The Arabidopsis defensin gene, AtPDF1.1, mediates defence against Pectobacterium carotovorum subsp. carotovorum via an iron-withholding defence system

Hsiao, Pao-Yuan; Cheng, Chiu-Ping; Koh, Kah Wee; Chan, Ming-Tsair

doi:10.1038/s41598-017-08497-7

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Published: 23 August 2017

The Arabidopsis defensin gene, AtPDF1.1, mediates defence against Pectobacterium carotovorum subsp. carotovorum via an iron-withholding defence system

Pao-Yuan Hsiao^1,2,
Chiu-Ping Cheng¹,
Kah Wee Koh¹ &
…
Ming-Tsair Chan^1,2,3

Scientific Reports volume 7, Article number: 9175 (2017) Cite this article

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Abstract

Plant defensins (PDFs) are cysteine-rich peptides that have a range of biological functions, including defence against fungal pathogens. However, little is known about their role in defence against bacteria. In this study, we showed that the protein encoded by ARABIDOPSIS THALIANA PLANT DEFENSIN TYPE 1.1 (AtPDF1.1) is a secreted protein that can chelate apoplastic iron. Transcripts of AtPDF1.1 were induced in both systemic non-infected leaves of Arabidopsis thaliana plants and those infected with the necrotrophic bacterium Pectobacterium carotovorum subsp. carotovorum (Pcc). The expression levels of AtPDF1.1 with correct subcellular localization in transgenic A. thaliana plants were positively correlated with tolerance to Pcc, suggesting its involvement in the defence against this bacterium. Expression analysis of genes associated with iron homeostasis/deficiency and hormone signalling indicated that the increased sequestration of iron by apoplastic AtPDF1.1 overexpression perturbs iron homeostasis in leaves and consequently activates an iron-deficiency-mediated response in roots via the ethylene signalling pathway. This in turn triggers ethylene-mediated signalling in systemic leaves, which is involved in suppressing the infection of necrotrophic pathogens. These findings provide new insight into the key functions of plant defensins in limiting the infection by the necrotrophic bacterium Pcc via an iron-deficiency-mediated defence response.

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Introduction

Organisms have evolved multiple defence strategies for protection against pathogen attack, many of which are triggered by environmental cues. One such strategy is the innate immune response, which utilizes Toll-like or pattern-recognition receptors to perceive specific pathogen-associated molecular patterns (PAMPs) that are derived from pathogens to trigger the defence signalling pathways. In plants, this process involves the activation of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling¹ pathways and the biosynthesis of defence molecules, such as antimicrobial peptides (AMPs)^{2, 3}, which cause membrane disruption, pore formation, and lead to cell lysis and death⁴. Based on sequence similarity, disulphide bond patterns and characteristic structural folds, AMPs can be categorized into several groups, including defensins, thionins, hevein-like peptides, knottin, α-hairpinin and lipid transfer proteins (LTPs)^5,6,7.

Defensins constitute a well-studied group of relatively small AMPs (34–54 amino acids) with cysteine-rich motifs, which are present in a taxonomically broad range of multicellular eukaryotic organisms⁸. Unlike animal defensins which mainly possess anti-bacterial properties, plant defensins (PDFs), which have a highly conserved cysteine composition but are highly variable in their primary sequence⁸, have primarily been associated with protection against fungi^{5, 8, 9} and heavy metal stress^{10, 11}. In contrast to the well-established actions of plant PDFs in limiting fungal infection, little is known about their roles and modes of action against bacteria⁵.

To date, more than 300 defensin-like (DEFL) genes have been identified in the model plant Arabidopsis thaliana ¹². AtPDF1.1 was originally described as encoding a seed-localized protein¹³, but its expression was later reported to be induced in leaves infected with the fungus Blumeria graminis f. sp. hordei ^{14, 15}.

An association between PDFs and metal homeostasis was also indicated by our preliminary data, which showed that AtPDF1.1 expression was induced upon iron overloading but not iron deficiency (Fig. S1B,D). Iron is essential for most living organisms and is involved in many biological processes¹⁶. In plants, intracellular iron is maintained at a very low level and excess iron is sequestered and stored in the apoplast and organelles, including the vacuole and plastid, to reduce iron toxicity¹⁷. Accordingly, iron homeostasis in plants is tightly controlled at the transcriptional and posttranslational levels^{18, 19}. For example, Fe-deficiency-induced transcription factor 1 (FIT1) has been shown to interact with other iron homeostasis responsive transcription factors, such as basic helix-loop-helix 38 and 39 (bHLH38 and bHLH 39), to activate the expression of FERRIC CHELATE REDUCTION OXIDASE 2 (FRO2) and IRON-REGULATED TRANSPORTER 1 (IRT1) in A. thaliana ²⁰.

Iron is also important for bacterial pathogens as it is required for their metabolism, enzymatic functions and pathogenesis. In order to acquire iron, plant pathogens utilize host iron-containing compounds such as heme²¹, transferrin, and lactoferrin, or synthesize siderophores to compete for iron uptake with their hosts²². Moreover, genes associated with immunity and iron homeostasis are also induced in response to treatment with the iron chelators ethylenediamine- di(o-hydroxyphenylacetic acid) (EDDHA) and desferrioxamine (DFO), suggesting that iron sequestration activates an immune response²³. Together these findings demonstrated the existence of a relationship between perturbation of iron distribution and immunity in plants.

