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The Arabidopsis thaliana Mediator subunit MED8 regulates plant immunity to Botrytis Cinerea through interacting with the basic helix-loop-helix (bHLH) transcription factor FAMA

  • Xiaohui Li ,

    Roles Data curation, Formal analysis, Funding acquisition, Writing – original draft

    lixiaohui@nbu.edu.cn

    Affiliation Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ningbo, Zhejiang, China

  • Rui Yang,

    Roles Conceptualization, Methodology, Resources, Writing – review & editing

    Affiliation Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ningbo, Zhejiang, China

  • Haimin Chen

    Roles Conceptualization, Funding acquisition, Methodology, Project administration, Writing – review & editing

    Affiliation Key Laboratory of Applied Marine Biotechnology, Ningbo University, Ningbo, Zhejiang, China

Abstract

The Mediator complex is at the core of transcriptional regulation and plays a central role in plant immunity. The MEDIATOR25 (MED25) subunit of Arabidopsis thaliana regulates jasmonate-dependent resistance to Botrytis cinerea through interacting with the basic helix-loop-helix (bHLH) transcription factor of jasmonate signaling, MYC2. Another Mediator subunit, MED8, acts independently or together with MED25 in plant immunity. However, unlike MED25, the underlying action mechanisms of MED8 in regulating B. cinerea resistance are still unknown. Here, we demonstrated that MED8 regulated plant immunity to B. cinerea through interacting with another bHLH transcription factor, FAMA, which was previously shown to control the final proliferation/differentiation switch during stomatal development. Our research demonstrates that FAMA is also an essential component of B. cinerea resistance. The fama loss-of-function mutants (fama-1 and fama-2) increased susceptibility to B. cinerea infection and reduced defense-gene expression. On the contrary, transgenic lines constitutively overexpressing FAMA showed opposite B. cinerea responses compared with the fama loss-of-function mutants. FAMA-overexpressed plants displayed enhanced resistance to B. cinerea infection and increased expression levels of defensin genes following B. cinerea treatment. Genetic analysis of MED8 and FAMA suggested that FAMA-regulated pathogen resistance was dependent on MED8. In addition, MED8 and FAMA were both associated with the G-box region in the promoter of ORA59. Our findings indicate that the MED8 subunit of the A. thaliana Mediator regulates plant immunity to B. cinerea through interacting with the transcription factor FAMA, which was discovered to be a key component in B. cinerea resistance.

Introduction

B. cinerea is a ubiquitous pathogen that causes gray mold disease on more than 200 host plants and results in crop losses of up to 20% globally [1]. As a typical necrotrophic pathogen, B. cinerea can produce a variety of cell wall-degrading enzymes, phytotoxic metabolites, and cell death elicitors to destroy host cells and induce necrosis [2, 3]. In order to defend B. cinerea attack, plants have evolved a complex immune system including changes in ion fluxes, synthesis of the defense related hormones, and transcriptional reprogramming [47]. The precise transcriptional regulation of a wide range of genes encoding diverse molecules is pertinent in determining plant resistance and susceptibility to B. cinerea infection [1]. Recent studies have indicated that the Mediator complex plays an important role in the transcriptional process underpinning plant immunity to bacterial and fungal infection.

Mediator is a conserved multisubunit complex which connects the transcription factors located in the promoter regions of protein-coding genes to the RNA polymerase II (Pol II) at the transcription start site in eukaryotes [8]. The Arabidopsis Mediator complex contains 21 conserved and 6 plant-specific subunits [9]. A number of mediator subunits play critical roles in a variety of signaling pathways including growth and development, response to biotic and abiotic stress, and cell life activities such as noncoding RNA processing, adjusting the stability of DNA and proteins, and secondary metabolism [1024]. Among them, MED8, MED16, MED18, MED21, MED25, and CDK8 play significant roles in plant immunity to necrotrophic pathogens [10, 11, 16, 17, 20, 21]. MED16 together with transcription factor WRKY33 were found to be critical to basal resistance against another devastating necrotrophic fungal plant pathogen in agriculture, Sclerotinia sclerotiorum [25]. MED18 interacts with the transcription factor YIN YANG1 to suppress the disease susceptibility genes glutaredoxins GRX480 and GRXS13, and thioredoxin TRX-h5 to mediate plant immunity to B. cinerea [20]. MED21 interacts with the A. thaliana RING E3 ligase HUB1, and MED21 RNAi plants are highly susceptible to A. brassicicola and B. cinerea infection [10]. CDK8 was found to regulate cuticle development by interacting with the transcription factor WAX INDUCER1, and the cdk8 mutant exhibited enhanced resistance to B. cinerea [21]. MED25 physically associates with the transcription factor MYC2 in the promoter regions of MYC2 target genes and exerts a positive effect on MYC2-regulated gene transcription during JA-dependent plant immunity [16]. As with MED25, MED8 is a regulator of JA-dependent plant immunity. The med8 mutant exhibited an F. oxysporum resistance phenotype and had increased susceptibility to A. brassicicola [11]. In addition, the expression level of PDF1.2 was slightly lower in both untreated and MeJA-treated med8 plants than in untreated and MeJA-treated wild-type plants. This suggests that MED8 is important to JA-dependent plant immunity. The med8med25 double mutant exhibited stronger defense than either of the single mutants, suggesting that MED8 and MED25 probably affect JA-dependent plant immunity signaling by independent and additive mechanisms[11].

FAMA, a basic helix-loop-helix (bHLH) transcription factor, was first reported to control the final proliferation/differentiation switch during stomatal development [2630]. Three bHLH transcription factors including SPEECHLESS (SPCH), MUTE, and FAMA were found to regulate stomatal differentiation that proceeds through a series of steps originating from meristemoid mother cells [3136]. SPEECHLESS is required for the first asymmetric ‘entry’ division into the stomatal lineage and is involved in promoting the asymmetric ‘amplifying’ divisions of meristemoids [37]. MUTE is essential for the termination of the stem cell-like asymmetric division activity and promotion of differentiation [38]. The FAMA transcription factor regulating the later stages of stomatal development is necessary to prevent further mitotic division of the guard mother cell after the single division that normally gives rise to a guard cell pair, and promotes guard cell fate [30]. In addition to its function in stomatal development, FAMA is an essential component for the differentiation of myrosin cells. Myrosin cell development and the biosynthesis of the myrosinases THIOGLUCOSIDE LUCOHYDROLASE1 (TGG1) and TGG2 were changed in both fama mutant and FAMA-overexpressed plants. FAMA expression in myrosin cells is independent of SPCH and MUTE [39, 40].

In this study, a yeast two-hybrid assay was performed to identify the potential transcription factors interacting with MED8. FAMA was identified as a potential partner of MED8 in plant immunity towards B. cinerea and was found to positively influence plant resistance to B. cinerea infection. The evidence showed that both FAMA and MED8 could be recruited to the G-box region in the promoter of the pathogen-related gene ORA59. In addition, we revealed that FAMA and MED8 functioned in the same pathway of plant immunity to B. cinerea based on genetic evidence. Our study not only elucidates the molecular mechanisms underlying MED8-regulated plant immunity to B. cinerea, but also extends our understanding of the biological functions of FAMA in regulating plant immunity.

Materials and methods

Plant materials and growth conditions

Arabidopsis thaliana ecotype Col-0 was used as the wild-type (WT). The mutant lines used are listed in the accession numbers section at the end of the methods. Homozygous lines including med8, med25, fama-1, and fama-2 were identified using the primers provided at http://signal.salk.edu/tdnaprimers.html and were used in the experiments described below. med8med25, med8fama-2, ProMED8: MED8-GFP/fama-2, and ProFAMA: FAMA-GFP/med8 were generated by crossing the parental single homozygous lines. The resulting F2 segregating progenies were genotyped to identify homozygous plants.

To obtain seeds, A. thaliana plants were grown in Murashige and Skoog (MS) medium [41] or sterile soil in plastic trays at 22°C with a 16-h-light/8-h-dark photoperiod (light intensity 120 μM photons m−2 s−1) as previously described [16]. For B. cinerea inoculation, A. thaliana plants were grown in sterile soil at 22°C with a 12-h-light/12-h-dark photoperiod as previously described [42]. Nicotiana benthamiana was grown in sterile soil under a 16-h-light (28°C)/8-h-dark (22°C) photoperiod.

