Accepted Manuscript High REDOX RESPONSIVE TRANSCRIPTION FACTOR1 levels result in accumulation of reactive oxygen species in Arabidopsis thaliana shoots and roots Mitsuhiro Matsuo, Joy Michal Johnson, Ayaka Hieno, Mutsutomo Tokizawa, Mika Nomoto, Yasuomi Tada, Rinesh Godfrey, Junichi Obokata, Irena Sherameti, Yoshiharu Y. Yamamoto, Frank-D. Böhmer, Ralf Oelmüller PII:

S1674-2052(15)00179-3

DOI:

10.1016/j.molp.2015.03.011

Reference:

MOLP 114

To appear in:

MOLECULAR PLANT

Received Date: 1 February 2015 Revised Date:

17 March 2015

Accepted Date: 19 March 2015

Please cite this article as: Matsuo M., Michal Johnson J., Hieno A., Tokizawa M., Nomoto M., Tada Y., Godfrey R., Obokata J., Sherameti I., Yamamoto Y.Y., Böhmer F.-D., and Oelmüller R. (2015). High REDOX RESPONSIVE TRANSCRIPTION FACTOR1 levels result in accumulation of reactive oxygen species in Arabidopsis thaliana shoots and roots. Mol. Plant. doi: 10.1016/j.molp.2015.03.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT High REDOX RESPONSIVE TRANSCRIPTION FACTOR1 levels result in accumulation of reactive oxygen species in Arabidopsis thaliana shoots and roots Mitsuhiro Matsuo*1,2, Joy Michal Johnson*1, Ayaka Hieno3, Mutsutomo Tokizawa3, Mika Nomoto4, Yasuomi Tada4, Rinesh Godfrey5,6, Junichi Obokata2, Irena

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Sherameti1, Yoshiharu Y. Yamamoto3, Frank-D. Böhmer5, Ralf Oelmüller1**

Institute of Plant Physiology, Friedrich-Schiller University Jena, Dornburger Str. 159, 07743 Jena, Germany

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Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan

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Faculty of Applied Biological Sciences, Gifu University, Yanagido 1-1, Gifu City, 5011193, Japan

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Center for Gene Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 4648601, Japan

5

Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena

Molecular Cardiology, Department of Cardiovascular Medicine, University Hospital

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University Hospital, Jena, Germany

Münster, 48149 Münster, Germany

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(*) even contribution

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(**) corresponding author; email: [email protected] Abbreviations: ABA, abscisic acid; ET, ethylene; JA, jasmonic acid; LL, low light; ML, medium light; HL, high light; LD, long day; SD, short day; oe, overexpressor; PAMP, pathogen-associated molecular pattern; ROS, reactive oxygen species; RRTF1, REDOX RESPONSIVE TRANSCRIPTION FACTOR1; SA, salicylic acid; WT, wildtype Running title: RRTF1 overexpression stimulates ROS accumulation Short summary: Analysis of overexpressor- and knockout-plant of Redox Responsive Transcription Factor1 (RRTF1) revealed that RRTF1 stimulates ROS accumulation in abiotic- and biotic-stress responses of Arabidopsis thaliana. In

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we

showed

that

RRTF1

expression

is

positively

regulated

by

WRKY18/40/60 and that a GCC-box like element functions as RRTF1 cis-element. Our results suggest that WRKY18/40/60-RRTF1 transcription circuit should have a

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role to amplify ROS under stress condition in plant.

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ACCEPTED MANUSCRIPT ABSTRACT Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H2O2, as well as biotic and abiotic induced redox signals. RRTF1 is highly conserved in angiosperms, however the physiological role remains

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elusive. Here we show that inactivation of RRTF1 restricts and overexpression promotes reactive oxygen species (ROS) accumulation in response to stress. Overexpressor (oe) lines are impaired in root and shoot development, light sensitive and susceptible to Alternaria brassicae infection. These symptoms are diminished by the beneficial root endophyte Piriformospora indica which reduces ROS accumulation

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locally in roots and systemically in shoots, and by antioxidants and ROS inhibitors which scavenge ROS. More than 800 genes were detected in mature leaves and

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seedlings of RRTF1 oe lines, ~ 40% of them have stress-, redox-, ROS regulated-, ROS scavenging-, defense-, cell death- and senescence-related functions. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box like sequences in the promoter of RRTF1-responsive genes. Upregulation of RRTF1 by stress stimuli as well as H2O2 requires WRKY18/40/60. RRTF1 is co-regulated with the phylogenetically related RAP2.6, which contains a

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GCC-box like sequence in its promoter, but RAP2.6 oe lines do not accumulate higher ROS levels. RRTF1 also stimulates systemic ROS accumulation in distal non-

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stressed leaves. We conclude that the elevated levels of the highly conserved RRTF1 induce ROS accumulation in response to ROS and ROS-producing abiotic

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and biotic stress signals.

Key words: abiotic stress, biotic stress, reactive oxygen species, H2O2, REDOX RESPONSIVE TRANSCRIPTION FACTOR1, RAP2.6, RRTF1 promoter binding sites

INTRODUCTION

Plants and many other organisms constantly produce reactive oxygen species (ROS) in chloroplasts, mitochondria, peroxisomes and other sites of the cell because of their metabolic processes such as photosynthesis and respiration. ROS accumulation is particularly enhanced under environmental constrains and excess ROS production leads to the activation of cell death programs or senescence (Apel and Hirt, 2004; 3

ACCEPTED MANUSCRIPT Foyer and Noctor, 2009; Miller et al., 2010; Tripathy and Oelmüller, 2012). Antioxidant enzyme systems and antioxidants such as ascorbate and glutathione detoxify the ROS (Asada, 2006; Dietz et al., 2006) and stabilize the redox poise in the different cellular compartments (cf. Asada, 2000). The entire cellular ROS gene network comprises more than 180 genes in Arabidopsis (Mittler et al., 2004; Mehterov et al.

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2012).

ROS also activate specific signaling pathways which counteract ROS-induced cell damage, activate responses to abiotic and biotic stresses or control developmental processes (Jaspers and Kangasjärvi, 2010; Swanson and Gilroy, 2010; Torres,

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2010). Pathogen or herbivore attack (Torres, 2010), treatment of plants with pathogen-associated molecular patterns (PAMPs; Mersmann et al., 2010), wounding

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(Torres and Dangl, 2005), or abiotic stress such as heat (Miller et al., 2009), ozone (Vahisalu et al., 2010) or salt (Torres and Dangl, 2005) induce an oxidative burst, in which plasma membrane-bound NADPH oxidases (respiratory burst oxidase homologues, RBOHs) and cell wall peroxidases release H2O2 into the apoplast (Torres and Dangl, 2005). RBOHs are important for defense responses and hormonal signaling (Marino et al., 2012), and are synergistically activated by

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phosphorylation, binding of Ca2+ to their EF hand motifs and S-nitrosylation (Ogasawara et al., 2008, Yun et al., 2011). Elevated ROS levels in the apoplast are

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toxic to microbes and play a profound role in mediating rapid, long-distance, cell-tocell propagating signals by the formation of a ROS wave (Miller et al., 2009; Mittler et

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al., 2011). Other well characterized ROS-induced responses are closure of stomata (Wang and Song, 2008) and regulation of cell expanse (Carol and Dolan, 2006).

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The existence of many interconvertible ROS species makes it difficult to distinguish between cytotoxic and signaling events which are induced by a particular ROS. ROS with signaling functions are H2O2, singlet oxygen (1O2), hydroxyl radical (OH.) and superoxide anion radical (O2.-) and a given ROS and its location affect its role in signaling (Wagner et al., 2004; Gadjev et al., 2006; Laloi et al., 2006; Mehterov et al., 2012). Genetic manipulations of ROS generation / scavenging and drug applications have been used to study the effects of particular ROS species. 1O2 is preferentially synthesized in plastids (op den Camp et al., 2003). Paraquat treatment leads to O 2.(and subsequently H2O2) production by an electron flow from photosystem I to oxygen. Peroxisomal catalases detoxify photorespiratory H2O2 and CAT2-deficient plants accumulate H2O2 in the peroxisomes (Vanderauwera et al., 2005). It is 4

ACCEPTED MANUSCRIPT believed that ROS production in a particular cellular compartment can have impacts on ROS levels and signaling in other locations (Gadjev et al., 2006). Stress-induced ROS-activating responses have to occur rapidly, and should decay when the stress disappears. Here we report on RRTF1, an APETALA2/ethylene response transcription factor (TF) (AP2/ERF). RRTF1 expression in the nucleus is

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controlled by signals from the plastids (Khandelwal et al., 2008), induced by jasmonic acid (JA) in a COI1-dependent manner (Wang et al., 2008) and repressed by WRKY40, which binds to the W-box in the RRTF1 promoter (Pandey et al., 2010). Khandelwal et al. (2008) have shown that RRTF1 is a component of a core redox

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signaling network that includes EDS1 and WRKY33. However, why such diverse signals regulate RRTF1 expression and how this is related to RRTF1 function, is not

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understood. We demonstrate that RRTF1 expression is rapidly and transiently stimulated by various ROS species and ROS-generating biotic and abiotic signals, and that the protein stimulates ROS accumulation in response to these stimuli. Inactivation of RRTF1 restricts and overexpression promotes ROS accumulation in response to biotic and abiotic stress signals. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box and GCC-box like

RESULTS

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sequences in the promoter of RRTF1-responsive genes.

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RRTF1 expression is rapidly up-regulated under stress Expression of RRTF1 was rapidly upregulated in response to light stress on plastids.

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Shifting Arabidopsis seedlings from continuous low (cLL, 30 µmol m-2 s-1) to high light (cHL, 200 µmol m-2 s-1) for 3 h resulted in an ~4-fold upregulation of the RRFT1 mRNA level in the leaves, and pretreatment of the leaves with DCMU prior to exposure to cHL completely prevented the induction (Table 1A; cf. Khandelwal et al., 2008). The RRTF1 mRNA levels dropped rapidly when the HL-exposed seedlings were transferred back to LL (Table 1B). Database search uncovered that application of methyl viologen, an electron donor of photosystem I, and singlet oxygen generated in the plastids stimulates RRTF1 expression (Toufighi et al., 2005). Therefore, different stress signals from the plastids control RRTF1 expression in the nucleus (cf. Khandelwal et al., 2008). RRTF1 expression is also upregulated when the leaves or roots are exposed to 10 mM H2O2 (Table 1C). Publically available data showed that 5

ACCEPTED MANUSCRIPT RRTF1 is induced by abiotic stress like salt, drought, cold, UV-B light, heat and osmotic stress (Toufighi et al., 2005; Gadjev et al. 2006; Figure S1), as well as the stress-related phytohormones ABA (Matsui et al., 2008) and JA (Wang et al., 2008). Furthermore, infection of the leaves with spores of the fungi Alternaria brassicae, Botrytis cinerea, the necrotrophic bacteria Pseudomonas syringae pv. tomato

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DC3000 and P. syringae pv. phaseolicola stimulated RRTF1 expression (Figure S1). Similar results were obtained after application of the PAMPs flg22, chitin or a toxin preparation from A. brassicae to the leaves (Table 1). The PAMP-induced responses were not prevented by DCMU and are therefore independent of the photosynthetic

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electron flow. In contrast to necrotrophs, the hemibiotrophic oomycete Phytophthora infestans and the biotrophic powdery mildew fungi did not stimulate or even repressed RRTF1 expression (Toufighi et al., 2005, Pandey et al., 2010, Figure S1).

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Cocultivation of Arabidopsis seedlings with the beneficial root-colonizing endophytic fungus Piriformospora indica resulted initially in a slight increase in the RRTF1 mRNA levels in roots and, to a lesser extent, in shoots. After 4 d of cocultivation, the fungus repressed the RRTF1 mRNA level in both roots and shoots (Figure S1, cf. Discussion). Taken together, stress and/or redox signals from plastids, different ROS species, abiotic stress and pathogens stimulate RRTF1 expression, while biotrophic

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and hemibiotrophic fungi and the beneficial fungus P. indica do not affect or repress

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RRTF1 expression, as soon as the symbiotic interaction is stable. RRTF1 belongs to the subgroup Xb of the ERF/AP2 TF family (Nakano et al., 2006). The protein is

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highly conserved in angiosperms and present in the major crop plants (Figure S2). These results suggest that RRTF1 plays an important role in a broad range of biotic

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and abiotic stress responses in higher plants. 35S-RRTF1 overexpressor (oe) lines were light sensitive RRTF1 was expressed under the control of the 35S promoter (35S-RRTF1) in Arabidopsis and three T3 plants with different RRTF1 mRNA levels (oe18, oe20 and oe32) were compared to the knock-out line rrft1 and the wild-type (WT). When the seedlings were grown under continuous middle light (cML) for 10 days in Petri dishes, the RRTF1 mRNA levels in the leaves of the oe18, oe20 and oe32 seedlings were 7- , 13- and 20-fold higher compared to the level in the WT control. In the rrtf1 knockout line, RRTF1 is not functional because of a T-DNA insertion into the conserved AP2/ERF DNA binding domain (Figure S3) and no RRTF1 transcripts could be detected in the knock-out line as described in Khandelwal et al. (2008) 6

ACCEPTED MANUSCRIPT (Figure 1A, Figure 5). The oe lines showed a strong phenotype in cML. After 14 days, symptoms of photoinhibition and ultimately photobleaching were clearly visible and they increased with increasing RRTF1 expression levels (Figure 1B). Photobleaching was lower under continuous low light (cLL) and higher under continuous high light (cHL). After 18 days on Hoagland medium in cML in square plates, the

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characteristics of the RRTF1-dependent phenotype is even more obvious and oe32 seedlings were completely white. The oe leaves were wrinkled and narrow and the roots were shorter, again in an RRTF1 mRNA concentration-dependent manner (Figure 1C, left). Therefore, the photosensitivity of the seedlings increased with

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increase in RRTF1 expression and light intensity, and this had severe consequences for the performance of the plants.

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The oe18 and oe20 seedlings were characterized in more detail. While oe seedlings performed relatively well under cLL, the chlorophyll content was reduced compared to WT and rrtf1 seedlings (Figure S4A). Under cLL, cML and cHL, the photosynthetic parameters ´quantum yield of photosystem II´ (PSII) (ΦPSII), ´photochemical´ (qP), ´non-photochemical quenching´ (NPQ), and ´maximum quantum yield of PSII´ (Fv/Fm) (Figure S4A) of oe were decreased with increasing light intensity and RRTF1

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levels. Thus, the efficiency of the photosynthetic electron transport (ΦPSII, qP), the ability of heat dissipation of photochemical energy (NPQ), and the ratio of functional

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PSII to total PSII (Fv/Fm) was impaired by photoinhibition in the chloroplasts of oe. oe20 seedlings suffered more than oe18 seedlings. The rrtf1 seedlings did not show

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a visible phenotype under these growth conditions when compared to the WT seedlings (Figures 1B-C). After transfer from cLL to cML for 2 days, the stems and emerging young leaves of oe18 and oe20 seedlings accumulated large amounts of

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anthocyanin, while rrtf1 seedlings grown under the same conditions accumulated less anthocyanin compared to WT (Figure S4B). Taken together, overexpression of RRTF1 impaired photosynthesis and other plastid functions and induced stress responses in a light-dependent manner. Fourteen day-old cLL-grown WT, rrtf1, oe18, oe20 and oe32 seedlings were transferred to soil. Almost all oe plants died after 3-8 weeks under cML, but the oe18 and oe20 plants survived under long-day (LD; 16 h L: 8 h D) ML and even better under short-day (SD; 8 h L: 16 h D) ML conditions (Figure 2). Moreover, the biomass and seed weights were significantly decreased with increasing levels of RRTF1 expression. Interestingly, the biomass and seed weights for rrtf1 and WT did not differ 7

ACCEPTED MANUSCRIPT (Figure 2). Thus, the oe lines were sensitive to light stress and the survival rate increased with increasing dark incubation periods and lower RRTF1 levels. 35S-RRTF1 lines were highly susceptible to A. brassicae infections Since the RRTF1 mRNA level was strongly upregulated after A. brassicae infection

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(Figure S1) and treatment of the leaves with the A. brassicae toxin preparation (Table 1), we tested whether overexpression of RRTF1 protected the plants against the pathogen. However, when WT, rrtf1, oe18 and oe20 leaves were infected with A. brassicae spores, the disease symptom development was much faster in oe than WT or rrtf1 leaves (Figure 3). Quantified data based on “Percentage Disease Index”

expression

level

(Figure

S5).

Therefore,

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confirmed that the disease development increased with increase of the RRTF1 the

long-term

and

constitutive

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overexpression of RRTF1 in all tissues weakens the plants to an extent that they become more sensitive to abiotic (HL) and biotic (A. brassicae infection) stress than WT and rrtf1 plants.

RRTF1 overexpression promoted ROS accumulation in leaves and roots We noticed that staining of the oe with 3,3´-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) was stronger than staining of WT or rrtf1 plants (Figure S6). This

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suggested that RRTF1 stimulates H2O2 and ROS accumulation. Quantitative

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measurements with the Amplex Red reagent (Molecular Probes) confirmed that the H2O2 levels in leaves increased with increasing RRTF1 mRNA levels and light

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intensity (Figure 4A). Similar results were obtained with the substrate 5-(and-6)carboxy-2´,7´-difluorodihydro-fluoresceindiacetate

(carboxy-H2DFFDA)

(Molecular

Probes) which determines the total ROS level (Figure 4B). In all studies, the rrtf1

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knock-out line accumulated less H2O2/ROS than the WT (Figures 4A-B). The H2O2/ROS levels in dark-adapted leaves of oe seedlings were only marginally higher when compared to the levels in WT and rrtf1 (Figures 4A-B). This suggests that elevated ROS levels in the oe leaves requires light stress, and is not caused by higher RRTF1 expression alone. In contrast, the ROS levels in oe roots were higher irrespective of the light conditions (Figures 4C-D; cf. Discussion). Therefore, several independent assay systems showed that RRTF1 promotes stress-induced H2O2/ROS accumulation. P. indica protected the oe against stress by reducing the ROS level Since beneficial microbes including the root-colonizing endophyte P. indica 8

ACCEPTED MANUSCRIPT (Baltruschat et al., 2008; Vadassery et al., 2009) stimulate antioxidant systems to counteract ROS-induced damage, we tested whether the stress-exposed oe perform better when the roots were colonized by P. indica. As shown in Figures 1C and 5A for seedlings grown under different conditions (Hoagland medium cML and MS medium LD ML), the roots of the oe seedlings cocultivated with P. indica are longer, the

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leaves are bigger and contain more chlorophyll and they are better protected against photoinhibition. P. indica also protected the oe leaves against A. brassicae infections and the disease development was visibly reduced and/or retarded (Figure 5B). Therefore, the endophyte protects the leaves of the oe lines systemically against

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abiotic (HL) and biotic (A. brassicae infection) stress.

The H2O2/ROS levels in roots and shoots of P. indica-colonized rrtf1, WT and oe

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seedlings were reduced and the degree of the reduction increased with the increase in the H2O2/ROS levels present in the tissues (Figure 5C). This was associated with the upregulation of the mRNA levels for ROS scavenging enzymes (Figure 5D) and the downregulation of RRTF1 in the wild-type (Figure 5E, cf. Discussion). Since the mRNA levels of ROS scavenging enzymes, such as the Fe2+-superoxide dismutase1 (FSD1), Cu2+-superoxide dismutase2 (CSD2), monodehydroascorbate reductase2

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(MDAR2), dihydroascorbate reductase5 (DHAR5) and ascorbate peroxidase1 (APX1) were not only upregulated in roots (not shown) but also in shoots of P. indica-treated

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seedlings (Figure 5D), the endophyte protected the aerial parts systemically against photoinhibition (Figures 1C and 5A) and disease development (Figure 5B). We

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propose that the constantly high H2O2/ROS levels weaken the oe seedlings so that they become sensitive to abiotic and biotic stress. Better performance of P. indicatreated oe seedlings supports the conclusion that elevated H2O2/ROS levels are

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responsible for the higher stress sensitivity of oe lines, and that the protective function of the fungus is caused by reducing the ROS levels. N-acetyl-L-cysteine (NAC) and diethyldithiocarbamate sodium salt (DDCNa) rescued the oe phenotypes Application of 1 mM NAC, an antioxidant which increases cellular pools of free radical scavengers (Joo et al., 2001; Baek et al., 2004), or of 1 mM DDCNa, a ROS inhibitor (Yokawa et al., 2011) also rescued the growth and light sensitivity phenotype of the oe lines (Figure 6A). The oe leaves were greener and the roots longer. Furthermore, the NAC- and DDCNa-exposed oe seedlings were also more resistant to A. brassicae infection than the WT seedlings (Figure 6B). Quantitative measurements 9

ACCEPTED MANUSCRIPT confirmed that NAC and DDCNa significantly reduced H2O2 and ROS levels in the roots and leaves of the oe seedlings (Figure 6C). The recovery effect was better with NAC than with DDCNa. This supports the results with P. indica that elevated H2O2 and ROS levels in the oe lines are responsible for the observed phenotypes.

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RRTF1 regulates stress-, senescence- cell death-, defense- and ROS-related genes

To identify RRTF-regulated genes, microarray analysis of oe18 was examined (Table S1-1 to S2). In the mature leaves of 5 weeks old oe18 plants grown under SD and ML conditions, 588 genes were up-regulated and 231 genes were down-regulated

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compared to wild type plants (Table S1-1). Gene ontology (GO) classification of the 819 up- or down-regulated genes revealed that 40% of the up-regulated genes and

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35% of the down-regulated genes code for defense- and stress-related proteins, senescence, or cell death proteins. Mapman categorization of the 819 up- or downregulated genes showed that they code for transcription factors, e.g. APETALA2/ETresponsive element binding proteins (AP2/EREBP), MYB, WRKY, basic helix-loophelix and C2H2 zinc finger family members were most frequently detected (Table S11, Figure S7). In all Mapman categories, genes with ROS-related functions were

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present (Table S1-1). These results suggest that RRTF1 regulates a broad-spectrum of genes involved in stress responses and ROS-related functions.

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Since the adult oe18 plants were retarded in growth and thus might have secondary effects, we also examined 14-day old oe18 seedlings grown under low light (LL)

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condition. They did not show visible symptoms of photodamage and the photosynthetic parameters did not differ significantly from those of WT and rrtf1

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seedlings, although the H2O2/ROS levels were marginally higher in oe18 and lower in rrtf1 leaves and roots compared to the WT (Figure S8 A-D). A comparative transcriptome analysis of shoots and roots of rrtf1, oe18 and WT seedlings detected 863 RRTF1-regulated genes. Compared to WT, 210 genes were upregulated and 164 genes downregulated at least 2-fold in oe18 shoots, whereas 222 genes were upregulated and 191 genes downregulated in oe18 roots. Likewise, compared to WT, 78 genes were up- and 11 genes downregulated in rrtf1 shoots and 137 genes were up- and 50 genes downregulated in rrtf1 roots (Table S1-2, Figure S7). 41% of the detected genes code for defense- and stress-related proteins, senescence or cell death proteins. Again, genes for transcription factors were most frequently detected. For a few genes of the microarray the results were confirmed by real-time and qRT10

ACCEPTED MANUSCRIPT PCR analyses (Figure S9). Comparative analysis of RRTF1-regulated genes in mature leaves, shoots and roots of oe18 seedlings showed that most of them are uniquely in each dataset, implicating that RRTF1 may regulate diverse age- and organ-specific biological processes (Figure 7). 178 genes were commonly in at least two datasets and 19 genes were

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present in all datasets. 74% of them code for proteins with stress-related functions and 58% with redox-related functions. ROS marker genes characterized in Mehterov et al. (2012) were remarkably enriched in this dataset compared to all other data combinations (Figure 7).

metabolism

(At3g47380)

(Mehterov

et

al.

