Abstract Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione-based antioxidant defense.

Citation: McLaughlin JE, Bin-Umer MA, Widiez T, Finn D, McCormick S, Tumer NE (2015) A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin. PLoS ONE 10(6): e0130204. https://doi.org/10.1371/journal.pone.0130204 Academic Editor: Els JM van Damme, Ghent University, BELGIUM Received: February 24, 2015; Accepted: May 17, 2015; Published: June 9, 2015 This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This is a cooperative project No. 59-0206-1-121 supported by the United States Department of Agriculture in cooperation with the United States Wheat and Barley Scab initiative and by the National Institutes of Health Grant S10RR025424 to Nilgun E. Tumer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Infection of small grain cereals such as wheat and barley with Fusarium graminearum or Fusarium culmorum causes Fusarium head blight (FHB), also known as scab, which is one of the most important diseases of cereals worldwide. FHB results in yield reductions and in the contamination of grain with heat stable trichothecene mycotoxins, the most prominent being deoxynivalenol (DON) [1–3]. Trichothecenes present a significant food safety risk and health hazard to humans and animals [4], and controlling their accumulation in small grains remains a huge challenge. Strict limits are set for DON levels in flour products and in malting barley and the crop is rejected by the industry if these limits are exceeded. Since the re-emergence of FHB in the USA in 1993, epidemics and associated mycotoxin contamination have been reported in wheat in different production regions [5]. Breeding for resistance to FHB has been challenging due to the potency and persistent nature of the mycotoxins, the need to introgress major resistance loci from exotic germplasm, and the multigenic nature of resistance [1,6]. Major quantitative trait loci (QTLs) like the Fhb1 region identified on the short arm of chromosome 3B from the wheat cultivar Sumai 3 are rare and the resistance of Fhb1 is dependent on the genetic background of the cultivar [6,7]. Control of FHB by the application of fungicides has been only partially effective [8]. Trichothecene mycotoxins function as virulence factors during pathogenesis of F. graminearum on wheat [9,10]. Gene disruption mutants of F. graminearum, which lack the trichothecene biosynthesis gene, trichodiene synthase (∆tri5), are less virulent [9,10]. Hence resistance to trichothecenes is considered to be an important aspect of resistance to FHB. Partial resistance to FHB in wheat is characterized by either inhibition of initial infection (type I) or inhibition of the spread of infection (type II). DON has been shown to promote the spread of F. graminearum in the rachis of wheat [11]. In barley however, the spread of the infection within the spike is inhibited due to type II resistance [11]. Therefore, strategies that prevent initial infection and inhibit spread of the disease are needed for effective resistance against FHB in wheat and barley. Trichothecenes target ribosomal protein L3 at the peptidyltransferase center in S. cerevisiae and inhibit peptidyltransferase activity of eukaryotic ribosomes [12–14]. Expression of a modified ribosomal protein L3 provided enhanced resistance to DON in tobacco and to FHB in wheat [15–17]. Additional studies demonstrated that increasing resistance of wheat to DON enhances resistance to FHB, indicating that genes that confer resistance to DON reduce the impact of FHB [18,19]. Trichothecenes are reported to have diverse roles in the cell that are not limited to the inhibition of cytosolic protein synthesis [20,21]. DON targets the innate immune system and activates ribotoxic stress, resulting in upregulation of cytokine gene expression in mammalian cells [22]. Trichothecene exposure in humans and animals can cause immunosuppression, anorexia, emesis, growth retardation and in large doses, death [20]. In plants DON causes oxidative stress damage by increasing reactive oxygen species (ROS) levels [21]. DON treatment of wheat induced hydrogen peroxide synthesis and caused an increase in programmed cell death [23]. Expression of ROS-related genes such as glutathione-S-transferase, superoxide dismutase, and peroxidase were altered in wheat or barley in response to DON or F. graminearum [24–27]. Hydrogen peroxide enhances DON production by Fusarium [28,29]. Moreover, upon infection with F. graminearum carbohydrate and nitrogen metabolism of the plant is affected [30]. Plant nitrogen metabolism is redirected towards the formation of polyamines, which induce DON production by F. graminearum [31]. We previously carried out a genome-wide screen of the S. cerevisiae deletion collection against trichothecin (Tcin), a type B trichothecene in the same class as DON, and identified mitochondrial translation inhibition and mitochondrial fragmentation as novel mechanisms of trichothecene toxicity [32]. Tcin inhibited translation in isolated mitochondria from yeast, demonstrating that mitochondrial translation is a direct target of trichothecene mycotoxins [33]. We demonstrated a vital role for mitochondrial oxidative stress in trichothecene sensitivity and mitophagy in elimination of trichothecene-damaged mitochondria [34]. Here we screened an activation tagged population of Arabidopsis thaliana against trichothecin (Tcin), a type B trichothecene in the same class as DON and identified a gain-of-function mutant in which two closely linked non-specific lipid transfer protein (nsLTP) genes were overexpressed. Overexpression of AtLTP4.4 in Arabidopsis or in S. cerevisiae enhanced resistance to Tcin. Tcin treatment increased reactive oxygen species (ROS) production and caused an imbalance of the redox state in wild type Arabidopsis and yeast. Glutathione content was higher and ROS levels were attenuated in both systems when AtLTP4.4 was overexpressed.

Discussion Activation tagging has not previously been used to isolate genes that can confer resistance to mycotoxins. In this study we screened activation-tagged Arabidopsis lines transformed with a T-DNA vector containing four copies of the CaMV 35S enhancer sequences and identified a mutant, trr1, which displayed enhanced resistance to Tcin. Analysis of trr1 revealed that the T-DNA, which contained tandem (4X) CaMV 35S enhancers upregulated expression of two tightly linked downstream genes AtLTP4.4 (AT5G55450) and AtLTP4.5 (AT5G55460) over 10-fold (Fig 1C). The two genes, which encode previously uncharacterized nsLTPs, show 48% nucleotide sequence identity and are arranged in tandem duplication repeats on chromosome 5. Arabidopsis contains 49 nsLTP genes, which are classified into nine types based on phylogenetic analysis [36]. AtLTP4.4 and AtLTP4.5 belong to type IV nsLTPs, which include 5 members on chromosome 5 [36]. Although other nsLTP genes were located near AtLTP4.4 and AtLTP4.5, only AtLTP4.4 and AtLTP4.5 were overexpressed in trr1, allowing functional characterization of these two genes without interference from other family members. The nsLTPs are small basic proteins characterized by a conserved eight cysteine motif backbone [42]. The Cys residues form 4 disulfide bonds that stabilize a hydrophobic cavity, which can bind fatty acid chains and potentially other small molecules [42]. The physiological function of nsLTPs is not well understood. The nsLTPs have been shown to exchange lipids between membranes and artificial vesicles in vitro [43]. There is evidence that they are involved in diverse biological processes, such as cutin biosynthesis, anther development, stress resistance and different signaling pathways [43], suggesting that they may be involved in intracellular lipid trafficking. They are generally secreted and associated with the cell wall [42]. We show here that AtLTP4.4:GFP localized not only to the cell wall and apoplast, but also colocalized with the chloroplast and possibly with the ER (Fig 3), providing evidence for intracellular localization of AtLTP4.4:GFP. Although nsLTPs have antimicrobial activity and are thought to be important components of defense against bacterial and fungal pathogens, their mechanism of action is not well understood. Pepper nsLTPs show structural similarity to elicitins from fungal pathogens [44]. Overexpression of these proteins was shown to activate systemic acquired resistance (SAR)-mediated signaling pathways [45]. The DIR1 gene of Arabidopsis, which encodes another type IV nsLTP (AtLTP 4.1) [36], was identified in a screen of mutants deficient in SAR, but not in local defense responses [46]. DIR1 overexpression had little or no effect on resistance to bacterial infection in Arabidopsis [47]. Although DIR1 belongs to the same group as AtLTP4.4 and AtLTP4.5, we did not see improved resistance to Tcin in the DIR1 overexpressing Arabidopsis line, DD5E [47]. Moreover, overexpression of another type IV nsLTP from Arabidopsis, AtLTP1.1 failed to produce resistance to Tcin in yeast (Table 1). These results indicate that there are functional differences between different members of the nsLTP gene family and that AtLTP4.4 and AtLTP4.5 have a function specific to the trichothecene response. AtLTP4.4 was expressed at a higher level in transgenic Arabidopsis than AtLTP4.5 and Arabidopsis plants overexpressing AtLTP4.4 showed a higher level of resistance. Yeast expressing AtLTP4.4 also showed a higher level of resistance to different trichothecenes compared to yeast expressing AtLTP4.5. The observed resistance to type A (T-2 and DAS) and type B (Tcin and DON) trichothecenes associated with AtLTP4.4 expression in yeast indicated that resistance was not specific to plants, suggesting a conserved cellular protection mechanism. Previous results indicated that impairment of mitochondrial function due to trichothecene treatment causes an increase in ROS in yeast [33]. Mitochondria not only accumulate ROS, they are also a target of Tcin-induced ROS because Tcin induced mitochondrial ROS damages mitochondrial membranes in yeast [33]. Several studies suggest that chloroplasts are a major source of ROS in plants [48,49]. Confocal microscopy analysis of Arabidopsis and tobacco leaves treated with trichothecenes showed that ROS stain is associated with the chloroplast, the cell wall, and the apoplast after treatment with DON or Tcin. Increasing the dose of Tcin or DON intensified the staining of chloroplasts. Overnight treatment with a high dose of DON (240 μM) damaged chloroplasts as does the treatment with a low concentration of paraquat (S3E and S3F Fig), suggesting that the chloroplast plays an important role in trichothecene sensitivity both as a target of the toxin and as a source of ROS generation. The damage from Tcin appeared to be light-dependent (S4B Fig) similar to Paraquat, which is toxic only in plants exposed to light [50]. These results suggest that Tcin induces photooxidative stress by either directly targeting the chloroplast or by promoting damage to the chloroplast. Application of DON to barley detached leaf segments induced bleaching in plants exposed to light [51]. As observed in our study, bleaching did not occur in samples exposed to dark. However, experimental evidence suggested that damage to the plasmalemma was the primary effect of the DON treatment. Hence, the authors concluded that the light-induced damage to the chloroplast represented a secondary effect [51]. Further evidence for the effect of light on plant responses to trichothecenes has been provided using Arabidopsis cell cultures and wheat plants exposed to DON [52]. This study showed that light plays a role in viability and induction of defense responses due to DON exposure, but did not address the direct effect of DON on the chloroplast [52]. Evidence that mycotoxins can damage chloroplasts was observed with fumonisin B1, which functions in a light-dependent manner to induce ROS production in chloroplasts, chloroplast damage, and ultimately cell death in Arabidopsis [53]. The protective effect of overexpressing the cysteine-rich AtLTP4.4 could be due to the reduction of membrane damage mediated by trichothecene-induced ROS accumulation. Glutathione is the primary defense compound against oxidative damage to membranes [39]. It reduces hydroperoxide groups on phospholipids and other lipid peroxides through glutathione-S-transferases [39]. We found that overexpression of AtLTP4.4 resulted in increased glutathione levels, suggesting the involvement of AtLTP4.4 in the regulation of cellular redox homeostasis. Exogenous addition of glutathione and other antioxidants enhanced resistance to Tcin while the addition of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, increased sensitivity to the toxin, suggesting that Tcin resistance is mediated by glutathione. A significant increase in the total glutathione level was observed in Arabidopsis and yeast overexpressing AtLTP4.4, and both systems showed improved resistance to Tcin. In contrast, a similar increase in the total glutathione level was not observed in yeast overexpressing AtLTP4.5, consistent with the lower level of trichothecene resistance compared to yeast expressing AtLTP4.4. Due to its reducing power, glutathione has essential functions in plant development, defense response