DNA constructs and plant transformation

The promoters of MED8 and FAMA were amplified with the listed primers (S1 Table). Enzyme-digested PCR products were cloned into the same site of the pCAMBIA1300 vector to generate ProMED8: pCAMBIA1300 and ProFAMA: pCAMBIA1300, respectively. Full-length coding sequences of MED8 and FAMA were amplified with the listed primers (S1 Table) and the enzyme-digested PCR products were ligated with ProMED8: pCAMBIA1300 and ProFAMA: pCAMBIA1300 to generate ProMED8: MED8-GFP and ProFAMA: FAMA-GFP. All primers used for DNA construct generation are listed in S1 Table.

The constructs of ProMED8: MED8-GFP and ProFAMA: FAMA-GFP were transformed into Agrobacterium strain GV3101 (pMP90), which was used for the transformation of the A. thaliana plants via the floral dip method [43]. Transformants were selected based on their resistance to hygromycin (25 μg/mL). Homozygous T3 or T4 transgenic seedlings were used for phenotyping and molecular characterization.

Yeast two-hybrid (Y2H) assays

Y2H assays were performed to assess the interaction of the transcription factors MYC2 and FAMA with MED8 and MED25. Full-length coding sequences of MED8 and MED25 were amplified using the listed primers for Y2H assays (S1 Table). Enzyme-digested PCR products were cloned into the same site of pGBKT7. Full-length MYC2 and FAMA were also amplified with the listed primers (S1 Table) and cloned into pGADT7. Matchmaker GAL4 two-hybrid systems (Clontech, Mountain View, California, USA) were used following the manufacturer’s instructions. Constructs for testing the interactions were co-transformed into the yeast strain Saccharomyces cerevisiae AH109. Transgene presence was confirmed by growth on an SD/-Leu/-Trp plate. To assess protein interactions, the transformed yeasts were suspended in liquid SD/-Leu/-Trp to OD = 1.0. Five microliters of suspended yeast were placed into the wells of 96-well plates containing SD/-Ade/-His/-Leu/-Trp medium. The interactions were observed after 3 d of incubation at 30°C. The experiments were performed in triplicate.

Co-immunoprecipitation (Co-IP) assays

The full-length coding sequence of MED8 was amplified using Gateway-compatible primers (S1 Table). The PCR product was cloned using pENTR Directional TOPO cloning kits (Invitrogen, Carlsbad, USA) and then recombined with the binary vector PGWB5 (35S promoter, C-GFP) to generate the 35Spro:MED8-GFP construct. The full-length coding sequence of FAMA was also cloned into the pGWB14 vector (35S promoter, C-3HA) to generate the 35Spro: FAMA-HA construct. The constructs of 35Spro:MED8-GFP and 35Spro: FAMA-HA were transformed into Agrobacterium strain GV3101 (pMP90). Then agrobacterial strains carrying constructs of 35Spro:MED8-GFP and 35Spro:FAMA-HA were co-infiltrated into Nicotiana benthamiana leaves. Agrobacterial strains carrying constructs of GFP-myc were used as a control. The infiltrated parts of N. benthamiana leaves were harvested and then ground in liquid nitrogen and re-suspended in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 0.2% Nonidet P-40, 0.6 mM PMSF, and 20 μMMG132 with Roche protease inhibitor cocktail). After protein extraction, 20 μL protein G plus agarose (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the 2-mg extracts to reduce nonspecific immunoglobulin binding. After 1 h of incubation, the supernatant was transferred to a new tube. GFP antibody-bound agarose beads (MBL) were then added to each reaction for 1 h at 4°C. The precipitated samples were washed at least three times with the lysis buffer and then eluted by adding 1×SDS protein loading buffer with boiling for 5 min. Total and immunoprecipitated proteins were analyzed by immunoblotting using anti-HA and anti-GFP antibodies.

Chromatin immunoprecipitation (ChIP)-PCR assays

ProFAMA: FAMA-GFP, ProMED8: MED8-GFP, ProFAMA: FAMA-GFP/med8, ProMED8: MED8-GFP/fama-2 or wild type seedlings were grown in MS medium. For B. cinerea treatment, 5-d-old seedlings of the above materials were sprayed with 0.5×105 to 1.0×105 spores/mL of B. cinerea over a period of 36 h, after which 1.5 g of inoculated entire plants were collected. Additionally, 1.5 g of uninoculated seedlings were collected as a control. The collected seedlings were cross-linked in 1% formaldehyde, and their chromatin isolated [44]. A GFP antibody (Abcam, Cambridge, UK) was used to immunoprecipitate the protein-DNA complex, and the precipitated DNA was purified using a PCR purification kit (Qiagen, Hilden, Germany) in preparation for real-time quantitative (RT-qPCR) analysis. The ChIP experiments were performed three times. Chromatin precipitated without an antibody constituted the negative control, while the isolated chromatin prior to precipitation was used as an input control. Primers used for ChIP-PCR are listed in S1 Table.

RNA extraction and gene expression analyses

To quantify the FAMA transcript levels in the fama mutants, total RNA was extracted from 2-week-old seedlings grown in MS medium. To evaluate the expression level of FAMA in FAMA overexpression plants (OE3 and OE7), total RNA was extracted from 6-d-old entire plants grown in MS medium. For the quantitative analysis of the expression levels of pathogen-responsive genes in fama mutants plants, total RNA was extracted from 3-week-old seedlings sprayed with B. cinerea as described below. For the quantitative analysis of the expression levels of pathogen-responsive genes in med8, med25, FAMA overexpression (OE3 and OE7) and OE-7/ med8 plants, total RNA was extracted from 5-week-old seedlings sprayed with B. cinerea as indicated (using TRIzol [Invitrogen] reagent). For small-scale RNA isolation, total RNA was extracted using the RNAqueous kit (Ambion, Foster City, California, USA). cDNA was prepared from 2 μg of total RNA with Superscript III reverse transcriptase (Invitrogen) and quantified with a cycler apparatus (Roche 480) using the SYBR Green kit (Takara, Japan) according to the manufacturer’s instructions. Expression levels of target genes were normalized to ACTIN7. The RT-qPCR experiments were performed three times. The statistical significance was evaluated by Student’s t-test. Primers used for RT-qPCR are listed in S1 Table.

Disease assays

Botrytis cinerea strain B05-10 was cultured on 2×V8 (36% V8 juice, 0.2% CaCO3, 2% Bacto-agar) and incubated at 22°C [45]. In the detached leaf disease assays of the fama mutants, a single 2.5 μL drop of a suspension of 0.5×105 spores/mL in 1% Sabouraud Maltose Broth buffer was placed onto the detached leaves of fama mutants and Col-0 plants that had been grown for 3 weeks in growth chambers as described earlier. The detached leaves were placed in transparent trays, which were sealed to maintain high humidity. Disease lesion diameter was measured after 2 d. For the whole plant disease assays of the fama mutants, the two mutants plants along with the Col-0 plants were inoculated by spraying with a suspension of 0.5×105 spores/mL in 1% Sabouraud Maltose Broth buffer. After inoculation, the plants were sealed with a transparent cover to maintain high humidity. Fungal hyphal staining and fungal growth were measured after 2 d. For the detached leaf disease assays for med8, med25, OE-3, OE-7, and OE-7/ med8 plants, a single 2.5 μL drop of a suspension of 2.5×105–3×105 spores/mL in 1% Sabouraud Maltose Broth buffer was placed onto the detached leaves of plants grown for five weeks. Once again, high humidity was maintained by sealing the transparent trays. Disease lesion diameter was measured after 3 d. For the whole plant disease assays, the above plants along with the Col-0 plants were inoculated by spraying with a suspension of 2.5×105–3×105 spores/mL in 1% Sabouraud Maltose Broth buffer. After inoculation, the plants were sealed with a transparent cover to maintain high humidity. Fungal hyphae staining and fungal growth was assessed after 3 d. All disease assays were repeated at least in triplicate.

Fungal hyphal staining with trypan blue

In order to visualize the fungal hyphae after B. cinerea inoculation, whole leaf mounts were stained with lactophenol-trypan blue (10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, 10 mg of trypan blue, dissolved in 10 mL of distilled water) as previously described [46]. Whole leaves were boiled for approximately 2 min in the staining solution and then decolorized in chloral hydrate (2.5 g of chloral hydrate dissolved in 1 mL of distilled water) for at least 30 min. They were then mounted in chloral hydrate and viewed under a compound microscope equipped with interference or phase-contrast optics.