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The list contains a gene for a pectin methyl esterase inhibitor, genes involved in H2O2 2012),

related

to

flavonoid,

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phenylpropanoid and JA biosynthesis and signaling (At1g43160, At3g48520, At3g55970 and At5g05600) (Fujita et al. 2007; Heitz et al. 2012, Lee et al. 2007; Nakatsubo et al., 2008, Mehterov et al. 2012), the 2-oxoglutarate-Fe2+-dependent oxygenase gene (At3g49620), the cytochrome P450 gene CYP94A1 (At3g48520), the gene for the O-methyl transferase At1g21100, as well as stress-related transcription factor genes, such as for RAP2.6 (At1g43160), ZAT10 (At1g27730) and the bHLH-

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type transcription factor At1g10585. Genes for stress responses and redox-related functions were enriched in the group commonly detected in two data sets.

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Interestingly, they included also H2O2-producing class III peroxidase genes (At2g37130, At5g64120) and a proline oxidase/dehydrogenase gene (At3g30775)

downregulated

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(Funck et al., 2010). For the gene group uniquely detected in each data set, genes

involved

in

ROS

scavenging

or

those

coding

for

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photosynthesis-related proteins were detected in oe18. These data suggest that ROS-related genes are major targets of RRTF1, and this is consistent with the results of higher H2O2/ROS levels in the oe lines. Based on studies by Vanderauwera et al. (2005), Gadjev et al. (2006) and Mehterov et al. (2012), the ROS regulated genes can be classified as those preferentially regulated by H2O2, O2.-, 1O2 or as common ROS marker genes in green tissue. To test whether specific ROS marker genes were regulated in oe18, the published microarray data were compared with our data sets. Analysis of the genes present in at least two microarray data sets revealed that twenty-seven ROS marker genes (Mehterov et al., 2012) were differentially regulated in oe18 relative to the WT (Table 11

ACCEPTED MANUSCRIPT 2; Table S2), 3 of them are H2O2 marker genes, 3 of them are O2.- marker genes, 4 of them are 1O2 marker genes and 17 are common ROS marker genes. No obvious preference for any of the ROS species could be detected among the ROS regulated

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genes in the oe line.

RRTF1 and RAP2.6

The similar regulation of RRTF1 and RAP2.6 in our expression profiles (Table S1-1, S1-2), the close phylogenetic relationship of the two AP2/EREBP TFs (Figure S2; Nakano et al. 2006; Dietz et al., 2010), the observations that both are responsive to

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the same stress hormones and abiotic stresses, and that their oe lines are smaller under greenhouse conditions and light sensitive (Krishnaswamy et al., 2011)

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prompted us to test whether both proteins have similar or related functions. When seedlings overexpressing either RRTF1 or RAP2.6 were grown under identical conditions, the RRTF1 oe lines were more light-sensitive than the RAP2.6 oe lines (Figure 8A). Furthermore, we did not detect elevated H2O2 or ROS levels in the RAP2.6 oe lines (Figure 8B). Finally, the RAP2.6 mRNA level increased with increase in RRTF1 overexpression, but the RRTF1 mRNA level did not increase with increase

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in RAP2.6 overexpression (Figure S10). However, the RRTF1 mRNA level was strongly upregulated in the rap2.6 insertion line in roots and in particular in shoots

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(Figure S10). This suggests that RRTF1 expression is either upregulated to compensate for the loss of RAP2.6 expression, or rap2.6 plants are stressed and

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therefore upregulate RRTF1 expression. In conclusion, RRTF1 and not RAP2.6 is responsible for the elevated ROS accumulation in the oe lines.

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Regulation of RRTF1 expression Since RRTF1 is known to be regulated by WRKY transcription factors (Pandey et al., 2010), we tested the regulation of RRTF1 in response to A. brassicae infection, HL, H2O2 application and P. indica colonization in the wrky18 wrky40 wrky60 background. Stimulation of RRTF1 expression by HL and H2O2 was almost completely prevented in the triple wrky knock-out line. Stimulation after A. brassicae infection was strongly reduced in the mutant, while the % repression of RRTF1 expression by P. indica was comparable for WT and mutant seedlings (Figure 9A). This suggests that full stimulation of RRTF1 expression by A. brassicae, HL and H2O2 requires WRKY18, 40 and/or -60, while the RRTF1 repression by P. indica is independent of the WRKY 12

ACCEPTED MANUSCRIPT transcription factors (Figure 9B).

RRTF1 is involved in systemic stress responses Arabidopsis WT and rrtf1 plants were grown on soil for 4 weeks under SD ML conditions, before half part of the plant were exposed to cHL, the other parts were

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covered with aluminum foil. After 3 and 6 days, we observed a systemic, ~4-fold upregulation of the RRTF1 mRNA in the distal, shaded leaves of WT plants (Table 3). Also the ROS level increased ~2-3-fold in the shade-exposed leaves of WT plants. For rrtf1 plants, the ROS level in the distal leaves increased ~10% (Table 3). When

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RRTF1-responsive genes (Myb122, CML38, PDF1.2, WRKY40, cf. Table S1-1) were analysed 3 days after exposure of the plants to cHL, they were significantly upregulated in the shaded leaves of WT, but not rrtf1 plants (Table 3). After 18 days,

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the number of leaves with severe visible lesions in photosynthesis and chlorophyll accumulation in the shaded areas was 6 times higher in WT than in rrtf1 plants (Table 3). The ROS level in the shaded WT leaves was twice as high as the level in the shaded rrtf1 leaves (Table 3). When senescence-associated ROS-regulated genes (SEN1, SRG1, SAG21) and the stress-related chalcone synthase (CHS) gene were analysed, none of them were significantly upregulated in shaded leaves of rrtf1

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leaves, while they were significantly higher in the shaded leaves of WT leaves (Table

stress responses.

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3). These findings suggest that RRTF1 participates in the systemic regulation of

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Identification of RRTF1 binding cis-element Among the RRTF1-responsive genes identified in the microarray analyses, RAP2.6

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was significantly repressed in shoots of rrtf1 seedlings and strongly induced in the shoots and roots of seedlings and leaves of mature oe18 plants (Tables S1-1 and S12). Vogel et al. (2014) reported that the RAP2.6 transcripts accumulate within 1 hour after induction of RRTF1 in high light stress. These data suggest that RRTF1 regulates RAP2.6 expression, presumably at the transcriptional level. Therefore, we analyzed the cis-elements in the RAP2.6 promoter to identify target sequences of RRTF1. Octamer-based cis-element prediction as previously described (Yamamoto et al, 2011) for promoters of RRTF-regulated genes from mature leaves of oe18 uncovered putative RRTF1-binding sites with peaks of high relative appearance ratios (RAR > 3.0) (Figure 10A). We have chosen the top 6 peaks and performed in vitro RRTF1-DNA binding experiments for validation (red box in Figure 10A). The 13

ACCEPTED MANUSCRIPT analysis revealed strong binding of RRTF1 to the probe F region (-109/-60) in the RAP2.6 promoter (Figure 10B). The positive probe F contains two predicted target sites of RRTF1 (probe F, Figure 10D). Subsequently, we examined which of the two elements is the binding site of RRTF1 by competition assays as shown in Fig. 10 (Panels C and D). The positive signal of the probe F without competitors did not show

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any significant reduction in the presence of a non-specific competitor (probe C), and dropped by addition of probe F as a competitor, demonstrating sequence-specific binding of RRTF1. The competitor activity of the probe F remained with the m1 mutant, but disappeared with the m2 mutant (Figure 10D). These results indicate that

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RRTF1 binds to the downstream predicted site of the probe F, that is TGACGGCT (underlined in Figure 10D). This region contains a GCC box-like sequence (AGCCGTCA in the complementary strand), which has similarity to a RRTF1 binding

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sequence determined by the protein-binding microarray analysis (PBM) of FrancoZorrilla et al. (2014), suggesting that the GCC box-like sequence is a cis-element of RRTF1-mediated stress response in the RAP2.6 promoter. To identify additional RRTF1 binding sites, we searched for octamer sequences with GCCGCC and GCCGTC sequences. The former hexamer, called GCC-box, is predicted as RRTF1binding sequence from the PBM analysis of Franco-Zorrilla et al. (2014). The later is

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part of the RRTF1 binding sequence in the RAP2.6 promoter identified in this study.

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Both hexamers are overrepresented in genes which are co-expressed with RRTF1 (Franco-Zorrilla et al., 2014). Our search detected 4 new GCC-box type RRTF1

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binding sequences and showed that the AGCCGTCA, but not the GCC-box sequence, should be a dominant cis-element in promoters of genes which strongly responded to RRTF1 (Figure 10E). Moreover, we surveyed how many RRTF1-

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responsive genes possess these RRTF1 binding sequences in their promoters. 11.9 % (45/379), 13.8% (57/413) and 11.2% (92/819) of RRTF1-responsive genes in the shoots and roots of the seedlings and mature leaves of the oe plants, respectively, contained the RRTF1 binding sequences within their 3 kb-promoter regions (Tables S1-1 and S1-2). This is not significantly different from the distribution of this ciselement in all Arabidopsis promoters, because the microarray identified genes which could be directly regulated by RRTF1 and those which respond to the altered redox situation and elevated ROS level in the oe line. However, if we assume that expression-relevant cis-elements are located in the vicinity of the transcription start site (500 bp upstream of the ATG codon) and that genes which respond directly to 14

ACCEPTED MANUSCRIPT RRTF1 are stronger regulated (>4 –fold) than those which are regulated due to secondary effects, then 5.8% (10/172) of the RRTF1-responsive genes of mature leaves contain the identified cis-elements in the 500 bp promoter region, while only 1.6 % (548/33602) of all genes and of those genes which do not respond to RRTF1 overexpression (528/33602) contains these cis-elements. The higher percentage

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values of RRTF1 responsive genes were also obtained from the data of shoots (3.8%, 2/53) and roots (5.1%, 3/59) of young seedlings. These results demonstrate that GCC-box type RRTF1 binding sequences are over-represented in strongly regulated

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RRTF1-responsive genes.

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DISCUSSION

RRTF1 regulation and RRTF1-stimulated ROS accumulation We demonstrate that the highly conserved RRTF1 (Figure S2) is rapidly and transiently (Table 1B) upregulated in response to abiotic (HL) and biotic (A. brassicae infection) stresses. Various ROS-inducing abiotic stresses (Jaspers and Kangasjärvi, 2010), ozone which promotes apoplastic H2O2 formation (Vahisalu et al., 2010;

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Wrzaczek et al., 2010), necrotrophic fungi (Table 1) which generate H2O2 (Heller and Tudzynski, 2011), PAMPs such as flg22 and chitin which generate O 2.- and

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subsequently H2O2 (Nürnberger et al., 1994; Lamb and Dixon, 1997) and the stressrelated and ROS-inducing phytohormones JA and ABA (Matsui et al., 2008; Wang et

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al., 2008, Jiang et al., 2011) stimulate RRTF1 expression. RRTF1 is also upregulated by H2O2 (Table 1). High RRTF1 mRNA levels are detectable in flu mutant in the light

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which accumulates 1O2 in plastids (op den Camp et al., 2003, Gadjev et al., 2006), in mutants which accumulate ROS due to genetic manipulation of ROS scavenging systems in plastids, mitochondria, peroxisomes or the cytosol (Gadjev et al., 2006; Balazadeh et al., 2012; Mehterov et al., 2012), and in the JUNGBRUNNEN1 oe, which accumulates H2O2 due to the overexpression of a H2O2-inducible NAC TF (Wu et al., 2012). Thus, RRTF1 is induced by different types of ROS from different cellular compartments. Many ROS marker genes, genes with ROS related and ROS scavenging functions and genes for redox processes are differentially regulated in the oe18 and rrtf1 material (Tables 1-1 and S1-2, Figure S9). ROS are signaling molecules for cell-tocell communication, and RRTF1 participates in systemic responses to various stress 15

ACCEPTED MANUSCRIPT signals (Table 3; Rossel et al., 2007; Miller et al., 2009). The ROS signal is spread systemically and activates stress responses in distal not yet stress exposed areas. Table 3 demonstrates that RRTF1 may be a crucial regulator of the systemic response. Plant cells communicate via plasmodesmata and recent studies suggest the participation of ROS in the regulation of plasmodesmata transport (Benitez-

synthesis

or

by

repressing

the

antioxidant

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Alfonso et al., 2011). RRTF1 may stimulate ROS accumulation by stimulating their metabolism.

