Repression of the Auxin Response Pathway Increases Arabidopsis Susceptibility to Necrotrophic Fungi
a Centro de Biotecnología y Genómica de Plantas (CBGP), Departamento de Biotecnología-UPM, ETS Ingenieros Agrónomos, Avda Complutense, E-28040 Madrid, Spain
b Department of Plant–Microbe Interactions, Max Planck Institute for Plant Breeding Research, Carl-von-Linné Weg 10, 50829 Cologne, Germany
1 To whom correspondence should be addressed. E-mail antonio.molina{at}upm.es, fax 34-91-3363985.
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Abstract |
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In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axr1, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skp1–Cullin–F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGT1b (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgt1b mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGT1b in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgt1b axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant–pathogen compatibility is established.
Key Words: plant defense • innate immunity • necrotrophic fungi • auxin signaling • proteasome
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plants protect themselves from pathogen infection through a combination of constitutive and induced defenses (Holt et al., 2003; Chisholm et al., 2006). The effectiveness of induced resistance relies in large part on perception of pathogen-derived molecules by plant receptors (Chisholm et al., 2006; Nürnberger and Kemmerling, 2006). Plant–pathogen recognition triggers the biosynthesis of phytohormones such as salicylic acid (SA), ethylene (ET), and jasmonic acid (JA) (Glazebrook, 2005; Ferry et al., 2004). The balance and interplay of these hormone systems has a pivotal role in the expression of resistance to particular pathogens and pests (Glazebrook, 2005; Ferry et al., 2004). For example, genetic evidence in Arabidopsis shows that extensive cross-talk between ET, JA, and SA signaling pathways determines resistance to different types of pathogens. Most apparent are cooperative and antagonistic interactions between SA and JA/ET signaling that affect local and systemic resistance responses (Berrocal-Lobo et al., 2002; Thomma et al., 1998, 1999; Laurie-Berry et al., 2006; Truman et al., 2007). Also, many plant pathogens are themselves able to produce phytohormones during infection that can interfere with host developmental processes and defense responses (Robert-Seilaniantz et al., 2007).
Plant resistance to necrotrophic fungi such as Plectosphaerella cucumerina and Botrytis cinerea is genetically complex (Llorente et al., 2005) in contrast to monogenic gene-for-gene resistance that often conditions resistance to biotrophic fungi, oomycetes (e.g. Hyaloperonospora parasitica) or bacteria (e.g. Pseudomonas syringae; Holt et al., 2003). Multiple hormone pathways were found to contribute to Arabidopsis resistance to necrotrophic fungi, since the ein2-5, coi1-1, sid2-1 mutants impaired, respectively, in ET, JA, and SA signaling, and NahG transgenic lines that are blocked in SA accumulation, were more susceptible than wild-type (WT) plants to P. cucumerina and B. cinerea (Thomma et al., 1998, 1999; Berrocal-Lobo et al., 2002; Ferrari et al., 2003). In contrast to the known contributions of SA, ET, and JA to plant disease resistance, the roles of other hormones such as abscisic acid (ABA), auxin and brassinosteroids in plant defense are less well defined (Robert-Seilaniantz et al., 2007). ABA appears to have multiple sites of action in plant–pathogen interactions (Mauch-Mani and Mauch, 2005). An ABA-mediated pathway promoted susceptibility in Arabidopsis infection with virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000; de Torres et al., 2007), but increased resistance to the soil-borne pathogens Ralstonia solanacearum and Pythium irregulare (Adie et al., 2007; Hernández-Blanco et al., 2007). ABA was also found to negatively regulate plant resistance to certain necrotrophic fungi, since ABA-deficient (e.g. aba2) and ABA-signaling mutants (e.g. abi1) were more resistant to these pathogens than WT plants (Audenaert et al., 2002; AbuQamar et al., 2006; Hernández-Blanco et al., 2007). Brassinosteroids can also affect the induction of plant defences. For example, treatment of rice or tobacco with brassinolide triggered enhanced resistance to different biotrophic fungi (Nakashita et al., 2003).
A negative effect of auxin signaling on plant resistance to biotrophic pathogens was recently described (Navarro et al., 2006; Wang et al., 2007). Auxin regulates many processes during plant development through direct interaction with TIR1-like F-box receptor proteins (Quint and Gray, 2006). Auxin binding to SCFTIR1 leads to enhanced removal of members of the AUX/IAA family of transcriptional factor (TF) repressors by the SCF (Skp1–Cullin–F-box) E3-ubiquitin ligase proteasome pathway (Gray et al., 2001; Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). The degradation of AUX/IAA proteins allows activation of Auxin Response Factors (ARFs) and the expression of auxin-responsive genes (Hagen and Guilfoyle, 2002). There is an increasing body of evidence that some plant pathogens either produce auxin themselves or increase plant auxin biosynthesis upon infection to manipulate host developmental processes (Manulis et al., 1998; Glickmann et al., 1998; Maor et al., 2004; Vandeputte et al., 2005). Repression of auxin-responsive genes also occurs upon plant treatments with the bacterial elicitor flg22 or the SA functional analog benzothiadiazole (BTH) (Navarro et al., 2006; Wang et al., 2007). Flg22 triggered the up-regulation of a canonical microRNA (miR393) that targets auxin receptors, thereby contributing to the down-regulation of auxin signaling (Navarro et al., 2006). Increasing the auxin response through overexpression of the TIR1 auxin receptor rendered plants more susceptible to PstDC3000 and, conversely, attenuation of auxin signaling through miR393 overexpression increased resistance to bacteria (Navarro et al., 2006). These results show that repression of auxin signaling is part of a bacterial-induced plant immune response. Notably, SA treatment caused a stabilization of AUX/IAA repressor proteins and inhibition of the auxin response, suggesting that SA contributes to a general repression of the auxin pathway (Wang et al., 2007). Consistent with this view, the axr2-1 mutant that is impaired in auxin responses restricted growth of virulent P. syringae pv. maculicola 4326 compared to WT plants (Wang et al., 2007).