Transient expression assay in N. benthamiana leaves

The transient expression assays were performed in N. benthamiana leaves as previously described [47]. For split-luciferase complementation (Split-LUC) assay, MED8 was cloned into vector pCAMBIA1300-nLUC, and FAMA was cloned into vector pCAMBIA1300-cLUC. Primers are summarized in S1 Table. For transcriptional activation assays, the ORA59 promoter was amplified using Gateway-compatible primers. The PCR products were cloned by pENTR Directional TOPO cloning kits (Invitrogen) and recombined with the binary vector pGWB35 to generate the reporter construct ORA59pro: LUC. The FAMA effector construct was 35Spro: FAMA-GFP (35Spro: FAMA). We used a low-light cooled CCD imaging apparatus (NightOWL II LB983 with indigo software) to capture the LUC image and to assess luminescence intensity. The leaves were sprayed with 0.5 mM luciferin and placed in darkness for 3 min prior to luminescence detection.

Microscopy

For two-channel fluorescence imaging using GFP and propidium iodide (PI) fluorescence filter sets, 6-d-old fresh seedlings of OE-3 and OE-7 were immersed in 2mg/mL PI solution for 5 min and then rinsed briefly with water before visualization with the Nikon microscope.

Accession numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: MED25 (AT1G25540), ORA59 (AT1G06160), MED8 (At2g03070), PDF1.2 (At5g44420), ERF1 (AT3G23240), PR1 (AT2G14610), ACTIN7 (At5g09810), FAMA (AT3G24140). The following mutant lines were used: med8 (At2g03070, SALK_092406), med25 (AT1G25540, SALK_129555), fama-1 (AT3G24140, SALK_100073), fama-2 (AT3G24140, SALK_049126).

Results

MED8 affects plant immunity to B. cinerea via a pathway other than MED25

Previous research demonstrated that med8 and med25 mutants influence jasmonic acid (JA)-induced pathogen resistance by independent and additive mechanisms [11]. Chen et al. [16] also reported that MED25 regulates JA-dependent pathogen resistance by interacting with the transcription factor MYC2. However, the details regarding the role of MED8 in resistance to B. cinerea remain unknown. To elucidate the role of MED8 in B. cinerea resistance, the disease phenotype of the T-DNA insertion (SALK_092406; [11]; [16]) mutant of MED8 needed to be verified. We thus used detached leaf disease assays to rapidly evaluate the disease phenotype in med8 mutant plants in comparison to the WT plants. Typical disease lesions were observed 3 d post-inoculation (dpi). The lesions on the med8 mutant plant leaves were larger than the WT plants at 3 dpi, and an approximately 38% increase in lesion size was observed (Fig 1A and 1B). Similar enhanced susceptibility was observed in the med25 mutant plants, and MED8 also possessed an additive effect with MED25 in pathogen susceptibility (Fig 1A and 1B) [11]. Pathogen responsive gene expression was altered in the med8 mutant. Two marker genes ERF1 and PDF1.2 [48], considered to be associated with the plant response to pathogen infection [49, 50], were selected for comparison of expressional changes in the med8 plants with the WT plants following B. cinerea infection. No significant differences in expression were observed in the two pathogen marker genes in the uninfected med8 mutant compared with the WT plants, indicating that the pathogen responsive genes were not constitutively expressed in the med8 mutant. In comparison to the mock-treated plants, the expression levels of ERF1 and PDF1.2 increased significantly following B. cinerea infection. Interestingly, the expression levels of ERF1 and PDF1.2 in the med8 mutant plants were remarkably reduced in comparison to the WT plants. Additionally, the expression levels of ERF1 and PDF1.2 in the med25 mutant plants were significantly reduced compared to that in the med8 mutant plants (Fig 1C and 1D). This indicates that MED25 plays a more important role in the expression of ERF1 and PDF1.2 than MED8. Furthermore, the med8med25 double mutant exhibited lower ERF1 and PDF1.2 expression levels compared to the med8 or med25 single mutant, which suggests that MED8 had an additive effect with MED25 in inducing ERF1 and PDF1.2 expression. These findings are consistent with previous results whereby the expression levels of pathogen-induced defense genes in med8med25 double mutant plants were lower than those in med8 or med25 single mutants [11]. Ultimately, the results demonstrate that MED8 affects plant immunity to B. cinerea is a similar manner to MED25. The stronger effect of the med8med25 double mutant on pathogen resistance than either of the single mutants suggests that the med8 and med25 mutations are likely to affect pathogen resistance by independent and additive mechanisms.

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Fig 1. med8 and med25 mutants affect plant resistance to B. cinerea by independent and additive means.

(A) (B) Disease symptoms and lesion sizes on the leaves of the B. cinerea-infected WT, med8, med25, and med8med25 at 3 d. The disease assay was performed by drop inoculation of B. cinerea on the leaves of soil-grown plants. The infected leaves were photographed and bar = 4 mm (A). Average values and SEM from relative values obtained in four biological replicates were plotted on the graph (B). A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated at least four times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test). (C) (D) RT-qPCR analysis of ERF1 and PDF1.2 RNA levels in the WT, med8, med25, and med8med25 leaves of soil-grown plants at 36 hpi after inoculation with B. cinerea. Expression of ERF1 and PDF1.2 was normalized against the constitutively expressed Actin7. Average values and SEM from relative values obtained in four biological replicates were plotted on the graph. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.g001

MED8 and FAMA have a direct physical interaction

Previous research demonstrated that MED25 could interact with the transcription factor MYC2 to act as part of the general transcriptional machinery in regulating JA-triggered gene expression [16]. However, the results from the yeast two-hybrid assay indicated no interaction between MED8 and MYC2 (Fig 2A). This corroborates previous results whereby MED8 and MED25 influenced JA-mediated pathogen resistance by independent and additive mechanisms [11], leading us to speculate that the interaction of another potential partner with MED8 might be involved in pathogen resistance. To test this, we used the Y2H system to identify potential factors that could interact with MED8. The full-length MED8 was fused to the Gal4 DNA binding domain of the bait vector (BD-MED8). After screening, three independent clones encoding FAMA, which plays an indispensable role in plant stomatal development [30], were identified by prototrophy for His and Ade. The full-length coding sequence (CDS) of FAMA was introduced into the prey vector (AD-FAMA), and the bait and prey vectors were co-transformed into yeast for reconstructing the protein–protein interaction (Fig 2B). A further split-luciferase complementation (Split-LUC) assay was performed in N. benthamiana leaves to validate the Y2H assay results. As illustrated in Fig 2C, the co-expression of nLUC-tagged MED8 with cLUC-FAMA produced detectable luciferase activity, confirming the results of the Y2H assay. In contrast, the co-expression of nLUC/cLUC, nLUC/cLUC-FAMA, or MED8-nLUC/cLUC resulted only in background luciferase signals (Fig 2C). In addition to the Split-LUC assays, MED8-FAMA interaction was verified by Co-IP assays using N. benthamiana total protein (Fig 2D). Taken together, these results indicate that MED8 interacts with FAMA proteins in the plant cell, implying that MED8 might regulate pathogen resistance by interacting with FAMA.

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Fig 2. MED8 can interact with FAMA, but not with MYC2.

(A) A Y2H assay was used to detect the interactions of MED8 with the MYC2 protein. Yeast cells co-transformed with pGADT7-MYC2 (preys) and pGBKT7-MED8 (baits) were selected and subsequently grown on yeast synthetic dropout lacking Leu and Trp (SD/-2) as a transformation control, or on selective media lacking Ade, His, Leu, and Trp (SD/-4) to test protein interactions. The pGADT7-MYC2 (preys) and pGBKT7-MED25 (baits) interaction constituted a positive control. pGADT7-MYC2 co-transformed with the pGBDT7 vector, and pGBKT7-MED8 or pGBKT7-MED25 co-transformed with the pGADT7 vector were included as controls. (B) A Y2H assay used to detect the interactions of MED8 with the FAMA protein. Yeast cells co-transformed with pGADT7-FAMA (preys) and pGBKT7-MED8 (baits) were selected and subsequently grown on yeast synthetic dropout lacking Leu and Trp (SD/-2) as a transformation control, or on selective media lacking Ade, His, Leu, and Trp (SD/-4) to test protein interactions. pGADT7-FAMA co-transformed with the pGBDT7 vector, and pGBKT7-MED8 co-transformed with the pGADT7 vector were included as controls. (C) Split-luc assays showing that MED8 can interact with FAMA in N. benthamiana leaves. Three biological replicates were performed, and similar results were obtained. (D) Co-IP assays were used to verify the interaction of MED8 with FAMA in N. benthamiana leaves. Protein extracts from N. benthamiana leaves infiltration with both 35Spro:FAMA-HA and 35Spro:MED8-GFP (FAMA-HA MED8-GFP) or 35Spro:GFP-myc (FAMA-HA GFP-myc) was immunoprecipitated (IP) with the GFP antibody, and immunoprecipitated proteins were analyzed by immunoblotting using anti-HA and anti-GFP antibodies. The experiments were repeated three times, with similar results.

