Arabidopsis thaliana plants differentially modulate auxin biosynthesis and transport during defense responses to the necrotrophic pathogen Alternaria brassicicola

Authors

  • Linlin Qi,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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    • These authors contributed equally to this work.

  • Jiao Yan,

    1. The State Key Laboratory of Crop Biology, Agronomy College, Shandong Agricultural University, Taian 271018, China
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    • These authors contributed equally to this work.

  • Yanan Li,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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  • Hongling Jiang,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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  • Jiaqiang Sun,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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  • Qian Chen,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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  • Haoxuan Li,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
    2. College of Agriculture, Nanjing Agricultural University, Nanjing 201195, China
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  • Jinfang Chu,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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  • Cunyu Yan,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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  • Xiaohong Sun,

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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  • Yuanjie Yu,

    1. The State Key Laboratory of Crop Biology, Agronomy College, Shandong Agricultural University, Taian 271018, China
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  • Changbao Li,

    1. Vegetable Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
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  • Chuanyou Li

    1. State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing 100101, China
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Author for correspondence:
Chuanyou Li
Tel: +86 10 64865313
Email: cyli@genetics.ac.cn

Summary

  • Although the role of auxin in biotrophic pathogenesis has been extensively studied, relatively little is known about its role in plant resistance to necrotrophs.
  • Arabidopsis thaliana mutants defective in different aspects of the auxin pathway are generally more susceptible than wild-type plants to the necrotrophic pathogen Alternaria brassicicola. We show that A. brassicicola infection up-regulates auxin biosynthesis and down-regulates the auxin transport capacities of infected plants, these effects being partially dependent on JA signaling. We also show that these effects of A. brassicicola infection together lead to an enhanced auxin response in host plants.
  • Application of IAA and MeJA together synergistically induces the expression of defense marker genes PDF1.2 (PLANT DEFENSIN 1.2) and HEL (HEVEIN-LIKE), suggesting that enhancement of JA-dependent defense signaling may be part of the auxin-mediated defense mechanism involved in resistance to necrotrophic pathogens.
  • Our results provide molecular evidence supporting the hypothesis that JA and auxin interact positively in regulating plant resistance to necrotrophic pathogens and that activation of auxin signaling by JA may contribute to plant resistance to necrotrophic pathogens.

Introduction

During the long history of evolution, plants have evolved a battery of complex mechanisms to effectively ward off different kinds of pathogen. The effectiveness of plant defense responses depends on proper recognition of the intruders. Two layers of recognition have been defined. First, plants are equipped to recognize a diverse range of pathogen-associated molecular patterns (PAMPs) through surface pattern-recognition receptors (PRRs), resulting in pattern-triggered immunity (PTI) (Jones & Dangl, 2006), which is sufficient to resist nonpathogenic microbes. Secondly, to counteract pathogens that are capable of inhibiting PTI by delivering virulence effector proteins into host cells, plants have also evolved resistance (R) genes that recognize specific pathogen effectors, resulting in effector-triggered immunity (ETI) (Jones & Dangl, 2006). Downstream of these surveillance systems, plants activate appropriate defense responses through crosstalk between specific hormonal signaling pathways (Chung et al., 2008; Koornneef & Pieterse, 2008).

Based on their lifestyles, plant pathogens can be classified into biotrophs and necrotrophs. Biotrophs are pathogens that derive nutrients from living host tissues, while necrotrophs are pathogens that derive nutrients from dead or dying cells (Glazebrook, 2005). Upon pathogen attack, it is therefore essential that plants activate the appropriate defense response according to the pathogen type. It is generally believed that salicylic acid (SA)-mediated resistance is effective against biotrophs and hemi-biotrophs (Thomma et al., 2001; Glazebrook, 2005; Loake & Grant, 2007), whereas jasmonic acid (JA)- and ethylene (ET)-mediated responses are predominantly effective against necrotrophs (Glazebrook, 2005; Lorenzo & Solano, 2005; Broekaert et al., 2006; Balbi & Devoto, 2008; Pieterse et al., 2009). In addition to SA, JA and ET, several growth-related hormones, including auxin (Kazan & Manners, 2009), abscisic acid (ABA) (Mauch-Mani & Mauch, 2005; Adie et al., 2007; De Torres-Zabala et al., 2007; Mohr & Cahill, 2007), gibberelins (GAs) (Achard et al., 2008; Navarro et al., 2008; Yang et al., 2008), cytokinins (CKs) (Siemens et al., 2006; Igari et al., 2008) and brassinosteroids (BRs) (Krishna, 2003; Kemmerling et al., 2007; Shan et al., 2008), have also been implicated in plant defense, but their significance has been less well studied (Bari & Jones, 2009). An ever-growing body of evidence reveals the existence of extensive crosstalk between hormonal pathways, which provides plants with the capability to fine-tune their defense against a particular lifestyle of pathogen (Robert-Seilaniantz et al., 2011). One of the best characterized examples of defense-related hormonal crosstalk is the antagonistic interaction between the SA-signaled resistance to a biotroph and the JA-signaled resistance to a necrotroph (Koornneef & Pieterse, 2008). However, crosstalk between the so-called ‘defense hormones’ and ‘growth hormones’ during plant defense is still largely unknown.

