidopsis seedlings is accompanied by the transient upregulation of the message level for an atypical receptor protein, LRR1 (e.g. Shahollari et al., 2007), and this response is not detectable in the roots of etr1, ein2 and ein3/eil1 seedlings (Fig. 3a). Similarly, four other genes with unrelated functions, which are upregulated in the roots of WT seedlings 2 d after cocultivation with the fungus, did not respond to the fungus in etr1, ein2 and ein3/eil1 seedlings (shown exemplarily for ein3/eil1; Fig. 3b). Thus, ET signalling is involved in the upregulation of these P. indica marker genes and these responses are downstream of EIN3/EIL1. By contrast, the message levels for the two genes Germin2 and CaM-BP, which are below detectability in uncolonized roots and strongly upregulated in the presence of the fungus, are still upregulated in etr1, ein2 and ein3/eil1 seedlings (shown for ein3/eil1; Fig. 3b). Apparently, these genes are expressed independently of EIN3/EIL1. Taken together, there are differential requirements for ET signalling for subsets of P. indica-regulated genes.

Figure 3.

 Gene expression analyses in the roots of wild-type (WT), etr1, ein2 and ein3/eil1 Arabidopsis seedlings, either cocultivated with Piriformospora indica (+) or mock treated (−). (a) Relative mRNA levels for LRR1 in roots of WT, etr1, ein2 and ein3/eil1 seedlings. The mRNA level for WT at t = 0 was set as 1.0 and all other values are expressed relative to it. Nine-day-old seedlings were cocultivated with P. indica for 12 d. The mRNA levels for uncolonized (−P. indica) and colonized (+P. indica) seedlings are given. The mRNA levels for colonized (dots) and uncolonized (*) etr1 (red) and ein2 (blue) seedlings are only given for day 3. After RT-PCR, the intensities of the bands on an agarose gel were quantified as described in Materials and Methods. Based on four independent experiments (bars, ± SE). (b) Regulation of P. indica-induced marker genes in the roots of WT and ein3/eil1 seedlings. RT-PCR with RNA from WT and ein3/eil1 roots mock treated (−) or cocultivated with P. indica (+) for 2 d. Genes code for 2-nitropropane dioxygenase (At5g64250); homeodomain transcription factor (At2g35940); nitrate reductase 2 (At1g37130); uclacyanin protein (STELLA, At3g60270); germin type 2 (At5g38910); calmodulin-binding protein (At5g26920); actin 3 (At3g53750). Representative of four independent experiments. (c) Regulation of defence genes in the roots of WT, etr1, ein2 and ein3/eil1 seedlings. RT-PCR with RNA from WT and mutant roots mock treated with P. indica (−) or cocultivated with the fungus (+) for 2 d. Representative of four independent experiments. Quantitative data are given in Table S3 (see Supporting Information).

Genes involved in plant defence are moderately upregulated in the roots of P. indica-colonized etr1, ein2 and ein3/eil1 seedlings  The PDF1.2 and PR-1PR-5 mRNA levels were not upregulated in WT roots in the presence of the fungus (Fig. 3c; quantified data in Table S3, see Supporting Information). However, in etr1, ein2 and ein3/eil1 roots, a slight upregulation was observed for the PDF1.2, PR-1, PR-2 and PR-5, but not PR-3 and PR-4, mRNA levels (Fig. 3c, Table S3). Although the responses were low compared with infections with pathogenic fungi (cf. Berrocal-Lobo et al., 2002; Berrocal-Lobo & Molina, 2004), these observations indicate that ET perception and signalling repress even moderate defence responses induced by P. indica in WT seedlings. Thus, the lack of growth promotion in the ET mutant seedlings is accompanied by a mild defence activation.

Piriformospora indica inhibits the performance of adult etr1, ein2 and ein3/eil1 plants because of uncontrolled hyphal growth in the roots  Root colonization of Arabidopsis seedlings can be monitored by fluorescence emission at 595 nm, which originates from an as yet unidentified dye synthesized by the fungus (Peškan-Berghöfer et al., 2004). The increase in fluorescence during the cocultivation experiment indicates that root colonization and propagation of the fungus proceed during the experimental period. The increase in the fluorescence in etr1, ein2 and ein3/eil1 roots, calculated on the basis of fresh weight, was comparable for mutant and WT seedlings (data not shown). Furthermore, PCR-based fungal biomass estimation [PCR analysis for the translation elongation factor 1α gene (Pitef1α) from P. indica (Bütehorn et al., 2000) relative to the Arabidopsis actin gene as a reference] in roots demonstrates no difference from WT (Fig. 1d,e). Hence, recognition and infection by the fungus are not affected at the seedling stage, but rather the signal transduction leading to growth promotion is abrogated or even reversed in etr1, ein2 and ein3/eil1 seedlings.