Given that AtPDF1.1 confers plant tolerance to fungi and that our preliminary data revealed increased transcript levels of AtPDF1.1 following iron overloading, in this study, we provide evidence that apoplastic AtPDF1.1 perturbs iron homeostasis via iron chelation, resulting in activation of the iron-deficiency-induced ethylene signalling pathway. This response confers protection against infection by Pectobacterium carotovorum subsp. carotovorum (Pcc), a necrotrophic pathogen that causes bacterial soft rot (BSR) and serious production losses of a wide range of economically important crops worldwide²⁴.

Results

Transcript levels of AtPDF1s increase in response to iron treatment and Pcc infection

The overexpression of AhPDF1.1 in A. thaliana and yeast has been shown to enhance tolerance to zinc¹⁰. To evaluate whether PDF1 genes in A. thaliana are similarly involved in zinc response, the expression profiles of the orthologs of AhPDF1.1, namely AtPDF1.1, AtPDF1.2a and AtPDF1.3, were monitored during ZnSO₄ treatment. The results showed that the expression of AtPDF1.2a and AtPDF1.3, but not AtPDF1.1, was upregulated by ZnSO₄ treatment (Fig. S1A), whereas transcript levels of these three AtPDF1 genes were upregulated by treatment with other essential divalent ions, such as FeSO₄ and CuSO₄ (Fig. S1B,C). This suggested that AtPDF1.1 may be involved in iron and copper homeostasis, but not in zinc homeostasis. In contrast to iron overloading, the expression of AtPDF1 gene was not dramatically affected by iron deficient treatment (Fig. S1D).

To evaluate whether these three AtPDF1 genes are involved in defence against bacterial pathogens, their transcriptional profiles were monitored in A. thaliana infected with Pcc. Time-course studies revealed that all three AtPDF1 genes were upregulated at the infection site 1 hour post infection (hpi), with maximum expression occurring at 3 hpi for AtPDF1.2 and AtPDF1.3, and at 4 hpi for AtPDF1.1 (Fig. S2A). Analysis of non-inoculated leaves from infected plants revealed that the transcript levels of AtPDF1.1, but not AtPDF1.2 or AtPDF1.3, were induced systemically at 24 hpi (Fig. S2B). In order to investigate the protein level of AtPDF1.1, the AtPDF1.1:green fluorescent protein fusion protein (AtPDF1.1:GFP) under the control of the AtPDF1.1 promoter was used to distinguish from other native defensins. When transgenic A. thaliana plants expressing AtPDF1.1:GFP were infected with Pcc, maximum protein accumulation was observed at the local site of infection at 3–4 hpi (Fig. S2C). Furthermore, in addition to a protein band of ~36 kDa, which corresponds to the predicted size of the AtPDF1.1-GFP fusion protein, a protein of ~33 kDa was also dramatically accumulated at the local site of infection at 5 hpi and in systemic leaves at 24 hpi (Fig. S2C), suggesting that AtPDF1.1 may have undergone post-translational modification, and AtPDF1.1 expression is induced both locally and systemically upon Pcc infection. Along with this notion, analysis of the predicted AtPDF1.1 protein sequence using SignalP 4.1 software (http://www.cbs.dtu.dk/services/SignalP/) revealed the presence of a putative N-terminal secretory signal peptide (Fig. S3A), suggesting that the AtPDF1.1 protein may be targeted to the secretory pathway^{25, 26}, and the 33-kDa protein is likely resulted from the cleavage of the N-terminal signal. Consistent with this targeting prediction, the first identified plant defensin was reported to be a secreted protein released from germinated radish seeds²⁷.

AtPDF1.1 confers plant tolerance to Pcc infection

Since AtPDF1.1 transcript levels increased in response to Pcc infection (Fig. S2), we then investigated the role of AtPDF1.1 in defending against pathogenic bacteria. Suppression (RNAi) transgenic lines were generated, because AtPDF1.1 expression in the only available AtPDF1.1 T-DNA insertion mutant (SALK_029051) is unaltered compared to the wild-type plant. In addition, to determine whether the cellular localization of AtPDF1.1 is critical for its functions, transgenic plants overexpressing a truncated form of AtPDF1.1, lacking the N-terminal signal peptide (AtPDF1.1ΔSP) and AtPDF1.1 overexpression (OE) were also generated (Fig. S3B). Quantitative PCR (qPCR) analysis verified the success of AtPDF1.1 OE and RNAi in transgenic plants (Fig. S3C). The results showed that, compared with the empty vector (Ev) harbouring control plants, the AtPDF1.1 OE plants exhibited enhanced tolerance to Pcc infection, the AtPDF1.1ΔSP plants exhibited similar disease response, while the AtPDF1.1 RNAi plants exhibited increased susceptibility (Fig. 1). These results indicated that the tolerance to Pcc infection correlates well with the expression level of AtPDF1.1, and that the putative N-terminal secretory signal peptide, which could be a key determinant for the apoplastic location of AtPDF1.1, is crucial for its function in defence against Pcc infection.