Genes

for

extracellular/endomembrane type peroxidases, which might catalyze the production of hydrogen peroxide, are up-regulated in the oe lines (Tables S1-1, S1-2, and S2).

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Therefore, the activation of those peroxidase genes may be essential for systemic ROS signalling. ROS-producing enzymes which are not up-regulated in the oe lines

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(Tables S1-2, S2-5, 2-6) might be activated posttranscriptionally under stress. RRTF1 expression respond to different abiotic and biotic stresses and is particularly sensitive to chloroplast redox signals and chloroplast-derived singlet oxygen (Khandelwal et al., 2008; Gadjev et al., 2006; Foyer et al., 2014). Since the protein confers a general increase in resistance against pathogens and various abiotic stresses, triggers innate immune responses, controls cell death programs, is involved

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in systemic responses to biotic and abiotic stresses and activates stress-related phytohromone functions, it is a likely candidate to integrate incoming stress signals

therein).

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and to coordinate cross-tolerance responses (Foyer et al., 2014, and references

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The stress-sensitivity of oe (light: Figures 1B-C left, 2, 5A left, A. brassicae infection: Figures 3 and 5B) demonstrates that control of RRTF1 expression is important for the

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plants, since the oe accumulate harmful amounts of ROS even when exposed to mild stress conditions (Figures 4A-B). ROS accumulation in the oe lines occurs in the light and not or to a lesser extent in the dark (Figures 4A-B). Thus, elevated RRTF1 expression per se does not lead to higher ROS levels. Imbalances in the cellular redox homeostasis under unfavorable conditions triggers ROS accumulation (Schippers et al., 2012), and this might also lead to higher RRTF1 expression. It is not known how the cellular ROS level increases in the oe lines. Since RRTF1 is a transcription factor, we propose that the altered gene expression pattern is mediated in two steps: RRTF1 alters the expression of genes directly, which results in cellular imbalances and ultimately ROS accumulation. The elevated ROS level activates a secondary class of genes. Therefore, the microarray data contains genes which are 16

ACCEPTED MANUSCRIPT directly regulated by RRTF1 and those which response to the imbalanced ROS situation in the cell. Stress also induces the production of nitric oxide which regulates e.g. antioxidant enzymes at the level of activity and gene expression (Groß et al., 2013) and affects the cellular ROS homeostasis. Since ROS accumulation is lower in the rrtf1 knockout line and higher in the oe lines compared to the WT control (Fig. 4),

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we propose that RRTF1 promotes ROS accumulation under unfavorable conditions. Under low stress conditions, the insertion line performs as good as the WT, which suggests that ROS amplification is not required under these conditions. However, under extreme stress such as high light (600 µmol m-2 s-1; Khandelwal et al., 2008), a

activate appropriate stress responses.

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RRTF1-regulated genes

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rapid and transient establishment of local ROS maxima may require RRTF1 to

To identify genes which are regulated by RRTF1 over-expression, we compared the microarray sets of oe18 vs. WT vs. rrtf1 seedlings and mature leaves. The analysis did not discover any specificity/preference for one of the bioactive ROS species (Table 2) or for a cellular ROS generating compartment (Tables S1-1, S1-2, S2; cf. Gadjev et al., 2006; Wrzaczek et al., 2010). All datasets identified genes which are

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directly or indirectly regulated by H2O2/ROS. Depending on the organ, tissue, the developmental stage and age of the plant material, the genes are involved in different

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ROS-regulated processes. ROS inducing-, stress- and defense-related genes are preferentially regulated in oe18 and rrtf1 (Table S1-1 and S1-2). Genes for C2H2, zinc

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finger, AP2/ERF, Myb, WRKY TFs, oxygenases, dehydrogenases, phosphatases, cytochrome P450 enzymes, photosynthesis and defense are the most dominant

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class of regulated genes. Zat10 (At1g27730), ZAT12 (At5g59820) and JRG21 (At3g55970) respond to increased photooxidative stress (Rossel et al., 2007), and stress-induced Myb51 and WRKY40 (Xu et al., 2006; Dubois et al., 2013) are differentially regulated in oe18 (Table 2, S1-1 and S1-2). RAP2.6 (At1g43160), an AP2/EREBP TF which is phylogenetically related to RRTF1 (Nakano et al., 2006; Dietz et al., 2010), participates in ABA, salt, drought and osmotic stress responses and is responsive to JA, SA, ABA and ET (Zhu et al., 2010; Krishnaswamy et al., 2011). RRTF1 and RAP2.6 are part of an AP2/EREBP-linked gene network which coordinates the plant response to redox changes, organelle to nucleus retrograde signals and various stresses (Dietz et al., 2010; Vogel et al., 2014). Eleven (DREB21910, DREB-33760, DREB77640, RAP2.4d, RAP2.6, RAP2.8, TEM1, BAP1, ERF17

ACCEPTED MANUSCRIPT 17490, ERF-47230 and ERF-61600) of the proposed 21 network members are differentially regulated in oe18 and rrtf1 (Table S1-1 and S1-2). ROS, in particular 1O2, play a crucial role in plastid-to-nucleus signaling (Galvez-Valdivieso and Mullineaux, 2010). The integration of the plastid-derived ROS signals with those from various other stress-induced ROS signals through the amplification by RRTF1 suggests that

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there might be an intensive crosstalk. Sixteen nuclear-encoded genes involved in photosynthesis and light reaction were also differentially regulated in oe18 and rrtf1 material (Table S1-1 and S1-2), which again demonstrates the role of RRTF1 in interorganellar communication (cf. Foyer et al., 2014).

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Several of the identified MYB genes function in stress or defense responses, and they are related to ROS signaling and protect plastids from stress and photodamage

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(Saibo et al., 2009). MYB28, MYB29 and MYB76 are regulators of aliphatic glucosinolates (Sønderby et al., 2010) and MYB59 which is repressed by H2O2 to promote root hair growth (Mu et al., 2009) is downregulated in oe18 (Table S1-1). The regulated WRKY genes are involved in hormone-induced biotic and abiotic responses or related to ROS functions including cell death programs. WRKY18, WRKY40 and WRKY60 are induced in responses to ABA, abiotic stress (Chen et al.,

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2010) and microbial pathogens (Xu et al., 2006). H2O2-regulated WRKY53 control defense and leaf senescence (Miao and Zentgraf, 2010). This confirms that RRTF1

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interferes with the regulatory circuits of the WRKY TFs (cf. Khandelwal et al., 2008; Pandey et al., 2010; Table 2, Figure S7, Tables S1-1, S1-2). Finally, the JA- and ET-

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regulated PDF1.2 and SA-regulated PR-2 were differentially regulated in oe18 (Tables S1-2). Therefore, RRTF1 is also involved in JA/ET- and SA-mediated stress

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responses.

Several extracellular/endomembrane type class III peroxidase genes were induced by RRTF1 overexpression (Tables S1-1 and S1-2). Some class III peroxidases are implicated to generate hydrogen peroxide in apoplastic oxidative burst (Rouet et al., 2006; O’Brien et al., 2012; Welinder et al., 2002) and a mutant (kua1) de-repressing the expression of apoplastic peroxidases showed increased H2O2 levels in leaves and significant reduction in size (Lu et al., 2014), which is similar to the phenotype of the RRTF1 oes. Therefore, the RRTF1-induced peroxidases are likely ROS generators causing oxidative burst. Apoplastic H2O2 and class III peroxidases participate in cell wall remodeling (Bradley et al., 1992; Olson and Varner, 1993; Østergaard et al., 2000; O’Brien et al., 2012) and cell expansion processes (Lu et al., 18

ACCEPTED MANUSCRIPT 2014). Interestingly, in rrtf1 and oe18 plants, the expression of many genes related to cell wall modification and cell expansion, such as xyloglucan:xyloglycosyl transferase and expansin genes, were significantly altered (Tables S1-1 and S1-2). The concept that RRTF1 regulates apoplastic peroxidases to induce H2O2 signaling and cell-wall remodeling is further supported by the observation that the peroxidase genes

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induced in oe18, At2g37130, and At4g30170, possess RRTF1 binding sequence in their promoters. These genes might be the primary players in RRTF1-mediated ROS amplification processes.

Genes for other ROS producers such as the plasma membrane-localized NADPH

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oxidases, peroxisomal glycolate oxidases and the components of the photosynthetic electron transport chain were not upregulated, and some genes for the mitochondrial

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respiration (e.g. β and γ subunits of ATP synthase, cf. Table S1-1) were even downregulated in oe18. Among the ROS scavenging enzymes, a dehydroascorbate reductase, DHAR5 (At1g19570), was up-regulated in oe18 (Table S1-2), however another DHAR isozyme (DHAR2 – At1g75270), ascorbate peroxidase 4 (APX4 At4g09010) and

Fe superoxide

dismutase

3

(FSD3

– At5g23310)

were

downregulated in oe18 (Tables S1-1, S1-2). Moreover, the gene for ascorbate-

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synthesizing VTC2 protein (At4g26850) was significantly repressed in oe18, implicating the less ROS-scavenging capacity in RRTF1 oe plant. 12 glutaredoxins, 3

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thioredoxins and 10 Glutathione S-transferases were differentially expressed in oe18 and rrtf1 compared to WT. They are located in chloroplasts (At5g61440),

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mitochondria (At1g02920, At1g03850), nucleus (At2g41330, At1g64500, At5g17220), vacuole (At1g02920, At5g17220), apoplast, plasmodesma (At1g02920, At1g45145) and cytosol (e.g. At1g17180, At1g6992, At2g29460), suggesting that RRTF1

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overexpression should cause an altered redox network in diverse cellular compartments. GO data analysis detected many oxidative stress-responsive genes in oe18 (Tables S1-1, S1-2), and many of the identified genes are involved in oxidation processes. These observations are consistent with higher ROS levels in oe18 (Figure 4). In addition, senescence- and programmed cell death associated genes (At2g17850, At5g15410, At4g16860) are upregulated in oe18 (Table S1-2). Interestingly, OXI1 which has been identified as a H2O2-inducible serine kinase gene (Rentel et al., 2004) is induced in oe18 and repressed in rrtf1 roots (Figure S9, Table S1-2). Taken together, many RRTF1-regulated genes are directly or indirectly related to ROS 19

ACCEPTED MANUSCRIPT function or homeostasis. Upregulation of genes involved in pathogen defense in shaded distal leaves of cHL-exposed plants suggests that part of the produced ROS is released into the apoplast. We found that also stress related receptor like kinases (TIR-NBS-LRR, CLAVATA3, LRR protein kinases and G-protein coupled RLK) are differentially regulated in oe18 and rrtf1 material (Tables S1-1, S1-2). These RLKs

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are also regulated in response to ROS, PAMPs, light stress and ozone, which is ultimately converted to H2O2 in the apoplast (Wrzaczek et al. 2010, and references therein). Therefore, the above mentioned RLKs may be candidates to sense ROS through redox modification in its extracellular domain.