https://doi.org/10.1371/journal.pone.0193458.g002

fama loss-of-function mutants attenuate the defense response against B. cinerea

FAMA was previously shown to influence stomatal cell fate [30]. Our results demonstrated that it could interact with MED8, suggesting that FAMA is probably involved in plant immunity towards B. cinerea. In order to investigate the function of FAMA in pathogen resistance, the phenotypes of two fama loss-of-function mutants, fama-1 (SALK_100073) and fama-2 (SALK_049126), were evaluated. The mutation site of fama-1 is located in the first intron of the FAMA gene, while that of fama-2 is in the promoter region of FAMA (Fig 3A). Both of the mutants failed to accumulate FAMA transcripts (Fig 3B). The disease phenotypes of the mutants after inoculation with B. cinerea were evaluated. The fama mutants were shown to display severe defects in growth, and healthy leaves of the fama mutants were chosen to compare the inoculation phenotypes (S1 Fig). In the detached leaf disease assays, fama-1 and fama-2 displayed rapid spreading of the pathogen. Two days after inoculation with B. cinerea, fama-1 and fama-2 showed severe and typical disease phenotypes for B. cinerea, while the WT control plants only possessed small spots on their leaves (Fig 3C and 3D). Further whole plant disease assays were carried out to confirm the disease phenotype observed in the fama-1 and fama-2 plants. In the whole plant disease assays, the two mutants plants along with the WT plants were inoculated by spraying with B. cinerea spore suspension. The disease phenotypes were revealed by trypan blue staining of fungal hyphae and in planta fungal growth was analyzed by comparing the transcription of the B. cinerea actin gene BcActin as an indicator of fungal growth in planta. The results indicated that fama-1 and fama-2 displayed much denser fungal hyphal growth than that of WT. Furthermore, the RT-qPCR determination of fungal growth also showed that fama-1 and fama-2 were susceptible to B. cinerea (Fig 3E and 3F). To summarize, our disease experiments demonstrate that the fama loss-of-function mutants exhibit an enhanced susceptibility towards B. cinerea infection.

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Fig 3. fama mutants display increased susceptibility to B. cinerea infection.

(A) Gene organization of FAMA. T-DNA insertions are shown for fama-1 (SALK_100073) and fama-2 (SALK_049126). Closed boxes, exons; black solid lines, introns; red solid lines, 5’ UTR; The translational start sites (ATG) are shown as +1. (B) RT-qPCR of FAMA at 14 d after germination of fama-1 and fama-2, and their respective wild-type lines (Col-0) using Actin7 as a control. Error bars indicate 95% confidence intervals (n = 3). (C) (D) Disease symptoms and lesion sizes on the B. cinerea-infected WT, fama-1, and fama-2 leaves. (E) Trypan blue staining of B. cinerea fungal hyphae growing on leaves at 2 d. Bars = 100 μm. (F) Fungal growth on the B. cinerea-infected WT, fama-1, and fama-2 leaves. The disease assay was performed as indicated (see Results and Methods) on the leaves of soil-grown plants. Photos (C) were taken at 2 d. Bars = 2.5 mm. Fungal growth in planta was assumed by analyzing the transcript levels of the BcActinA gene by RT-qPCR using Actin7 as an internal control 2 d after inoculation (F). Average values and SEM from relative values obtained in three biological replicates were plotted on the graph (D) (F). A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated three times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.g003

FAMA-overexpressed plants exhibit enhanced resistance to B. cinerea infection

To further explore the role of FAMA in pathogen resistance, FAMA-overexpressed plants were generated and their immunity phenotypes toward B. cinerea infection were evaluated. Two FAMA overexpression lines OE-3 (ProFAMA: FAMA-GFP 3#) and OE-7 (ProFAMA: FAMA-GFP 7#) generated significantly higher FAMA transcript levels than WT as revealed by RT-qPCR (Fig 4A). FAMA was found to express in the stomatal guard cells as determined by fluorescence microscopy (Fig 4B), which is in accordance with previous results [30]. Detached leaf disease assays of B. cinerea in overexpressed lines and WT suggested that FAMA is a positive regulator of B. cinerea resistance, as evidenced by the significant alleviation of disease symptoms in the OE-3 and OE-7 lines and obvious disease symptoms in WT, including larger and expanding disease lesions beyond the inoculation site (Fig 4C and 4D). Fungal hyphae stained with trypan blue and RT-qPCR determination of fungal growth also indicated that the OE-3 and OE-7 lines were resistant to B. cinerea infection (Fig 4E and 4F). In combination, these data further demonstrate that FAMA positively influences plant immunity towards B. cinerea.

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Fig 4. FAMA overexpression plants enhance B. cinerea resistance.

(A) RT-qPCR of FAMA at 6 d after germination of FAMA overexpression lines (OE-3 and OE-7) and WT using Actin7 as a control. Error bars indicate 95% confidence intervals (n = 3). (B) Localization of FAMA in OE-3 and OE-7 leaves. Confocal imaging of transgenic A. thaliana plants expressing FAMA. Bars = 20 μm. (C) (D) Disease symptoms and lesion sizes on the B. cinerea-infected WT, OE-3, and OE-7 leaves. (E) Trypan blue staining of B. cinerea fungal hyphae growing on leaves at 3 d. Bars = 100 μm. (F) Fungal growth on the B. cinerea-infected WT, OE-3, and OE-7 leaves. The disease assay was performed as indicated (see Results and Methods) on the leaves of soil-grown plants. Photos (C) were taken at 3 d. Bars = 3 mm. Fungal growth in planta was assumed by analyzing the transcript levels of the BcActinA gene by RT-qPCR using Actin7 as an internal control 3 d after inoculation (F). Average values and SEM from relative values obtained in three biological replicates were plotted on the graph (D) (F). A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated three times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.g004

FAMA affects pathogen-induced plant defensin gene expression

The expression levels of three JA-induced plant defensin genes (ERF1, ORA59, and PDF1.2) were examined in FAMA mutants and overexpression lines. The results demonstrated that the mRNA levels were significantly reduced in the fama-1 and fama-2 plants compared to WT plants following B. cinerea inoculation. On the contrary, the expression of ERF1, ORA59, and PDF1.2 were increased in OE-3 and OE-7 overexpression plants at 36 h after inoculation with B. cinerea. Additionally, the basal levels of ERF1, ORA59, and PDF1.2 were also relatively lower in fama-1 and fama-2 plants in comparison with WT plants, but higher in OE-3 and OE-7 overexpression plants in comparison with WT plants (Fig 5). Furthermore, the expression of one salicylic acid (SA)-induced plant pathogenesis-related (PR1) gene was also examined in FAMA mutants and overexpression lines. In contrast to the three JA-induced plant defensin genes, the expression of PR1 was reduced in OE-3 and OE-7, but increased in the fama-1 and fama-2 plants before and after B. cinerea inoculation (S2 Fig). In combination, our gene expression results thus indicate that FAMA influences pathogen resistance at both basal and induced levels.

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Fig 5. FAMA affects the function of MED8 in regulating the transcriptional expression of pathogen-responsive genes.