Multiple auxin-resistant mutants have been isolated in Arabidopsis that define SCF ubiquitin-mediated protein degradation as a central component of auxin signaling (Gray et al., 1999, 2001). Ubiquitination of a target protein is operated by a multienzyme system consisting of ubiquitin-activating (E1), -conjugating (E2) and -ligating (E3) enzymes. Polyubiquitinated proteins are normally escorted to the 26S proteasome to be degraded (Devoto et al., 2003). Another Arabidopsis mutant, sgt1b, is defective in one of two highly related, functional SGT1 genes (SGT1a and SGT1b) (Azevedo et al., 2006). SGT1 proteins structurally resemble and behave as HSP90/HSC70 co-chaperones (Shirasu and Schulze-Lefert, 2003; Azevedo et al., 2006; Noël et al., 2007). Plant SGT1 also co-immunoprecipitated with the SCF structural subunit SKP1 and the COP9 signalosome (CSN) that regulates SCF ubiquitin-proteasome degradation (Azevedo et al., 2002; Liu et al., 2002). Consistent with SGT1 assisting SCF ubiquitin E3-ligase activities, Arabidopsis SGT1b was found to contribute to the auxin response controlled by SCFTIR1 and the JA response mediated by SCFCOI1 (Gray et al., 2001, 2003). SGT1b is also important for cell death-associated resistance responses to biotrophic oomycetes and hemi-biotrophic bacteria (Austin et al., 2002; Azevedo et al., 2006; Holt et al., 2005). However, it is unclear whether the role of SGT1b in resistance to these pathogens reflects an activity in SCF-mediated ubiquitination or a different co-chaperone function (Muskett and Parker, 2003; Noël et al., 2007).
Here, we explore the role of auxin in Arabidopsis resistance to necrotrophic fungi. We show that repression of auxin signaling either through mutations in the auxin pathway or by pharmacological interference with the auxin response impairs resistance to the necrotrophic fungi P. cucumerina and B. cinerea. We further provide evidence that ubiquitin-mediated proteolysis by the proteasome contributes to the restriction of the fungal diseases caused by P. cucumerina. The differential effects of sgt1b and auxin signaling mutants on resistance to the necrotroph P. cucumerina and the biotroph H. parasitica point to SCF-mediated ubiquitination being important for resistance to necrotrophic fungi, but contributing less to gene-for-gene resistance to biotrophic oomycetes.
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Resistance to Necrotrophic Fungi Is Reduced in Auxin Signaling Mutants
In order to test whether auxin signaling competence affects the response of Arabidopsis to necrotrophic fungi, we infected wild-type (WT) Columbia (Col-0) and Landsberg-erecta (Ler) plants and different auxin response mutants in these two accessions with the necrotrophic ascomycete fungus P. cucumerina. The mutants selected for the analysis are impaired in distinct components of the auxin signaling pathway: (1) the recessive mutant tir1-1 is defective in the F-box TIR1 protein that is one of four auxin-binding proteins (ABPs) expressed in Arabidopsis (Gray et al., 2001; Dharmasiri et al., 2005a, 2005b; Kepinski and Leyser, 2005); (2) the dominant axr2-1 mutant displays an enhanced stabilization of the auxin repressor transcriptional factor AXR2/IAA17 (Nagpal et al., 2000; Ramos et al., 2001); (3) the recessive aux1-3 mutant is blocked in AUX1, one of the multiple Arabidopsis auxin importers (Marchant et al., 1999; Woodward and Bartel, 2005); and (4) the mutants axr1-12 and axr6-1 are defective in AXR1/RUB1 and AXR6/CUL1 proteins, two components of different SCF E3-ubiquitin ligase complexes (Leyser et al., 1993; Hellmann et al., 2003; del Pozo et al., 2002). Ten-day-old seedlings of WT, P. cucumerina-susceptible Col-NahG transgenic plants (Berrocal-Lobo et al., 2002) and the auxin response mutants axr1-12, axr2-1, axr6-1, tir1-1, and aux1-3 grown on MS medium were sprayed with water (mock-treatment) or with a spore suspension of P. cucumerina (4 x 105 spores ml–1). Susceptibility to P. cucumerina was estimated as the percent reduction of plant fresh weight (FW) at 12 d post inoculation (dpi), as described previously (Berrocal-Lobo and Molina, 2004; Llorente et al., 2005). This method to determine susceptibility to necrotrophs is not distorted by the different starting sizes of the genotypes analyzed (Llorente et al., 2005). As shown in Figure 1A, the reduction in plant FW caused by fungal infection was higher in the axr1-12, axr2-1, and axr6-1 mutants than in WT plants. In axr1-12 and axr2-1, the loss of plant FW was as extreme as in the highly susceptible NahG line (Berrocal-Lobo et al., 2002). By contrast, the level of susceptibility of tir1-1 and aux1-3 mutants to P. cucumerina did not differ from corresponding WT seedlings (Figure 1A). The progression of fungal infection in the axr1-12, axr2-1, and axr6-1 mutants, as in the NahG line, correlated with the spread of necrosis in infected leaves that eventually consumed seedlings (Figure 1B and data not shown). In WT seedlings and the tir1-1 and aux1-3 mutants, disease symptoms were less pronounced, although a reduction in plant FW and wilting of leaves were detected (Figure 1B and data not shown). Auxin signaling affects many developmental processes and, consequently, auxin response mutants have alterations in growth and development (Woodward and Bartel, 2005). To exclude the possibility that the observed susceptibility to P. cucumerina of the auxin response mutants analyzed could be related to the age of seedlings or the growing conditions, we also tested the resistance of adult (4-week-old) plants grown on soil. Individual leaves of WT plants (Col-0 and Ler) and axr1-12, axr2-1, tir1-1, and aux1-3 mutants were drop-inoculated with a 5-µl suspension of P. cucumerina spores (2 x 106 spores ml–1) or water (control). After inoculation, the progression of infection was followed for 10 d, and a disease rating (DR; from 0, no symptoms, to 3, severe tissue maceration) was recorded daily (1–12 dpi). As shown in Figure 1C, the DR scores at a representative time point of 7 dpi were higher in the axr1-12 and axr2-1 mutants than in WT Col-0, whereas the DR scores of tir1-1 and aux1-3 were similar to those observed in corresponding WT Col-0 and Ler plants, respectively. These results show that the enhanced susceptibility of axr1-12 and axr2-1 mutants to P. cucumerina is independent of plant developmental stage or growing conditions.