against oxidative stress, including redox-homeostatic buffering and detoxification of heavy metals and xenobiotics [54]. Detoxification mechanisms against DON include conjugation to glucose and glutathione [24,55,56]. Detoxifying trichothecenes by overexpressing UDP-glucosyltransferase, an enyzme that converts DON to the less toxic DON-3-O-glucoside, has been shown to provide enhanced resistance in Arabidopsis, barley, and wheat [18,57]. Glutathione conjugates of DON have been identified in wheat, indicating that this is an important detoxification mechanism [24,55,56]. The thiol group of glutathione can react with the α,β-unsaturated ketone of type B trichothecenes and form a Michael adduct [58]. The trichothecene conjugates are known as masked mycotoxins since they are undetectable by conventional analytical techniques because their structure has been altered in the plant [59]. They may present a health risk because conversion back to a toxic form can occur upon consumption by animals or humans [59]. However, the glutathione conjugates cannot cross biological membranes and are not likely to revert back to the toxic form [59]. Hence during infection by F. graminearum, glutathione may be sequestered by conjugation to DON, affecting the oxidative status and the defense response of the host plant. We show here that enhancing glutathione content impacts plant resistance to trichothecenes and may offer a way to compliment other trichothecene detoxification methods. Emerging evidence suggests that redox sensitive Cys residues in proteins may function as oxidant sensors and can scavenge ROS [60]. Maspin (a serine protease inhibitor) which is rich in cysteine residues (8 in total, similar to nsLTPs) was found to function as an efficient ROS scavenger in mouse mammary cells [61]. Recently an Arabidopsis screen using paraquat identified an oxidative stress-induced bioreactive peptide, OSIP108, containing a ROS-scavenging cysteine [62]. The Cys groups in AtLTP4.4 may participate in maintaining the redox state by becoming oxidized by ROS. Our results demonstrate the general ability of AtLTP4.4 to impact the redox system of the cell and protect against trichothecene-mediated oxidative stress by maintaining redox homeostasis.

Materials and Methods Screening activation tagged lines and characterization of the Tcin resistant mutant The activation tagged Arabidopsis thaliana lines in Columbia (Col-0) background were generated by Agrobacterium transformation with the pSKI015 plasmid containing the modified T-DNA vector, which contains four copies of the cauliflower mosaic virus (CaMV) 35S enhancer [35], and were a kind gift from Dr. Brian Gregory. In vitro germination assays on solid media in petri plates (150 mM) were used to screen for mutants resistant to the toxin. The plates contained 1X Murashige and Skoog (Gamborg Modified) Basal Media (2.5 g/L) (Sigma M0404), 50 mM Ferric EDTA, and 0.6% Phytagel (Sigma P8169) and 4 μM Tcin. Resistance was scored 30 days after seeding the plates. Resistant mutants were planted in MetroMix 360 potting soil (Sun Gro Horticulture), and grown to maturity in a growth chamber maintained at 22°C (16 h light/8 hour dark). The trr1 was characterized by thermal asymmetric interlaced (TAIL) PCR [63]. The amplified band was sequenced and the genomic location of the insert was mapped using T-DNA express (http://signal.salk.edu/cgi-bin/tdnaexpress). Plant expression vectors The AtLTP4.4 and AtLTP4.5 coding DNA sequence (CDS) without the stop codon were amplified by RT-PCR and cloned into the entry vector pCR8/GW/TOPO (Invitrogen) to generate NT1618 and NT1619, respectively. AtLTP4.4 and AtLTP4.5 without the stop codon were cloned into the binary vector pEarleyGate 103 (BAR-35S-Gateway-GFP-His tag-OCS 3')[37] to generate NT1620 and NT1621, respectively. Agrobacterium (GV3101 MP90 strain) containing NT1620 and NT1621 was used to transform Arabidopsis [38]. Transformed seedlings were identified by plating out on MS agar media containing BASTA (25 μg/mL). Trichothecene mycotoxin Tcin, DAS, T-2, and DON were isolated and prepared as described previously [32,64]. Real-time PCR Quantitative Reverse Transcription PCR (qRT-PCR) using SYBR Green was used to quantify expression of genes in trr1. AtLTP4.4 expression was determined in the vector control, overexpression lines, and a knockout line (SALK_207859c, Col-0), which was obtained from the Arabidopsis Biological