As a growth regulator, auxin is involved in almost every aspect of plant growth and development (Friml et al., 2002a,b, 2003; Benkova et al., 2003; Friml, 2003). Recent studies have provided new insights into the role of auxin in plant defense. Similar to SA and JA, auxin appears to differentially affect resistance to different pathogen groups. For example, as a virulence strategy, many biotrophic pathogens have evolved mechanisms to disturb host defense by producing auxin-like molecules or directly impact the auxin pathway of the host plant (O’Donnell et al., 2003; Navarro, 2006; Wang et al., 2007; Kazan & Manners, 2009; Kidd et al., 2011). In response, host plants could employ hormone crosstalk as a direct defense mechanism to counteract pathogen-triggered perturbation of auxin signaling. Indeed, it was shown that inhibition of auxin signaling is part of the SA-signaled host defense to biotrophic pathogens (Navarro, 2006; Wang et al., 2007). These studies demonstrated that SA does not affect auxin biosynthesis, but particularly represses the expression of auxin receptor genes TIR1/AFBs (TRANSPORT INHIBITOR RESPONSE 1/AUXIN-SIGNALING F-BOX PROTEINs) to reduce the auxin response (Navarro, 2006; Wang et al., 2007).

In the context that SA-signaled resistance to biotrophs and JA-signaled resistance to necrotrophs can compromise one another, and that SA represses the auxin response during plant defense against biotrophic pathogens, it is of interest to investigate how JA may modulate the auxin pathway during plant defense against necrotrophic pathogens, in which JA plays a dominant role. Several studies have suggested that, in contrast to the well-recognized antagonistic crosstalk between SA and auxin during plant resistance to biotrophic pathogens, JA and auxin may interact positively in plant resistance to necrotrophic pathogens (Kazan & Manners, 2009). In this study, we investigate the role of auxin in the interaction between Arabidopsis thaliana plants and their necrotrophic pathogen Alternaria brassicicola. Given that the JA pathway plays a dominant role in regulating resistance of A. thaliana plants to A. brassicicola (Glazebrook, 2005), we pay special attention to the crosstalk between JA and auxin during this process. Alternaria brassicicola infection activates the transcription of a battery of auxin biosynthetic genes and therefore elevates auxin biosynthesis of host plants. Alternaria brassicicola infection also reduces protein levels of the PIN-FORMED (PIN) family of auxin transporters and therefore reduces auxin transport capacities. We show that the above-described effects of A. brassicicola infection together lead to an enhanced auxin response in the host plants. Our results provide molecular evidence supporting the hypothesis that JA and auxin interact positively in regulating plant resistance to necrotrophic pathogens.

Materials and Methods

Plant material and growth conditions

The Arabidopsis thaliana (L.) Heynh mutants and transgenic lines used in the study are in the Columbia (Col-0) background. Some of the plant material used has been previously described: asa1-1 (ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1), coi1-1 (CORONATINE INSENSITIVE 1), coi1-2, DR5::GUS (Sun et al., 2009), cyp79b2/b3 (CYTOCHROME P450, FAMILY 79, SUBFAMILY B, POLYPEPTIDE 2/3) (Zhao et al., 2002), axr2-1 (AUXIN RESISTANT 2) (Nagpal et al., 2000), doc1-1 (DARK OVER-EXPRESSION OF CAB 1) (Gil et al., 2001), PIN1::PIN1-GFP (Benkova et al., 2003), PIN2/3/4/7::PIN2/3/4/7-GFP (Blilou et al., 2005), AUX1::AUX1-YFP (Swarup et al., 2004), PIN1::GUS, PIN2::GUS (Friml et al., 2003), PIN3::GUS (Friml et al., 2002b), PIN4::GUS (Friml et al., 2002a), PIN7::GUS (Friml et al., 2003), AUX1::GUS (Marchant et al., 2002), HS::AXR3NT-GUS (Gray et al., 2001) and IAA28::IAA28-myc (Strader et al., 2008).

Seeds were surface-sterilized for 15 min in 10% bleach, washed four times with sterile water, and plated on half-strength Murashige and Skoog (MS) medium. Plants were stratified at 4°C for 2 d in darkness and then transferred to a phytotrone set at 22°C with a 16 h : 8 h, light : dark photoperiod (light intensity 120 μmol m−2 s−1).

Introduction of IAA28::IAA28-myc into asa1-1 mutants

To generate the IAA28::IAA28-myc transgenic line in the asa1-1 mutant background, a homozygous asa1-1 plant was crossed to a transgenic line harboring the IAA28::IAA28-myc construct to produce an F2 population. Putative IAA28::IAA28-myc/asa1-1 plants, which were homozygous for the asa1-1 mutation as well as the IAA28::IAA28-myc construct, were identified in F2 and then re-tested in F3 (i.e. in F3, 100% of seedlings showed no lateral root formation in the presence of 20 μM MeJA; 100% of seedlings showed uniform kanamycin resistance).

