TaMDHAR4, a monodehydroascorbate reductase gene participates in the interactions between wheat and Puccinia striiformis f. sp. tritici

Plant Physiology and Biochemistry 76 (2014) 7e16

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Research article

TaMDHAR4, a monodehydroascorbate reductase gene participates in the interactions between wheat and Puccinia striiformis f. sp. tritici Hao Feng a,1, Wei Liu a,1, Qiong Zhang b, Xiaojie Wang a, Xiaodong Wang a, Xiaoyuan Duan b, Feng Li a, Lili Huang a, Zhensheng Kang a, * a b

State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 October 2013 Accepted 20 December 2013 Available online 30 December 2013

Reactive oxygen species (ROS) in plants are induced in various cellular compartments upon pathogen infection and act as an early signal during plantepathogen interactions. Monodehydroascorbate reductase (MDHAR) is involved in plant disease resistance through the regulation of the ROS level via the ascorbate-glutathione (AsA-GSH) cycle. In this study, TaMDHAR4 was firstly isolated from wheat cultivar Suwon 11, and this protein exhibits high similarity to MDHAR proteins from other plant species. Bioinformatics analyses indicated that TaMDHAR4 contains typical structural features, such as mPTS-like sequences in the C-terminal extension and trans-membrane domain followed by five basic arginine residues (-RKRRR), which predicted that this protein may be localized in the peroxisome. qRT-PCR analyses demonstrated that TaMDHAR4 could be induced by various exogenous hormones, such as ABA, MeJA, and ETH. TaMDHAR4 is sharply down-regulated at 12 and 18 hpi only in wheat leaves challenged with Puccinia striiformis f. sp. tritici (Pst) race CYR23 and induced at 48 hpi with both Pst races CYR23 and CYR31. SOD and APX injection analyses demonstrated that TaMDHAR4 may be involved in the interaction between wheat and Pst through the regulation of its expression. Moreover, the knockdown of TaMDHAR4 through virus-induced gene silencing (VIGS) enhanced the wheat resistance to Pst by inhibiting sporulation in the compatible interaction. Histological observations also demonstrated that silenced wheat resulted in an increased proportion of necrotic area at the infection sites and suppressed Pst hypha elongation. The study provided novel insights into the molecular functions of TaMDHAR4 during plant epathogen interactions. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: AsA-GSH cycle Cell death Puccinia striiformis f. sp. tritici ROS VIGS Wheat

1. Introduction With the evolution of aerobic metabolic processes, such as respiration and photosynthesis, reactive oxygen species (ROS) are continuously produced in the mitochondria, chloroplasts, and peroxisomes of plant cells. Plants also produce ROS by activating various oxidases and peroxidases in response to certain environmental

Abbreviations: ABA, abscisic acid; APX, ascorbate peroxidase; AsA, ascorbate; BA, benzyladenine; CAT, catalase; CYR, Chinese yellow rust; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; ETH, ethylene; GAs, gibberellins; GPX, glutathione peroxidase; hpi, hours post inoculation; hpt, hours post treatment; HR, hypersensitive response; MDHA, monodehydroascorbate; MeJA, methyl jasmonate; ORF, open reading frame; PrxR, peroxiredoxin; Pst, Puccinia striiformis f. sp. tritici; qRT-PCR, quantitative real-time-PCR; ROS, reactive oxygen species; SA, salicylic acid; SOD, superoxide dismutase; VIGS, virus-induced gene silencing. * Corresponding author. Tel./fax: þ86 029 87080061. E-mail address: [email protected] (Z. Kang). 1 These two authors contributed to this work equally. 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.12.015

stimuli, including biotic and abiotic stresses (Dat et al., 2000; Torres et al., 2002). In recent years, dual roles of ROS have been reported: as toxic byproducts of aerobic metabolism (Mittler et al., 2004) and as key regulators of growth, programmed cell death, and hormone signaling (Foreman et al., 2003; Overmyer et al., 2003). Plants have evolved a series mechanism, including cellular enzymatic and nonenzymatic scavenging mechanisms, to remove excessive ROS. The major ROS-scavenging enzymes of plants include superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GPX), and peroxiredoxin (PrxR). SOD acts as the first line of defense against ROS by detoxifying superoxide to H2O2. APX, GPX, and CAT, subsequently, reduce H2O2 to H2O. GPX is more effective against phospholipid hydroperoxide, rather than hydrogen peroxide. Nonenzymatic antioxidants include the major cellular redox buffers ascorbate and glutathione (GSH), as well as tocopherol, flavonoids, alkaloids, and carotenoids. In many organisms, the ascorbate-glutathione cycle plays a major role in the protection of the organism against reactive oxygen

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species because it maintains high levels of ascorbate (AsA) in the different cell compartments (Asada, 1997). MDHAR and DHAR are the two enzymes of the ascorbate-glutathione cycle that maintain AsA in its reduced state. In 1981, Arrigoni et al. reported that MDHAR is necessary for the regeneration of AsA and that DHAR plays a secondary role (Arrigoni et al., 1981). The specific activity of MDHAR is 10-fold higher than that of DHAR in both the mitochondria and chloroplasts of tomato responding to salt stress (Mittova et al., 2000). In addition, the results of computer simulations based on known enzyme concentrations and properties in the chloroplast led to the conclusion that the majority of monodehydroascorbate is reduced by MDHAR (Polle, 2001). Thus, MDHAR plays an important role in maintaining the reduced pool of AsA to scavenging hydrogen peroxide as substrate. Plants live in a complex environment full of stresses from a variety of sources, such as unsuitable climatic conditions, pests, and pathogenic microorganisms. The role of MDHAR in the environmental stress resistance is of great interest. In general, MDHAR is selected as an assessment gene to determine the tolerance or resistance of a plant to environment stress by determining the activity of the MDHAR enzyme (Ali et al., 2005; Sharma and Dubey, 2005). In addition, several genes encoding MDHAR have been cloned from different plant species, such as Brassica campestris (Yoon et al., 2004), Arabidopsis (Lisenbee et al., 2005), and soybean (Leterrier et al., 2005). Since 1995, several studies have reported that MDHAR genes are regulated by abiotic stresses (Leterrier et al., 2005; Grantz et al., 1995; Eltelib et al., 2011). In a complex environment, plants are affected not only by abiotic stress but also by other organisms. Pathogens are a very serious element that impacts plant growth. The disease resistance and susceptibility of plants is governed by the timely recognition of the invading pathogens and the rapid activation of the host defense responses. Upon pathogen attack, the interaction between ROS and the AsA-GSH cycle generates compartment-specific redox signals that interfere with other signaling pathways to achieve a complex rearrangement of the primary metabolism from photo-assimilatory pathways into an emergency survival strategy (Scharte et al., 2005). For example, the activity of MDHAR was decreased when tobacco leaves were infected by the fungus Botrytis cinerea (Elzbieta and Maria, 2005). However, no systemic study on MDHAR in wheat, particularly in response to biotic stresses, has been reported. Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. The wheat yield can be greatly reduced or even completely destroyed during the severe epidemics. Pst evolved rapidly that is constantly generating new races with novel virulence. Thus, it is important to explore the molecular mechanism in the interactions between wheat and Pst. In the last several years, many genes have been identified to be related to wheat resistance/susceptibility to Pst (Ma et al., 2009; Wang et al., 2009). But few gene is reported in the regulation of the ROS level via the ascorbate-glutathione (AsA-GSH) cycle during the interactions between host and pathogens. In this study, an MDHAR gene was isolated from wheat and characterized in wheat in response to Pst. 2. Materials and methods 2.1. Plant materials and treatments The wheat (Triticum aestivum L.) cultivar ‘Suwon 11’ and two Pst races, CYR23 and CYR31, were employed in this study. Suwon 11, possessing the stripe rust resistance gene YrSu, responds with a typical HR-resistance to CYR23 (incompatible interaction) but is susceptible to CYR31 (compatible interaction). Wheat plants were grown following the procedures described by Kang and Li (1984).