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We tested whether the increased susceptibility of auxin signaling mutants extended to a different necrotrophic fungus, Botrytis cinerea. Ten-day-old seedlings of wild-type (Col-0 or Ler), axr1-12, axr2-1, tir1-1, and aux1-3 grown on MS medium were spray-inoculated with water (mock-treatment) or with a spore suspension of B. cinerea (5 x 104 spores ml–1). Disease susceptibility was measured as the percent plant decay at different dpi, as described previously (Llorente et al., 2005). The level of plant decay caused by B. cinerea infection progressed more rapidly in axr1-12 and axr2-1 mutants than in WT Col plants (Figure 2). By contrast, progression of the infection in tir1-1 and aux1-3 mutants did not differ significantly from that of WT plants (Figure 2). Thus, the auxin signaling mutants axr1-12 and axr2-1 exhibit enhanced susceptibility to two necrotrophic fungi. These results point to a potential role of the auxin response pathway in regulation of Arabidopsis resistance to these necrotrophic pathogens.
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Auxin affects many signaling processes (Woodward and Bartel, 2005). Thus, some auxin response mutants have been shown to be impaired either in JA (e.g. axr1 and axr6), or JA/ET (e.g. axr2) signaling pathways (Woodward and Bartel, 2005). Moreover, SA was found to inhibit the auxin response pathway (Wang et al., 2007). We therefore investigated whether the enhanced susceptibility of axr1-12 and axr2-1 mutants to necrotrophic fungi might be a consequence of impairment of SA or ET/JA defense. The expression patterns of PR1 and PDF1-2, marker genes of SA and JA/ET signaling, respectively (Glazebrook, 2005), were examined in WT and mutants after pathogen infection. Ten-day-old MS-grown seedlings of WT Col-0, and the axr1-12 and axr2-1 mutants were inoculated with water (mock) or with a spore suspension of P. cucumerina (4 x 105 spores ml–1) and total RNA extracted from seedlings at 3 and 5 dpi. The progression of the infection was followed in a proportion of seedlings to ensure the course of disease and hypersusceptibility of NahG, axr1-12, and axr2-1 mutants prior to Northern analysis (Figure 3A). Probing of a Northern-blot showed that expression of PR1 and PDF1.2 mRNAs was induced to the same or higher extent in the axr1-12 and axr2-1 mutants than in WT plants (Figure 3B). By contrast, induction of PR1 mRNA upon infection was impaired in NahG plants that are blocked in SA accumulation, whereas the expression profile of PDF1.2 mRNA in these plants was similar to that observed in the WT plants (Figure 3B).
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Two branches of the JA signaling pathway have been described to control antagonistically the response to pathogen infection and wounding (Lorenzo et al., 2004). The fine-tuning regulation of these responses depends on the balance of activation of ERF1 and MYC2 transcriptional factors (Lorenzo et al., 2004). Thus, MYC2 mutants (e.g. jin1) are impaired in the wound-responsive, JA-regulated signaling pathway, but show an enhanced activation of the JA/ET defensive pathway upon pathogen infection (Lorenzo et al., 2004). To exclude the possibility that the enhanced susceptibility observed in the axr1-12 and axr2-1 mutants was the result of a defect in the activation of the wound-responsive branch of JA signaling, the expression of two marker genes, TAT and LOX3, of this pathway (Lorenzo et al., 2004) was analyzed by qRT–PCR in these mutants and WT plants. As showed in Figure 3C, upon P. cucumerina infection, the expression of both genes was induced to a higher extent in the axr1-12 and axr2-1 mutants than in WT, probably reflecting enhanced fungal colonization observed in the mutants. These results suggest that activation of SA, JA/ET, and JA defensive pathways upon P. cucumerina fungal infection is not impaired in the axr1-12 and axr2-1 mutants. We therefore reasoned that the enhanced susceptibility of these mutants to P. cucumerina may be due to a more specific defect in the auxin response. The expression of the AXR1 and AXR2 genes in WT plants and the axr1-12 and axr2-1 mutants upon P. cucumerina infection was also tested by Northern-blot analysis but no significant changes in expression were observed compared to mock inoculated plants (data not shown).