Resource Center (ABRC). Glutamate-cysteine ligase (GSH1, AT4G23100) expression was measured from mature leaf tissue collected from the vector control and overexpression lines. The Comparative Ct method (∆∆Ct) method was used to quantity gene expression using Actin8 for normalization relative to the Col-0 control. Transient expression and confocal microscopy analysis Fluorescence of the lower epidermis of tobacco leaf discs was visualized with a Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss, Inc., Thornwood, NY, USA) two to three days after infiltration with AtLTP4.4:GFP and AtLTP4.5:GFP. GFP and chlorophyll were excited with a 488-nm argon laser and fluorescence was detected using 495–570 nm and 650–760 nm bandpass filters, respectively. Image data were analyzed using the ZEN 2010 software (Carl Zeiss, Inc.). Leaves from stably transformed Arabidopsis lines were visualized with a Zeiss LSM 710 laser scanning microscope as described above for the transient expression analysis in tobacco. Yeast Strains and Plasmids Yeast strain BY4743 (MATa/α, his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, LYS2/lys2Δ0, met15Δ0/MET15, ura3Δ0/ura3Δ0) was used as the background for all experiments. Gateway entry vector pDONR221 was purchased from Invitrogen while the destination vector pAG425-GAL1-ccdB-HA (plasmid# 14249) was purchased from Addgene (www.addgene.com). Standard gateway cloning procedure was followed to clone AtLTP4.4 and AtLTP 4.5 genes into the destination vector, pAG425-GAL1-ccdB-HA to generate NT1754 and NT1755, respectively. Measurement of ROS The quantification of H 2 O 2 in leaf extracts was performed using Amplex Red Ultra (Invitrogen-Molecular Probes) according to the manufacturer’s instructions. Fluorescence was measured with the BioTek Synergy 4 plate reader (excitation 530 nm, emission 590 nm). The concentration of H 2 O 2 in each sample was calculated using a standard curve. The standard curve was linear to at least 10 μM H 2 O 2 . ROS was visualized in leaf tissue by infiltrating the cell permeable fluorescent probe 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) using vacuum infiltration. Leaf disks were visualized with a Zeiss LSM 710 laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY, USA) as described above. Treated and untreated yeast cells were stained with 2',7'-dichlorfluorescein-diacetate (DCFH-DA) for ROS generation. Cells were analyzed using the Accuri C6 Flow Cytometer (Accuri Cytometers Inc., Ann Arbor, MI) as described previously [34]. GSH and GSSG quantification Reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) levels were determined using the GSH-Glo Glutathione Assay (Promega) according to the manufacturer’s instructions. Briefly, leaf tissue was quick frozen in liquid nitrogen and ground in an eppendorf tube in 250 μL of PBS (pH 7.4) containing 2 mM EDTA. The protein content of the lysate was determined by the Bradford assay. The lysate was diluted to ~0.05 mg/mL and a 15 μL aliquot was added to two adjacent wells in a 384-well white plate. To one well 1 mM TCEP was added to determine total (GSH and GSSG) glutathione concentration. 15 μl of GSH-Glo Reagent (Luciferin-NT and Glutathione S-Transferase added at a 1/100 ratio to GSH-Glo Reaction Buffer) was added to each sample and incubated at room temperature in the dark for 30 minutes. 15 μl reconstituted Luciferin detection reagent was then added and the sample was incubated for 15 minutes. Luminescence was determined using the BioTek Synergy 4 plate reader and glutathione levels were established by comparing data to a standard curve. For GSH/GSSH quantification for yeast, cultures were spun down, supernatant removed, quick frozen in liquid nitrogen and stored at -80°C. The yeast pellet was resuspended in 250 μL of PBS (pH 7.4) containing 2 mM EDTA and the cells were broken using glass beads with the MiniBeadBeater (Biospec Products). The protein content of the lysate was determined via UV absorbance using the BioTek Synergy4 spectrophotometer with the Take3 plate. The lysate was diluted to ~0.05 mg/mL concentration of protein and analyzed as described for the plant samples. Statistics The data are shown as means ± SEM and analyzed, where indicated, either by performing t-tests or by ANOVA using the Bonferroni algorithm to test differences between individual treatments. Origin 2015 (OriginLab) software was used for both graphing and for the statistical analysis.