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Regulation of RAP2.6 and RRTF1 is very similar under various biotic and abiotic stress conditions. RAP2.6 is upregulated in the roots and shoots of oe18 and

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downregulated in the shoots of rrtf1. Therefore, this gene appears to be directly regulated by RRTF1 and was identified as one of RRTF1 targets. RRTF1 and RAP2.6 are phylogenetically related and their expression responds to stress signals and stress hormones (Nakano et al., 2006, Dietz et al., 2010; Krishnaswamy et al., 2011). oe for both genes have a dwarf phenotype (Figure 2; Krishnaswamy et al., 2011) and flower earlier in the greenhouse, and young leaves are often wrinkled. To

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check the relation between the two TFs, we grew RAP2.6 oe (Krishnaswamy et al., 2011) under the same conditions as RRTF1 oe. Figure 8A demonstrates that only the

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RRTF1, but not the RAP2.6 oe shows a bleached phenotype. Furthermore, the RAP2.6 oe lines did not accumulate higher ROS levels (Figure 8B). This suggests

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that RAP2.6 alone does not control ROS accumulation. Our bioinfomatics analysis and in vitro DNA binding assay identified a GCC-box like

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sequence and 4 GCC-box sequences as RRTF1 binding motifs (Figure 10E). These cis-elements were detected in the promoter sequences of ~10% of the RRTF1 responsive genes in our microarray analyses (Tables S1-1, S1-2). The remnant of RRTF1 responsive genes are likely indirectly regulated, e.g. by other transcription factors, which were induced by RRTF1 or an altered cellular redox state as the consequence of RRTF1 expression. Many genes which respond to elevated RRTF1 level in the oe lines are involved in stress responses and redox regulation, for example, the STZ and RAP2.6 transcription factors mediate photooxidative, salt, drought and cold stress responses (Sakamoto et al., 2004; Rossel et al., 2007; Zhu et al., 2010), SIB1 is a transcription factor regulating the response to pathogen, SA and JA (Xie et al., 2010), and class III type peroxidases (At2g37130, At4g30170) are 20

ACCEPTED MANUSCRIPT involved in cell wall functions. Interestingly, a RRTF1 binding sequence exists also in the RRTF1 promoter. Since RRTF1 is transiently expressed, with a rapid increase of the mRNA level in high light (Vogel et al., 2014) or after JA treatment (Wang et al., 2008) followed by a fast decline, RRTF1 might be negatively auto-regulated (Figure 9B). These observations suggest that RRTF1 should regulate various stress

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responses not only via ROS production but also by the direct regulation of multiple stress-related transcription factors. Genes related to diverse metabolic and cellular processes, such as cell wall remodelling, were also identified as RRTF1 targets. Their participation in RRTF-induced physiological processes will be elucidated in

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future work.

Our analysis showed also that the GCC-box like sequence AGCCGTCA was more

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often used than the sequences with the GCC-box core element GCCGCC in strongly RRTF1-regulated genes (Figure 10E). This result was unexpected, because the PBM analysis with the recombinant RRTF1 predicted that the GCC-box is the best RRTF1 binding sequence (Franco-Zorrilla et al., 2014). The difference between our microarray based analysis and the in vitro analysis of Franco-Zorrilla et al. (2014) might indicate that cis-element recognition of RRTF1 is influenced by the intranuclear

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environment in vivo such as chromatin structure or other transcription factors. In vivo, the preference to the GCC-box like sequence seems to be an essential characteristic

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of RRTF1 to regulate the gene set identified in this study. While the canonical GCCbox is broadly recognized by other ERF family proteins (Franco-Zorrilla et al., 2014),

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it might not be adequate for RRTF1-specific gene regulation. The genes with the GCC-box like sequence may play key roles in RRTF1-mediated stress responses.

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RRTF1 regulation by microbes Database analyses (Figure S1) and infection studies with P. indica (Figures 5E and S1, Table 1) support the concept that biotrophs and beneficial microbes, which live and propagate in living host cells after establishing a stable interaction, either do not activate or repress RRTF1 expression. Interestingly, co-cultivation of Arabidopsis with P. indica results in an initial upregulation of the RRTF1 mRNA level, while the message level is downregulated during later stages of the symbiosis (Figure S1). Likewise, during early stages of mycorrizha formation, H2O2 is produced and this production declines as soon as a mutualistic interaction has been established (Fester and Hause, 2005). Apparently, during early phases of the interaction, the beneficial 21

ACCEPTED MANUSCRIPT symbiosis is not yet established and therefore RRTF1 expression is not yet repressed. Regulation of RRTF1 in P. indica-colonized plants occurs in roots and leaves (Fig. 5E) indicating a systemic effect of the fungus on RRTF1 expression. WRKY18, WRKY33, WRKY40 and WRKY60 are induced in response to H2O2/ROS and H2O2/ROS generating agents (https://www.genevestigator.com, and references

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therein). The transcript levels of WRKY18, -33, -40 and -60 are also differentially regulated in oe18 and rrtf1 material (Tables S1-1, S1-2, S2). ROS-generating stimuli from necrotroph infections stimulate RRTF1 expression, while biotrophic and mutualistic microbes repress RRTF1 expression. The rapid decline of the RRTF1

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mRNA level after the end of the stress ensures that H2O2/ROS accumulation is only transient.

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P. indica actively represses ROS accumulation by activating ROS scavenging genes. This is particularly striking for the oe lines. Figures 1C and 5 demonstrate that stressexposed oe lines can better survive in the presence of P. indica, and this is associated with a strong reduction of the ROS level. The slight reduction of ROS level in the stress-exposed rrtf1 in the presence of P. indica demonstrates that some reduction of ROS also occurs independently of RRTF1 (Figure 5C). Colonized WT

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seedlings perform better under stress because P. indica reduces the ROS level by activating ROS scavenging systems and by downregulating RRTF1 expression

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(Figures 5E and S1). The NAC and DDCNa experiments also demonstrate that the high H2O2/ROS levels in the oe lines is the reason for the stress sensitivity (Figures

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6). It is reasonable to assume that the continuously long-term high H2O2/ROS levels in all tissues of the oe lines weakens the plants, similar to results observed for other

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ROS overproducers, such as the flu mutant (Gadjev et al., 2006). Figure 9 demonstrates that activation of RRTF1 by abiotic (HL) and biotic (A. brassicae infection) stress requires WRKY18/40/60. The presence of multiple Wboxes in the RRTF1 promoter and their involvement in RRTF1 regulation (Pandey et al., 2011) is in line with the observation that these WRKYs regulate target genes in response to ABA as well as abiotic and biotic stresses (Xue et al., 2006, Chen et al., 2010; Jiang et al, 2011). WRKY18, WRKY40 and WRKY60 are also differentially regulated in the rrtf1 and oe18 lines (Tables S1-1, S1-2, S2) suggesting the existence of a regulatory network among the four TF. Downregulation of RRTF1 by P. indica is not directly controlled by the three WRKY TFs, because the repression is similar in WT and wrky18 wrky40 wrky60 seedlings (Figure 9A). The fungus, like 22

ACCEPTED MANUSCRIPT NAC and DDCNa, restricts ROS accumulation under stress, which results in lower RRTF1 expression (Figure 9B). In summary, RRTF integrates various stress signals and activates a variety of downstream cellular responses. No obvious preference for a particular stress perception or response is detectable. The microarray analyses suggest the

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participation of RRTF1 in the synergistic activation of stress pathways which do not need to be directly related to the incoming stress signal. Therefore, RRTF1 is a good candidate for the activation of cross-tolerance responses (cf. Pastori and Foyer, 2002; Foyer et al., 2014). We demonstrate that elevated RRTF1 levels in plants

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cause ROS accumulation in response to biotic and abiotic stress signals. Various ROS themselves stimulate RRTF1 expression, which suggests that RRTF1 amplifies

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ROS formation in response to stress which already generate basal levels of ROS. Stimulation of ROS production by RRTF1 might be important for a rapid and transient establishment of local ROS maxima to induce appropriate downstream responses. Upregulation of RRTF1 expression is mediated via WRKY18/40/60, and these TFs may be part of a regulatory circuit which coordinate the response to abiotic and biotic stress. Bioinformatic analyses and binding studies of RRTF1 to cis-elements in

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RRTF1-responive genes demonstrate that the TF also activates stress-responsive genes directly. The gene products and an alteration in the cellular redox homeostasis

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trigger further downstream responses. Constitutive upregulation of

RRTF1

expression has severe consequences since even minor stress induces ROS levels

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which are harmful for the oe. The tight regulation of RRTF1 suggests that the TF is important in controlling ROS accumulation. The nuclear location (not shown), domain structure (Figure S2), putative DNA-binding domains and putative interaction partners

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of RRTF1 suggest that the control of H2O2/ROS accumulation by RRTF1 starts with the transcriptional reprogramming of the cell.

METHODS Plant material and growth Arabidopsis thaliana Col-0 (for Fig. 10B, Ws) was used as WT control. The T-DNAinserted RRTF1 knockout mutant (SALK_150614) was obtained from NASC (http://arabidopsis.info/). Homozygote RRTF1 knockout plant was identified by PCR with gene specific primers (LP_SALK_150622: 5’-CGCGATGCTTTGTAGGAGTAG-3’, 23

ACCEPTED MANUSCRIPT RP_SALK_150622: 5’-TGTCAGGGTTTTTCCAGTGAC-3’) and T-DNA left border primer Lba1 (5’-TGGTTCACGTAGTGGGCCATCG-3’). The wrky triple knockout line was a gift from Dr. I. Somssich (MPI for Plant Breeding, Cologne, Germany). The seedlings were grown on MS medium with 1.37% sucrose (except for the Results in Table 1A and 1B, where sucrose was omitted) under cLL; 30 µmol m -2s-1,

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cML; 80 µmol m-2s-1 or cHL; 200 µmol m-2s-1 at 20˚C. Alternatively, growth occurred under SD or LD conditions in combination with the three different light intensities. For long-term experiments (18 days) in plates and root analyses, the seedlings were grown on Hoagland medium in square plates and vertical orientation.

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For experiments on soil, 14 days old seedlings grown on MS in cLL were transferred to pots containing garden soil and vermiculite (9:1; v/v) and cultivated under the given

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light regimes at 20°C, as outlined in the text or figure legends. Immediately after budding, tripod aracon tubes were placed on every plant to synchronize growth of the individual plants and to prevent cross pollination. After complete drying of the plants, seeds were harvested and the biomass was determined. Experiments with P. indica

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P. indica was cultured on Kaefer medium as described in Johnson et al. (2011). The cocultivation experiment with P. indica was done as described in Johnson et al. (2011,

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Method 2). Control seedlings were transfered to plates inoculated with a plaque without fungal hyphae. Plates were incubated at 20°C under the light regimes described in the text or figure legend. Spores of P. indica were harvested from 4

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week-old fungal plates and resuspended in sterile water at a concentation of 106-107

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ml-1. One ml of spore suspension was added to 1 l of MS or Hoagland media. Experiments with A. brassicae A. brassicae (FSU-3951, Jena Microbial Resource Center) was grown on PDA medium (pH 6.5) at 22°C, 12/12 h light/dark and 75% relative humidity in a temperature controlled growth chamber. After two weeks, the dense fungal mycelium sporulated heavily. The medium was removed by filtering through 4 layers of sterilized nylon membrane, and the mycelium with spores was washed 3 times with sterile H2O. The spores and mycelia were gently resuspended in 100 ml of sterile H2O and filtered through four layers of sterilized nylon membrane to remove the mycelia and hyphae. The spore density was adjusted to 10 5-106 ml-1, either by serial dilution or a Haemocytometer. For leaf infection, 14-day old seedlings were 24

ACCEPTED MANUSCRIPT transferred to fresh PNM plates with a sterilized nylon membrane and incubated at 20°C under LD ML conditions (for details see Johnson et al., 2011). After 48 h, 5 μl of the spore suspension was inoculated on to 6 leaves in the middle whorl per seedling and were incubated in a temperature controlled growth chamber as described earlier. After 3-7 days, progression of disease development was quantified as Percentage

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Disease Index (PDI) using standard disease intensity grades based on the number and area of leaves infected.

The competitive experiments with P. indica and A. brassicae on Arabidopsis seedlings were performed by cocultivating the roots with P. indica and inoculating the

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leaves with A. brassicae spores 48 h later. A. thaliana was cocultivated with P. indica on modified PNM medium as described in Johnson et al. (2011). After 48 h of

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cocultivation, the upper 6 leaves are inoculated with 5 μl of A. brassicae spore suspension (105-106 spores ml-1) and incubated as described earlier. The seedlings not treated with P. indica but inoculated with A. brassicae served as control. The toxin from A. brassicae was prepared according to Vidyasekaran et al. (1997). The toxin was further concentrated, lyophilised and resuspended in sterile H2O. Quantification of A. brassicae in infected leaves (Figure 3) was performed by real-

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time PCR for the AbreAtr1 gene marker (Guillemette et al. 2004) as described in

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Johnson et al. (2014).