Pharmacological Inhibition of Auxin Transport Leads to Increased Necrotrophic Infection
Extensive mining of publicly available Arabidopsis transcriptome data (www.genenvestigator.ch.org) revealed that a significant portion (
65%) of auxin signal transduction-related genes encoding AUX/IAA, ARFs, or TIR/ABP are down-regulated upon Arabidopsis infection with B. cinerea (Table S1). The repressed genes include the auxin receptor TIR1, the majority of characterized AUX/IAA genes encoding transcriptional repressors, such as AXR2/IAA7 and AXR3/IAA17, and a significant number of ARF-encoding genes (Hagen and Guilfoyle, 2002; Dharmasiri et al., 2005a, 2005b). These data are consistent with enhanced susceptibility of Arabidopsis axr1-12 and axr2-1 mutants to necrotrophic fungi observed here (Figures 1 and 2) and point to a general transcriptional repression of the auxin response that may contribute to disease development. To test this hypothesis, we treated WT, axr1-12, axr2-1, and NahG plants with different concentrations (1 and 5 µM) of the auxin transport inhibitor TIBA (2,3,5-Triiodobenzoic acid; Geldner et al., 2001). The plants were then sprayed with P. cucumerina spores (4 x 105 spores ml–1). The susceptibility of TIBA-treated and non-treated (control) plants was determined at 10 dpi. As shown in Figure 4A, there was a higher reduction in plant FW caused by fungal infection of WT plants treated with TIBA than in non-treated plants. The level of susceptibility of WT plants treated with 5 µM TIBA was similar to that observed in the control axr1-12 and axr2-1 mutants and NahG plants (Figure 4A). Significantly, TIBA treatment of axr1-12 and axr2-1 mutants and NahG plants did not result in further enhancement of susceptibility to P. cucumerina compared to untreated plants of the same lines (Figure 4A). These data show that loss of auxin transport phenocopies the hypersusceptibility of axr1-12 and axr2-1 mutants to necrotrophic fungi.
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The expression of PR1 and PDF1.2 mRNAs was analyzed on a Northern-blot of the non-treated (control) and TIBA-treated plants after mock inoculation or P. cucumerina infection. Upon fungal infection, the expression of PR1 in TIBA-pretreated WT plants was higher than in the control samples, whereas expression of PDF1.2 was lower in the TIBA-treated than control plants (Figure 4B). A similar pattern of expression of the PR1 and PDF1-2 genes was detected in the inoculated axr1-12 and axr2-1 mutants, irrespective of TIBA pretreatment (Figure 4B). As expected, induction of PR1 upon fungal infection was blocked in NahG plants (Figure 4B). However, a repression of fungal-induced expression of PDF1.2 was also observed in the NahG line (Figure 4B). The expression level of the JA-regulated genes TAT and LOX3 was also tested by qRT–PCR in WT plants and both genes were found to be induced upon P. cucumerina infection in TIBA-treated WT plants (data not shown). These data suggest that inhibition of auxin signaling by blocking auxin transport affects the activation of SA and JA/ET signaling pathways upon necrotrophic fungal infection. The enhanced expression of SA-regulated PR1 in TIBA-treated WT plants is in line with the proposed negative cross-talk between the SA and auxin signaling (Wang et al., 2007). We also tested the effect of exogenous treatments of WT plants with different concentrations (1–10 µM) of the natural auxin IAA before or after P. cucumerina inoculation, but no significant changes in plant susceptibility were detected (data not shown).
P. cucumerina Infection Leads to Stabilization of Heat Shock-Induced AXR3-GUS Protein
The enhanced susceptibility of axr1-12 and axr2-1 mutants (Figures 1 and 2) and TIBA-treated WT plants (Figure 4) to the necrotrophic fungi tested, as well as the global down-regulation of auxin response genes upon B. cinerea infection (Table S1), suggest that Arabidopsis infection with virulent necrotrophs causes a repression of auxin signaling. This repression could be in part mediated by reduced degradation of some AUX/IAA repressors (Quint and Gray, 2006; Navarro et al., 2006; Wang et al., 2007). To test this hypothesis, we examined expression of the reporter gene HS::AXR3NT–GUS encoding a fusion between the amino terminus (NT) of the auxin response repressor AXR3/IAA17 and GUS (β-glucuronidase) driven by a heat-shock (HS)-inducible promoter. After heat shock treatment, the stability of AXR3NT–GUS protein, measured as GUS activity, declines in IAA-treated plants whereas it continues to increase in SA-treated plants (Wang et al., 2007). Moreover, flg22 treatment of plants caused enhanced stability of AXR3NT–GUS protein compared to non-treated plants (Navarro et al., 2006). Leaves of 10-d-old WT (Col-0) and transgenic HS::AXR3NT-GUS seedlings grown in MS medium were mock-inoculated or inoculated with a spore suspension of P. cucumerina (4 x 105 spores ml–1). Two days later, the plants were subjected to a heat shock treatment (37°C for 2 h) and the level of AXR3NT–GUS fusion protein determined (Gray et al., 2001). As previously described, GUS activity was detected in the roots but not leaves of HS-treated HS::AXR3NT–GUS seedlings (Figure 5; Gray et al., 2001). Notably, GUS staining was detected in the leaves of HS::AXR3NT–GUS seedlings inoculated with P. cucumerina and HS-exposed, but not in the mock inoculated, HS-exposed seedlings (Figure 5). No GUS staining was detected in HS-exposed WT plants (Figure 5). These results suggest that a stabilization of the AXR3 transcriptional repressor protein occurs in leaves upon P. cucumerina inoculation that might contribute to inhibition of the plant auxin response.