By contrast, 7-wk-old adult mutant plants cocultivated with P. indica contained a significantly higher fluorescence compared with WT plants (data not shown), which was reflected by an increase in the fungal growth progression based on the Pitef1α/actin DNA ratio (Fig. 1f). We observed a two- to three-fold increase in the colonization of etr1 and ein2 plants and a three- to four-fold increase in the colonization of ein3/eil1 plants. This clearly indicates that the ET signalling components restrict root colonization during longer cocultivation periods in WT plants or, alternatively, there is differential ET response of plant developmental stages. Higher root colonization in adult plants might ultimately cause the mild defence response.

Response of seedlings and adult plants overexpressing ERF1 to P. indica

The ET responsive transcription factor ERF1 is involved in ET- and JA-mediated defence responses against pathogen attack (cf. van Loon et al., 2006) by inducing the expression of defence genes, such as PDF1.2 (Berrocal-Lobo et al., 2002; Lorenzo et al., 2003). The ERF1 mRNA level was not regulated by P. indica in the roots of WT, etr1, ein2 and ein3/eil1 seedlings (Table S4, see Supporting Information; and data not shown). ERF1 is a main target of EIN3 and a target transcription factor of ET and JA signalling in Arabidopsis. Because of potential redundancy in the large ERF transcription factor family, we, like others (cf. Lorenzo et al., 2003), used an ERF1 overexpressor line to investigate the role of ERF1 in the beneficial interaction between P. indica and Arabidopsis. 35S::ERF1 seedlings were impaired in their response to P. indica: promotion of shoot growth was reduced and root growth was completely abolished compared with uncolonized ERF1 seedlings (Fig. 4a). Furthermore, although the ERF1, PDF1.2, PR-1, PR-2 and PR-5, but not PR-3, PR-4 and ACS4, mRNA levels were higher in the shoots of the overexpressor line (data not shown, but the results are similar to those reported earlier; Lorenzo et al., 2003), we observed only a marginal upregulation of ERF1 mRNA in the 35S::ERF1 roots of the same seedlings (Fig. 4b, top and bottom; Table S4). Surprisingly, the expression of the defence genes PR-1, PR-2, PR-5 and PDF1.2, but not of PR-3, PR-4 and LOX1, was stimulated by the fungus in 35S::ERF1 roots, but no significant regulation was observed in WT roots (Fig. 4b top; Table S4). We conclude that defence responses become more efficiently activated against P. indica in 35S::ERF1 plants.

Figure 4.

 Response of 35S::ERF1 seedlings and plants to Piriformospora indica. (a) Increase in fresh weight of the shoots and roots of Arabidopsis WT and 35S::ERF1 seedlings cocultivated with P. indica for 10 d. Based on six independent experiments (bars, ± SE). (b) Top panel: mRNA levels for defence genes in the roots of WT and 35S::ERF1 seedlings cocultivated with the fungus (+) or mock treated (−) for 10 d. Bottom panel: ERF1 mRNA levels in the shoots and roots of WT and 35S::ERF1 seedlings grown under the same conditions. Quantified data are given in Table S4 (see Supporting Information). (c) WT and 35S::ERF1 plants on soil, either mock treated (−) or cocultivated with P. indica for 3 wk.

Adult 35S::ERF1 plants grown on soil showed a retarded growth compared with WT (Berrocal-Lobo et al., 2002; Berrocal-Lobo & Molina, 2004), and 6-wk-old plants were not larger in the presence of P. indica (Fig. 4c). In addition, the seed yield was not promoted by the fungus and was comparable with the yield of the uncolonized control. Root colonization was lower in adult 35S::ERF1 plants compared with WT (Fig. 1f). This suggests that the overexpression of ERF1 prevents the beneficial interaction between the two symbiotic partners, and that the loss of P. indica-mediated benefits in 35S::ERF1 may be caused by higher defence gene activation, as even moderately higher ERF1 mRNA levels suffice to stimulate some defence responses in colonized seedlings.

Discussion

The role of ET and ET signalling components in the P. indica– Arabidopsis interaction