Plasmid construction and plant transformation

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For the RRTF1 (At4g34410) oe construct p35S-RRTF1, the RRTF1 coding region was amplified from RRTF1 cDNA clone (DKLAT4G34410), obtained from Arabidopsis

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Biological Resource Center (http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc /abrchome.htm). The PCR product was cloned into pCR®8/GW/TOPO® TA vector (Invitrogen), and then the insert was integrated into a binary vector, pB2GW7 containing CaMV 35S promoter, using Gateway LR recombination reaction system (Invitrogen). p35S-RRTF1 was transformed into Arabidopsis Col-0 via Agrobacterium tumefaciens strain GV3101 with floral-dip method. T1 plants were selected by spraying 1-2 weeks old seedlings with 0.1% BASTA (Bayer, Germany). T2- and T3plant resistant to DL-phosphinothricin (50µM) (Sigma-Aldrich) on MS medium plate were used for physiological experiments. RNA analyses 25

ACCEPTED MANUSCRIPT Total RNA was isolated from shoot and roots with the RNeasy Plant Mini Kit (Qiagen). cDNA was synthesized from 1 µg total RNA with Omniscript RT Kit (Qiagen) and oligo (dT)20 in 20 µl reaction volume. Semi-quantitative and quantitative real-time PCR were done with gene specific primers (Table S3). Real-time quantitative RTPCR was performed using the iCycler iQ real-time PCR detection system and iCycler

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software version 2.2 (Bio-Rad). For the amplification of the PCR products, iQ SYBR Supermix (Bio-Rad) was used according to the manufacturer´s instructions in a final volume of 23 µl. The iCycler was programmed to 95°C 2 min, 35× (95°C 30 s, 55°C 40 s, 72°C 45 s), 72°C 10 min followed by a melting curve programme (55–95°C in

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increasing steps of 0.5°C). All reactions were repeated twice. The mRNA levels for each cDNA probe were normalized with respect to the GAPDHC message levels. Fold induction values were calculated with the ΔΔCP equation of Pfaffl (2001). The

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ratio of a target gene was calculated in the treated sample versus the untreated control in comparison to a reference gene. Microarray analysis

Total RNA was isolated from the cotyledons and roots of 14-day old seedlings grown on MS under SD LL conditions and mature leaves of 5 weeks old plant (for growth

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conditions, cf. Plant material and growth). The microarray hybridization was performed with the Arabidopsis Genome Array ATH1 (Affymetrix, USA) at the

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Kompetenzzentrum für Fluoreszente Bioanalytik, Regensburg, Germany, and are accessible under the accession numbers, GSE64783 and GSE64853. The signal

data

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hybridization

(http://mapman.gabipd.org/web/

were

analyzed

guest/robinsoftware)

with and

ROBIN

program

MapMan

program

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(http://mapman.gabipd.org/web/guest/ mapman). Statistical analysis for t-test and subsequent calculation of false discovery rate (FDR) was according to ROBIN program with the data of 3 biological independent experiments. DCMU treatment

The electron transport inhibitor DCMU (Sigma) was applied to the seedlings in Petri dishes by spraying 0.5 ml of a 5 µM solution to the leaves 90 min before transfer of the seedlings from LL to HL or before the application of the PAMPs. Control seedlings were treated with the solvent without DCMU. PAMP treatments with flg22, chitin or an A. brassicae toxin preparation Each leaf of 10-day old Arabidopsis seedlings were treated with 20 μl of 1 μM flg22, 26

ACCEPTED MANUSCRIPT or 1 µM chitin (crab shells, Sigma-Aldrich) or an A. brassicae toxin preparation and the control seedlings with 20 μl of sterile H2O. Quantitative H2O2 and ROS measurements and detection of ROS in roots Quantitative H2O2 measurement from leaves and roots were performed using the

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Amplex Red hydrogenperoxide/peroxidase assay kit (Molecular Probes) according to the manufacturer’s instructions (http://tools.invitrogen.com/content/sfs/manuals/mp 22188.pdf). Leaf sections of 0.5-1 mm width and root sections of 2-3 cm length were incubated in the reaction mixture for 10 min in dark at room temperature. The fluorescence intensity was quantified with a fluorescence microplate reader (TECAN

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Infinite 200) with an excitation at 540 nm and emission at 610 nm. H2O2 was used to prepare the standard curve. The reaction mixture without the substrate and plant

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material served as control.

ROS measurements from leaves and roots were performed using the substrate carboxy-H2DFFDA (Molecular Probes) according to the manufacturer’s instructions (https://tools.invitrogen.com/content/sfs/manuals/mp36103.pdf). The plant material was incubated in 20 μM carboxy-H2DFFDA prepared in KRPG buffer for 30 min in the dark. The fluorescence intensity was quantified with a fluorescence microplate reader

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(TECAN Infinite 200) with an excitation at 485 nm and emission at 530 nm. The

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reaction mixture without the substrate and plant material served as control. Stock solutions of the anti-oxidants and ROS inhibitors N-acetyl-L-cysteine (NAC)

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and diethyldithocarbamate sodium salt (DDCNa) (Sigma-Aldrich) were prepared in their respective solvents, filter-sterilized, diluted in sterile water before adding to the

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sterilized media before H2O2 and ROS measurements. ROS imaging in roots were performed by fixing their ends on a glass slide (Thermo Scientific, Germany) and by incubating the root sections with 20 μM carboxyH2DFFDA prepared in KRPG buffer for 30 min. The treated roots were washed 5 times with KRPG buffer without mechanical disturbances and immediately photographed using Axiocam MRC5 fluorescence microscope (Carl Zeiss, Germany).

Prediction of RRTF1 cis-element and in vitro RRTF1-DNA binding assay RRTF1 binding octamer sequences were predicted according to Yamamoto et al. (2011) using the microarray data of mature leaves (Table S1-1). Binding of RRTF1 to 27

ACCEPTED MANUSCRIPT DNA was detected as luminescence signals by using the AlphaScreen assay (PerkinElmer Japan, Tokyo). Biotinylated double-stranded DNA probes corresponding to the RAP2.6 promoter sequences -1000/-951 (Probe B), -812/-763 (Probe C), 424/-375 (Probe D), -362/-313 (Probe E), -109/-60 (Probe F) and to the -985/-936 fragment of the At3g49160 promoter (Probe A) were incubated with Flag-tagged

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RRTF1 synthesized in the wheat germ translation system (BioSieg Inc., Tokushima, Japan). Unpurified translation products of DNA probes with biotin at 5’ end of the forward strand (final concentration: 50 nM), and unbiotinylated DNA competitors (final concentration: 0 or 100 nM) were mixed in the reaction buffer in a total volume of 25

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μl in 384-well Optiwell™ microtiter plates (PerkinElmer Japan, Tokyo), and incubated for 20 min at room temperature (~23 °C). After the initial incubation, acceptor beads were diluted 1:40 in water and 4 μl of the dilution was added to each well, and

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incubated again for 10 min at room temperature. Donor beads were then added. After incubation in the dark for 3 hour at 23 ± 1°C, light emission was measured using the EnSpire reader (PerkinElmer Japan, Tokyo) and the data were analyzed with the EnSpire manager software. Detected AlphaScreen signals were normalized with those of the control samples with unbiotinylated DNA probes. The data are based on triplicate experiments, with averages and standard deviations (SEs). The Probes and

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competitor sequences are given in Table S4.

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The promoter sequences of RRTF1-response genes, which were detected in the microarray experiments (fold change ≥2 or ≥4), were acquired from the Arabidopsis sequence

database

(TAIR10_upstream_3000_20101028,

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upstream

http://www.arabidopsis.org). In these sequences, the RRTF1 cis-elements described in Fig. 10 (AGCCGTCA, CTGCCGCC, TGCCGCCA, CAGCCGCC, GCCGCCTT and

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their complementary sequences) were searched.

Miscellaneous

For quantification of photobleached leaf areas, the white areas of the leaf were quantified as pixel in PhotoshopTM on a digital photograph. Measurement of photosynthesis parameters and analysis of chlorophyll and anthocyanin contents were described in the Figure S4.

ACKNOWLEDGEMENTS 28

ACCEPTED MANUSCRIPT We like to thank Dr. N.N. Kav (University of Alberta, Canada) for the RAP2.6 oe lines; Dr. Imre Somssich (MPI Cologne, Germany) for the gift of the triple wrky knockout line, Prof. Scheel for providing flg22 and Sarah Mußbach and Claudia Röppischer for

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their excellent technical assistance.

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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1. Characterization of rrtf1 and the RRTF1 overexpressor lines oe18, oe20 and oe32. (A) RRTF1 RNA levels in the leaves of 10-day old seedlings grown on MS medium in cML. The RNA level of WT seedlings are set as 1.0 and the other levels are expressed relative to it. The qRT-PCR products of RRTF1 were normalized

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to those of GAPDHC as control. Based on 6 independent qRT-PCR experiments, bars represent SEs. (B) 14-day old rrtf1, WT, oe18, oe20 and oe32 seedlings which were grown under cLL, cML or cHL on MS medium. The bottom part shows false color images of the plates representing Fs/Fm values as described in Methods. (C)

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(left): rrtf1, WT, oe18, oe20 and oe32 seedlings grown on Hoagland plates for 18 days in cML and (right) amended with P. indica spores as described in Methods. The numbers on the bottom of the plate refer to the lengths of the roots in cm (n=40

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seedlings, values are means ± SEs from 4 independent experiments). Figure 2. The performance of RRTF1 overexpressors increase with longer darkincubation periods. Fourteen day-old WT, rrtf1, oe18 and oe20 seedlings, grown on MS medium in Petri dishes under cLL, were transferred to soil and grown under SD 3.and LD conditions for 6 weeks. Almost all oe18 and oe20 and all o32 plants died in

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cML after 6 weeks on soil and are not shown. The picture shows representative plants under SD or LD conditions. The table gives quantitative data (means ± SEs)

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based on four independent experiments with 14 plants per line in each experiment.

seeds.

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The dry weights were determined from the completely dried plants after harvesting of

Figure 3. The RRTF1 overexpressors are highly susceptible to A. brassicae

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infections. 14 day-old WT, rrtf1, oe18 and oe20 seedlings, grown in Petri dishes on MS medium under cLL, were transferred to sterile water-soaked Whatmann paper in Petri dishes, and the leaves were inoculated with 5 μl of spore suspension (105-106 spores ml-1) as described in Methods. Representative pictures were taken at 3 or 5 days after infection (dai) from 6 independent experiments with 20 seedlings per line in each experiment. Water was used as control and did not show any effect. After 5 days of co-cultivation, 13,4 ± 3,0 (oe20), 12,9 ± 3,2 (oe18) and 1,06 ± 0,22 (rrtf1) times more fungal DNA was found in the leaves of the transgenic seedlings than in the WT control (1,00 ± 0,16). Figure 4. ROS levels in the leaves (A and B) and roots (C and D) of 14-day old 39

ACCEPTED MANUSCRIPT rrtf1, WT, oe18, oe20 and oe32 seedlings. The seedlings were grown in Petri dishes on MS medium in either darkness, cLL, cML or cHL. The H2O2 (A) or ROS (B) levels were determined for the leaves as described in the Methods. (C) The roots were stained with carboxy-H2DFFDA for imaging of the ROS level. (D) Quantified data for H2O2 and ROS levels in the roots. Data are based on 6 independent

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experiments with 20 seedlings per treatment in each line. Mean values ± SEs are given. RFU, relative fluorescence unit; FW, fresh weight. For (A) and (B), all data are significantly different from the WT control with P < 0.01, except those marked with an asterisk (P < 0.5).

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Figure 5. Piriformospora indica rescues the light- and A. brassicae-induced stress phenotypes of the oe lines by reducing H2O2/ROS accumulation in

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leaves and roots. (A) 14-day old rrtf1, WT, oe18, oe20 and oe32 seedlings grown under LD ML on MS medium, either in the absence (- P. indica) or presence (+ P. indica) of P. indica. Representative pictures from 5 independent experiments with 10 replications in each treatment are shown. (B) 12-day old rrtf1, WT, oe18, oe20 and oe32 seedlings grown on MS medium under LD ML were transferred to sterile nylon membrane on PNM plates and each leaf was infected with 5 µl of an A. brassicae

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spore suspension (cf. Methods and Materials). Lower panels: False color images of the plates shown above representing Fs/Fm values as described in Methods.

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Representative pictures from 5 independent experiments with 10 replications. (C) H2O2/ROS levels in the leaves and roots of 14-day old rrtf1, WT, oe18, oe20 and

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oe32 seedlings grown under LD ML on MS medium in the absence (dark bars) or presence (light bars) of P. indica. Based on 5 independent experiments with 30

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seedlings per treatment. Mean values ± SEs are given. Significant differences between seedlings grown ± P. indica were determined by Student’s t-test (* P < 0.05; ** P < 0.01). (D) Relative mRNA levels for ROS scavenging enzymes in the leaves of seedlings which were treated as described under (B). For each panel, the mRNA level of WT seedlings infected with A. brassicae was taken as 1.0 (± SEs), and the other values are expressed relative to it. Based on 5 independent experiments with 30 seedlings in each line. Significant differences between seedlings grown ± P. indica were determined by Student’s t-test (* P < 0.05; ** P < 0.01). (E) Relative RRTF1 mRNA levels in the leaves and roots of 14-day old rrtf1, WT, oe18 and oe20 seedlings grown under LD ML on MS medium in the absence (dark bars) or presence

40

ACCEPTED MANUSCRIPT (light bars) of P. indica. Based on 4 independent experiments with 30 seedlings per line. Mean values ± SEs are given. In all cases, P > 0.1. Figure 6. The antioxidants N-acetyl cysteine (NAC) and diethyl dithiocarbamate sodium salt (DDCNa) recovered the ROS induced phenotype and A. brassicae susceptibility in oe seedlings by reducing H2O2/ROS accumulation. (A) rrtf1, WT,

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oe18, oe20 and oe32 seedlings were grown under LD ML on MS medium or MS medium supplemented with 1mM NAC or 1mM DDCNa for 19 days. (B) As in (A), except that each leaf was inoculated with 5 µl of A. brassicae spore suspension on day 14. Mock treatment was performed with sterile H2O. Representative pictures

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from 5 independent experiments with 10 replications for each experiment are shown. (C) ROS levels in the leaves and roots of 14-day old rrtf1, WT, oe18, oe20 and oe32

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seedlings grown with or without the antioxidants. The seedlings were grown under LD ML on MS medium supplemented with or without 1 mM NAC or 1 mM DDCNa. The total H2O2 or ROS levels were determined separately for the leaves and roots as described in Methods and Material. The data are based on 4 independent experiments with 20 seedlings per treatment. Mean values ± SEs are given. The asterisks indicate that the values for the control seedlings are significant different

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from those for NAC- and DDCNa-treated seedlings; determined by Student’s t-test (*

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P < 0.05; ** P < 0.01).

Figure 7. RRTF1-regulated genes. Venn diagram of the number of genes

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differentially regulated in oe18 mature leaves, as well as shoots and roots of seedlings. The numbers in brackets refer to genes with ROS related functions

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(Mehterov et al., 2012), they are presented in Table 2, Table S1 and Table S2. Figure 8. Comparison of RRTF1 and RAP2.6 lines. (A) rrtf1, WT, oe18, oe20 and oe32 were compared to rap2.6, WT and the RAP2.6 oe lines A6, A39 and A2. All seedlings were grown for 10 days under cLL before transfer to cML for 5 days. (B) Leaves and roots of these seedlings were harvested separately for H2O2/ROS determination as described in Methods. The data are means of 5 independent experiments with 40 seedlings each, bars represent SEs. Significant differences between RRTF1 and RAP2.6 seedlings were determined by Student’s t-test (* P < 0.05; ** P < 0.01). A6, A39 and A2 are in the Wasilewski (Ws) background and Ws WT was used as control.

41

ACCEPTED MANUSCRIPT Figure 9. Full regulation of RRTF1 by A. brassicae (Ab) infection, HL and H202, but not by P. indica (Pi) colonization requires WRKY18/40/60. (A) Ab: Shoots and roots of 14-day old WT and wrky18 wrky40 wrky60 seedlings grown under LD ML on MS medium were inoculated with an Ab spore suspension for 3 d. Mock treatment was performed with H2O. HL: Fourteen-day old WT and wrky18 wrky40 wrky60

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seedlings grown under LD ML on MS medium were exposed to HL for 24 h. Seedlings grown under LD ML served as control. H2O2: Fourteen-day old WT and wrky18 wrky40 wrky60 seedlings grown under LD ML on MS medium were treated with 10 mM H2O2 for 1 h. H2O served as control. Pi: Twelve-day old WT and wrky18

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wrky40 wrky60 seedlings grown under LD ML on MS medium were co-cultivated with Pi for 6 d. Mock treatment was performed with a KM plug. For all experiments, the

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relative RRTF1 mRNA levels were determined from the shoots and roots. The mRNA levels marked with § were set as 1.0 (± SEs) and the other values expressed relative to them. Based on 3 independent experiments. Significant differences for the untreated control seedlings were determined by Student’s t-test (* P < 0.05; ** P < 0.01).

(B) A model summarizing RRTF1 regulation. A. brassicae, HL and H2O2

require WRKY18, WRKY40 and/or WRKY60 to fully activate RRTF1 expression by

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binding to one or more W box(es) in the RRTF1 promoter. P. indica, NAC and DDCNa repress and RRTF1 amplifies H2O2 accumulation. We propose that RRTF1

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model (cf. Discussion).

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also represses the expression of its own gene, as shown in the upper section of the

Figure 10. Identification of RRTF1 binding sequence on RAP2.6 promoter. (A)

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Scanning of the RAP2.6 promoter. Promoter sequences which are overrepresented in the RRTF1-activated promoters of oe18 were assayed with the microarray data from mature leaves (cf. Methods), and candidate sites for RRTF1-regulated ciselements are shown as high RAR (vertical axis). Horizontal axis shows position from transcription start site (TSS) of RAP2.6 (Hieno et al., 2014). Red boxes represent loci used for in vitro DNA binding assays as probes. Red bar means threshold (RAR > 3), which were used for the prediction of the plant cis-element. (B) Identification of a RRTF1-binding locus on the RAP2.6 promoter by in vitro binding assay. Biotinylated DNA probes as shown were incubated with in vitro synthesized RRTF1 protein. Binding of RRTF1 to the DNA probe was detected as a luminescence signal by using 42

ACCEPTED MANUSCRIPT the AlphaScreen assay, and expressed as relative signal, i.e. a ratio of the signal with the biotinylated DNA probe to one with the corresponding unbiotinylated DNAs. Data derived from triplicate assays, and are shown as averages with standard deviations. (C) Competition assays to identify a RRTF1 target site within Probe F. Biotinylated probe and nonbiotinylated competitors as shown were incubated with RRTF1. Probe

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C is a non-specific competitor used in (B). Unmutated and mutated Probe F, as shown in (D) were also used as competitors. (D) Sequences of competitors. Underlined sequences are predicted RRTF1 target sites. m1 and m2 have mutation shown as lowercase letters. (E) RRTF1-binding sequence and the gene number

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possessing the sequence. RRTF1 binding sequences which fulfilled 3 criteria are presented: 1) overrepresented octamers in oe18-regulated genes (RAR > 3.0), 2) presence in promoters with fold changes > 5.0 in oe18-regulated genes, and 3)

(2014). GCC-box is underlined.

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sequences related to the RRTF1 binding motif reported by Franco-Zorrilla et al.

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Figure 11. A model that describes RRTF1 regulation.

Supporting Information Legends

Figure S1. RRTF1 expression in response to biotic and abiotic stimuli.

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Figure S2. Phylogenetic analysis of RRTF1. Figure S3. T-DNA insertion sites in rrtf1 knockout line

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Figure S4. Overexpression of RRTF1 causes chlorophyll bleaching and photoinhibition and RRTF1 stimulates anthocyanin accumulation. Figure S5. RRTF1 oe are highly susceptible to A. brassicae infection. Figure S6. NBT staining for ROS detection and DAB staining for H2O2 detection in seedlings of rrtf1, WT and oe18. Figure S7. Classification of RRTF1-regulated genes in mature leaf, seedlings shoot and seedling root. Figure S8. Seedlings used for microarray analysis. Figure S9. Differentially regulated genes in either the shoots or roots or both of rrtf1, WT and oe18 seedlings.

43

ACCEPTED MANUSCRIPT Figure S10. RRTF1 and RAP2.6 mRNA levels in the shoots and roots of rrtf1 and rap2.6 insertion lines, wild-type and RRTF1 and RAP2.6 oe lines. Table S1-1. Analysis of microarray data for mature leaves. Table S1-2. Analysis of microarray data for shoots and roots of the seedlings. Table S1-3. RRTF1 response genes commonly detected in at least two microarray

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data sets for mature leaf, seedlings shoot and seedlings root. Table S3. Primer list for RT-PCR.

Table S4. Probe- and competitor-sequences used in RRTF1-DNA binding

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experiment.

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ACCEPTED MANUSCRIPT TABLES Table 1. Regulation of RRTF1 by light and PAMPs (A) RRTF1 mRNA levels in the leaves of 10-day old Arabidopsis seedlings, grown on MS medium (without sucrose) and kept in LL, or after transfer from LL to HL for 3 h,

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or after application of PAMPs [flg22, chitin or an A. brassicae toxin preparation (toxAlt)] to the leaves for 3 h. DCMU was sprayed to the leaves 90 min before transfer to HL or application of the PAMPs.

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(B) After 3 h in HL, the seedlings were transferred back to LL and the RRTF1 mRNA level was measured over a period of 9 h. The qRT-PCR products of RRTF1 were compared with those of GAPDHC as control. The data represent mRNA levels

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relative to the LL + 3 h LL control, which was taken as 1.0. SEs are based on 6 independent experiments for (A) and (B). Significant differences to the data obtained for seedlings kept in LL were determined by Student’s t-test (* P < 0.05; ** P < 0.01).

(C) H2O2 activates RRTF1 in WT seedlings. 12-day old WT seedlings grown under

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LD ML on MS medium were treated with different concentrations of H2O2. After 3 h, shoots and roots were harvested separately and relative mRNA levels of RRTF1

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were determined. Control treatment was performed with sterile H2O. The qRT-PCR products of RRTF1 were compared with those of GAPDHC as control. The data

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represent mRNA levels relative to the control, which was taken as 1.0. SEs are based

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on 4 independent experiments.