AtPDF1.1 is secreted into the apoplast

To determine the subcellular localization of AtPDF1.1, we generated transgenic A. thaliana plants overexpressing GFP fused to the C-terminal of full-length AtPDF1.1-encoding gene (AtPDF1.1:GFP), or a truncated AtPDF1.1-encoding gene lacking either the signal peptide (AtPDF1.1ΔSP:GFP) or the mature protein region (AtPDF1.1(SP only):GFP) (Fig. 2A). A. thaliana lines harbouring the empty expression vector pH7FWG2 (Ev-GFP) were used as a negative control (Fig. 2A). Due to the presence of the ccdB and chloramphenicol-resistance (Cm^R) gene cassette upstream of the GFP-encoding gene in this vector, GFP transcripts are not translated into functional protein. Confocal microscope imaging of the plasmolysed epidermis of the leaves of transgenic plants revealed that the GFP signal was localized in the apoplastic region surrounding the plasma membrane of AtPDF1.1:GFP and AtPDF1.1(SP only):GFP plants (Fig. 2B–D and H–J), while AtPDF1.1ΔSP:GFP plants showed the GFP signal in the cytosol (Fig. 2E–G). We concluded that the mature form of AtPDF1.1 is localized in the apoplast, which is consistent with the localization of other plant defensins^{27, 28}.

To confirm that AtPDF1.1 is secreted, levels of the recombinant fusion protein were measured in the growth medium of suspension cells generated from transgenic plants (Fig. S4A) together with levels in protein lysates from whole seedlings, by western blot analysis with an anti-GFP antibody. Two GFP fusion protein bands were detected in the seedling lysates of AtPDF1.1:GFP and AtPDF1.1(SP only):GFP plants, suggesting that AtPDF1.1 with and without the N-terminal signal peptide were expressed and that AtPDF1.1(SP only):GFP (band 3 and 4) may increase molecular weight via certain post-translational modification (Fig. S4B). In contrast, a single GFP fusion protein band was observed in the protein lysate of AtPDF1.1ΔSP:GFP seedlings. When the proteins in the media of the suspension cells were analysed, only GFP fusion proteins containing the N-terminal signal peptide were detected in the supernatants of AtPDF1.1:GFP and AtPDF1.1(SP only):GFP suspension cell cultures, and no immunoreactive proteins were detected in the growth medium of AtPDF1.1ΔSP:GFP suspension cells (Fig. S4C). These data further suggest that the N-terminal signal peptide is responsible for targeting AtPDF1.1 to the apoplast. Because the apoplast is the initial site for Pcc infection²⁹, and the correct localization of AtPDF1.1 is critical for its function in defence against Pcc infection (Fig. 1), our results are consistent with the idea that apoplastic AtPDF1.1 can interfere with Pcc infection.

AtPDF1.1 localized to the apoplast is involved in iron distribution

The apoplast is a primary infection site for bacterial pathogens of plants, and pathogens such as P. syringae rely on extracellular nutrients³⁰. The iron status of A. thaliana has been reported to be critical for the pathogenicity of the necrotrophic bacterium Dickeya dadantii ³¹. Under host iron limiting conditions, bacteria produce siderophores to enhance their iron uptake³². Since AtPDF1.1 expression is responsive to both iron overloading (Fig. S1B) and Pcc infection (Fig. S2), we hypothesized that AtPDF1.1 may function as an iron chelator to limit the availability of iron for Pcc as part of an iron-withholding defence system³³. To determine whether apoplastic iron affects Pcc pathogenesis, exogenous iron was supplemented to plants and disease response was assessed. The results showed that extra iron supplement to the leaves increased the susceptibility of all transgenic plants to Pcc invasion (Fig. S5), supporting the importance of apoplastic iron for Pcc infection. However, even with extra iron supplement, the AtPDF1.1 OE plants still displayed the lowest susceptibility to Pcc infection, further supporting a positive role of AtPDF1.1 in plant defence against Pcc.

To evaluate whether AtPDF1.1 is involved in chelating/sequestering iron, we first determined whether it has the capacity to bind metal ions. In a metal affinity assay (Fig. S6), AtPDF1.1ΔSP showed binding affinity to iron ions. Since the resins derivatized from iminodiacetic acid exhibited different degrees of affinity to metal ions, with a preference for transition metals, such as cobalt (Co), nickel (Ni), copper (Cu) and zinc (Zn)^34,35,36, we deduced that fewer Fe³⁺-conjugated resins are available for AtPDF1.1ΔSP protein to bind. This may explain the relatively weak protein band detected, and so the intensity of detection signal does not correlate with the binding affinity between the metal ions and AtPDF1.1ΔSP protein. Overall, the data suggest that AtPDF1.1ΔSP protein is able to bind to iron ions.

