1. Volume 8, Issue 8, Pages 1253-1273 (August 2015)
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 
Molecular Plant 
Volume 8, Issue 8, Pages (August 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. 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 is set at 1.0 and the other levels are expressed relative to it. The qRT–PCR products of RRTF1 were normalized to those of GAPDHC as control. Based on six independent qRT–PCR experiments, bars represent SEs.
(B) 14-day-old rrtf1, WT, oe18, oe20, and oe32 seedlings 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 the Methods section.
(C) Left: rrtf1, WT, oe18, oe20, and oe32 seedlings grown on Hoagland plates for 18 days in cML; right: amended with P. indica spores as described in the Methods section. The numbers on the bottom of the plate refer to the lengths of the roots in centimeters (n = 40 seedlings, values are means ± SEs from four independent experiments).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

3. Figure 2
The Performance of RRTF1 Overexpressors Increase with Longer Dark Incubation Periods.
14-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 and LD conditions for 6 weeks. Almost all oe18 and oe20 and all o32 plants died in 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) based on four independent experiments with 14 plants per line in each experiment. The dry weights were determined from the completely dried plants after harvesting of seeds.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

4. Figure 3
The RRTF1 Overexpressors Are Highly Susceptible to A. brassicae 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 Whatman paper in Petri dishes, and the leaves were inoculated with 5 μl of spore suspension (105–106 spores ml−1) as described in the Methods section. Representative pictures were taken at 3 or 5 days after infection (dai) from six 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 cocultivation, 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).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

5. Figure 4
ROS Levels in the Leaves and Roots of 14-Day-Old rrtf1, WT, oe18, oe20, and oe32 Seedlings.
(A and B) 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 section.
(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 six independent 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).
Molecular Plant 2015 8, DOI: ( /j.molp )
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6. Figure 5
Piriformospora indica Rescues the Light- and A. brassicae-induced Stress Phenotypes of the oe Lines by Reducing H2O2/ROS Accumulation in 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 five 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 spore suspension (cf. Methods section). Lower panels: false color images of the plates shown above representing Fs/Fm values as described in the Methods section. Representative pictures from five independent experiments with 10 replications.
(C) H2O2/ROS levels in the leaves and roots of 14-day-old rrtf1, WT, oe18, oe20, and oe32 seedlings grown under LD ML on MS medium in the absence (dark bars) or presence (light bars) of P. indica. Based on five independent experiments with 30 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 that 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 five 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 (light bars) of P. indica. Based on four independent experiments with 30 seedlings per line. Mean values ± SEs are given. In all cases, P > 0.1.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

7. Figure 6
The Antioxidants NAC and DDCNa Recovered the ROS-Induced Phenotype and A. brassicae Susceptibility in oe Seedlings by Reducing H2O2/ROS Accumulation.
(A) rrtf1, WT, oe18, oe20, and oe32 seedlings were grown under LD ML on MS medium or MS medium supplemented with 1 mM NAC or 1 mM 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 from five 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 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 the Methods section. The data are based on four independent experiments with 20 seedlings per treatment. Mean values ± SEs are given. The asterisks indicate that the values for the control seedlings are significantly different from those for NAC- and DDCNa-treated seedlings; determined by Student's t-test (*P < 0.05; **P < 0.01). FW, fresh weight.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

8. Figure 7 RRTF1-Regulated Genes.
Venn diagram of the number of genes 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 (Mehterov et al., 2012); they are presented in Table 2, Supplemental Tables 1-1, 1-2, and 2.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

9. Figure 8 Comparison of RRTF1 and RAP2.6 Lines.
(A) rrtf1, WT, oe18, oe20, and oe32 were compared with 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 the Methods section. The data are means of five 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. FW, fresh weight.
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

10. Figure 9
Full Regulation of RRTF1 by A. brassicae (Ab) Infection, HL and H2O2, but Not by P. indica (Pi) Colonization Requires WRKY18/40/60.
−/+ 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 days. Mock treatment was performed with H2O. ML/HL: 14-day-old WT and wrky18 wrky40 wrky60 seedlings grown under LD ML on MS medium were exposed to HL for 24 h. Seedlings grown under LD ML served as control. −/+ H2O2: 14-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: 12-day-old WT and wrky18 wrky40 wrky60 seedlings grown under LD ML on MS medium were cocultivated with Pi for 6 days. Mock treatment was performed with a KM plug. For all experiments, the 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 three independent experiments. Significant differences for the untreated control seedlings were determined by Student's t-test (*P < 0.05; **P < 0.01).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions

11. Figure 10 Identification of RRTF1 Binding Sequence on RAP2.6 Promoter.
(A) Scanning of the RAP2.6 promoter. Promoter sequences that are overrepresented in the RRTF1-activated promoters of oe18 were assayed with the microarray data from mature leaves (cf. the Methods section), and candidate sites for RRTF1-regulated cis-elements are shown as high RAR (vertical axis). The horizontal axis shows the position from the 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 lines means threshold (RAR >3), which was used for the prediction of the plant cis-element.
(B) Identification of an 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 the AlphaScreen assay, and expressed as relative signal, i.e. a ratio of the signal with the biotinylated DNA probe to the signal with the corresponding unbiotinylated DNAs. Data were derived from triplicate assays and are shown as averages with standard deviations.
(C) Competition assays to identify an RRTF1 target site within probe F. Biotinylated probe and non-biotinylated competitors as shown were incubated with RRTF1. Probe 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 mutations shown as lowercase letters.
(E) RRTF1 binding sequence and the gene number possessing the sequence. RRTF1 binding sequences that fulfilled three 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) sequences related to the RRTF1 binding motif reported by Franco-Zorrilla et al. (2014). The GCC-box is underlined.
Molecular Plant 2015 8, DOI: ( /j.molp )
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12. Figure 11 A Model that Describes RRTF1 Regulation.
A. brassicae, HL, and H2O2 require WRKY18, WRKY40 and/or WRKY60 to fully activate RRTF1 expression by 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 also represses the expression of its own gene, as shown in the upper section of the model (cf. Discussion section).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions