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Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat [1]

['Cong Liu', 'State Key Laboratory Of Crop Stress Biology For Arid Areas', 'College Of Plant Protection', 'Northwest A F University', 'Yangling', 'Shaanxi', 'P. R. China', 'Yunqian Wang', 'Yanfeng Wang', 'Yuanyuan Du']

Date: 2022-10

Abstract Puccinia striiformis f. sp. tritici (Pst) secretes an array of specific effector proteins to manipulate host immunity and promote pathogen colonization. In a previous study, we functionally characterized a glycine-serine-rich effector PstGSRE1 with a glycine-serine-rich motif (m9). However, the mechanisms of glycine-serine-rich effectors (GSREs) remain obscure. Here we report a new glycine-serine-rich effector, PstGSRE4, which has no m9-like motif but inhibits the enzyme activity of wheat copper zinc superoxide dismutase TaCZSOD2, which acts as a positive regulator of wheat resistance to Pst. By inhibiting the enzyme activity of TaCZSOD2, PstGSRE4 reduces H 2 O 2 accumulation and HR areas to facilitate Pst infection. These findings provide new insights into the molecular mechanisms of GSREs of rust fungi in regulating plant immunity.

Author summary Pst secretes numerous effectors to modulate host defense systems. However, the mechanisms of these effectors, especially for glycine-rich or serine-rich effectors, remain obscure. In this study, we identified a new glycine-serine-rich effector, PstGSRE4, which exhibits unusual biochemical properties and is highly induced during early stages of infection. Transgenic expression of PstGSRE4-RNAi constructs in wheat significantly reduced virulence of Pst and increased H 2 O 2 accumulation in wheat. Overexpression of PstGSRE4 in wheat significantly increased virulence of Pst and reduced H 2 O 2 accumulation in wheat. PstGSRE4 was shown to target the ROS-associated regulatory factor TaCZSOD2, which was proved as a positive regulator of wheat immunity in this study. Further study revealed that PstGSRE4 inhibited the enzyme activity of TaCZSOD2 and thus compromises the host immune systems. This work reveals a novel strategy that rust fungi exploit to modulate host defense and facilitate pathogen infection.

Citation: Liu C, Wang Y, Wang Y, Du Y, Song C, Song P, et al. (2022) Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat. PLoS Pathog 18(7): e1010702. https://doi.org/10.1371/journal.ppat.1010702 Editor: Jie Zhang, Institute of Microbiology, Chinese Academy of Sciences, CHINA Received: January 26, 2022; Accepted: June 23, 2022; Published: July 26, 2022 Copyright: © 2022 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was financially supported by the National Key Research and Development Program of China to JG (2021YFD1401000) (http://www.most.gov.cn/), the National Natural Science Foundation of China to JG (32172381 and 31972224) (https://www.nsfc.gov.cn/), Key Research and Development Program of Shaanxi to JG (2021ZDLNY01-01) (https://kjt.shaanxi.gov.cn/), Natural Science Basic Research Program of Shaanxi to JG (2020JZ-13) (https://kjt.shaanxi.gov.cn/) and the 111 Project from the Ministry of Education of China to ZK (B07049) (http://www.moe.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction In nature, plants are exposed to a variety of biotic and abiotic stresses, including the invasion of numerous pathogenic microorganisms. In their interactions, plants and pathogens confront processes of defense and pathogenicity and co-evolve. Upon pathogen infection, pattern recognition receptors (PRRs) in plants recognize the pathogen-associated molecular pattern (PAMP) and activate PAMP-triggered immunity (PTI) to form the first level of defense [1]. Pathogens have formed a large number of virulence factors during the long-term evolution with the host, and successfully infect and colonize the host by acting on the host plant cells [1]. Effectors, as a type of very important virulence factors, are secreted from the pathogen into the host primarily to inhibit the host’s defense response, and thus cause host plant susceptibility. In addition, when certain