Expression of ERF1, ORA59, and PDF1.2 was examined by RT-qPCR in Col-0, fama-1, fama-2, OE-3, and OE-7 plants following inoculation of B. cinerea. Average values and SEM from relative values obtained in three biological replicates were plotted on the graph. A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated three times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.g005

FAMA and MED8 work together in pathogen resistance

The above results demonstrate that MED8 and FAMA are required for plant immunity against B. cinerea infection (Fig 1A; Fig 3C; Fig 4C). We also found that FAMA could directly interact with MED8 (Fig 2B), implying that FAMA and MED8 might operate in the same pathogen resistance pathway. To test this hypothesis, the med8fama-2 double mutant was constructed. The pathogen inoculation results showed that MED8 and FAMA acted in the same plant immunity pathway during B. cinerea infection (S3 Fig). We also crossed the previously characterized ProFAMA: FAMA-GFP transgene into the med8 mutant background and investigated the associated effects in pathogen resistance. Confirming our previous observation, med8 was susceptive to B. cinerea infection, while OE-7 showed enhanced resistance to B. cinerea (Fig 6A and 6B). However, the effects of FAMA overexpression on pathogen resistance were completely blocked by the med8 mutant (Fig 6A and 6B), indicating that FAMA-activated pathogen resistance requires MED8. Expression of the plant defensin genes ERF1 and PDF1.2 further supported that FAMA-mediated resistance against B. cinerea depends on MED8 (Fig 6C and 6D).

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Fig 6. FAMA-activated defense responses are MED8 dependent.

(A) (B) Disease symptoms and lesion sizes of the B. cinerea-infected WT, med8, OE-7, and OE-7/med8 leaves at 3 d. The disease assay was performed by drop inoculation of B. cinerea on the leaves of soil-grown plants. The infected leaves were photographed and bar = 4 mm (A). Average values and SEM from relative values obtained from three biological replicates were plotted on the graph (B). A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated in triplicate, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test). (C) (D) RT-qPCR analysis of ERF1 and PDF1.2 RNA levels in the WT, med8, OE-7, and OE-7/med8 leaves of soil-grown plants at 36 hpi after inoculation with B. cinerea. Expression of ERF1 and PDF1.2 was normalized against the constitutively expressed Actin7. Average values and SEM from relative values obtained in three replicates were plotted on the graph. A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated three times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.g006

MED8 and FAMA bind the G-box region of the promoter of ORA59

ERF1 and PFD1.2 constitute downstream genes in plant immunity to B. cinerea. The RT-qPCR results showed that the expression levels of ERF1 and PFD1.2 changed significantly in fama mutants and FAMA-overexpressed plants. The ability of MED8 and FAMA to directly occupy the ERF1 or PFD1.2 promoter was also assessed. ChIP assays using ProMED8: MED8-GFP and ProFAMA: FAMA-GFP plants and anti-GFP antibodies could not detect any significant occupation of MED8 and FAMA in the promoters of ERF1 and PFD1.2. We then evaluated whether ORA59, which occupies the promoter of PFD1.2 [51], could be the direct target of MED8 and FAMA. The ChIP assays indicated that MED8 and FAMA had bound to the G-box region in the promoter of ORA59, which is also the occupation site of MYC2 at steady state [16]. We also found that B. cinerea inoculation resulted in a marked increase in the binding of MED8 and FAMA within 36 h (Fig 7B and 7C), while the non-G-box region in the promoter of ORA59 exhibited little MED8 and FAMA enrichment neither at steady state nor after B. cinerea inoculation (S4 Fig). These data suggest that ORA59 might constitute the direct target of MED8 and FAMA.

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Fig 7. FAMA and MED8 can occupy the G-box region in the promoter of ORA59.

(A) Schematic diagram of the promoter regions of ORA59. The black line represents the promoter region of the gene. The black box on the line indicates the putative G-box cis-elements (CACGTG) of the ORA59 promoter. The region between the two coupled-arrowheads (red line) indicates the DNA fragments used for the ChIP-PCR. The translational start sites (ATG) are shown as +1. (B) FAMA could occupy the G-box region in the promoter of ORA59, and MED8 affects the recruitment of FAMA to the promoter of ORA59. The ProFAMA: FAMA-GFP and ProFAMA: FAMA-GFP /med8 transgenic seedlings were used in ChIP using an anti-GFP antibody (Millipore). ProFAMA: FAMA-GFP and ProFAMA: FAMA-GFP /med8 seedlings were inoculated with B. cinerea for varying lengths of time (0 and 36 h) before cross-linking. The “No Ab” immunoprecipitates served as negative controls. The ChIP signal was quantified as the percentage of total input DNA by RT-PCR. Average values and SEM from relative values obtained in four biological replicates were plotted on the graph. The ChIP assay was repeated at least four times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test). (C) MED8 could occupy the G-box region in the promoter of ORA59, and FAMA affects the recruitment of MED8 to the promoter of ORA59. The ProMED8: MED8-GFP and ProMED8: MED8-GFP /fama-2 transgenic seedlings were inoculated with B. cinerea for varying lengths of time (0 and 36 h) before cross-linking. The “No Ab” immunoprecipitates served as negative controls. The ChIP signal was quantified as the percentage of total input DNA by RT-PCR. Average values and SEM from relative values obtained from four biological replicates were plotted on the graph. The ChIP assay was repeated at least four times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.g007

We also assessed the interaction between FAMA and MED8 during binding the promoter of the target gene ORA59. For this purpose, we crossed the previously characterized ProFAMA: FAMA-GFP transgene into the med8 mutant background (ProFAMA: FAMA-GFP/med8). In ProFAMA: FAMA-GFP/med8 plants, the recruitment of FAMA to the promoter of ORA59 was severely reduced whether inoculated with B. cinerea or not (Fig 7B). This result indicated that MED8 could influence the recruitment of FAMA to the promoter of ORA59. We also assessed whether FAMA could influence the recruitment of MED8 to the promoter of ORA59. We crossed the ProMED8: MED8-GFP transgene into the fama-2 mutant background (ProMED8: MED8-GFP / fama-2) and discovered that the recruitment of MED8 to the promoter of ORA59 was also reduced in ProMED8: MED8-GFP / fama-2 plants, demonstrating that the recruitment of MED8 to the promoter of ORA59 is FAMA-dependent (Fig 7C).

FAMA directly activates the expression of ORA59

ChIP-PCR assays using ProFAMA: FAMA-GFP plants indicated that FAMA had bound to the promoter of ORA59. To test whether FAMA could directly activate the expression of ORA59, we used a transient assay to compare the activatory effect of FAMA on the expression of ORA59pro: LUC reporters containing the ORA59 promoter fused with the LUC gene. The co-expression of ORA59pro: LUC with 35Spro: FAMA led to an obvious increase in luminescence intensity, indicating that 35Spro: FAMA activated the expression of ORA59pro: LUC. In contrast, expression of the empty LUC vector with 35Spro: FAMA or ORA59pro: LUC alone resulted in little luciferase signal (Fig 8A and 8B). These results imply that FAMA is required for the expression of ORA59.

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Fig 8. FAMA can activate the expression of ORA59.

(A) Transient expression assays showing that the overexpression of FAMA could activate ORA59 expression. Representative images of N. benthamiana leaves 72 h after infiltration are shown. The bottom panel indicates the infiltrated constructs. Bars = 2.5 mm. (B) Quantitative analysis of luminescence intensity in (A). Average values and SEM from relative values obtained in four biological replicates were plotted on the graph. The transient expression assay was repeated at least four times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.g008

Discussion

The transcriptional regulation of gene expression plays a central role in plant immunity [52, 53]. Upon pathogenic infection, plant cells trigger genome-wide transcriptional reprogramming [54, 55]. Increasing efforts are being made to elucidate the functions of the Mediator complex, which interacts with RNA polymerase during gene transcription in plant immunity. For example, the Arabidopsis WRKY33 transcription factor-activated transcription of PDF 1.2 and ORA59 depends on mediator subunit MED16 for resistance to the pathogenic fungus Sclerotinia sclerotiorum [25]. Similarly, in this study, we provide evidence that MED8 regulates Botrytis resistance through interaction with FAMA transcription factor.

MED8 in combination with MED25 was initially reported to be involved in the defense response of plants to a variety of biotic stresses by independent and additive mechanisms [11]. The knockdown of NtMed8, which is homologous to the MED8 subunit of the Arabidopsis Mediator complex, caused abnormal development of the vegetative and floral organs in tobacco [56]. A later study found that MED8 could regulate organ size in Arabidopsis [57]. Recently, MED8 was shown to be necessary for the transcriptional regulation of genes associated with cell elongation and cell wall composition in response to cell wall defects and in sugar-responsive gene expression [58]. These results suggest that the Arabidopsis MED8 subunit has multiple roles in the development and stress response of plants. However, in contrast to the Arabidopsis Mediator subunit MED25, the molecular mechanisms underlying the diverse functions of MED8 are largely unclear.