To further evaluate the iron binding affinity of the AtPDF1.1 protein in planta, transgenic plants were grown under hydroponic conditions and their iron acquisition capabilities were compared. As shown in Fig. 3A, Perls staining revealed a high abundance of iron ions in the intercellular regions of the root epidermis of AtPDF1.1 OE plants, but only weak staining was observed in the Ev, AtPDF1.1ΔSP and AtPDF1.1 RNAi transgenic plants. Furthermore, sections of roots and leaves from transgenic plants were subjected to Perls/diaminobenzidine (DAB)/H₂O₂ staining and then examined by confocal microscopy to spot regions with iron deposits. Consistent with previous studies of A. thaliana wild-type plants³⁷, the apoplastic regions of the cortex of roots were stained in all sections (Fig. 3B), indicating extracellular iron accumulation. However, compared to the Ev, AtPDF1.1ΔSP and AtPDF1.1RNAi transgenic plants, more heavily stained zones were observed in the apoplast of the root epidermis and cortex of AtPDF1.1 OE plants. Similarly, substantial amounts of iron aggregates were observed in the leaf apoplast of AtPDF1.1 OE plants (Fig. 3C), while only low levels of iron aggregates were present in the Ev, AtPDF1.1ΔSP and AtPDF1.1 RNAi plants. Since the amounts of iron aggregates accumulated in the apoplast, where AtPDF1.1 is localized (Fig. 2), correlated well with the expression levels of AtPDF1.1 (Fig. 3) and AtPDF1.1 can bind to iron (Fig. S6), we projected that AtPDF1.1 can chelate/sequester iron.

We next determined whether apoplastic sequestration of iron by AtPDF1.1 might affect the intracellular iron content. The results showed that AtPDF1.1 OE and AtPDF1.1 RNAi plants, respectively, had the highest and lowest apoplastic iron content in the roots and leaves (Fig. S7A,B), and that the levels of apoplastic iron were inversely correlated with intracellular iron content (Fig. S7C,D). Given that roots are the site for iron uptake³⁸, we concluded that the binding of iron by AtPDF1.1 in the root apoplast might limit the amount of intracellular iron, hence accounting for the low levels of iron in the root and leaves of the AtPDF1.1 OE plants.

We next evaluated the effect of Pcc infection on systemic iron content. As shown in Fig. S7A and C, Pcc infection caused a decrease in root apoplastic iron content and an increase in root intracellular iron content. In contrast, an increase in leaf apoplastic iron content after Pcc infection was observed in all transgenic plants, except the AtPDF1.1 OE lines (Fig. S7B), while the leaf intracellular iron content was not significantly changed (Fig. S7D). AtPDF1.1 overexpression seemed to restrict iron in leaf apoplasts without significantly affecting the leaf intracellular iron content. Because Pcc infection is mainly confined to leaves during the duration of our experiments, these results suggested that Pcc infection results in root apoplastic iron depletion and induces a change in root-to-shoot translocation of iron.

When transgenic plants were grown under long-term-hydroponic conditions, slight chlorosis was observed in the leaves of the AtPDF1.1 OE plants, but chlorophyll content was higher and leaves were greener in the AtPDF1.1 RNAi plants (Fig. S8). Iron is involved in the formation of δ-aminolevulinic acid, which is required for chlorophyll formation, and loss of chlorophyll is associated with a lack of iron availability³⁹. Since the iron distribution of the leaf was aggregated in the apoplast of AtPDF1.1 transgenic OE plants (Figs 3C and 7B), these results are consistent with the observation that the AtPDF1.1 OE plant had lower chlorophyll content than the others (Fig. S8).

Chelation of iron in the leaf by apoplastic AtPDF1.1 induces an iron deficiency response

Given that the apoplastic iron content is positively associated with AtPDF1.1 expression level, we next examined whether the activation of the regulatory pathways involved in iron homeostasis correlated with AtPDF1.1 expression. Analysis of the transcriptional profiles of the iron homeostasis transcription factor genes, FIT1, bHLH38, bHLH39, bHLH100 and bHLH101 ⁴⁰ showed that, under non-infected condition, these genes were significantly upregulated in the leaves of AtPDF1.1 OE plants, and, with the exception of FIT1, bHLH38 and bHLH101, were downregulated in the AtPDF1.1 RNAi plants, (Fig. 4). Similarly, transcript levels of the iron-deficiency-responsive genes, IRT1, FRO2, NAS1 and NAS4 ^{41, 42} were higher and lower, respectively, in the roots of AtPDF1.1 OE and AtPDF1.1 RNAi plants compared with the Ev plants (Fig. 4). This suggests that the high levels of AtPDF1.1 protein present in the apoplast result in a perturbation of iron homeostasis and consequently a state of intracellular iron deficiency.

When plants were subjected to Pcc infection, transcript levels of the iron homeostasis transcription factor genes, bHLH38, bHLH39 and bHLH100 and the iron-deficiency-responsive genes, IRT1, FRO2, and NAS4, were increased in the systemic leaves or roots of all transgenic plants (Fig. S9). This indicated that Pcc infection induces an iron deficiency response that is independent of AtPDF1.1 transcript levels.