Supporting Information S1 Fig. AtLTP4.4:GFP expression in the roots of transgenic Arabidopsis plants. Confocal microscopy analysis of root cuttings from wild type Arabidopsis Col-0 (A) and Arabidopsis line #16 overexpressing AtLTP4.4:GFP (B). https://doi.org/10.1371/journal.pone.0130204.s001 (TIF) S2 Fig. Transient expression of AtLTP4.4:GFP and AtLTP4.5:GFP in tobacco leaves. (A) Mock treated tobacco leaf with autofluorescent (red) chloroplasts. (B) Tobacco leaf infiltrated with Agrobacterium containing AtLTP4.4:GFP. (C) Tobacco leaf infiltrated with Agrobacterium containing AtLTP4.5:GFP. https://doi.org/10.1371/journal.pone.0130204.s002 (TIF) S3 Fig. DON induces ROS accumulation in wild type tobacco plants. (A) Mock infiltration of tobacco leaves with buffer. (B) Infiltration of tobacco leaves with 10 μM DON for 24h, (C, D and E) infiltration of tobacco leaves with 240 μM DON for 24h. Chloroplast membrane damage is shown in (E) after 240 μM DON treatment for 24h resulting in disorganized chlorophyll autofluorescence. (F) Treatment with 600 nM paraquat for 2h. Colocalization of the DCF fluorescence (green) with chloroplasts (red) is indicated by yellow. https://doi.org/10.1371/journal.pone.0130204.s003 (TIF) S4 Fig. Exogenous antioxidants enhance the tolerance of Arabidopsis (Col-0) to Tcin. (A) Wild type Arabidopsis (Col-0) leaves were treated with 2 mM vitamin C, vitamin E, or para-amino benzoic acid (PABA) either alone or together with 8 μM Tcin for 48h and photographed. (B) Wild type Arabidopsis (Col-0) seedlings were treated with 4 μM or 40 μM Tcin and grown in 16h light/8h dark cycle or in continuous dark cycle for 6 days and photographed. https://doi.org/10.1371/journal.pone.0130204.s004 (TIF) S1 Table. Primers used for qRT-PCR analysis of trr1 and wild type Arabidopsis (Col-0). Primer pair sequences used to amplify At5G55440 and upstream and downstream flanking genes and the housekeeping gene Act8 are shown. https://doi.org/10.1371/journal.pone.0130204.s005 (TIF)

Acknowledgments We would like to acknowledge Dr. Brian Gregory for supplying activating tagged seed, Ms. Emily Salmon-Denikos for help with the TAIL PCR analysis, Dr. Nicole Cathala for the 35S:GFP (Col-0) transgenic line, Dr. Robin Cameron for the DIR1 overexpressing Arabidopsis line, Drs. Tom Leustek and Naveen Joshi for discussions and technical help with the glutathione quantification assay, Dr. Hugo Dooner for critical reading of the manuscript and members of the Tumer laboratory for insightful discussions and technical advice.

Author Contributions Conceived and designed the experiments: NET JEM. Performed the experiments: JEM MAB TW DF. Analyzed the data: JEM NET. Contributed reagents/materials/analysis tools: SM. Wrote the paper: NET JEM.