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Role of SCF (Skp1–Cullin–F-box) E3-Ubiquitin Ligase Complexes in Restriction of Fungal Necrotroph Infection
An effective Arabidopsis auxin response relies on the removal of AUX/IAA family of TF repressors through auxin-stimulated binding by SCFTIR1 complexes and targeting to the ubiquitin–proteasome pathway (Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). The enhanced susceptibility to necrotrophic fungi of the axr2-1 mutant that has a stabilized AXR2 repressor protein, and of the axr1-12 and axr6-1 mutants that are impaired in two components (AXR1/RUB1 and AXR6/Cullin1) of the SCF E3-ubiquitin ligase complexes and are defective in IAA/AUX degradation (Gray et al., 2001; Hellmann et al., 2003), suggest that ubiquitin-mediated protein degradation is an important component of plant resistance to necrotrophs. Since Arabidopsis SGT1b, encoding a molecular co-chaperone, contributes to the auxin response controlled by SCFTIR1 (Gray et al., 2001, 2003), we explored whether mutations in SGT1b also affect resistance to P. cucumerina. Alongside, we tested the responses of an Arabidopsis rar1 mutant lacking a SGT1/HSP90-interactor RAR1 (Shirasu and Schulze-Lefert, 2003) that is important for many R gene-mediated resistance responses to P. syringae bacteria and the obligate biotrophic oomycete H. parasitica (Muskett et al., 2002; Tornero et al., 2002; Holt et al., 2005), and a rar1 sgt1b mutant combination (Muskett et al., 2002). Ten-day-old MS-grown seedlings of the rar1-10 and sgt1b-1 single mutants and rar1-10 sgt1b-1 double mutant in accession Ler were sprayed with a suspension spore of P. cucumerina (4 x 105 spores ml–1). As shown in Figure 6A, the reduction in plant FW caused by fungal infection was similar in the mutants and WT Ler. The response of an sgt1a-1 mutant defective in the second Arabidopsis SGT1 gene SGT1a (Azevedo et al., 2006) was also similar to its WT parental accession Ws-0 (Figure 6A). Thus in contrast to AXR1, AXR6, and AXR2, SGT1 and RAR1 do not help to restrict necrotrophic fungal infection. We then tested resistance of the Col-0 double mutants rar1-21 axr1-3 and sgt1beta3 axr1-3 to P. cucumerina (sgt1beta3 is phenotypically a null sgt1b mutant in Col-0 that expresses a truncated SGT1b protein; Gray et al., 2003; Noël et al., 2007). No significant differences were observed in the reduction of plant FW compared to the highly susceptible axr1-3 single mutant (Figure 6B). Therefore, while the activities of AXR1 and AXR2 which are intimately linked to SFC E3 ubiquitin ligase functions are needed for full restriction of necrotroph infection, SGT1b and RAR1 do not appear to contribute to SCF-related processes in resistance to these pathogens. By contrast, isolate-specific (gene-for-gene) resistance mediated by RPP4 to the obligate oomycete pathogen H. parasitica is strongly disabled in rar1 and moderately compromised in sgt1b mutants (Holt et al., 2005). We therefore tested whether the mutants disabling SCF E3-ubiquitin ligase activities affect RPP4-mediated resistance. Wild-type, axr1-3, axr1-12, axr2-1, and axr6-1 mutants were spray inoculated with conidiospores (1 x 104 spores mL–1) of H. parasitica isolate Emwa1 (recognized by RPP4; Holt et al., 2005) and the extent of pathogen infection measured by the production of conidiospores on leaves at 5 dpi. As shown for axr1-3 (Figure 7A), none of the mutants tested displayed increased susceptibility to H. parasitica. This was confirmed by monitoring the extent of pathogen hyphal growth in leaves stained with lactophenol-trypan-blue (Figure 7B). As expected, we observed a strong impairment of RPP4 resistance in rar1-21 and a weak defect in sgt1beta3 single mutants (Figure 7A) that correlated with increased colonization of leaves compared to WT Col-0 (Figure 7B). We then tested the phenotypes of the rar1-21 axr1-3 and sgt1beta3 axr1-3 double mutants in response to H. parasitica Emwa1 inoculation and observed an increase in susceptibility to H. parasitica in sgt1beta3 axr1-3 at the level of sporulation (Figure 7A) and oomycete colonization of leaf tissues (Figure 7B). The genetic dispensability of AXR1 and AXR2 in RPP4 resistance to H. parasitica suggests that SCF-mediated ubiquitination does not play a major role in the restriction of biotrophic pathogens, in contrast to its measurable contribution to resistance to necrotrophic fungi. The requirement for SGT1b in oomycete resistance and the genetic additivity of sgt1beta3 axr1-3 mutants in susceptibility point to a minor but detectable contribution of AXR1 to oomycete resistance that is only seen if SGT1b function is disabled.
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Inhibition of Proteasome Activity Impairs Resistance to P. cucumerina
We took a pharmacological approach to test whether proteasome-mediated protein degradation plays a role in plant resistance to necrotrophs. Ten-day-old MS-grown seedlings of WT (Col-0), axr1-12, axr2-1, and NahG were treated for 24 h with water (control) or different sub-lethal concentrations (2 and 10 µM) of the proteasome inhibitor MG132 (Ramos et al., 2001). Plants were then transferred to MG132-free MS plates and either mock-inoculated or infected with P. cucumerina (4 x 105 spores ml–1). Susceptibility was then determined at 10 dpi. The reduction in plant FW caused by fungal infection was higher in WT seedlings treated with 10 µM MG132 than in control (untreated) seedlings and the extent of susceptibility was similar to that observed in control (untreated) axr1-12, axr2-1, or NahG seedlings (Figure 8). As with the auxin transport inhibitor (TIBA; Figure 4), treatment of the axr1-12, axr2-1, and NahG seedlings with MG132 did not lead to enhanced susceptibility to P. cucumerina compared to untreated seedlings (Figure 8). These results indicate that proteasome activity is required for effective mobilization of Arabidopsis resistance to P. cucumerina.