For the exogenous hormone treatments, two-week-old plants were sprayed separately with 100 mM abscisic acid (ABA), 100 mM gibberellins (GAs), 2 mM salicylic acid (SA), 100 mM methyl jasmonate (MeJA), 100 mM ethylene (ETH), and 100 mM benzyladenine (BA). The wheat seedlings treated with the various exogenous hormones, as well as the control plants (0.1% ethanol), were sampled at 0, 2, 6, 12, and 24 h post treatment (hpt). Freshly collected urediospores of CYR23 and CYR31 were inoculated onto the surface of the primary leaves of wheat with a paintbrush. Similarly, the control plants were mock-inoculated with sterile water. All of the plants were maintained in the dark for 24 h with 100% relative humidity and subsequently transferred to a growth chamber with a 16-h/8-h light/dark photoperiod at 16  C. The inoculated leaves were harvested at 0, 12, 18, 24, 48, 72, and 120 h post inoculation (hpi). For the SOD and APX injection treatments, wheat seedlings inoculated with CYR23 were maintained in the dark for 10 h with 100% relative humidity and subsequently transferred to a growth chamber with 1-h lighting. Then, 500 mg/ml SOD and 300 mg/ml APX were infiltrated into the leaves through the stomata with a syringe. The control plants were mock-injected with MES buffer in the same manner. When the waterlogging disappeared, the wheat seedlings were placed in the dark. At 16 hpi, the wheat leaves were excised and stained with DAB as described by Wang et al. (2007). Transparent leaf segments were sampled at 24 hpi and examined with an Olympus BX-51 microscope (Olympus Corp., Tokyo). The treated leaves with SOD and APX were excised at 0, 6, 12, 24, and 36 hpt. All samples were quickly frozen in liquid nitrogen and stored at 80  C. Three independent biological replications were performed for each treatment and each time course. 2.2. RNA extraction and cDNA synthesis The total RNA was extracted from each pool sample using the TrizolÔ Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. DNase I was used to remove any residual genomic DNA. The integrity of the total RNA was assessed by formamide denaturing gel electrophoresis, and the concentration was measured using a NanoDropÔ 1000 spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized using the GoScript Reverse Transcription system (Promega, Madison, WI, USA) following the manufacture’s protocol. All of the cDNA samples were 20-fold diluted with sterile water as a template in the qRT-PCR analyses. 2.3. cDNA cloning and sequence analyses of TaMDHAR4 An EST sequence fragment from the cDNA database of wheat challenged with Pst was used as the query sequence to screen the wheat EST database in GenBank. Homologous sequences were downloaded and assembled using the CAP3 Sequence Assembly Program (http://pbil.univ-lyon1.fr/cap3.php/) and the ORF Finder (http://www.ncbi.nlh.nih.gov/gorf/gorf.html). According to the assembled sequence, the specific primers MDHAR-F and MDHAR-R (Appendix S1) were designed using Primer Premier 5.0 to amplify the open reading frame (ORF) of TaMDHAR4 with the following procedures: 95  C for 4 min, 35 cycles of 95  C for 1 min, 60  C for 1 min, and 72  C for 2 min, and then 72  C for 10 min. For PCR reaction, a total 25 ml volume contained 1 ml of template (10 dilution of the first strand cDNA), 0.5 ml of each primer (10 mM), and a PCR Mix (0.3 ml of 5U/ml Tag DNA polymerase, 2.5 ml of 10 Taq buffer, 0.5 ml of 10 mM dNTP, 2.0 ml of 25 mM MgCl2, and 17.7 ml of ddH2O). The PCR products were subcloned into the pGEM-T Easy

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Vector (Promega, Madison, WI, USA) and sequenced with an ABI PRISM 3130XL Genetic analyzer (Applied BioSystems, USA). The amino acid sequence was analyzed using the InterProScan (http://www.ebi.ac.uk/cgi-bin/iprscan/), PSORT (http://psort.ims. utokyo.ac.jp/form.html), TargetIP (http://cbs.dtu.dk/services/ SignalP/), Compute pI/MW (http://web.expasy.org/compute_pi/), ProtParam (http://www.expasy.org/tools/pi_tool.html). Phylogenetic comparison was processed using DNAMAN version 5.2.2 programs (Lynnon Biosoft, Quebec, Canada). 2.4. Quantitative real-time PCR The expression profiles of TaMDHAR4 after different treatments (exogenous hormones and Pst) were detected through qRT-PCR analyses. The specific primers: Q-MDHAR-F and Q-MDHAR-R were listed in Appendix S1. To ensure gene-specific amplification, the previous primers were used to amplify the TaMDHAR4 fragment through regular PCR. The resulting 217-bp PCR fragment was amplified, and the expected TaMDHAR4 fragment was then confirmed through sequence analyses. A 107-bp fragment of the wheat translation elongation factor 1 alpha-subunit (TEF1) mRNA (GenBank: "http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? cmd¼Retrieve&db¼Nucleotide&list_uids¼170775&dopt¼GenBank &RID¼UEWCU5MZ016&log$¼nucltop&blast_rank¼100") was amplified with primer pair Q-EF-F and Q-EF-R (Appendix S1) as an internal reference for the qRT-PCR analyses. All of the quantitative PCR analyses were performed in a CFX96Ô Real-Time System (BioRad) using SYBR Green I (Invitrogen) to detect the double-strand cDNA synthesis. The real time-PCR reaction was performed in a total reaction mixtures composed of 12.5 ml of 2 SYBR Premix Ex TaqÔ (Takara), 2.0 ml of 20 first-strand cDNA, 0.2 mM of each primer, and 10.1 ml of sterile water. Thermal conditions were 95  C for 3 min followed by 40 cycles of 95  C for 10 s, 60  C for 10 s, and then 72  C for 30 s. The melting curves were obtained immediately after the completion of the qRT-PCR to detect primer dimerisation and other artifacts of amplification. The results were analyzed with OOct the 2 method (Livak and Schmittgen, 2001). Three independent biological replications and three technical replicates were performed for each treatment. 2.5. Vector construction The plasmids used for VIGS are based on the constructs described by Holzberg et al. (2002). The two RNA-derivative constructs BSMV-g5V (526 bpe662 bp) and BSMV-g3V (1441 bpe 1537 bp) were created by replacing BSMV:gPDS with the target sequences, The 136- and 97-bp target sequences were derived from upstream and downstream of the TaMDHAR4 sequence, respectively. The T-TaMDHAR4-g5V and T-TaMDHAR4-g3V vectors were digested with the restriction endonucleases PacI and NotI and cloned into the similarly digested BSMV:gPDS. 2.6. In vitro transcription of viral RNAs Capped in vitro transcripts were prepared from three linearized plasmids that contained the tripartite BSMV genome (Holzberg et al., 2002) using the mMessage mMachine T7 in vitro transcription kit (Ambion, Austin, TX), according to the manufacturer’s instructions. The transcription products were diluted 30-fold for inoculation into plants. Then, 0.5 ml of each of the in vitro transcription reactions were combined together and with 9 ml of FES buffer, which was contributed to rob inoculation with BSMV constructs in the seedling plants (Pogue et al., 1998). The mixture was then applied to the plants through rub inoculation.