Our investigation of the function of MED8 in Botrytis resistance focused on its interplay with FAMA; a basic helix-loop-helix (bHLH) transcription factor that acts a key component in stomatal development [30]. Our speculation that MED8 regulates plant immunity via its interaction with FAMA is based on the following findings. Firstly, MED8 interacted directly with FAMA (Fig 2B–2D), suggesting that MED8 and FAMA might function together in the same pathway. Secondly, a novel function for FAMA in plant immunity to B. cinerea was revealed in this study (Figs 3C and 4C), which indicated that MED8 could be recruited by FAMA during B. cinerea infection. Thirdly, genetic analyses revealed that MED8 affected the functioning of FAMA in the regulation of pathogen resistance and the expression of pathogen-responsive genes (Fig 6A–6F). Most importantly, we discovered that MED8 could be recruited to the promoter region of the FAMA target gene (Fig 7A–7C). Based on the above results, our study presents a new FAMA-MED8 mediated Botrytis resistance pathway. As shown in Fig 9, FAMA will bind the G-box region in the promoter of ORA59 when the pathogen signals are perceived by plant cells. FAMA will then recruit MED8 to the promoter of its target and activate the expression of downstream defensin genes, such as PDF1.2. The above-described mode of action of the Arabidopsis MED8 together with FAMA in regulating Botrytis resistance is similar to that of the Arabidopsis Mediator subunit MED25 in regulating MYC2-mediated transcription. It has been shown that in the presence of JA, MED25 is recruited to the promoter regions of MYC2 targets and, through direct interaction with MYC2, positively influences MYC2 transcriptional regulation [16]. The Mediator complex is considered to connect gene-specific transcription factors with RNA polymerase machinery to regulate gene expression [59]. Chen et al., showed that MED25 mediated the recruitment of the Pol II subunit to the promoter of MYC2 targets [16]. Wang et al., also demonstrated that MED16 played a key role in JA/ ethylene (ET)-induced recruitment of RNAPII to PDF1.2 and ORA59 [25]. As a subunit of the Mediator complex, we speculate that MED8 is likely to recruit Pol II to the promoter of the FAMA target and regulate its expression. However, this speculation requires further experimental verification. Collectively, these results illustrate that MED8 might act as a coactivator of FAMA in regulating Botrytis resistance.

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Fig 9. A model of FAMA-MED8 mediated signaling pathway in pathogen resistance.

Once pathogen signals are perceived by plant cells, FAMA will recruit MED8 to the G-box region in the promoter of FAMA targets. MED8 then recruits pol II to the promoter of FAMA target and activates downstream defensin genes. This signaling pathway is independent of the MYC2-MED25 signaling pathway in pathogen resistance.

https://doi.org/10.1371/journal.pone.0193458.g009

The necrotrophic pathogen B. cinerea can infect the host via cuticle penetration. In addition to the enzymolysis holes caused by necrotrophic pathogens, pathogens may enter the plant via the stomata, which serve as passive ports of entry during infection [60]. Therefore, it follows that defective stomata could significantly prevent pathogen infection, while increased stomatal numbers could promote pathogen infection. However, the FAMA mutation associated with defective stomata exhibits reduced resistance to B. cinerea infection, whereas the overexpression of FAMA results in a stoma-in-stoma (SIS) phenotype, characterized by the asymmetrical division of guard cells, which confers increased resistance to B. cinerea infection. This implied that the positive role of FAMA in B. cinerea resistance might be stomata-independent. Accordingly, we discovered that the expression of defense genes was significantly affected in FAMA mutated or overexpression plants. Additionally, FAMA was found to occupy the promoter of ORA59 along with MED8 upon B. cinerea infection, suggesting that FAMA regulates B. cinerea resistance through affecting defense gene expression other than the reason of developmental alteration. This differs slightly from that observed in NtMEK2. Under dexamethasone induction, GVG-Nt-MEK2DD plants exhibited no stomatal differentiation on the cotyledon epidermis, but were resistant to B. cinerea infection [61, 62]. These results suggest a complex relationship between stomatal development and B. cinerea resistance.

Our study revealed for the first time that FAMA functions in plant immunity by mediating the expression of defense genes. FAMA occupies the promoter of ORA59 along with MED8 upon B. cinerea infection. However, it was not able to directly occupy the promoter of PDF1.2. Similar results were observed with the MYC2 transcription factor in the JA pathway. MYC2 mediates JA-mediated B. cinerea resistance by regulating the expression of the MYC2-target gene, ORA59 [51]. However, MYC2 has not been found to directly occupy the promoter of PDF1.2 in any of the relevant literature, which suggests that transcription factors regulate gene expression at different levels. MYC2, as a core transcription factor in the JA pathway, mediates two branches of the JA pathway by targeting different downstream transcription factors. MYC2 can occupy the promoters of NAC019 and NAC055 to positively regulate JA-mediated root growth and insect resistance, and also occupy the promoter of ORA59 to negatively regulate JA-mediated pathogen resistance [51, 63]. Similarly, FAMA can occupy the promoters of the transcription factor bHLH090 in myrosin cell development and ORA59 in pathogen resistance [40]. This indicates that the upstream core transcription factors could regulate different specific downstream transcription factors to mediate specific pathways. Although both MYC2 and FAMA can bind the promoter of ORA59 to regulate B. cinerea resistance, they display opposite phenotypes in B. cinerea resistance [64]. Song et al., showed that MYC2 interacted with EIN3 to attenuate the transcription of ORA59 and repress ET-enhanced B. cinerea resistance [65]. Conversely, our results showed that FAMA could induce the expression of ORA59 and positively regulate the transcription of PDF1.2. An assessment of the genetic relationship between FAMA and MYC2 in B. cinerea resistance is warranted.

Our data demonstrate that MED8 regulates plant immunity towards B. cinerea by interacting with the transcription factor FAMA. Both MED8 and FAMA could occupy the G-box region in the promoter of ORA59 following B. cinerea inoculation (Fig 7A–7C). However, several questions regarding the mechanisms of action of MED8 and FAMA in pathogen resistance remain unanswered. Further identification of the genes regulated by both MED8 and FAMA should elucidate the molecular mechanisms and the signaling pathways involved in the regulation of pathogen resistance by MED8 and FAMA. Particularly, the identification of genes regulated by both MED8 and FAMA in other signaling pathways should enhance our understanding of the functions of MED8 and FAMA. Elucidating the genetic relationships of FAMA and MYC2 is important for clarifying the molecular mechanisms of FAMA in plant immunity toward B. cinerea.

Supporting information

S1 Fig. The morphological phenotypes of indicated mutants and the transgenic lines.

Photographs of Col-0, fama-1, fama-2, OE-3, OE-7, med8, and med25 plants were taken three to four weeks after being grown on soil without B. cinerea inoculation. Bars = 1 cm.

https://doi.org/10.1371/journal.pone.0193458.s001

(TIF)

S2 Fig. FAMA affects the transcriptional expression of SA-induced PR1 gene.

Expression of PR1 was examined by RT-qPCR in Col-0, fama-1, fama-2, OE-3, and OE-7 plants following inoculation of B. cinerea. Average values and SEM from relative values obtained in four biological replicates were plotted on the graph. A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated at least four times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.s002

(TIF)

S3 Fig. med8fama-2 displays increased susceptibility to B. cinerea infection.

(A) (B) Disease symptoms and lesion sizes on the B. cinerea-infected WT, med8, fama-2, and med8fama-2 leaves at 2 days.

The disease assay was performed by drop inoculation of B. cinerea on the leaves of soil-grown plants. The infected leaves were photographed and bar = 2.5 mm (A). Average values and SEM from relative values obtained from three biological replicates were plotted on the graph (B). A minimum of 10 leaves for each genotype was used for each biological replicate, and the disease assay was repeated three times, with similar results. The mean values followed by different letters represent significant differences (P< 0.01, Student’s t-test).

https://doi.org/10.1371/journal.pone.0193458.s003

(TIF)

S4 Fig. FAMA and MED8 specifically bind the G-box region in the promoter of ORA59.

(A) Schematic diagram of the promoter regions of ORA59. The black line represents the promoter region of the gene. The black box on the line and red line with the letter “a” indicates the putative G-box cis-elements (CACGTG) of the ORA59 promoter; the red line with letter “b” indicates the non-G-box region of the ORA59 promoter. The regions of “a” and “b” indicate the DNA fragments used for ChIP-PCR. The translational start sites (ATG) are shown as +1.