Pathogen treatments of plants

The A. brassicicola infection assays were performed as described previously, with slight modifications (Mengiste, 2003). Alternaria brassicicola strain MUCL20297 was grown on potato dextrose agar for 12 d, and spores were suspended in distilled water. The fungal spore density was adjusted to 5 × 105 spores ml−1. For treatments of plants in petri dishes, 4 ml of spore suspension was sprayed onto seedlings in each petri dish. After incubation for different times, the samples were taken for analysis. To test the susceptibility of plants to A. brassicicola infection, we followed the method described previously (Veronese et al., 2006). The disease assay was performed with detached leaves of 4-wk-old plants in petri dishes containing 0.8% agar to maintain high humidity. A single 5-μl spore suspension was deposited on each detached leaf. Inoculated leaves were kept in a phytotrone set at 22°C with a 16 h : 8 h light : dark photoperiod (light intensity 120 μmol m−2 s−1). After incubation for 4 d, average lesion size and disease severity were analyzed to evaluate susceptibility. Disease severity was represented as the percentage of leaves showing different ranges of lesion size. Pathogen growth was evaluated as cutA/iASK (CUTINASE A/ARABIDOPSIS THALIANA SHAGGY-RELATED KINASE 11) using quantitative PCR (qPCR) according to the method previously described (Gachon & Saindrenan, 2004). For each sample, 10 leaves with visible lesions were collected 2 d after inoculation for DNA extraction and qPCR analysis. For treatments with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), 12-d-old seedlings were sprayed with 4 ml of bacterial suspension (108 cfu ml−1) in 10 mM MgCl2. Samples were taken for analysis after incubation for 36 h.

GUS staining and quantification

Histochemical staining for GUS activity in transgenic plants was performed as described previously (Jefferson et al., 1987). Briefly, whole seedlings were immersed in GUS staining buffer (100 mM sodium phosphate, pH 7.0, 0.1 mM EDTA, 0.5 mM ferricyanide, 0.5 mM ferrocyanide, 0.1% Triton X-100, and 1 mM X-Gluc), and were incubated at 37°C from 30 min to 2 h. Chlorophyll was cleared from plant tissues by immersing them in 70% ethanol. Individual representative seedlings were photographed. Quantification of GUS activity was performed using the 4-MUG (4-Methylumbelliferyl-β-D-glucuronide) assay as described previously (Cao et al., 1994).

Quantitative reverse transcription–polymerase chain reaction (qRT-PCR)

RNA isolation and quantitative RT-PCR were performed as previously described (Sun et al., 2009). The primers used in the qRT-PCR are listed in Supporting Information Table S1.

IAA measurement

For quantification of the free IAA concentration, the following method was used. Briefly, 200 mg of plant tissues was homogenized and extracted for 24 h with cold methanol after the deuterium-labeled internal standard 2H2-IAA (CDN Isotopes Inc., Quebec, Canada) had been added. Purification was performed on an Oasis Max solid phase extract cartridge (150 mg/6 cm3; Waters, Massachusetts, USA); IAA and 2H2-IAA were eluted with 5% formic acid in methanol (v/v). The eluent was dried with nitrogen gas and reconstituted in water/methanol (20 : 80, v/v), then subjected to a UPLC-MS/MS system consisting of an Acquity Ultra Performance Liquid Chromatography (Acquity UPLC; Waters) and a triple quadruple tandem mass spectrometer (Quattro Premier XE; Waters). At least three biological replicates were analyzed for each treatment.

Confocal laser microscopy analysis

The GFP and yellow fluorescent protein (YFP) fluorescences were imaged under a Leica confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) according to our previous report (Sun et al., 2011).

Polar auxin transport assay

Polar auxin transport in roots was assayed as previously described, with minor modifications (Lewis & Muday, 2009). Briefly, 5-d-old seedlings grown vertically on plates were transferred to medium containing 50 μM JA for 16 h. Then seedlings were transferred to half-strength MS medium and the apical 1-cm ends of the roots were cut for analysis. A small volume of MS medium containing 1.25% agar and 100 nM 3H-IAA was used for droplet preparation. For basipetal transport, agar droplets containing 3H-IAA were placed onto the root tips and incubated for 6 h in the dark at room temperature. For each sample, 15 root segments of 5-mm length from the base ends were collected into 2.0 ml of scintillation fluid, and the radioactivity was determined using a Perkin Elmer (Massachusetts, USA) 1450 Microbeta scintillation counter. For root apical transport analysis, the droplets were placed onto the base ends and incubated for 12 h in the dark at room temperature. For each sample, 15 root segments of 5-mm length from the root tips were placed into 2.0 ml of scintillation fluid, and the radioactivity was determined as described above.