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DISCUSSION |
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Auxin affects many aspects of development and growth in healthy plants (Woodward and Bartel, 2005; Quint and Gray, 2006). The auxin response has emerged more recently as an important factor in promoting P. syringae infection of Arabidopsis (Navarro et al., 2006) that can be repressed at multiple levels by the defence hormone SA (Wang et al., 2007). We present evidence here that supports a role of SCF-mediated ubiquitination and, more specifically, auxin signaling in promoting resistance to fungal necrotrophs (Figure 9). We find that the auxin signaling mutants axr1, axr2, and axr6, which are impaired in auxin-mediated AUX/IAA degradation through SCFTIR1-targeted ubiquitination (Gray et al., 2001; Hellmann et al., 2003), allow more infection of leaves by the necrotrophic fungi P. cucumerina and B. cinerea (Figures 1, 2, and 9). The defective degradation of AUX/IAA transcriptional repressor proteins in auxin signaling mutants affect the activation of Auxin Response Factors (ARFs) and the expression of auxin-responsive genes (Hagen and Guilfoyle, 2002; Figure 9). Both TIBA inhibition of auxin transport (Geldner et al., 2001) and MG132 proteasome activity (Ramos et al., 2001) phenocopy the signaling defects of axr1-12 and axr2-1 mutants, leading to an enhanced susceptibility to necrotrophic fungi, such as P. cucumerina (Figures 4, 8, and 9). Also, infection by virulent necrotrophic fungi, such as P. cucumerina, causes increased stabilization of heat shock-induced AXR3–GUS protein (Figure 5) that is normally destabilized through SCFTIR1-targeted ubiquitination (Gray et al., 2001), and a global down-regulation of auxin response genes (Table S1), suggesting that Arabidopsis infection with virulent necrotrophs causes a repression of auxin signaling (Figure 9). The contrasting effects of auxin on the progression of disease caused by hemi-biotrophic bacteria (Navarro et al., 2006; Wang et al., 2007) and necrotrophic fungi (this work) suggest that auxin signaling is an integral component of the complex hormone network that modulates plant responses to different pathogens and pests (Glazebrook, 2005; Ferry et al., 2004).
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The enhanced susceptibility of auxin response mutants to infection by necrotrophs suggests that auxin perception and/or signaling might connect closely to the modulation of plant cell death programs, since colonization by necrotrophs is aided by factors that promote cell death (Govrin and Levin, 2000). A challenge is to distinguish direct from indirect hormone effects. For example, the axr1 and axr2 mutants are known to be impaired also in JA/ET signaling pathways (Tiryaki and Staswick, 2002; Woodward and Bartel, 2005), and the enhanced susceptibility of axr1 to different isolates of the soil-borne pathogen Pythium spp. correlated with a defect in JA signaling (Tiryaki and Staswick, 2002). Analysis of other JA pathway response mutants, such as coi1-1 and jar1-1, also showed that JA plays a key role in Arabidopsis resistance to Pythium sp., P. cucumerina, and B. cinerea (Adie et al., 2007; Berrocal-Lobo et al., 2002; Thomma et al., 1998, 1999). Moreover, SA can negatively regulate auxin signaling (Wang et al., 2007). Importantly, in our study, we found that the expression patterns of PR1 and PDF1-2 that are marker genes of the SA and JA/ET signaling pathways, respectively (Glazebrook, 2005), and of TAT and LOX3 that are marker genes of the wound-inducible, JA-regulated signaling pathway (Lorenzo et al., 2004), were not impaired in the axr1-12 and axr2-1 mutants upon P. cucumerina infection (Figure 3). These results suggest that the enhanced susceptibility of axr1 and axr2 plants to necrotrophic fungi is unlikely to be accounted simply by repression of the SA, JA/ET, or JA defense pathway. Enhanced and reduced expression of PR1 and PDF1.2, respectively, in TIBA-treated WT plants (Figure 4) are consistent with a proposed negative cross-talk between SA and both the auxin and ET/JA pathways (Thomma et al., 1998; Wang et al., 2007). A recent comprehensive transcriptomic analysis of auxin response in Arabidopsis has revealed that auxin regulates in a complex manner genes associated with the biosynthesis, catabolism, and signaling pathways of other phytohormes (Paponov et al., 2008). The characterization of this complex signaling interaction that determines the fine control of plant resistance to pathogens is a future challenge in the plant immunity field.