(B) FAMA could occupy the G-box region in the promoter of ORA59, but not the non-G-box region in the promoter of ORA59. The ProFAMA: FAMA-GFP transgenic seedlings were used in ChIP using an anti-GFP antibody (Millipore). ProFAMA: FAMA-GFP seedlings were inoculated with B. cinerea for varying lengths of time (0 and 36 h) before cross-linking. The “No Ab” (no antibody) immunoprecipitates served as negative controls. The ChIP signal was quantified as the percentage of total input DNA by real-time PCR. Three biological replicates were performed and identical results were obtained. Standard deviations were calculated from 3 technical replicates.

(C) MED8 could occupy the G-box region in the promoter of ORA59, but not the non-G-box region in the promoter of ORA59. The ProMED8: MED8-GFP transgenic seedlings were used in ChIP using an anti-GFP antibody (Millipore). ProMED8: MED8-GFP seedlings were inoculated with B. cinerea for varying lengths of time (0 and 36 h) before cross-linking. The “No Ab” (no antibody) immunoprecipitates served as negative controls. The ChIP signal was quantified as the percentage of total input DNA by real-time PCR. Three biological replicates were performed and identical results were obtained. Standard deviations were calculated from 3 technical replicates.

https://doi.org/10.1371/journal.pone.0193458.s004

(TIF)

Acknowledgments

We thank Chuanyou Li (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for providing pGWB5 and pGWB17 plasmids. We also thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