Western blot

Ten-day-old IAA28::IAA28-myc seedlings in the Col-0 or asa1-1 background were sprayed with 4 ml of spore suspension (5 × 105 spores ml−1) of A. brassicicola. Samples were harvested after incubation for different times. Total proteins were extracted from whole seedlings using NP-40 buffer. Protein samples were quantified using the Bradford method. Approximately 100 μg of protein was heated with loading buffer for 5 min at 100°C and subjected to 10% SDS-PAGE. Electrophoresed proteins were transferred to PVDF membranes following standard protocols. Membranes were incubated with anti-myc antibodies (1 : 2000; Abmart, Shanghai, China) and then with goat radish peroxidase-conjugated secondary antibodies (1 : 4000; Abmart) for the detection of immunoreactive bands with the ECL kit from Amersham Biosciences.

Results

Auxin-related mutants are more susceptible to A. brassicicola infection than wild type

Repression of auxin signaling by the SA pathway was recently shown to contribute to plant resistance to biotrophic pathogens (Navarro, 2006; Wang et al., 2007). To determine how JA may interact with auxin in regulating plant defense responses to necrotrophic pathogens, we infected a series of auxin-related A. thaliana mutants with A. brassicicola, a model fungus with a typical necrotrophic lifestyle (Glazebrook, 2005). Our pathogen infection assays showed that, while the wild type (WT) Col-0 plants were resistant to A. brassicicola, the coi1-2 mutants, which contain a point mutation of the jasmonate receptor gene COI1 (Xu et al., 2002), were highly susceptible to this pathogen (Fig. 1). These results are consistent with previous observations showing that the COI1-dependent JA signaling pathway is required for defense responses of A. thaliana plants to A. brassicicola (Penninckx et al., 1996, 1998; Glazebrook, 2005). Interestingly, the asa1-1 (Sun et al., 2009) and cyp79b2 cyp79b3 (Zhao et al., 2002) mutants, which have been shown to be defective in auxin biosynthesis, were more susceptible to A. brassicicola infection than WT (Fig. 1a–d). The doc1-1 mutant, which harbors a mutation in the calossin-like protein BIG that is required for polar auxin transport (Gil et al., 2001), was also more susceptible to A. brassicicola than WT (Fig. 1a–d). Similar to the auxin biosynthesis- and transport-related mutants, the axr2-1 mutant, which contains a point mutation that stabilizes the auxin signaling repressor AXR2/IAA7 (Nagpal et al., 2000), was more susceptible to A. brassicicola infection than WT (Fig. 1a–d). These results indicate that the auxin pathway plays a positive role in regulating plant resistance to A. brassicicola. In the context that the JA pathway plays a dominant role in regulating plant defense responses to A. brassicicola (Glazebrook, 2005), our results support the hypothesis that JA and auxin interact positively in regulating plant defense responses to necrotrophic pathogens (Kazan & Manners, 2009).

Figure 1.

Auxin-related mutants are more susceptible to Alternaria brassicicola infection than wild-type Arabidopsis thaliana plants. (a) Leaves of drop-inoculated Col-0 and auxin-related mutant plants 4 d after infection. Leaves from 4-wk-old plants were detached and inoculated with a 5-μl drop of A. brassicicola spore suspension (5 × 105 spores ml−1). Shown are representative images 4 d after infection. The experiment was repeated three times with similar results. (b) Graphical representation of disease severity caused by A. brassicicola infection 4 d post inoculation. Disease severity is represented as the percentage of leaves showing different ranges of lesion size. Data values represent one of three independent experiments that gave similar results. (c) Average lesion size measured 4 d after infection. Data values represent one of three independent experiments with similar results. Error bars represent SD. *, P < 0.05; **, P < 0.01; Student’s t-test. (d) Pathogen growth evaluated as cutA/iASK (CUTINASE A/ARABIDOPSIS THALIANA SHAGGY-RELATED KINASE 11) in the detached leaves with different genotypes 2 d after drop-inoculation. Leaves with visible lesions were collected 2 d after drop-inoculation for DNA extraction and cutA/iASK was measured to evaluate pathogen growth conditions in different genotypes using qPCR. Data points represent averages ± SD from triplicate reactions. The experiment was repeated three times with similar results.

Alternaria brassicicola infection activates the expression of auxin biosynthetic genes and elevates free IAA concentrations