We did not observe increased susceptibility to necrotrophs in the auxin response mutants tir1-1, aux3-1, and sgt1beta3 (Figures 1, 2, and 6). The lack of phenotype in tir1-1 and aux3-1 may be explained by functional redundancy in Arabidopsis. TIR1 is one of four Arabidopsis auxin binding proteins identified (Dharmasiri et al., 2005a, 2005b; Kepinski and Leyser, 2005) and AUX1 is one of several auxin efflux importers expressed in the Arabidopsis genome (Woodward and Bartel, 2005). Hence, the auxin resistance phenotypes of these single mutants are comparatively weak (Woodward and Bartel, 2005). It is possible that sgt1b defects in SCF E3-ubiquitin ligase functions are also compensated for by the presence of SGT1a, which, although less penetrant, has intrinsic SGT1 activity (Azevedo et al., 2006). However, the partial loss of RPP4 resistance observed in sgt1b mutants to the obligate biotrophic pathogen H. parasitica combined with genetic dispensability of AXR1, AXR2, and AXR6 in resistance to this pathogen and an opposite trend in these mutants in response to P. cucumerina suggests different mechanisms are being engaged in defence responses to biotrophs and necrotrophs. Since, in yeast and plants, SGT1 has multiple sites of action inside the cell (Azevedo et al., 2006; Noël et al., 2007; Dubacq et al., 2002; Catlett and Kaplan, 2006), we think that an SGT1 function other than assisting SCF E3-ubiquitin ligases predominates in R gene-triggered resistance to oomycetes. Such an SGT1 activity might be as a co-chaperone in combination with HSP90 in protein complex assembly and/or maturation or influence the HSC70 chaperone machinery in controlling protein steady-state levels or localization (Azevedo et al., 2006; Noël et al., 2007). The additive loss of RPP4 resistance observed in the sgt1beta3 axr1-3 double mutant (Figure 7) may signify a role for SGT1-assisted SCF-mediated ubiquitination in the absence of R gene-triggered immunity, after a certain degree of plant–oomycete compatibility is established. By contrast, SCF-mediated ubiquitination and proteolysis, that do not genetically engage SGT1b, are clearly important for resistance to fungal necrotrophs.
The positive contribution of auxin signaling to defense against necrotrophic fungi may render this pathway vulnerable to manipulation by pathogens and it is notable that Arabidopsis infection with virulent necrotrophic fungi such as B. cinerea (Table S1) or P. cucumerina (C. Sánchez-Rodriguez and A. Molina, unpublished results) cause a general down-regulation in the expression of auxin response genes (Figure 9). Repression of auxin-regulated genes was also described in the interaction between Nicotiana benthamiana and B. cinerea (El Oirdi and Bouarab, 2007). A down-regulation of auxin response genes may, in part, be mediated by the stabilization of auxin transcriptional factor repressors such as AXR3 (Figure 5) or AXR2 that, in turn, would result in reduced activities of ARF TFs (Hagen and Guilfoyle, 2002; Figure 9). During necrotroph infection of wild-type plants, we think it is likely that a moderate suppression of the auxin response by necrotrophs is countered by endogenous promotion of the auxin response that limits pathogen colonization. The results presented here and those published previously (Wang et al., 2007; Navarro et al., 2006) emphasize the fine control of plant defences to necrotrophic and biotrophic pathogens through the differential engagement and balance of hormone response systems.
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Biological Materials and Growth Conditions
The Arabidopsis wild-type accessions used in this study were Col-0, Ler, and Ws-0. The auxin response mutants axr1-12, axr1-3, axr2-1, axr6-1, and tir1-1 (in Col-0 background) and aux1-3 (in Ler background) have been described previously (Pickett et al., 1990; Leyser et al., 1993; Ruegger et al., 1998; Nagpal et al., 2000; Hellmann et al., 2003). The mutants rar1-10, sgt1b-1, and rar1-10 sgt1b-1 (in Col-0), and sgt1a-1 (in Ws-0) have been reported previously (Muskett et al., 2002; Azevedo et al., 2006). Double mutant lines were made between rar1-21 (Tornero et al., 2002) or sgt1beta3 (Gray et al., 2003) and axr1-3 (Lincoln et al., 1990) all in accession Col-0, by crossing the single mutants and selecting homozygous double mutant combinations in F2 progeny through allele-specific PCR (primer combinations are available on request). The HS::AXR3NT–GUS plants were kindly provided by Dr Mark Estelle (Indiana University, IN, USA).
Plants were grown in growth chambers under a 10 h light/14 h dark photoperiod, 70% relative humidity, 22°C day and 20°C night temperatures, and a light intensity of 150 µE m–2 s–1, as described previously (Berrocal-Lobo et al., 2002; Llorente et al., 2005). For plant growth on soil, seeds were sown in pots containing a mixture of organic substrate and vermiculite (3:1 v/v) and irrigated with water once a week (Berrocal-Lobo et al., 2002). For plants growth on Murashige-Skoog (MS) medium, seeds were surface-sterilized and sown on plates containing MS medium solidified with 0.8% (w/v) phytoagar (Sigma), as reported previously (Llorente et al., 2005).
The fungal pathogens Plectosphaerella cucumerina and Botrytis cinerea were kindly provided by Dr B. Mauch-Mani (University of Fribourg, Switzerland) and Dr R. Raposo (INIA, Spain), respectively. Spores from P. cucumerina and B. cinerea were collected as reported (Berrocal-Lobo et al., 2002; Berrocal-Lobo and Molina, 2004). Hyaloperonospora parasitica isolate (Emwa1) was maintained on the genetically susceptible Arabidopsis accession Ws-0, as described (Feys et al., 2005).