References

  1. 1. Mengiste T. Plant Immunity to Necrotrophs. Annu Rev Phytopathol. 2011;50: 267–294.
  2. 2. Laluk K and Mengiste T. Necrotroph Attacks on Plants: Wanton Destruction or Covert Extortion? The Arabidopsis Book 8: e0136. 2010.
  3. 3. Friesen TL, Faris JD, Solomon PS, Oliver RP. Host-specific toxins: effectors of necrotrophic pathogenicity. Cellular Microbiol. 2008;10: 1421–1428.
  4. 4. Hahlbrock K, Bednarek P, Ciolkowski I, Hamberger B, Heise A, Liedgens H, et al. Non-self recognition, transcriptional reprogramming, and secondary metabolite accumulation during plant/pathogen interactions. Proc Natl Acad Sci USA. 2003;100: 14569–14576. pmid:12704242
  5. 5. Jones JD and Dangl JL. The plant immune system. Nature. 2006;444: 323–329. pmid:17108957
  6. 6. Boller T and Felix G. A Renaissance of Elicitors: Perception of Microbe-Associated Molecular Patterns and Danger Signals by Pattern-Recognition Receptors. Annu Rev Plant Biol. 2009;60: 379–406. pmid:19400727
  7. 7. Fu ZQ and Dong X. Systemic Acquired Resistance: Turning Local Infection into Global Defense. Annu Rev Plant Biol. 2013;64: 839–863. pmid:23373699
  8. 8. Conaway RC and Conaway JW. Function and regulation of the Mediator complex. Curr Opin Genet Dev. 2011;21: 225–230. pmid:21330129
  9. 9. Bäckström S, Elfving N, Nilsson R, Wingsle G, Björklund S. Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 subunit. Mol Cell. 2007;26: 717–729. pmid:17560376
  10. 10. Dhawan R, Luo H, Foerster AM, Abuqamar S, Du HN, Briggs SD, et al. HISTONE MONOUBIQUITINATION1 interacts with a subunit of the mediator complex andregulates defense against necrotrophic fungal pathogens in Arabidopsis. Plant Cell. 2009;21: 1000–1019. pmid:19286969
  11. 11. Kidd BN, Edgar CI, Kumar KK, Aitken EA, Schenk PM, Manners JM, et al. The mediator complex subunit PFT1 is a key regulator of jasmonate-dependent defense in Arabidopsis. Plant Cell. 2009;21: 2237–2252. pmid:19671879
  12. 12. Kim YJ, Zheng B, Yu Y, Won SY, Mo B, Chen X. The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana. EMBO J. 2011;30: 814–822. pmid:21252857
  13. 13. Caillaud MC, Asai S, Rallapalli G, Piquerez S, Fabro G, Jones JD. A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex. PLoS Biol. 2013;11: e1001732. pmid:24339748
  14. 14. Hemsley PA, Hurst CH, Kaliyadasa E, Lamb R, Knight MR, De Cothi EA, et al. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes. Plant Cell. 2014;20: 9812–9841.
  15. 15. Cevik V, Kidd BN, Zhang P, Hill C, Kiddle SJ, Denby KJ, et al. MEDIATOR25 Acts as an Integrative Hub for the Regulation of Jasmonate-Responsive Gene Expression in Arabidopsis. Plant Physiol. 2012;160: 541–555. pmid:22822211
  16. 16. Chen R, Jiang HL, Li L, Zhai QZ, Qi LL, Zhou WK, et al. The Arabidopsis mediator subunit MED25 differentially regulates jasmonate and abscisic acid signaling through interacting with the MYC2 and ABI5 transcriptionfactors. Plant Cell. 2012;24: 2898–2916. pmid:22822206
  17. 17. Zhang X, Wang C, Zhang Y, Sun Y, Mou Z. The Arabidopsis mediator complex subunit16 positively regulates salicylate-mediated systemic acquired resistance and jasmonate/ethylene-induced defense pathways. Plant Cell. 2012;24: 4294–4309. pmid:23064320
  18. 18. Bonawitz ND, Soltau WL, Blatchley MR, Powers BL, Hurlock AK, Seals LA, et al. REF4 and RFR1, subunits of the transcriptional coregulatory complex mediator, are required for phenylpropanoid homeostasis in Arabidopsis. J Biol Chem. 2012;287: 5434–5445. pmid:22167189
  19. 19. Zhang X, Yao J, Zhang Y, Sun Y, Mou Z. The Arabidopsis Mediator complex subunits MED14/SWP and MED16/SFR6/IEN1 differentially regulate defense gene expression in plant immune responses. Plant J. 2013;75: 484–497. pmid:23607369
  20. 20. Lai ZB, Schluttenhofer CM, Bhide K, Shreve J, Thimmapuram J, Lee SY, et al. MED18 interaction with distinct transcription factors regulates multiple plant functions. Nat Commun. 2014;5: 3064. pmid:24451981
  21. 21. Zhu YF, Schluttenhoffer CM, Wang PC, Fu FY, Thimmapuram J, Zhu JK, et al. CYCLIN-DEPENDENT KINASE8 differentially regulates plant immunity to fungal pathogens through kinase dependent and independent functions. Plant Cell. 2014;26: 4149–4170. pmid:25281690
  22. 22. Li W, Yoshida A, Takahashi M, Maekawa M, Kojima M, Sakakibara H, et al. SAD1, an RNA polymerase I subunit A34.5 of rice, interacts with Mediator and controls various aspects of plant development. Plant J. 2015;81: 282–291. pmid:25404280
  23. 23. Zou JJ, Li XD, Ratnasekera D, Wang C, Liu WX, Song LF, et al. Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-mediated signaling and H2O2 homeostasis in stomatal guard cells under drought stress. Plant Cell. 2015;27: 1445–1460. pmid:25966761
  24. 24. Chhun T, Chong SY, Park BS, Wong EC, Yin JL, Kim M, et al. HSI2 repressor recruits MED13 and HDA6 to down-regulate seed maturation gene expression directly during Arabidopsis early seedling growth. Plant Cell physiol. 2016;57: 1689–1706. pmid:27335347
  25. 25. Wang C, Yao J, Du X, Zhang Y, Sun Y, Rollins JA, et al. The Arabidopsis mediator complex subunit16 is a key component of basal resistance against the necrotrophic fungal pathogen sclerotinia sclerotiorum. Plant Physiol. 2015;169: 856–872. pmid:26143252
  26. 26. Lee E, Lucas JR, Goodrich J, Sack FD. Arabidopsis guard cell integrity involves the epigenetic stabilization of the FLP and FAMA transcription factor genes. Plant J. 2014;78: 566–577. pmid:24654956
  27. 27. Chen L, Guan L, Qian P, Xu F, Wu Z, Wu Y, et al. NRPB3, the third largest subunit of RNA polymerase II, is essential for stomatal patterning and differentiation in Arabidopsis. Development. 2016;143: 1600–1611. pmid:26989174
  28. 28. Hachez C, Ohashi-Ito K, Dong J, Bergmann D. Differentiation of Arabidopsis guard cells: analysis of the networks incorporating the basic helix-loop helix transcription factor, FAMA. Plant Physiol. 2011;155: 1458–1472. pmid:21245191
  29. 29. Matos JL, Lau OS, Hachez C, Cruz-Ramírez A, Scheres B, Bergmann DC. Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMA-RELATED module. Elife. 2014;3: e03271.
  30. 30. Ohashi-Ito K and Bergmann DC. Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell. 2006;18: 2493–2505. pmid:17088607
  31. 31. Pillitteri LJ and Torii KU. Breaking the silence: three bHLH proteins direct cell-fate decisions during stomatal development. Bioessays. 2007;29: 861–870. pmid:17691100
  32. 32. Serna L. Emerging parallels between stomatal and muscle cell lineages. Plant Physiol. 2009;149: 1625–1631. pmid:19201912
  33. 33. Tricker PJ, Gibbings JG, Rodríguez López CM, Hadley P, Wilkinson MJ. Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development. J Exp Bot. 2012;63: 3799–3813. pmid:22442411
  34. 34. Torii KU, Kanaoka MM, Pillitteri LJ, Bogenschutz NL. Stomatal development: three steps for cell-type differentiation. Plant Signal Behav. 2007;2: 311–313. pmid:19704632
  35. 35. Peterson KM, Rychel AL, Torii KU. Out of the mouths of plants: The molecular basis of the evolution and diversity of stomatal development. Plant Cell. 2010;22: 296–306. pmid:20179138
  36. 36. Kanaoka MM, Pillitteri LJ, Fujii H, Yoshida Y, Bogenschutz NL, Takabayashi J, et al. SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation. Plant Cell. 2008;20: 1775–1785. pmid:18641265
  37. 37. MacAlister CA, Ohashi-Ito K, Bergmann DC. Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature. 2007;445: 537–540. pmid:17183265
  38. 38. Pillitteri LJ, Sloan DB, Bogenschutz NL, Torii KU. Termination of asymmetric cell division and differentiation of stomata. Nature. 2007;445: 501–505. pmid:17183267
  39. 39. Li M and Sack FD. Myrosin idioblast cell fate and development are regulated by the Arabidopsis transcription factor FAMA, the auxin pathway, and vesicular trafficking. Plant Cell. 2014;26: 4053–4066. pmid:25304201
  40. 40. Shirakawa M, Ueda H, Nagano AJ, Shimada T, Kohchi T, Hara-Nishimura I. FAMA is an essential component for the differentiation of two distinct cell types, myrosin cells and guard cells, in Arabidopsis. Plant Cell. 2014;26: 4039–4052. pmid:25304202
  41. 41. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473–497.
  42. 42. Liao CJ, Lai Z, Lee S, Yun D, Mengiste T. Arabidopsis HOOKLESS1 Regulates Responses to Pathogens and Abscisic Acid through Interaction with MED18 and Acetylation of WRKY33 and ABI5 Chromatin. Plant Cell. 2016;28: 1662. pmid:27317674
  43. 43. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium‐mediated transformation of Arabidopsis thaliana. Plant J. 1998;16: 735–743. pmid:10069079
  44. 44. Zhu JY, Sun Y, Wang ZY. Genome-wide identification of transcription factor-binding sites in plants using chromatin immunoprecipitation followed by microarray (ChIP-chip) or sequencing (ChIP-seq). In: Wang ZY., Yang Z. (eds) Plant Signalling Networks. Methods in Molecular Biology (Methods and Protocols), 876. Humana Press; 2011.
  45. 45. Zheng ZY, Abuqamar S, Chen ZX, Mengiste T. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J. 2006;48: 592–605. pmid:17059405
  46. 46. Koch E and Slusarenko A. Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell. 1990;2: 437–445. pmid:2152169
  47. 47. Chen Q, Sun J, Zhai Q, Zhou W, Qi L, Xu L, et al. The basic helix-loop-helix transcription factor MYC2 directly represses PLETHORA expression during jasmonatemediated modulation of the root stem cell niche in Arabidopsis. Plant Cell. 2011;23: 3335–3352. pmid:21954460
  48. 48. Penninckx IA, Eggermont K, Terras FR, Thomma BP, De Samblanx GW, Buchala A, et al. Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid independent pathway. Plant Cell. 1996;8: 2309–2323. pmid:8989885
  49. 49. Boter M, Ruíz-Rivero O, Abdeen A, Prat S. Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes Dev. 2004;18: 1577–1591. pmid:15231736
  50. 50. Lorenzo O, Chico JM, Sánchez-Serrano JJ, Solano R. JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell. 2004;16: 1938–1950. pmid:15208388
  51. 51. Zhai QZ, Yan LH, Tan D, Chen R, Sun JQ, Gao L, et al. Phosphorylation-coupled proteolysis of the transcription factor MYC2 is important for jasmonate-signaled plant immunity. PloS Genet. 2013;9(4):e1003422 pmid:23593022
  52. 52. Li B, Meng X, Shan L, He P. Transcriptional regulation of pattern-triggered immunity in plants. Cell Host Microbe. 2016;19: 641–650. pmid:27173932
  53. 53. Birkenbihl RP, Kracher B, Somssich IE. Induced Genome-Wide Binding of Three Arabidopsis WRKY Transcription Factors during Early MAMP-Triggered Immunity. Plant Cell. 2016. http://doi.org/10.1105/tpc.16.00681.
  54. 54. Abuqamar S, Chen X, Dhawan R, Bluhm B, Salmeron J, Lam S, et al. Expression profiling and mutant analysis reveals complex regulatory networks involved in Arabidopsis response to Botrytis infection. Plant J. 2006;48: 28–44. pmid:16925600
  55. 55. Thilmony R, Underwood W, He SY. Genome-wide transcriptional analysis of the Arabidopsis thaliana interaction with the plant pathogen Pseudomonas syringae pv. tomato DC3000 and the human pathogen Escherichia coli, O157:H7. Plant J. 2006;46: 34–53. pmid:16553894
  56. 56. Wang F, Wei H, Tong Z, Zhang X, Yang Z, Lan T, et al. Knockdown of NtMed8, a Med8-like gene, causes abnormal development of vegetative and floral organs in tobacco (Nicotiana tabacum L.). Plant Cell Rep. 2011;30: 2117–2129. pmid:21744120
  57. 57. Xu R, Li Y. The Mediator complex subunit 8 regulates organ size in Arabidopsis thaliana. Plant signaling & behavior. 2012;7: 182–183.
  58. 58. Seguela-Arnaud M, Smith C, Uribe M C, May S, Fischl H, Mckenzie N, et al. The Mediator complex subunits MED25/PFT1 and MED8 are required for transcriptional responses to changes in cell wall arabinose composition and glucose treatment in Arabidopsis thaliana. BMC plant biol. 2015;15: 215. pmid:26341899
  59. 59. Nozawa K, Schneider TR, Cramer P. Core Mediator structure at 3.4 Å extends model of transcription initiation complex. Nature. 2017;545: 248–251. pmid:28467824
  60. 60. Hsieh TF, Huang JW, Hsiang T. Light and Scanning Electron Microscopy Studies on the Infection of Oriental Lily Leaves By Botrytis Elliptica. Eur J Plant Pathol. 2001;107: 571–581.
  61. 61. Wang H, Ngwenyama N, Liu Y, Walker J, Zhang S. Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinase in Arbidopsis. Plant Cell. 2007;19: 63–73. pmid:17259259
  62. 62. Ren D, Liu Y, Yang KY, Han L, Mao GH, Glazebrook J, et al. A Fungal-Responsive MAPK Cascade Regulates Phytoalexin Biosynthesis in Arabidopsis. P Natl Acad Sci USA. 2008;105: 5638.
  63. 63. Bu QY, Jiang HL, Li CB, Zhai QZ, Zhang J, Wu XY, et al. Role of the Arabidopsis thaliana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses. Cell Res. 2008;2008: 756–767.
  64. 64. Dombrecht B, Xue GP, Sprague SJ, Kirkegaard JA, Ross JJ, Reid JB, et al. MYC2 differentially modulates diverse jasmonatedependent functions in Arabidopsis. Plant Cell. 2007;19: 2225–2245. pmid:17616737
  65. 65. Song S, Huang H, Gao H, Wang J, Wu D, Liu X, et al. Interaction between MYC2 and ETHYLENE INSENSITIVE3 modulates antagonism between jasmonate and ethylene signaling in Arabidopsis. Plant Cell. 2014;26: 263. pmid:24399301