The finding that auxin biosynthesis mutants are more susceptible to A. brassicicola than WT suggests that, as a defense mechanism, A. thaliana plants may activate auxin biosynthesis in response to A. brassicicola infection. To test this hypothesis, we monitored the expression of several auxin biosynthetic genes following A. brassicicola infection. As shown in Fig. 2(a–d), A. brassicicola infection substantially increased the transcript levels of ASA1, ASB1 (ANTHRANILATE SYNTHASE BETA SUBUNIT 1), CYP79B2 and CYP79B3 in a time-dependent manner. Given the established role of ASA1 (Sun et al., 2009), ASB1 (Stepanova et al., 2005) and CYP79B2/CYP79B3 (Zhao et al., 2002) in auxin biosynthesis, we reasoned that A. brassicola infection could lead to an elevation of the auxin concentrations of host plants. Indeed, our auxin measurement assays revealed that A. brassicicola infection significantly increased the free IAA concentration as early as 24 h after inoculation in our time course experiment (Fig. S1), and the increase became dramatic at 36 h after infection (Fig. 2e). Significantly, the A. brassicicola-induced increase in free IAA concentrations was largely abolished by the asa1-1 mutation, implying that A. brassicicola-induced IAA biosynthesis largely depends on the ASA1 gene (Fig. 2e). Our recent work demonstrated that the defense hormone JA, which plays a dominant role in plant resistance to A. brassicicola infection, was also able to activate the expression of ASA1, ASB1, CYP79B1 and CYP79B2 and, as a result, led to elevated free IAA concentrations (Sun et al., 2009). We speculate that the JA pathway may be important for A. brassicicola-induced elevation of free IAA concentrations in host plants. Indeed, the A. brassicicola-induced increase of free IAA concentrations was substantially impaired in the coi1-1 mutant (Fig. 2e), confirming that the JA pathway plays an important role in the A. brassicicola-induced increase of free IAA concentrations in the host plants. The fact that the A. brassicicola-induced increase of free IAA concentrations was not completely blocked by the coi1-1 mutation suggests the existence of COI1-independent responses in mediating A. brassicicola-induced auxin elevation. Together, these results support the hypothesis that activation of auxin biosynthesis is part of the JA-mediated defense response to the necrotrophic pathogen A. brassicicola. By contrast, our parallel experiments indicated that infection with the hemi-biotrophic pathogen Pst DC3000 or treatment with SA did not significantly increase free IAA concentrations (Fig. S2).

Figure 2.

Alternaria brassicicola infection activates the expression of auxin biosynthetic genes and elevates free IAA concentrations in Arabidopsis thaliana. (a–d) Up-regulation of the expression of auxin biosynthesis genes in plants upon pathogen infection. Ten-day-old seedlings were sprayed with a 4-ml spore suspension (5 × 105 spores ml−1) of A. brassicicola. Samples were harvested at the different time-points for RNA extraction and qRT-PCR analysis. The transcript levels of the target genes were normalized to the ACTIN7 expression, and error bars represent the SD of triplicate reactions. The experiment was repeated three times with similar results. hpi, hours post-inoculation. (e) Increase of free IAA concentrations upon pathogen infection was partly dependent on ASA1 (ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1) and COI1 (CORONATINE INSENSITIVE 1). Twelve-day-old seedlings were infected with A. brassicicola (closed bars) or mock inoculated (open bars) for 36 h and free IAA concentrations were measured. Data points represent averages ± SD from three experiments. This experiment was conducted for two biological replicates, yielding similar results. *, P < 0.05; **, P < 0.01; Student’s t-test.

JA treatment down-regulates PIN protein levels and reduces polar auxin transport capacities

Unique among phytohormones, auxin is transported from cell to cell in a directional manner to create an asymmetric distribution in specific tissues. Polar auxin transport (PAT) mainly involves the PIN-FORMED (PIN) family of auxin efflux carriers and the AUX1/LAX (AUXIN RESISTANT 1/LIKE AUX1) family of auxin influx carriers. Polar localization and plasma membrane abundance of PIN proteins determine the direction and rate of intercellular auxin flow (Vanneste & Friml, 2009). In this context, we asked whether A. brassicicola infection affects the general PAT capacities of host plants. Using transgenic plants containing the promoters of PINs combined with PIN-GFP protein fusions, we examined whether A. brassicicola infection affects the levels of PIN-GFP fusions. Unexpectedly, A. brassicicola infection led to the generation of strong background fluorescence in roots of host plants, which made observation impossible. To circumvent this difficulty, we instead examined the effect of JA on the expression of PIN genes at both the transcription and protein levels. Using transgenic plants containing the promoter of PIN genes fused with the GUS reporter, we showed that JA up-regulates the expression of PIN1::GUS, PIN2::GUS, PIN3::GUS and PIN4::GUS, but down-regulates the expression of PIN7::GUS (Fig. S3a). These results, together with the qRT-PCR assays (Fig. S3b), revealed that JA up-regulates the transcription of PIN1/2/3/4, but down-regulates the transcription of PIN7. However, we observed that JA substantially reduces the levels of PIN-GFP protein fusions including PIN1::PIN1-GFP, PIN2::PIN2-GFP, PIN3::PIN3-GFP, PIN4::PIN4-GFP and PIN7::PIN7-GFP, suggesting a general reduction effect of JA on PIN protein levels (Fig. 3a,b). Interestingly, however, we did not find obvious JA-related effects on the protein levels of the AUX1::AUX1-YFP fusion, even though JA substantially up-regulated AUX1::GUS levels (Figs 3a,b, S3).

Figure 3.