Plant Infection with Pathogens
Four-week-old soil-grown plants or 10-d-old MS medium-grown plants were used for the experiments with necrotrophic fungi. Inoculation of MS medium-grown plants with P. cucumerina and B. cinerea was done by spraying MS plates containing plants with 1.5 ml of 4 x 105 and 5 x 104 spores ml–1, respectively (Berrocal-Lobo and Molina, 2004; Llorente et al., 2005). Mock inoculations were done with sterile water containing an equivalent amount of glycerol to the fungal spore suspension used for infection (< 0.01%). Disease caused by P. cucumerina was determined by measuring the percent fresh weight reduction (FW ± standard error) caused by the fungal infection (Berrocal-Lobo and Molina, 2004). Progression of B. cinerea infection was followed macroscopically by viewing the disease symptoms and the percent decay at different dpi was estimated. At least 15 plants per genotype were inoculated in multiple independent repeats. For inoculation of soil-grown plants with P. cucumerina, three leaves per plant were drop inoculated with 5 µl of a spore suspension (4 x 105 spores ml–1) of the fungus or with water. After inoculation, plants were kept under the same growth conditions and the average disease rating (± SD) was measured at different dpi, as reported (Llorente et al., 2005). Disease rating (DR) was: 0, no symptoms; 1, chlorosis; 2, necrosis; 3, severe tissue maceration. A minimum of 20–30 plants per genotype were inoculated in at least three independent experiments, and the DR means and standard deviations estimated at different dpi.
Inoculation with H. parasitica was done by spraying 2-week-old plants grown on soil with a conidiosphore suspension (1 x 104 spores ml–1) of isolate Emwa1. The extent of pathogen sporulation was quantified as described before (Feys et al., 2005). Lactophenol trypan-blue staining of leaves was done, as described (Feys et al., 2005). Statistical analysis of the data in disease resistance experiments was performed using a two-tailed Student's t-test assuming equal variances, with 
= 0.05.
Plant Pharmacological Treatments
Treatments with the auxin transport inhibitor TIBA (2,3,5-Triiodobenzoic acid, Sigma-Aldrich, USA), IAA (3-Indolacetic acid, Sigma-Aldrich) or the proteasome inhibitor MG132 (Z-Leu-Leu-al, Sigma-Aldrich) were performed on 10-d-old MS-grown seedlings. For the TIBA and IAA treatments, seedlings were transferred to MS plates containing different concentrations of the chemicals and, 24 h later, they were sprayed with a suspension (4 x 105 spores mL–1) of P. cucumerina or water, and the reduction plant FW was determined. For MG132 experiments, 10-d-old MS-grown seedlings were sprayed with water or different concentrations of MG132 (2 or 10 µM). After 5 h, plants were transferred to chemical-free MS plates, inoculated with a suspension (4 x 105 spores mL–1) of P. cucumerina or water and the reduction in plant FW was determined as previously indicated.
Gene Expression Analysis
For Northern-blot analyses, total RNA was purified and blotted on Hybond-N+ membranes (Amersham, UK), as reported (Berrocal-Lobo et al., 2002). Probes were labeled with 50 µCi of -32P-dATP. The PR1, PDF1.2, and β-tubulin probes and the hybridization conditions have been previously described (Berrocal-Lobo et al., 2002). The AXR1 probe (201 pb) was amplified using the oligonucleotides 5'-GTTTGTTCCGATGTTGGGG-3' and 5'- GGTCTCAGCTGAACTGTC-5'. The AXR2 probe (221 pb) was amplified using the oligonucleotides 5'-CGTTTGCGCATTATGAAGGG-3' and 5'-CTGCCCTATATACCCAT-3'. At least 12 plants per genotype were inoculated in each experiment performed and the experiment repeated at least twice. The expression of 61 auxin regulated genes (Hagen and Guilfoyle, 2002) upon pathogen and hormone treatment was analyzed using the Genevestigator Meta-Analyzer Tools (www.genevestigator.ethz.ch/at/). qRT–PCR analyses were performed, as described previously (Hernandez-Blanco et al., 2007). Ubiquitin (UBQ, AT5G25760) expression was used to normalize the transcript level in each sample. Oligonucleotides used for cDNA amplification were designed with Primer Express (version 2.0; Applied Biosystems): At1g17420 (LOX3), 5'-GCGGAGATTGTTGAAGCGTTT-3' and 5'-GCCCCACACCTATTTCTACGGT-3'; At2g24850 (TAT), 5'-TTCGCAAATACGATCTTCTCCC-3' and 5'-GTTGATGATTACCATTGCGACG-3'; UBQ (At5g25760), 5'-AAAGGACCTTCGGAGACTCCTTACG-3' and 5'-GGTCAAGAATCGAACTTGAGGAGGTT-3'.
Reporter Gene Activity Assay
Wild-type (Col-0) and HS::AXR3NT–GUS plants were grown on MS plates, as indicated above. Ten-day-old seedlings were mock treated with water or inoculated with a suspension (2 x 106 spores mL–1) of P. cucumerina spores. After 48 h, plants were heat shocked at 37°C for 2 h. GUS activity was assayed by staining seedlings in 5-bromo-4chloro-3indolyl-beta-D-glucoronic acid solution, as previously described (Cao et al., 1994).
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Supplementary Data are available at Molecular Plant Online.
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This work was supported by the MEC (Grants BIO2003–4424 and BIO2006–00488 to A.M.). P.M. and J.P. were supported by The Alexander von Humboldt Foundation and DFG ‘Sonderforschungsbereich’ grant 635. B.R. is a Profesor Ayudante from the Universidad de Salamanca (Spain).
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Acknowledgements |
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We thank Dr Xinnian Dong for helpful discussions of the manuscript and sharing unpublished results and Dr Ken Shirasu for providing sgt1a mutant seed. F. Llorente was a postdoctoral Fellow of the Comunidad de Madrid (CM, Spain). A. Sánchez-Vallet and C. Sánchez-Rodriguez were recipients of Ph.D. fellowships from the Ministerio de Educación y Ciencia (MEC) of Spain.
No conflict of interest declared.
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