As the growth of etr1, ein2 and ein3/eil1 is not promoted or even inhibited by the fungus (Fig. 1), and as colonized etr1 and ein3/eil1 mutants flower later, produce less seed and activate defence responses to some extent (Fig. 3c), the beneficial interaction between P. indica and Arabidopsis requires ET signalling components. The manipulation of ET production appears to be less important, at least at the seedling stage, as eto1 insertion and overexpression lines show a WT response to the fungus (Fig. 2). The strongest phenotype is observed for etr1 and ein3/eil1, for which growth is inhibited by the fungus, whereas ein2 plants grow like uncolonized plants in the presence of the fungus (Fig. 1). Considering that the ein2 mutant exhibits the strongest ET-insensitive phenotype of all the Arabidopsis ET mutants isolated so far (Schaller & Kieber, 2001; The Arabidopsis Book), it appears that fungal signals interfere preferentially with ET perception and/or targeted transcription factors rather than the ET signalling component EIN2. The reason for this is unclear; however, in all independent experiments, both ein2 seedlings and adult plants respond better to the fungus than do etr1 and ein3/eil1 seedlings and plants (Fig. 1a,b). Root colonization is at least one crucial aspect that is controlled by ET signalling components (Fig. 1d–f). In adult plants, the absence of ET signalling components results in uncontrolled hyphal growth, which is probably caused by a long-term unbalanced interaction between the two symbionts. Overexpression of ERF1 also disturbs the beneficial interaction (Fig. 4a,c), and the stimulation of root growth is completely inhibited in 35S::ERF1 seedlings (Fig. 4a). As root colonization is reduced compared with WT in adult plants (Fig. 1f), it appears that the slightly elevated ERF1 mRNA level in 35S::ERF1 roots (or signals from the leaves) restricts fungal growth and makes the roots more sensitive to the fungus to activate defence genes (Fig. 4b). Thus, ET signalling components and, in particular, ET-targeted transcription factors are required to balance the beneficial and nonbeneficial traits in the symbiosis and to maintain a long-term harmony between P. indica and A. thaliana. Interestingly, the growth of etr1 and ein3/eil1 is inhibited rather than promoted by the fungus, suggesting that inactivation of ET signalling causes a (partial) shift from mutualism to parasitism.

The promotion of leaf growth by the root-colonizing endopyhte is also reduced in the ET signalling plants (Figs 1a,b,4a,c), suggesting that the information flow from the roots to the leaves is impaired. In addition to secondary effects caused by a less efficient nutrient supply to the aerial parts in mutants with smaller roots, ET signalling components may also play a direct role in information transfer. ET, together with JA, for instance, is a crucial component for induced system resistance, which also plays a role in the P. indica–Arabidopsis interaction (Stein et al., 2008). Although resistance against pathogens and the stimulation of the beneficial traits studied here are not necessarily regulated through the same mechanisms and/or signalling components, it cannot be excluded that ET components may participate in different aspects of root–shoot communication.

Recently, Barazani et al. (2007) have shown that Sebacina vermifera, a growth-promoting Sebacinaceae fungus closely related to P. indica, promotes the growth and fitness of Manduca sexta-attacked Nicotiana attenuata plants by inhibiting ET biosynthesis. Thus, the response of ET and ET signalling in N. attenuata caused by the Sebacinaceae fungus appears to differ from Arabidopsis. This is not surprising considering that quite diverse and opposite functions of ET, ET signalling components and ET-targeted transcription factors have been described even for the same organism. For instance, ET can support or repress plant defence responses to microbes (cf. El-Kazzaz et al., 1983; Brown & Lee, 1993; van Loon & Pennings, 1993; Berrocal-Lobo et al., 2002; Diaz et al., 2002; van Loon et al., 2006), and the overexpression of ERF2 and ERF4 results in opposite disease-resistance phenotypes towards infection with the necrotrophic pathogen Fusarium oxysporum in Arabidopsis (McGrath et al., 2005).

EIN3, EILs and ERF signalling

EIN3 and EIL1 are important for the beneficial interaction between Arabidopsis and P. indica. In the absence of ET, EIN3 is continuously degraded with the help of two F-box proteins, EBF1 and EBF2, which recruit EIN3 to an E3 complex for polyubiquitination (Guo & Ecker, 2003; Potuschak et al., 2003; Yanagisawa et al., 2003; Gagne et al., 2004). EIN3/EIL1 bind to ET-response elements in the upstream region of other (secondary) transcription factor genes, such as ERFs. We did not observe higher ERF mRNA levels in P. indica-colonized Arabidopsis roots, suggesting that they might be regulated post-transcriptionally. ERFs initiate cascades containing transcriptional activators and repressors (Fujimoto et al., 2000; Ohta et al., 2001). Differential activation of these factors by P. indica might be crucial in determining whether the plant response is beneficial or pathogenic (Fig. 4). We have demonstrated that ERF1 is a crucial transcriptional regulator for the beneficial interaction. Overexpression of ERF1 under the control of the 35S promoter results only in a minor elevation of the ERF1 mRNA level in the roots (Fig. 4b), in contrast with the situation in leaves, in which the ERF1 mRNA level is high (e.g. Lorenzo et al., 2003; Table S4). However, the low elevation of the ERF1 mRNA level in the roots (or signals from the leaves) suffices to trigger quite efficient defence gene activation when the roots are exposed to P. indica (Fig. 4b, Table S4), and prevents the beneficial interaction (Fig. 4a,c). As more than one ERF gene is activated by EIN3/EIL1, it is also possible that the manipulation of just one of the ERFs results in an unbalanced response pattern to P. indica. Therefore, it is also difficult to compare the response of the ein3/eil1 and 35S::ERF1 lines to P. indica.