JA treatment down-regulates PIN-FORMED (PIN) protein levels and reduces polar auxin transport capacities in Arabidopsis thaliana. (a) Effect of JA on the expression of PIN1::PIN1-GFP, PIN2::PIN2-GFP, PIN3::PIN3-GFP, PIN4::PIN4-GFP, PIN7::PIN7-GFP and AUX1::AUX1-YFP. Five-day-old seedlings containing the indicated markers were transferred to medium without (MS) or with 20 μM JA (JA) for 1 d and GFP/YFP expression was monitored. Images shown are representative of at least three independent experiments. Bars, 50 μm. (b) Quantification of GFP/YFP fluorescence by image analysis of confocal sections as described in (a). Open bars, control; closed bars, JA. Data shown are the mean ± SD for 20 seedlings and are representative of at least three independent experiments. (c) JA reduces the relative acropetal auxin transport capacities in roots. (d) JA reduces the relative basipetal auxin transport capacities in roots. For (c) and (d), 5-d-old seedlings were transferred to medium without (control) or with 50 μM JA (JA) for 16 h and then root segments of 1-cm length were used for auxin transport analysis. Each sample included 15 root segments and at least three samples were examined for each independent experiment. Data shown are mean ± SD and are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; Student’s t-test.

To determine whether the JA-mediated reduction of PIN protein levels may eventually affect the root PAT capacities, we examined the effect of JA on PAT in both acropetal and basipetal directions, two distinct auxin transport flows spatially separated in the root (Tanaka et al., 2006). Measurement of the transportation of locally applied 3H-IAA indicated that, upon JA treatment, a c. 40% reduction of auxin transport capacities occurred in both acropetal and basipetal directions (Fig. 3c,d). Together, our results indicate that JA generally reduces the levels of PIN proteins and therefore negatively modulates auxin transport in the root. It is worth noting that, because of the above-described technical difficulties, we failed to determine the impact of pathogen infection on PIN protein levels. Considering the dominant role of JA in regulating plant resistance, it is reasonable to envisage that A. brassicicola infection also modulates PIN proteins and PAT capacities in a similar manner to JA.

Alternaria brassicicola infection affects the asymmetric auxin distribution in planta

An important feature of auxin action is its spatiotemporal asymmetric distribution (also known as gradient distribution), the formation and maintenance of which depend on local auxin biosynthesis (Zhao, 2010) and directional intercellular transport (Vanneste & Friml, 2009). The effects of A. brassicicola on auxin biosynthesis and PAT could eventually lead to altered auxin distribution in host plants. Using a transgenic plant containing the auxin-responsive DR5::GUS construct as a reporter, we found that the DR5::GUS expression was greatly enhanced and expanded in both leaves and roots of pathogen-inoculated plants (Fig. 4a). Quantification of GUS activity in whole seedlings gave similar results (Fig. 4b). Consistently, expression of the auxin-responsive genes, including GH3-like and IAA2, was also significantly increased after pathogen inoculation (Fig. 4c,d).

Figure 4.

Alternaria brassicicola infection enhances the Arabidopsis thaliana auxin response and alters the asymmetric auxin distribution in planta. (a) Enhanced expression of DR5::GUS reporter upon pathogen infection. Ten-day-old DR5::GUS seedlings were either mock-treated with water for 48 h or infected with A. brassicicola for different times and GUS staining was then performed. The experiment was repeated three times with similar results, and representative images are shown. hpi, hours post-inoculation. Bars, 2 mm. (b) Quantification of GUS activity after pathogen infection. Data points represent mean ± SD from triplicate reactions. The experiment was repeated three times with similar results. (c, d) Up-regulation of the expression of auxin-responsive genes GH3-like and IAA2 after infection with A. brassicicola for 48 h. The transcript levels of GH3-like and IAA2 were normalized to the ACTIN7 expression, and error bars represent the SD of triplicate reactions. The experiment was repeated three times with similar results.

Alternaria brassicicola infection activates auxin responses of host plants

Auxin exerts its transcriptional regulation through Skp1-Cullin-F-box (SCF)TIR1/AFBs-mediated ubiquitination and degradation of auxin signaling repressor AUX/IAA proteins (Gray et al., 2001). To investigate whether A. brassicicola infection may eventually lead to increased auxin responses of host plants, we examined the degradation efficiency of AUX/IAA repressors in response to pathogen infection. For these experiments, we used the reporter HS::AXR3NT-GUS, which contains a fusion between the coding sequences of the amino terminus (NT) of the auxin signaling repressor IAA17 and the GUS-encoding uidA gene driven by a heat shock-inducible promoter (HS) (Gray et al., 2001). As shown in Fig. 5(a–e), the AXR3NT-GUS fusion protein clearly accumulated after heat shock, but such accumulation was greatly reduced in the seedlings pre-infected with A. brassicicola for 24 h. qRT-PCR assays showed that transcriptional induction of GUS after heat shock was not significantly affected by the pathogen infection (Fig. 5f). These results indicated that infection with A. brassicicola did not affect the expression of the AXR3NT-GUS fusion at the transcription level, but strongly enhanced the degradation of the AXR3NT-GUS fusion protein, suggesting that this pathogen indeed activates auxin signaling of infected host plants. Next, using transgenic plants carrying IAA28-myc, a c-myc epitope-tagged version of the AUX/IAA repressor IAA28 (Strader et al., 2008), we examined accumulation of the IAA28-myc fusion proteins in response to pathogen infection. As expected, IAA28-myc levels were drastically reduced upon pathogen infection in WT plants (Figs 5g, S4), suggesting an enhancement of auxin response in these plants. Importantly, pathogen-induced IAA28-myc degradation was greatly reduced in asa1-1 mutants (Fig. 5g), suggesting that A. brassicicola infection-induced enhancement of auxin responses greatly depends on ASA1-mediated auxin biosynthesis.

Figure 5.

Degradation of AUX/IAAs proteins in Arabidopsis thaliana upon Alternaria brassicicola infection. (a–d) Representative images showing the degradation of AXR3NT-GUS after pathogen infection. Ten-day-old seedlings containing HS::AXR3NT-GUS were either mock-treated with water or sprayed with 4 ml of spore suspension (5 × 105 spores ml−1). After 24 h of incubation, GUS activity was determined immediately or after 2 h of heat shock at 37°C. Shown are representative images of three independent experiments. Bars, 5 mm. (e) Quantification of the GUS activity in (a–d). Open bars, no heat shock; closed bars, heat shock. Data points represent mean ± SD from three triplicate reactions. The experiment was repeated three times with similar results. (f) Analysis of the GUS transcript levels in (a–d) with qRT-PCR. The transcript levels of GUS were normalized to the ACTIN7 expression, and error bars represent the SD of triplicate reactions. The experiment was repeated three times with similar results. Bars as in (e). (g) Degradation of IAA28-myc upon pathogen infection was partly dependent on ASA1 (ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1). Ten-day-old IAA28::IAA28-myc seedlings in the Col-0 or asa1-1 background were infected with A. brassicicola for different time periods. Proteins from whole seedlings were extracted for immunoblotting using anti-myc antibodies. The experiments were repeated three times with similar results.

Auxin enhances JA-induced expression of defense-related genes

It has long been reported that A. brassicicola infection leads to increased JA concentrations in A. thaliana (Penninckx et al., 1996, 1998). We showed here that A. brassicicola infection also leads to increased free IAA concentrations (Fig. 2e). Concomitant elevation of JA and auxin concentrations by A. brassicicola infection leads us to the hypothesis that these two hormones might interact with each other to contribute to plant resistance. To test this hypothesis, we examined the expression of several necrotrophic defensive marker genes after seedlings had been treated with 50 μM MeJA, 25 μM IAA, or both. As shown in Fig. 6(a), MeJA treatment led to a 20-fold increase of PDF1.2 expression, which reached a maximum 36 h after treatment; IAA alone did not have a significant effect on PDF1.2 expression; however, when both were applied together, the induction of PDF1.2 expression was greatly enhanced. Similarly, auxin substantially enhanced the induction effect of MeJA on the expression of HEL (hevein-like protein or PR4), another JA-inducible defense-related gene in A. thaliana (Fig. 6b). These results suggest that enhancement of JA-dependent defense may be part of the auxin-mediated defense mechanism involved in resistance to necrotrophic pathogens.

Figure 6.

Auxin enhances JA-induced expression of defense-related genes in Arabidopsis thaliana. Twelve-day-old seedlings were respectively treated with 50 μM MeJA (blue lines), 25 μM IAA (black lines), or 50 μM MeJA and 25 μM IAA together (red lines) for different times. Samples were taken at the indicated time to determine the expression level of (a) PDF1.2 (PLANT DEFENSIN 1.2) and (b) HEL (HEVEIN-LIKE) using qRT-PCR. The transcript levels of PDF1.2 and HEL were normalized to the ACTIN7 expression, and error bars represent the SD of triplicate reactions. The experiment was repeated three times with similar results.

Discussion

Phytohormones play important roles in regulating plant resistance to various biotic stresses. Recent studies indicate that, in addition to the well-recognized defense-related hormones (SA, JA and ET), growth-related hormones such as auxin, ABA, GA, CK and BR are also implicated in plant defense responses to pathogen infections (Bari & Jones, 2009). Accumulating evidence has revealed that, like SA and JA, the growth hormone auxin differentially affects plant resistance to different lifestyles of pathogens (reviewed in Kazan & Manners, 2009). Recent evidence has revealed an antagonistic crosstalk between SA and auxin in regulating plant resistance to biotrophs (Navarro, 2006; Chen et al., 2007; Wang et al., 2007). It is generally considered that down-regulation of auxin signaling is part of the SA-mediated disease-resistance mechanism. Recent molecular genetic studies have advanced our understanding of the molecular mechanisms of plant resistance to necrotrophs, in which JA plays a dominant role (Mengiste, 2003; Veronese et al., 2006; Kidd et al., 2009; Laluk et al., 2011; Kazan & Manners, 2011). Importantly, emerging evidence reveals that, in addition to JA, auxin is also involved in plant defense against necrotrophs (reviewed in Kazan & Manners, 2009). For example, the axr1 mutant, which harbors a mutation of a shared component of auxin and JA signaling, was more susceptible than WT to the necrotrophic pathogen Pythium irregulare (Tiryaki & Staswick, 2002). It was shown 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 Plectosphaerella cucumerina and Botrytis cinerea (Llorente et al., 2008). Together, these results support the hypothesis that JA and auxin interact positively in regulating plant resistance to necrotrophs (reviewed in Kazan & Manners, 2009). However, the molecular details underlying crosstalk between JA and auxin in regulating plant defense against necrotrophic pathogens remain unexplored.