Treatment (A) LL + 3 h LL LL + 3 h HL LL + DCMU + 3 h HL LL + 3 h flg22 (in LL) LL + 3 h flg22 (in HL) LL + DCMU + 3h flg22 (in HL) LL + 3 h chitin (in LL) LL + 3h chitin (in HL)

relative RRTF1 mRNA levels 1.0 ± 0.2 4.3** ± 0.6 1.1 ± 0.2 5.9** ± 0.2 6.1** ± 0.8 5.9** ± 0.9 4.9** ± 0.5 5.5** ± 0.7 45

ACCEPTED MANUSCRIPT 5.4** ± 0.7 5.1** ± 0.5 5.3** ± 0.5 5.0** ± 0.4

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4.3** ± 0.6 3.3** ± 0.6 1.6* ± 0.4 1.1 ± 0.5 0.8 ± 0.4 0.9 ± 0.2 1.0 ± 0.1 2.1 ± 0.2 3.2 ± 0.4 4.5 ± 0.3 1.0 ± 0.1 1.9 ± 0.1 2.4 ± 0.2 3.2 ± 0.4

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LL + DCMU + 3h chitin (in HL) LL + 3 h toxAlt (in LL) LL + 3 h toxAlt (in HL) LL + DCMU + 3h toxAlt (in HL) (B) LL + 3 h HL LL + 3 h HL +0.5 h LL LL + 3 h HL +1.0 h LL LL + 3 h HL +1.5 h LL LL + 3 h HL +3.0 h LL LL + 3 h HL +9.0 h LL (C) LD ML + 3 h water (root) LD ML + 3 h 1 mM H2O2 (root) LD ML + 3 h 5 mM H2O2 (root) LD ML + 3 h 10 mM H2O2 (root) LD ML + 3 h water (shoot) LD ML + 3 h 1 mM H2O2 (shoot) LD ML + 3 h 5 mM H2O2 (shoot) LD ML + 3 h 10 mM H2O2 (shoot)

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Table 2. RRTF1-regulated ROS marker genes ROS marker genes (Mehterov et al., 2012) regulated in oe18 with difference in signal

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log ratio >1 relative to WT controls. Genes commonly detected in at least two microarray data sets of mature leaves, seedlings shoots and seedlings roots were analysed.

No.

1 2 3 4

ROS marker genes

Class of ROS

Annotation (TAIR)

Location (TAIR)

At1g43160

ROS

RAP2.6

nucleus

At4g17490

O2.-

ATERF6

nucleus

At1g10585

ROS

basic helix-loop-helix (bHLH) family

At1g27730

ROS

ZAT10 - salt tolerance zinc family

cytoplasm, nucleus nucleus 46

ACCEPTED MANUSCRIPT 5

ZAT12 - C2H2-type zinc finger intracellular, family nucleus

At5g59820

ROS

6 7 8

At3g55980 At1g18570

ROS 1 O2

At1g77450

ROS

NAC032 - NAC domain containing nucleus protein 32

9 10

At1g80840

ROS

WRKY40

At5g42380

ROS

CML37 - calmodulin like 37

11 12

At5g54490

ROS

PBP1 - pinoid-binding protein 1

At1g35230

H2O2

AGP5 - arabinogalactan protein 5

13 14

At1g61340

ROS

F-box family protein

At3g49620

H2O2

2-oxoglutarate/iron-dependent oxygenase

15 16 17 18 19

At4g24570

DIC2 - dicarboxylate carrier 2

At1g21100

ROS O2.ROS O2.-

At5g20230

H2O2

SAG14

At2g40000

1

ortholog of sugar beet HS1 PRO-1 mitochondrion 2

At2g41640

1

At1g69890

ROS

Protein of unknown function

At1g05340

ROS

unknown protein

24 25 26 27

O-methyltransferase

D

O2

Glycosyltransferase protein

At1g76600

ROS

unknown protein

At2g18690

ROS 1 O2 ROS

unknown protein

At3g10020 At3g10930

unknown protein unknown protein

nucleus cytoplasm, nucleus nucleus plasma membrane nucleus

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Copper transport protein family

O2

nucleus nucleus

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VQ motif-containing protein

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22 23

MYB51

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21

At5g52760

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20

At2g22880

SZF1 - salt-inducible zinc finger 1

family

cytoplasm mitochondrion nucleus nucleus cytosol, nucleus plasma membrane, vacuole

61 extracellular

region nucleus cytoplasm, nucleus nucleolus, nucleus chloroplast nucleus mitochondrion

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ACCEPTED MANUSCRIPT Table 3. RRTF1 is involved in systemic effects. WT and rrtf1 seedlings were kept in Petri dishes for 2 weeks in cLL before transfer to pots in SD ML conditions for 4 weeks. Leaves of WT and rrtf1 plants were then harvested for ROS and mRNA measurements (t = 0) and these values were set as 1.0 ± SEs. The plants were then exposed to cHL for 3 d, 6 d or 18 d, whereas half of

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the plants was exposed to cHL (light exposed leaf) and the other half was covered with aluminum foil (distal shaded leaf). At the end of the experiment the light exposed and distal shaded leaf material was harvested for ROS and RNA measurements and the values were expressed relative to the values obtained for the material at t = 0.

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Based on 5 independent experiments with 14 plants per treatment, relative errors (SEs) are given. The SE of the proportion is the sum of the individual SEs.

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Photodamaged leaves: absolute numbers are given. A photodamaged/-bleached leaf is defined as a leaf with > 20% white areas. Significant differences between distal shaded leaves and light-exposed leaves were determined by Student’s t-test (* P < 0.05; ** P < 0.01).

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WT (light exposed leaf)

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Treatment

Parameter (fold induction)

RRTF1 mRNA level

8.2 ± 1.2

6 d cHL

RRTF1 mRNA level

5.1 ± 0.9

3 d cHL

ROS level

3.9 ± 1.2

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3 d cHL

6 d cHL

ROS level

3.2 ± 1.2

3 d cHL

MYB122 mRNA level

3.7 ± 0.6

3 d cHL

CML38 mRNA level

2.7 ± 0.5

3 d cHL

PDF1.2 mRNA level

3.1 ± 0.3

3 d cHL 18 d cHL

WRKY40 mRNA level photodamaged leaves (absolute numbers)

WT (distal shaded leaf)

rrtf1 (light exposed leaf)

rrtf1 (distal shaded leaf)

4.4** ± 0.7

-

-

-

-

1.9 ± 0.5

1.1* ± 0.3

1.6 ± 0.3

1.0 ± 0.3

1.2 ± 0.3

1.0 ± 0.3

1.9 ± 0.3

1.3 ± 0.2

2.4 ± 0.5

1.2* ± 0.4

3.6** ± 0.6 2.3* ± 1.0 2.3* ± 0.8 2.0** ± 0.4 2.2* ± 0.4 1.9** ± 0.3

2.6 ± 0.4

2.0 ± 0.4

2.0 ± 0.4

1.2 ± 0.3

10.0 ±0.3

6.1** ± 0.4

14.6 ± 0.6

0.9** ± 0.1

48

ACCEPTED MANUSCRIPT 2.0 ± 0.3

18 d cHL 18 d cHL 18 d cHL 18 d cHL 18 d cHL

ROS level SEN1 mRNA level SRG1 mRNA level SAG21 mRNA level CHS mRNA level

2.5 ± 0.4 2.2 ± 0.3 2.0 ± 0.3 4.3 ± 0.5 3.1 ± 0.4

1.4* ± 0.4 2.1 ± 0.3 1.9 ± 0.3 2.0 ± 0.3 3.5 ± 0.7 3.2 ± 0.4

-

-

1.2 ± 0.2 1.5 ± 0.3 1.4 ± 0.2 2.2 ± 0.4 2.0 ± 0.4

1.0 ± 0.2 1.1 ± 0.2 1.0 ± 0.2 0.9* ± 0.2 1.1* ± 0.1

RI PT

RRTF1 mRNA level

AC C

EP

TE

D

M AN U

SC

18 d c HL

49

AC C

EP

TE

D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

______ 2.5cm

RI PT

long day

Survival in soil (%) Seed yield (mg plant-1)

Fig. 2

rrtf1

AC C

WT

EP

TE

D

M AN US C

short day

Dry weight (mg plant-1)

oe18

oe20

WT

short day

rrtf1

oe18

oe20

____ 2.5cm

long day

WT

rrtf1

oe18

oe20

WT

rrtf1

oe18

oe20

100 ± 0

100 ± 0

64 ± 3

43 ± 3

100 ± 0

100 ± 0

46 ± 2

32 ± 2

123 ± 8

132 ± 6

33 ± 4

20 ± 3

114 ± 4

128 ± 4

47 ± 3

32 ± 3

480 ± 14

485 ± 14

243 ± 10

186 ± 9

415 ± 15

448 ± 15

179 ± 7

113 ± 5

RI PT M AN US C

+ A. brassicae 3 dai

- A. brassicae 3 days of water

______ 1cm _____ 1cm

+ A. brassicae 5 dai

rrtf1

oe18

oe20

AC C

WT

EP

TE

D

- A. brassicae 5 days of water

Fig. 3

2,5

Dark

RI PT

cLL cML

2,0

cHL 1,5

1,0

**

0,5

M AN US C

μMol H2O2 mg-1 leaf FW

A

0,0 rrtf1

35

RFU mg-1 leaf FW x 103

B

WT

30 25 20 15

*

10

*

*

oe18 oe18

oe20 oe20

oe32 oe32

TE

D

WT WT

EP

C

oe32

**

0 rrtf1 rrtf1

oe20

*

*

*

5

oe18

AC C

rrtf1

D

(RFU mg-1

ROS root FW x 103)

H2O2 (µM mg-1 root FW)

WT rrtf1 6582 ± 361 0.58 ± 0.04

oe18 WT 8494 ± 706 0.91 ± 0.05

oe20

oe18 13761 ± 1253 1.71 ± 0.12

oe32

oe20 17151 ± 1185 2.32 ± 0.19

oe32 20036 ± 1921 3.32 ± 0.19

Fig. 4 A-D

EP

C

AC C TE D

RI PT

B

SC

M AN U

A D

E

Figure 5

AC C EP TE D

C

SC

M AN U

B

RI PT

A

Figure 6

RI PT

M AN US C

Up-regulated in oe18 compared to WT Mature leaves

676(14)

73(7)

51(9)

D

19(7) 35(5)

308(13)

EP

TE

252(23)

Seedlings-roots

AC C

Seedlings-shoots

Fig. 7

AC C EP TE D

B

SC

M AN U

RI PT

A

Figure 8

4

12

**

**

4

**

3

2

6

*

** **

0,8

8

3

§

1,2 1,0

10

2

RI PT

5

M AN US C

Relative RRTF1 mRNA levels

SHOOT

0,6

§

4

0,4

1

1

2

§

0

0 WT

0,2 0,0

0

WT

wrky

wrky

WT

§ WT

wrky

wrky

20

12

**

**

16

D

10

6

8

TE

12 6

8

2

§

*

4

0

0

EP

4

§

AC C

Relative RRTF1 mRNA levels

ROOT

1,2

**

5

1,0

4

0,8

3

0,6

2

§

1

*

** **

0,4

0,2

§ 0

-AbWT+Ab

-Ab wrky+Ab

MLWTHL

MLwrkyHL

-H2O2 WT+H2O2

WT

wrky18/40/60

WT

wrky18/40/60

WT

0,0

-H2O2wrky+H2O2

wrky18/40/60

-PiWT+Pi

WT

-Piwrky+Pi

wrky18/40/60

Fig. 9

Probe position

B

C

D

E

F

5 4 3 2 1 0 -1000

-800

-600

-400

-200

100 80

0

SC

60 40 20 0 Probe A

M AN U

Relative AlphaScreen Signals

Position from TSS of RAP2.6 [b]

B

RI PT

Relative appearance ratio (RAR)

A

Probe B

AT3G49160

Probe C

Probe D

Probe E

Probe F

Probe F

Probe F

AT1G43160

100 80

40 20 0

Biotinylated probe

-

Probe F

Probe F

Probe F

Probe F

-

Probe C

Probe F

Probe F m1 Probe F m2

EP

Competitor

Probe F: TTATGTCCATTTCCCACGTGTCACTATTTGTATGACGGCTAGAGAAAGAC

AC C

D

D

60

TE

C

Relative AlphaScreen Signals

(RAP2.6)

m1 m2

: ----------ggaaacatgt-----------------------------: -------------------------------cgtcattagc---------

E

Figure 10

RI PT M AN US C

RRTF1

GCC box

W box

RRTF1

WRKY18/40/60

HL

H2 O 2

amplification

P. indica, NAC, DDCNa

AC C

EP

TE

D

A. brassicae

RRTF1

Fig. 11

High REDOX RESPONSIVE TRANSCRIPTION FACTOR1 Levels Result in Accumulation of Reactive Oxygen Species in Arabidopsis thaliana Shoots and Roots.

Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H2O2, as well as biotic- and abiotic-induced r...
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