avirulence effectors from the pathogen are directly or indirectly recognized by plant disease-resistant proteins, the plant immune system is strongly activated to induce the host cell hypersensitive response (HR), which has been termed effector-triggered immunity (ETI) [2]. Therefore, the effectors of a pathogen have a dual function of virulence and avirulence, which is not only an important weapon of pathogenicity, but also an important target of the plant immune system. Both PTI and ETI include the induction of reactive oxygen species (ROS), a key component of the defense system [3,4]. The sharp increase of ROS is a common manifestation when plants are confronted with various pathogens, indicating that ROS play a vital role in the process of plant resistance to pathogens. ROS burst is generally defined as a rapid production of high levels of ROS in response to external stimuli [5]. Superoxide radicals (O 2 -) and hydrogen peroxide (H 2 O 2 ) are considered important ROS in response to biotic stress [6]. During plant-pathogen interaction, penetration of pathogen into host plasma membrane triggers the early O 2 − burst by an NADPH oxidase, then they are rapidly converted to H 2 O 2 by dismutation [5]. Most of the data seem to indicate that the major ROS building the oxidative burst is H 2 O 2 , with possible participation of O 2 −. On the one hand, O 2 - and H 2 O 2 are directly toxic to pathogens. For instance, the accumulation of O 2 - or H 2 O 2 caused by Pseudomonas syringae pv. tabaci significantly decreased the number of bacteria in Nicotiana benthamiana, and then the number of bacteria significantly increased following the addition of SOD or other reactive oxygen scavengers [7]. On the other hand, H 2 O 2 can also act as signaling molecules to directly or indirectly activate the expression of resistance genes and defense genes. H 2 O 2 can induce the increase of antioxidant enzyme activity in plants to resist the invasion of pathogens. Exogenous H 2 O 2 can induce a significant increase in glutathione S-transferase (GST) transcription in soybean suspension cells, and H 2 O 2 scavengers can prevent this effect [8]. In addition, H 2 O 2 also participate in the lignification of cell walls and the cross-linking of proteins to cell walls to strengthen plant cell walls against pathogen invasion. After infection with diseased substances, synthesis of H 2 O 2 was observed in lignification sites of plant tissues [7]. H 2 O 2 can also induce the occurrence of plant HR response. A large number of experiments proved that exogenous H 2 O 2 can induce HR in cells of Arabidopsis thaliana [9]. Interestingly, many studies have shown that effector proteins can control the host immune response by interfering with the host ROS signaling pathway [10–13]. Understanding the mechanism of effectors regulating ROS-related targets will increase our knowledge of molecular mechanisms underlying the interaction between plants and phytopathogens, and provide a theoretical foundation to achieve durable disease resistance. Superoxide dismutase (SOD) is an important component of the antioxidant enzyme system and is widely distributed in microorganisms, plants and animals. It catalyzes superoxide anion (O 2 −) radical disproportionation to produce O 2 and H 2 O 2 , and plays an important role in the balance between oxidation and oxidation resistance [14]. Based on their metal cofactors, protein folds, and subcellular distribution, SODs are mainly categorized as CuZnSODs, FeSODs, and MnSODs [15]. A previous study indicated that infection of grape with grapevine fanleaf virus caused the accumulation of ROS and activated its enzyme defense system, including SOD [16]. Among the isoenzymes of SOD in sunflower, the expression of CuZnSOD under biological stress is the most affected, indicating that CuZnSOD is the main antioxidant defense enzyme [17]. When the CuZnSOD gene in tomato chloroplasts was transferred into two N. benthamiana strains, it enhanced the resistance to anthrax by changing the expression of the antioxidant enzyme [18]. In the Phaseolus vulgaris-Uromyces appendiculatus