Regulation of defence-related genes in response to P. indica

Defence genes become mildly activated in P. indica-colonized roots of ET mutants and of the 35S::ERF1 line. In both cases, this leads to a loss of the benefits for the plant (ein2; 35S::ERF1), or even to reduced growth and biomass production relative to uncolonized controls (etr1; ein3/eil1). The reason for defence gene activation in the two types of mutant might be different: ET signalling mutants appear to be less protected and thus activate defence responses against P. indica, or the defence response cannot be repressed. As the defence response is already detectable in young seedlings (Fig. 3c), in which the roots are not yet overcolonized, they must be activated via an ET-independent signalling pathway. The elevated level of ERF1 mRNA in the overexpression line might establish a situation in which the roots respond more sensitively to even low levels of defence-activating signals from the beneficial fungus. Both cases demonstrate that the control of defence gene activation plays a crucial role in the establishment and/or maintenance of the beneficial interaction, and that perturbation of ET signalling affects the interaction, probably because beneficial and pathogenic phases in the symbiosis require a fine-tuned balance of ET signalling.

PR-1, PR-2 and PR-5 are upregulated in response to SA signalling, whereas PDF1.2 and PR-3 expression is mainly controlled by the JA/ET pathway. ERF1 is activated via the ET/JA signalling pathway, which activates defence genes mainly directed against necrotrophic pathogens, whereas biotrophic pathogens are countered more efficiently via the SA pathway (Thomma et al., 1998, 1999). The constitutive expression of ERF1 confers resistance to several necrotrophic fungi, but reduces the tolerance against Pseudomonas syringae in Arabidopsis, suggesting negative crosstalk between the ET and SA signalling pathways (Berrocal-Lobo et al., 2002). Interestingly, 35S::ERF1 seedlings and plants also do not respond to P. indica. Considering that both SA- and JA/ET-regulated defence genes respond to the fungus in the ET signalling mutants, it is unlikely that the activation is pathway specific. Rather, we propose that the less protected ET signalling mutants express a more general stress response when exposed to P. indica.

Piriformospora indica-mediated growth promotion and higher seed yield in Arabidopsis appears to be independent of JA

In addition to ET, JA is an important signalling molecule in plant–microbe interactions and often acts in concert with ET in activating the expression of defence responses (cf. Penninckx et al., 1996; Pozo et al., 2005; Beckers & Spoel, 2006; Chague et al., 2006). JA is a regulator of ERF1 and PDF1.2 (Stintzi & Browse, 2000; Brown et al., 2003; Wasternack, 2007), and plays a role in the response to beneficial microorganisms, either through the induction of systemic resistance or in the establishment of a beneficial association of the symbiotic partners (cf. Pozo et al., 2005). Finally, JA, in combination with ET, is a potential candidate for the transduction of information from the colonized roots to the aerial parts of the the plants (Van Wees et al., 2008). To test whether JA is required for P. indica-mediated growth promotion and higher seed yield in Arabidopsis, we analysed the insertion line jar1 (Staswick et al., 2002), the double insertion line aoc3/4 (insertions in the allene oxide cyclase genes 3 and 4, which are highly expressed in roots; Delker et al., 2006) and opr3 (cf. Wasternack, 2007). Seedlings and adult plants of all three mutant lines responded to P. indica and produced more seeds than uncolonized controls (Fig. S1, see Supporting Information). Furthermore, the roots of Arabidopsis lines expressing the uidA gene under the control of either the AOC3 or AOC4 promoter regions, which are highly active in roots (Delker et al., 2006), did not show higher GUS activity in the presence of the fungus compared with the control (data not shown). This suggests that JA is not required for P. indica-induced growth promotion and higher seed yield in Arabidopsis. Stein et al. (2008) have shown that P. indica root colonization reduces powdery mildew conidia in WT. Two JA signalling mutants were nonresponsive to P. indica, suggesting that the fungus confers resistance reminiscent of induced systemic resistance through JA signalling. This suggests different JA requirements for different P. indica-mediated processes.

Acknowledgements

We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, Dr J. Ecker for the ein3-1 and ein3-1/eil1-1 seeds, Dr K. Wang for the 35S::eto1-481 line, and Dr Wasternack for the JA lines. This work was supported by the Sonderforschungsbereich 604, Deutsche Forschungsgemeinschaft (Oe133/19-1, LE2321/1-2), Bundesministerium für Bildung und Forschung (IND 03/013), the Alexander-von-Humboldt-Foundation (Bonn) and the International Max Planck Research School Jena.

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