Ferric chelate reductase (FCR) is required to reduce Fe³⁺ to Fe²⁺ in the rhizoshere to allow iron uptake into the cell, and its activity is highly activated in response to iron deficiency⁴³. As shown in Fig. S10, FCR activity was the highest and lowest, respectively, in the roots of AtPDF1.1 OE and AtPDF1.1 RNAi plants, suggesting that the AtPDF1.1 OE plants were experiencing greater iron deficiency. In addition, FCR activity was further enhanced by Pcc infection, further confirming that Pcc infection can lead to an iron deficiency response (Fig. S10). The observation that Pcc infection caused an increase in FCR activity in all transgenic plants was consistent with the decreased root apoplastic iron content and the increased root intracellular iron content, respectively (Fig. S7A,C).

A defence response is activated upon the induction of an iron deficiency response

To examine whether iron aggregation in the apoplast increased as a result of Pcc infection, both locally infected and systemic leaves were harvested at 4 and 24 hpi and subjected to Perls/DAB/H₂O₂ staining analysis. In the absence of Pcc infection, iron aggregates were only detected in the leaves of the AtPDF1.1 OE plants. When plants were infected with Pcc, enhanced accumulation of iron aggregates was observed in the local and systemic leaf tissues of all transgenic plants, particularly near to the vascular bundles (Fig. S11). This suggested that Pcc infection induced iron accumulation both locally and systemically, and that the accumulation of iron aggregates was further enhanced by the presence of AtPDF1.1. AtPDF1.1-mediated enhancement on Pcc-induced iron accumulation would account for the overall perturbation of iron homeostasis and elicitation of the iron deficiency response observed in transgenic lines (Figs S9 and S10).

Studies by Alia Dellagi et al.³³ revealed that the sequestration of iron by synthetic iron chelators or bacterial siderophores can induce the expression of genes associated with iron homeostasis and the subsequent activation of immune responses, including callose deposition, hydrogen peroxide production and a SA-mediated response²³. The transcriptional profiles of JA/ET- and SA-responsive genes were evaluated to investigate whether the AtPDF1.1-mediated activation of iron homeostasis genes (Fig. 4) also triggered a plant defence response²³. As shown in Fig. 5A, expression of the JA/ET-responsive genes, ERF1, ERF2, PR3, PR4 and AtPDF1.2a, was upregulated in the AtPDF1.1 OE plants. However, the expression of VSP2 and MYC2, which are in the MYC-branch of the JA response pathway⁴⁴, was not induced in any of transgenic plants. This suggests that ET signalling, but not JA signalling, may respond to altered AtPDF1.1 expression. In contrast, most of the tested genes associated with the SA signalling pathway (WRKY46, WRKY53 and WRKY70 and PR1) were vaguely downregulated and upregulated, respectively, in the AtPDF1.1 OE and AtPDF1.1 RNAi plants (Fig. 5B). Upon Pcc infection, genes associated with the JA/ET responses were upregulated in all transgenic plants, with the highest induced levels in the AtPDF1.1 OE plants (Fig. S12A). In contrast, the expression profiles of genes associated with the SA response were inconsistent and not correlate with AtPDF1.1 expression levels (Fig. S12B). These results suggest that AtPDF1.1 action and Pcc infection promote the JA-/ET-mediated signalling. Since the JA/ET signalling pathway is associated with defence against necrotrophic pathogens, including Pcc ⁴⁵, these results may explain the enhanced tolerance to Pcc infection in the AtPDF1.1 OE plants (Fig. 1), but not to the hemibiotrophic bacterium Pst DC3000 (Fig. S13).

The state of iron deficiency leads to the induction of ethylene biosynthesis in the roots^{46, 47}. Since iron deficiency response occurred in the leaves and roots of AtPDF1.1 OE plants (Figs 4 and S10), we evaluated whether the state of iron deficiency induced by AtPDF1.1 overexpression also led to activation of ethylene signalling in the roots. As shown in Fig. 6, the ET-biosynthesis genes, SAM1, ACS6 and ACO2 (Fig. 6A) and ET-signalling genes, ETR1 and EIN2 (Fig. 6B) were expressed at higher levels in APDF1.1 OE plants compared to the other transgenic plants. The activation of ET signalling in the AtPDF1.1 OE plants may explain why the JA-/ET-responsive ERF1/2 and their downstream genes, but not the JA-responsive MYC2 and VSP2, were upregulated (Fig. 5A)⁴⁸. Furthermore, the expression of JA/SA biosynthesis genes was not significantly affected by AtPDF1.1 (Fig. 6C), but the expression of ET/JA biosynthesis genes and ET signalling genes in the roots was increased at varied levels after Pcc infection (Fig. S14). To further verify whether ethylene signalling is involved in AtPDF1.1-mediated tolerance to Pcc, the ethylene inhibitor 1-methylcyclopropene (1-MCP) was applied to transgenic plants, followed by inoculation with Pcc. With the 1-MCP treatment, the soft rot symptom caused by Pcc was enhanced in all of transgenic plants, including the AtPDF1.1 OE plants (Fig. S15). This result further points to the involvement of ET signalling in AtPDF1.1-mediated tolerance to Pcc infection.