interaction, the expression of CuZnSOD was increased greatly during the incompatible interaction [19]. Recently, in the study of barley-powdery mildew interaction, loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of copper-zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in Rom1 re-established HvSOD1 levels [20]. In the study of rice-Magnaporthe oryzae interaction, different SODs in miR398b regulated resistance to rice blast disease, and miR398b increased total SOD activity to upregulate the H 2 O 2 concentration and thereby improve disease resistance [21]. However, there have been no reports on phytopathogenic effectors targeting and regulating CuZnSODs from plants to suppress host immune response. Among the diseases caused by rust fungi, the diseases on Gramineae and Leguminosae seriously threaten the safety of food production in China and throughout the world [22]. Stripe rust is one of the most serious diseases of wheat in the world [23]. Wheat has evolved resistance genes to protect against disease. However, Pst constantly mutates to overcome these resistance genes, and the effectors contributed significantly to the virulence diversity of Pst [24]. Due to the importance of effector proteins in the interaction between pathogens and plants, more and more attention has been paid to the study of effector proteins. Recently, stripe rust effector Pst18363 has been reported to stabilize a negative regulator of wheat defense, TaNUDX23, which suppresses ROS accumulation and facilitates Pst infection [25]. Another stripe rust effector, Pst_12806, is translocated into chloroplasts and perturbs photosynthesis, avoiding triggering cell death and supporting pathogen survival on living plants [26]. In several organisms, glycine- or serine-rich proteins have been shown to participate in RNA splicing, metabolism and signal transduction [27,28]. Pathogen effectors with a high content of glycine or serine could potentially modify the host’s metabolism or signal transduction [29]. In Pst, a glycine-serine-rich effector protein PstGSRE1 containing a glycine-serine-rich motif (m9) has been shown to disrupt the nuclear localization of TaLOL2 and suppress ROS-mediated cell death induced by TaLOL2, thus compromising host immunity [29]. However, the mechanisms of glycine-serine-rich effectors, remain obscure, and further investigation is required. In this study, we characterized a new glycine-serine-rich effector protein PSTCY32_07414 (alias PstGSRE4), which lacks the m9-like motif, targets a wheat copper zinc superoxide dismutase TaCZSOD2. PstGSRE4 is required for full virulence of Pst in wheat. Further analyses showed that TaCZSOD2 is a positive regulator of wheat resistance to Pst, and PstGSRE4 reduces H 2 O 2 accumulation by inhibiting the activity of TaCZSOD2 to facilitate Pst infection. Our results provide new insights into the molecular mechanisms of glycine-serine-rich effectors of rust fungi regulating host immunity.

Materials and methods Plant materials and fungal Strains In this study, we used wheat cultivar Suwon11 (Su11), Fielder and N. benthamiana. Wheat cultivar Su11 is highly susceptible to CYR31 and CYR32 and highly resistant to CYR23 [53]. Wheat seedlings were planted, inoculated with Pst and maintained in accordance with the procedures and conditions described previously [54]. Wheat cultivar Fielder was used for transgenic transformation. Co-immunoprecipitation (CoIP) analysis was performed in four-leaf tobacco seedlings permeated with agrobacterium tumefaciens GV3101. Freshly collected urediniospores of Pst race CYR23 were obtained from the leaves of wheat cultivar Thatcher (MX169), while CYR31 and CYR32 were obtained from Suwon11. The wheat cultivars Su11 and MX169 were grown at 16°C in an artificial climate chamber. Plasmid construction The PstGSRE4 gene was cloned using complementary DNA from Pst CYR32. Full-length TaCZSOD genes were cloned from Su11. The amplicons were prepared using the appropriate restriction enzymes (S4 Table) and ligated into pBINGFP2 (a plasmid containing green fluorescent protein, GFP) for transient expression in