Here, we address this question by investigating the interaction between A. thaliana and A. brassicicola, a pathogen with a necrotrophic lifestyle. We show that, similar to the JA signaling mutant coi1-2, the asa1-1 and cyp79b2/b3 mutants, which have been shown to be defective in auxin biosynthesis, are generally more susceptible than WT to A. brassicicola infection. These results support the hypothesis that auxin biosynthesis is important for resistance to necrotrophs. It is noteworthy that, in addition to affecting auxin biosynthesis, mutations of ASA1 (Thomma et al., 1999; Zhao, 2010; Stotz et al., 2011) and CYP79B2/CYP79B3 (Thomma et al., 1999; Glawischnig et al., 2004; Nafisi et al., 2007; Stotz et al., 2011) also impair the biosynthesis of camalexin and indolic glucosinolates (IGs), which are important for plant defense against pathogens. We therefore could not rule out the possibility that the susceptibility of asa1-1 and cyp79b2/b3 partially results from defective camalexin and IG accumulation in these mutants. Further evidence supporting the hypothesis that the auxin pathway is important for resistance to A. brassicicola came from our finding that the doc1-1 and axr2-1 mutants, which are defective in auxin transport and signaling, respectively, are more susceptible than WT to A. brassicicola infection. Together, these results are consistent with the hypothesis that JA interacts positively with auxin during plant resistance to necrotrophs.

In the context that SA has a minor, if any, effect on auxin biosynthesis, but selectively represses the expression of the auxin receptor gene during plant resistance to biotrophs (Navarro, 2006; Wang et al., 2007), we designed a set of experiments to examine the effects of JA on different aspects of the auxin pathway (i.e. biosynthesis, transport, distribution and signaling) during plant resistance to A. brassicicola infection. First, A. brassicicola infection elevates free IAA concentrations of host plants through activating the expression of a battery of auxin biosynthetic genes; Secondly, JA differentially modulates the expression of PIN genes at the transcription and the protein levels and, eventually, alters the general PAT capacities of host plants; Thirdly, A. brassicicola infection affects the asymmetric auxin distribution in planta. Fourthly, A. brassicicola infection activates auxin responses of host plants, as evidenced by accelerated degradation of AUX/IAAs of infected plants. Finally, we showed that auxin substantially enhanced JA-induced expression of defense-related genes. Together, these results substantiate the proposal that JA and auxin interact positively in regulating plant resistance to necrotrophs and support the possibility that enhancement of JA-dependent disease resistance may be part of the auxin-mediated defense mechanism against necrotrophic pathogens. However, in agreement with a previous report (Llorente et al., 2008), auxin-related mutants did not show compromised PDF1.2 expression after A. brassicicola infection (Fig. S5), suggesting that their susceptibility is unlikely to be accounted for simply by repression of the JA defense pathway. It is possible that auxin contributes to necrotrophic resistance also through some undiscovered mechanisms that are independent of the JA pathway.

Of interest is our finding that A. brassicicola infection leads to degradation of AUX/IAA proteins, indicating that A. brassicicola infection activates the auxin signal transduction pathway. This, together with the finding that the axr2-1 mutant is more susceptible than WT to A. brassicicola infection, supports the hypothesis that SCF ubiquitination pathway-mediated degradation of AUX/IAA proteins contributes to plant resistance to A. brassicicola. These results are consistent with a previous observation showing that SCF-mediated protein degradation is important for plant resistance to the necrotrophic pathogens P. cucumerina and B. cinerea. Alternatively, A. brassicicola infection-induced protein turnover could also be explained by the fact that the JA- and auxin-mediated protein degradation pathways are directly connected through AXR1, a shared component of SCFTIR1 and SCFCOI1 (Tiryaki & Staswick, 2002). However, our data showing that A. brassicicola-induced degradation of IAA28-myc is greatly reduced in the asa1-1 mutant (Fig. 5g) do not favor this hypothesis. In summary, our data support the hypothesis that, in response to A. brassicicola infection, JA promotes ASA1-dependent auxin biosynthesis which, in turn, activates auxin signaling and contributes to plant resistance.

Acknowledgements

We thank Mark Estelle, Bonnie Bartel and Daoxin Xie for kindly providing seeds used in this study. This work was supported by grants from the Beijing Natural Science Foundation (Grant number 6120001), the Ministry of Agriculture of China (2011ZX08009-003-001) and the National Natural Science Foundation of China (31030006, 30970255, 31170260).

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