Discussion

Iron is an essential nutrient for hosts and pathogens²¹ and so the iron status of a plant can affect the pathogenesis of pathogens³¹. One of the first lines of defence against bacterial infection is to sequester and withhold iron in an iron-storage compartment, limiting bacterial growth and pathogenicity⁴⁹. In this study, we found that transcription levels of AtPDF1.1 are elevated during Pcc infection (Fig. S2) and that the AtPDF1.1 protein is secreted into the apoplast to sequester iron (Figs 2 and 3 and S11). We propose that this process serves as a key defence mechanism, limiting iron availability and thereby compromising the pathogenesis of Pcc. This hypothesis was supported by the increased sensitivity to Pcc invasion of the AtPDF1.1 OE plants after iron supplementation (Fig. S5).

The state of iron deficiency in AtPDF1.1 OE plants leads to the induction of ET signalling in the roots (Fig. 6)^{46, 47}, which can then activate the ERF branch, but not the MYC branch, of the JA/ET-mediated response in systemic leaves (Fig. 5). Since the JA/ET signalling pathway is involved in the defence against Pcc ^{44, 45}, the ability of AtPDF1.1 to systemically induce an iron-deficiency-mediated ET response would be one of its functions in defending off Pcc. The fact that blocking ET signalling led to increased susceptibility to Pcc infection (Fig. S15) further reinforces the involvement of ET signalling in AtPDF1.1-mediated tolerance to Pcc infection.

Our study showed that AtPDF1.1 could chelate iron (Figs 3 and S11) and its overexpression caused an iron deficiency response (Fig. 4), which correlates well with the promoted tolerance to Pcc infection (Figs 1 and S5). These results mirror previous studies which demonstrated that iron deficiency triggers defence responses and prompts resistance against pathogenic microbes. For instance, Koen et al.⁵⁰ showed that treatment of β-aminobutyric acid (BABA), a nonprotein amino acid able to chelate iron, led to a transient iron deficiency response which is linked with a protective effect against microbial pathogens. In addition, siderophore pyoverdine, which is produced by the beneficial bacterium Pseudomonas fluorescens, restored the growth of A. thaliana under iron-deficient conditions by repressing defence pathways and activating iron acquisition/redistribution signalling⁵¹, further demonstrating the link between iron deficiency and immune responses.

During Pcc infection, genes associated with both JA/ET- and SA-mediated defence responses are activated⁵². In this study, we showed that plants ectopically expressing AtPDF1.1 had pre-existing protection against Pcc infection (Fig. 1) and exhibited an iron deficiency state (Figs 4 and S8). In addition, the iron-deficiency-mediated JA/ET response is further enhanced upon Pcc infection (Fig. S12A), suggesting that AtPDF1.1 has a synergistic effect on the Pcc-mediated iron deficiency response, and is capable of amplifying the ET defence response. Pcc infection also led to decreased iron content in root apoplasts (Fig. S7A) and activated JA/ET signalling (Fig. S12A). Therefore, exposing plants to AtPDF1.1 prior to pathogen infection may have a protective and amplifying effect on their disease defence responses.

SA-mediated defence responses are involved in preventing infection by the hemibiotrophic bacterial pathogen Pst DC3000⁵³. Bacterial siderophores induce an iron-deficiency-mediated SA immune response to defend against Pst DC3000 in A. thaliana by restricting Fe³⁺ availability in the leaf cell wall. This restriction results in a perturbation of iron distribution and activates an iron deficiency response²³. Although AtPDF1.1 also has an iron-sequestration action, and is capable of inducing an iron-deficiency-mediated response. The expression of AtPDF1.1 does not affect defence against Pst DC3000 infection (Fig. S13) nor the activation of genes involved in SA signalling (Fig. 5B). Interestingly, siderophore pyoverdine produced by the beneficial bacterium Pseudomonas fluorescens has an opposite effect, in which pyoverdine promoted Arabidopsis growth under iron-deficient conditions by repressing defence pathways, including SA and abscisic acid, leading to increased susceptibility to Botrytis cinerea ⁵². Taken together, these results indicate that the iron-deficiency-mediated signalling triggered by AtPDF1.1 specifically enhances the ET-mediated defence response. The differences in the immune response pathways activated by the AtPDF1.1 protein and siderophores may be due to their different properties. First, the cysteine-rich AtPDF1.1 utilizes sulphur (S) atoms as an electron donor to bind Fe²⁺ and Fe³⁺ ions (Fig. S6B), whereas siderophores predominantly chelate Fe³⁺ via their oxygen (O) ligands^{22, 23, 54, 55}. In addition, AtPDF1.1 is induced systemically during Pcc infection (Fig. S2B), whereas siderophores are not proteins and are secreted by pathogens at the infection site²².

The fact that AtPDF1.1 overexpression resulted in high levels of apoplastic iron accumulation (Fig. 3), which was accompanied by a decrease in intracellular iron content, and reduced AtPDF1.1 expression gave an opposite effect (Fig. S7) suggests that the chelation of iron by AtPDF1.1 in the apoplast influences intracellular iron levels. It has been reported that coumarin phenolic compounds and nicotianamine can chelate apoplastic iron ions and are required for iron uptake from roots^{41, 56, 57}. We postulate that high levels of AtPDF1.1 proteins present in the apoplast may compete for iron ions with coumarin phenolic compounds and nicotianamine, resulting in the sequestration of iron in the apoplast (Figs 3 and S11). In addition, siderophore-mediated-upregulation of AtFer1 has been identified as an iron-withholding defence against the proliferation of the necrotrophic bacterium D. dadantii ³³. We propose that AtPDF1.1 may similarly compete with bacterial siderophores for iron binding and thereby mediate an iron-withholding defence against Pcc invasion.

In addition to its known antifungal function⁵⁸, this study shows that AtPDF1.1 possesses an iron-withholding defence function to limit the growth of the necrotrophic bacterial pathogen Pcc via the activation of the ET defence pathway (Fig. 7). During Pcc infection, signals induced by the pathogen may trigger the translocation of iron ions from the root apoplast to the leaf apoplast for bacterial utilization and proliferation. As a defence strategy, plants elevate AtPDF1.1 expression, and apoplast-localized AtPDF1.1 chelates iron, thereby iron homeostasis is perturbed to activate an iron deficiency response in the leaf. In addition, this may potentially lead to the activation of ET signalling and biosynthesis in the roots, and subsequently trigger an ET-mediated defence response in systemic leaves to counteract the further attack of pathogens^{45, 59}. The pre-existing ET defence response mediated by AtPDF1.1 in the AtPDF1.1 OE plants may account for the increased tolerance to Pcc infection, while suppressing AtPDF1.1 expression reduces defence against Pcc infection.

Methods

Plant materials and growth conditions

The growth conditions and selection of Arabidopsis are described in Method S1.

Generation of transgenic plants

The constructions of AtPDF1.1 OE (OE), AtPDF1.1ΔSP (ΔSP), AtPDF1.1 RNAi, AtPDF1.1:GFP, AtPDF1.1ΔSP:GFP, AtPDF1.1 (SP only):GFP and Arabidopsis transformation are described in Method S2.

Gene expression analysis following metal treatments and Pcc infection

Ten-day old seedlings of Ev plants were cultured on ½ MS medium supplemented with 600 μM ZnSO₄, 600 μM FeSO₄, 75 μM CuSO₄ or 300 μM ferrozine (Aldrich,160601) for 0 to 5 days. Four-week old soil-grown Ev plants were punctured with a 5 ml syringe needle and inoculated with or without 3 μl liquid culture of 5 × 10⁶ cfu ml⁻¹ of Pcc (three rosette leaves/plant) and local and systemic leaves were harvested at 1–5 hours or 4–24 hours post inoculation (hpi). Total RNA was isolated using the Trizol RNA isolation reagent (Invitrogen), according to the manufacturer’s instructions. cDNAs were synthesized using the M-MLV reverse transcriptase cDNA synthesis kit (Promega) according to the supplier’s instructions. Actin2 (AT3G18780) or UBC21 (AT5G25760) were used as internal normalization controls. qPCR was performed as described by Ko et al.⁶⁰. The primers used to analyse AhPDF1s have already been described by Shahzad et al.¹¹. Primers used for gene expression studies are listed in Table S2.

Plant pathogen inoculation and disease response assay

For the Pcc infection assay, bacteria were grown at 28 °C overnight in Luria-Bertani (LB) medium (Difco, 244620). Three rosette leaves from each four-week old soil-grown plant were punctured with a 5 ml syringe needle and inoculated with 3 μl liquid culture of 5 × 10⁶ cfu ml⁻¹ of Pcc or water (control). Bacterial soft rot symptoms were evaluated periodically. The maximal lesion sizes (diameter; mm) on leaves were recorded at 24 hpi using the protocol described by Li et al.⁶¹. For the P. syringae pv. tomato (Pst) DC3000 infection assay, the bacteria were grown at 28 °C overnight in King’s B medium containing 50 μg ml⁻¹ rifampicin⁶². Plants were sprayed with liquid culture of 2 × 10⁸ cfu mL⁻¹ of Pst DC3000 containing 0.02% Silwet L-77 or 10 mM MgSO₄ (control). Disease symptoms were evaluated periodically and in planta bacterial CFU counts in leaves were measured at 3 dpi.

Subcellular protein localization

Ten-day-old transgenic plants were immersed in 1× phosphate-buffered saline (pH 7.4; MDBio) for 30 min to induce plasmolysis and to stabilize EGFP fusion proteins in the apoplast^{63, 64}. The true leaves were removed and imaging was performed using a Zeiss confocal microscope LSM 710 (Zeiss Axio Observer Z.1) with a Plan-Apochromat 100×/1.40 Oil DIC M27 objective lens. For detection of GFP signals, a 488 nm excitation wavelength and a 500–550 nm emission wavelength were used.

Detection of secreted proteins

Suspension cells of transgenic plants were induced as described by May and Leaver⁶⁵. The culture media were harvested weekly and combined. Each 200 ml of culture media was centrifuged at 8,000 g for 10 min to remove debris, and filtered through Miracloth (Calbiochem) and two layers of Whatman grade 1 qualitative filter paper. The filtrates were lyophilized using a freeze dryer (Uniss), and dissolved in 10 ml milli-Q H₂O and then desalted and concentrated to 10-fold volume using Vivaspin 6 centrifugal filters (Sartorius). The protein concentrations in the different samples were measured by protein assay dye reagent (Bio-Rad [5000006]) and adjusted to the same value with SDS sample buffer⁶⁶ and 20 µg protein per sample was used for immunoblot analysis.

Immunoblot analysis

Experimental procedures are described in Method S3.

Metal binding assay

The experimental design and procedure are described in Method S4.

Detection of iron distribution

For Perls staining, roots were vacuum infiltrated with Perls staining solution containing equal volumes of 4% (v/v) HCl and 4% (w/v) K-ferrocyanide for 15 min and incubated at room temperature for 30 min⁶⁷. The formation of blue pigments, denoting iron accumulation, was observed using light microscopy. For Perls/DAB/H₂O₂ staining, leaves and roots were vacuum infiltrated with fixation solution and dehydrated as described by Roschzttardtz et al.⁶⁸. Embedding and sectioning were performed as described by Lin et al.⁶⁹. Sections for infected leaves (7 mm from the inoculation site), systemic leaves and roots were subjected to Perls/DAB/H₂O₂ staining⁶⁸ and imaging using a Zeiss LSM 710 confocal microscope fitted with an EC Plan-Neofluar 20×/0.50 M27 lens, under transmitted light (bright field).

Measurement of metal concentration

The experimental design and procedure are described in Method S5.

FCR activity assays

FCR activity was measured in four-week-old transgenic plants grown in hydroponic medium. Three leaves from each plant were punctured and inoculated with 3 μl liquid culture of 5 × 10⁶ cfu ml⁻¹ of Pcc or water (control). The roots were harvested at 24 hpi and a modified FCR activity assay was performed as previously described⁷⁰.

Iron infiltration and treatment of ethylene inhibitor

The 2.5 mM MES buffer, pH 5.6 containing with or without FeSO₄ was infiltrated into the Arabidopsis leaves using 1 ml syringes without a needle, and followed by inoculation of Pcc 1 hour after iron infiltration.

Four-week-old soil-grown transgenic plants were treated with ethylene inhibitor, 1-methylcyclopropene (1-MCP, Lytone Enterprise, one tablet containing 2 mg 1-MCP was dissolved in 15 ml milli-Q H₂O) in a 150 L sealed chamber for 24 hours, followed by inoculation with Pcc.

Accession numbers

The sequences of genes used in this article were obtained from The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/index.jsp). The accession numbers of the genes are described in Method S6.

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Acknowledgements

We are grateful to research assistants; Mr. Chin-Lin Chung at the Confocal Microscope Core Facility, Biotechnology Experimental Center in Southern Taiwan for his assistance with confocal microscopy; Ms. Fu-Hui Wu, Mr. Chen-Tran Hsu and Ms. Shu-Chen Shen at the Transformation Core Facility and the Confocal Microscope Core Facility, respectively, of the Agricultural Biotechnology Research Center, Academia Sinica, Taiwan for their assistance with the transformation and confocal microscopy; Dr. Kuo-Chen Yeh at the Agricultural Biotechnology Research Center, Academia Sinica, Taiwan for providing comments on the manuscript; and to Ms. Miranda Loney for English editing and providing comments and suggestions. We thank PlantScribe (www.plantscribe.com) for editing this manuscript. This work was supported by the Academia Sinica (to M.T.C.) and the Ministry of Science and Technology, Taiwan (103-2311-B-002-010-, 104-2311-B-002-009, 105-2311-B-002-002, to C.P.C.).

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Institute of Plant Biology, National Taiwan University, Taipei, 10617, Taiwan
Pao-Yuan Hsiao, Chiu-Ping Cheng, Kah Wee Koh & Ming-Tsair Chan
Academia Sinica Biotechnology Center in Southern Taiwan, Tainan, 74145, Taiwan
Pao-Yuan Hsiao & Ming-Tsair Chan
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 11529, Taiwan
Ming-Tsair Chan

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Pao-Yuan Hsiao
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P.Y.H. performed the experiments, interpreted data, generated figures and wrote the main part of the manuscript; C.P.C. and M.T.C. conceived and supervised the project, contributed to the writing and were involved in data interpretation; K.W.K. assisted in writing the manuscript and data interpretation; M.T.C. wrote the manuscript with contributions from all the authors.

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Hsiao, PY., Cheng, CP., Koh, K.W. et al. The Arabidopsis defensin gene, AtPDF1.1, mediates defence against Pectobacterium carotovorum subsp. carotovorum via an iron-withholding defence system. Sci Rep 7, 9175 (2017). https://doi.org/10.1038/s41598-017-08497-7

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Received: 09 May 2017
Accepted: 10 July 2017
Published: 23 August 2017
DOI: https://doi.org/10.1038/s41598-017-08497-7

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