tobacco, and pEDV6 for transient expression in wheat as well as pSUC2T7M13ORI (pSUC2), pEGX4T-1-GST, pET32a-His with the ClonExpress II One Step Cloning Kit (Vazyme Biotechnology, Nanjing, China). For VIGS analysis, specific cDNA segments of PstGSRE4 and TaCZSODs were predicted by siRNA finder software Si-Fi and then inserted into BSMV-γ carriers with NotI and PacI restriction sites [55]. For Co-IP assays, coding sequences of PstGSRE4 were ligated into pBINGFP2, and TaCZSOD2 and TaCZSOD1 were ligated into pICH86988 (a plasmid containing HA-tag), respectively. For Y2H assays, the coding sequences of PstGSRE4 and TaCZSODs were separately prepared using appropriate restriction enzymes (S4 Table) and ligated into pGADT7 and pGBKT7 vectors. The PstGSRE4 was ligated into pCAMBIA3301 for overexpression in wheat by Agrobacterium-mediated transformation, and the TaCZSOD2 was ligated into CUB for overexpression in wheat by Agrobacterium-mediated transformation. qRT-PCR analysis To assay expression levels of PstGSRE4, urediniospores and leaves of wheat Suwon11 infected with CYR32 at 6, 12, 18, 24, 36, 48, 72, 120, 168, 216 and 264 h post-inoculation (hpi) were harvested. To analyze the transcript levels of TaCZSOD2, leaves of Su11 inoculated with CYR23 and CYR31 at 0, 6, 12, 24, 48, 72, 96, 120 hpi were sampled. RNA of all samples was extracted with the Quick RNA isolation Kit (Huayueyang Biotechnology, China, Beijing, 0416-50GK). Approximately 2 μg of the total RNA were also used for reverse transcription using RevertAid First Strand cDNA Synthesis Kit (MNI, K1622). qRT-PCR on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) was performed in a 25-μl reaction mixture containing 12.5 μl of LightCycler SYBR Green I Master Mix, 2 μl of diluted cDNA (1:5), 8.9 μl of distilled H 2 O, 0.8 μl of forward primer (10 mM) and 0.8 μl of reverse primer (10 mM). The primers used are listed in S4 Table. Real-time PCR data were analyzed by the comparative 2-ΔΔCT method to quantify relative gene expression [25]. The expression levels of PstGSRE4 and TaCZSOD2 were normalized to PstEF1 and TaEF-1α, respectively. Each sample was analyzed in three biological replications, and each PCR analysis included three technical repeats. The statistical significance was evaluated by Student’s t-test. Yeast signal sequence trap system To validate the function of the predicted signal peptide of PstGSRE4, the yeast signal sequence trap system was used as described previously [56]. The predicted signal peptide sequence of PstGSRE4 was cloned into vector pSUC2T7M13ORI (pSUC2) using the specific primers (S4 Table) and then transformed into the invertase mutant yeast strain YTK12 [57]. To test the secretion function of the recombinant plasmid, positive clones were selected from the CMD-W medium then transferred to YPRAA medium to determine whether the recombinant plasmid had secretory function. In addition, invertase enzymatic activity was detected by the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble red colored 1,3,5-triphenylformazan (TPF) according to procedures and conditions described previously [58]. Agrobacterium tumefaciens infiltration assays The sequence encoding PstGSRE4 without the signal peptide (PstGSRE4(ΔSP)) was ligated into pGR107 carrier to construct the agrobacterium recombinant plasmid PVX-PstGSRE4-HA. The Avr1b gene from Phytophthora sojae and eGFP-HA were used as controls (S4 Table). A. tumefaciens cultures were prepared as described previously [59]. Resuspended A. tumefaciens cultures carrying each effector gene or eGFP at a final OD 600 of 0.2 and 10 mM MgCl 2 buffer were infiltrated into the leaves of 4-week-old N. benthamiana using a syringe without a needle. After 24 h, A. tumefaciens cultures for delivery of Bax or Pst322 at a final OD 600 of 0.2 were also infiltrated into the same site of N. benthamiana leaves. Expression of genes in all infiltration sites was detected by immunoblot three days after infiltration. Symptoms were monitored and recorded from 3 to 8 d after infiltration. Three independent biological replicates were conducted for each experiment. Yeast two hybrid (Y2H) assay TaCZSODs was constructed into pGBKT7 as bait, while PstGSRE4(ΔSP) was constructed into pGADT7 as prey (S4 Table). Then they were co-transformed into yeast strain AH109, plated on SD-Trp-Leu and SD-Trp-Leu-His medium, and cultured at 30°C for 3 to 5 d. The monoclonals grown on SD-Trp-Leu-His were selected and diluted with water, then the interactions were confirmed by growth on the SD-Trp-Leu-His-Ade medium containing X-α-gal. Bacterial T3SS-mediated overexpression in wheat plants pEDV6-PstGSRE4(ΔSP), pEDV6-TaCZSOD2 were transformed into P. fluorescens strain EtHAn by electroporation. pEDV6-RFP was used as a control. Infiltration into wheat leaves was performed according to the method described previously [60]. The involvement in Pst pathogenicity or host defense response was tested by challenging the second leaves in pEDV6-PstGSRE4-inoculated wheat plants with Pst avirulent race CYR23 after 24 h. For determination of H 2 O 2 measurements, according to the previously described method [61], the inoculated leaves were sampled at 24 and 48 hpi and determined by 3–3’diaminobenzidine (DAB) staining. To examine the suppression of callose deposition, pEDV6-, pEDV6-PstGSRE4- and pEDV6-RFP-inoculated wheat plants were sampled at 48 hpi. Leaf samples were stained with 0.05% aniline blue in 67 mM K 2 HPO 4 (pH 9.0) overnight in darkness [29]. Leaves were rinsed in water and mounted in 50% glycerol and examined under an Olympus BX-53 fluorescence microscope (Olympus Corporation, Tokyo, Japan) using a DAPI filter. Images were acquired using a constant setting with 1000-ms exposure time. The number of callose deposits was quantified using ImageJ software [62]. Glutathione S-transferase (GST) pull-down assay PstGSRE4(ΔSP) and TaCZSOD2/TaCZSOD1 were separately ligated into pGEX-4T-1 and pET22b/pET32a through enzyme digestion and ligation. Vectors were transformed into E. coli BL21 cells for protein expression. The corresponding protein was expressed and purified according to the prokaryotic expression procedure. GST-pull down kit (Thermo, Shanghai, China, UB281159) was used to validate the protein interactions in vitro. Another protein was detected by Western blot analysis. Horseradish peroxidase (HRP)-conjugated anti GST-Tag rabbit polyclonal antibody (Cwbiotech, cat. no. CW0144M) and HRP conjugated anti His-Tag mouse monoclonal antibody (Cwbiotech, cat. no. CW0285M) were used for Western blots. Co-immunoprecipitation assays PstGSRE4(ΔSP) and TaCZSOD2/TaCZSOD1 were ligated into pBINGFP2 and pICH86988 carriers, respectively. In addition, agrobacterium-mediated transient gene expression technology was used to co-express the above combinations in N. benthamiana. At 48 h after agroinfiltration, 100 μL of co-injected leaf proteins were extracted as the control (Input). Twenty μL of GFP Trap beads were added to the remaining extracts and incubated for 1 h, and centrifuged at 12000g at 4°C for 1min. After removal of the supernatant, the beads in 60 uL volume of wash buffer were mixed with 20 uL of loading buffer, and heated at 100°C for 5min. Precipitated proteins and crude proteins (Input) were detected by immunoblotting with an anti-GFP antibody (#A02020; Abbkine, Wuhan, China) and an anti-HA antibody (Beyotime, AF5057). Activity assays of CuZnSOD PstGSRE4-15bs, TaCZSOD2-15bs/TaCZSOD1-15bs and GFP-15bs were expressed in vitro by a prokaryotic expression system, then diluted to the same concentration after purifying by His-tag Purification Resin (BeyoGold, P2210) according to the protocol of manufacturer. The activity of TaCZSOD2/TaCZSOD1 was determined by nitroblue tetrazolium (NBT) reaction [63] in different combinations. The 3 mL reaction mixture contained 39 mM L-methionine 1.5 mL, 225 μM nitroblue tetrazolium (NBT) 0.3 mL, 8 μM riboflavin (dissolve in 30 μM EDTA-Na 2 buffer) 0.3 mL, 10 μL purified enzyme and 50 mM potassium phosphate buffer (pH 7.8) 890 μL. The reaction was initiated by illuminating the reaction mixture for 20 min, and photochemically produced superoxide reacted with NBT. Absorbance of formazan, the product of NBT reduction, was then recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme that caused 50% of the maximum inhibition of NBT reduction. These experiments were repeated three times. A standard curve of protein concentration was obtained with bovine serum albumin as standard [64]. In vivo, we determined the activity of CuZnSOD by using the CuZnSOD assay kit (colorimetry) (Jian Cheng, Nanjing, China, A001-4-1) according to the protocol of manufacturer. Weigh 0.2 g plant tissue sample accurately, add 4 times volume homogenate medium according to mass(g)-volume(ml) ratio of 1:4, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 3500 rpm for 10 min, take supernatant for assay. Take 0.1 ml 20% homogenate supernatant, add 0.2 ml homogenate medium (equals to 3 times dilution), mix sufficiently, take 3 samples of different volumes (10 μl, 30 μl, 50 μl), do pre-test according to operation table in order to determine optimal sample volume. Curve appears direct proportion while inhibition percentage is between 15–55%. Take the tube which inhibition percentage is between 45% to 50% as optimal sample volume. Use xanthine and xanthine oxidase reaction system to produce superoxide anion radicals (O 2 −), the latter will oxidate hydroxylamine to form nitrite, appears prunosus color under effect of chromogenic agent, its absorbance can be measured by visible range spectrophotometer. If sample to assay contains SOD, then it has a narrow spectrum depressant effect for superoxide anion radicals, as result, absorbance in sample tube will be lower than absorbance in contrast tube, SOD activity can be calculated by formula. MnSOD and FeSOD loss activity in pretreated samples and CuZnSOD activity keeps stable. These experiments were repeated three times. Barley stripe mosaic virus (BSMV)-mediated silencing Based on the cloned PstGSRE4 and TaCZSOD2 genes, non-conserved regions were analyzed, and Premier Primer 5.0 was used to design gene silencing vector primers. According to previously described methods [65], two fragments of PstGSRE4 or TaCZSOD2 were cloned and inserted into BSMV to produce BSMV:PstGSRE4-1/2as, BSMV:TaCZSOD2-1/2as. The wheat phytoene desaturase gene (PDS) was silenced as a positive control. BSMV:α and BSMV:β were mixed with BSMV:γ or recombinant γ-gene, in 1:1:1, and then the appropriate amount of FES buffer (2.613g dipotassium phosphate, 1.877 g glycine, 0.5 g sodium pyrophosphate, 0.5 g diatomite, 0.5 g porphyritic soil, 50 ml constant volume, 20 min sterilization by autoclaving) was added. Each independent experiment set FES buffer as a negative control, BSMV:γ as a blank control and BSMV:γ-TaPDS as positive controls for about 10 d to observe the symptoms of virus infection. After 10 to 14 d following inoculation, Pst races CYR23 and CYR31 (fresh urediniospores were collected from the infected leaves of Su11 that were grown at 16°C in artificial climate chamber) were separately inoculated on the fourth leaf of wheat plants, which were placed in a dark and high humidity environment at 12°C for 24 h, then grown in a normal 16/8 h light-dark cycle. The fourth leaves were sampled at 24, 48 or 120 hpi for assessment of silencing efficiency and histological observation. The phenotypes of the fourth leaves were photographed at 12 d after inoculation with Pst. These experiments were repeated three times. Cytological observations of fungal growth and host response The observation of necrotic death area hyphae and H 2 O 2 detection assay were performed as previously described [26]. Leaf segments were fixed and decolorized in a mixture of acetic acid/ethanol (1:1) for 3 d. Autofluorescence of mesophyll cells was observed to determine necrotic death area using epifluorescence microscopy (excitation filter, 485 nm; dichromic mirror, 510 nm; barrier filter, 520 nm). H 2 O 2 accumulation was detected by staining with DAB (Amresco, Solon, OH, USA). Hyphae were stained with WGA conjugated to Alexa-488 (Invitrogen, Carlsbad, CA, USA) and observed under blue-light excitation (excitation wavelength 450–480 nm, emission wavelength 515 nm). Only the site where an appressorium had formed over a stoma was considered to be a successful penetration. The H 2 O 2 accumulation, necrotic areas, hyphal length, and hyphal areas were observed with a BX-53 microscope (Olympus) and calculated using DP-BSW software. Western blotting Proteins were separated by SDS-PAGE. Gels were blotted onto a PVDF membrane (Merck Millipore, Burlington, MA, USA) with transfer buffer at 64V for 2h. Membranes were blocked for 1 h at room temperature, followed by washing. The antibodies—anti-GFP (1:2,000; #A02020; Abbkine, Wuhan, China), anti-RFP (1:2,000; #A02120; Abbkine), anti-His (1:3,000; #A02050; Abbkine), or anti-GST (1:2,000; #A02030; Abbkine)—were added and incubated at 4°C overnight, followed by three washes. Membranes were then incubated with goat anti-mouse antibody (ab6789; Abcam), or goat anti-rabbit (ab205718; Abcam) at a ratio of 1:10,000 in the blotting buffer at room temperature for 2 h. After three washes, membranes were incubated with chemiluminescence HRP substrate (#WBKLS0100, Merck Millipore) for 5 min, and then visualized by excitation at 780 or 800 nm. Determination of the accumulation of O 2 − and H 2 O 2 In vivo, we determined the content of O 2 − by using the O 2 − assay kit (SA-2-G, Comin Biotechnology, Suzhou, China) according to the protocol of manufacturer. Weigh 0.1 g inoculated leaves in different hours accurately, add 10 times 65 mM phosphate buffer (pH 7.8) according to mass (g)- volume(mL) ratio of 1:10, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 10000 g for 20 min, take supernatant for assay. The 900 μL reaction mixture contained 0.5 mL homogenate, 0.4 mL 10 mM hydroxylamine solution, 37°C for 20 min. Then add 0.3 mL 17mM 4-aminobenzenesulfonic acid and 0.3 mL 7mM α-naphthylamine, 37°C for 20 min. Then add 0.5 mL 1 chloroform, centrifugate at 8000 g for 5 min, take 1 mL supernatant for assay. Recorded absorbance at 530 nm against a distilled water blank. We determined the content of H 2 O 2 by using the H 2 O 2 assay kit (A064-1-1, Comin Biotechnology, Suzhou, China) according to the protocol of manufacturer. Weigh 0.1 g inoculated leaves in different hours accurately, add 10 times propanone according to mass (g)—volume (mL) ratio of 1:10, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 8000 g for 10 min, take supernatant for assay. The 1.3 mL reaction mixture contained 1 mL homogenate, 0.1 mL titanic sulfate solution and 0.2 mL ammonium water, centrifugate at 8000 g for 5 min, take sediment for assay. Then add 1 mL sulphuric acid solution to dissolve the sediment, let stand for 10 min at the room temperature. Recorded absorbance at 415 nm against a distilled water blank. These experiments were repeated three times. Oxidative burst measurement Leaves from 6-wk-old WT and TaCZSOD2-knockdown or TaCZSOD2-overexpression transgenic lines were sliced into 10 mm2 discs, and maintained overnight in water in a 96-well plate. Then, the leaf discs were treated with 200 μL of solution containing 8nM chitin (hexa-N-acetyl-chitohexaose), 20 μg/ml peroxidase (Sigma-Aldrich) and 20 nM luminol. Luminescence was recorded for 30 min using a multiscan spectrum. Each data point consisted of six replicates. These experiments were repeated three times. Phylogenetic relationship analysis Multiple alignment was performed by Muscle in MEGA6.0 [66]. The phylogenetic relationship was inferred based on the multiple alignment in MEGA6.0 by the Maximum Likelihood (ML) method based on LG model with bootstrap 1000. The unrooted tree was performed by Interactive Tree of Life (IToL) Version 3.2.3 (http://itol.embl.de/). Statistical analyses Statistical analyses of each treatment were performed with the statistical software version package of IBM SPSS Statistics 21 (IBM SPSS Statistics, IBM Corporation, Armonk, New York, USA).

Acknowledgments We thank Professor Larry Dunkle for editing the manuscript and Professor Daolong Dou for helpful suggestions. We thank Qiong Zhang, Fengping Yuan, Hua Zhao and Xiaona Zhou of State Key Laboratory of Crop Stress Biology for Arid Areas for their technical support.

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