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2.7. BSMV-VIGS-mediated TaMDHAR4 gene silencing After two-week vernalization, seedlings of Suwon 11 were sown and placed in a growth chamber under the following maintained conditions: 16  2  C with supplemental light 16 h per day and watered as needed. The entire second leaf surface of a two-leaf wheat seedling was inoculated with different recombinant BSMV transcripts (BSMV-g, BSMV-g5V, and BSMV-g3V) by gently sliding the pinched fingers from the base to the tip five times. The mock seedlings were inoculated with FES buffer. The seedlings were subsequently placed in a growth chamber with high humidity at 25  2  C. Fourteen days later, freshly collected urediniospores of Pst pathotypes CYR23 and CYR31 were inoculated onto the surface of the fourth leaves of wheat with a paintbrush. The control plants were mock-inoculated in the same manner without spores. After inoculation, all of the plants were maintained in the dark for 24 h with 100% relative humidity and subsequently transferred to a growth chamber at 15  C with a 16-h/8-h light/dark photoperiod. Wheat leaves were excised at 0, 24, 48, and 120 hpi, quickly frozen in liquid nitrogen, and stored at 80  C. Three independent biological replications were performed for each treatment. 2.8. Histological observation of fungal growth and host response The fourth leaves of wheat challenged with Pst were sampled at 24, 48, and 120 hpi and decolored as previously described (Wang et al., 2007). Transparent leaf segments were examined with an Olympus BX-51 microscope (Olympus Corp., Tokyo) to perform infection site statistics. A total of 50 infection sites were examined in each of five randomly selected leaf segments per treatment. The hyphae length and the proportion of the necrotic area were calculated using the DP-BSW software. The SPSS software was used to calculate the standard deviations and to perform a paired sample t-test for the statistical analyses. 3. Results 3.1. Full-length cDNA clone and sequence analyses of TaMDHAR4 Based on the partial candidate MDHAR gene sequence from the cDNA database of wheat challenged with Pst, initiation codon was in the context of AGGATGG, which is consistent with the Kozak consensus A/GXXATGG (Kozak, 1987). To verify the in silico cloned sequence, the specific primers were designed to amplify the candidate wheat MDHAR gene by RT-PCR. After cloning and sequencing, a 1570-bp cDNA fragment, which was identical to the in silico cloned sequence, was obtained. The longest ORF was 1431 bp and encoded a protein of 477 amino acids that shared 92%, 85%, and 85% homology with the MDHAR proteins from Brachypodium distachyon (GenBank: LOC100824698), Oryza sativa (GenBank: Os02g0707100), and Zea mays (GenBank: ACG34163), respectively (Appendix S2). The phylogenetic tree constructed from the deduced amino acid sequence and the six MDHAR genes in Arabidopsis thaliana showed that the candidate gene was highly homologous with AtMDHAR4 (GenBank: AT3G27820, Appendix S3). Hence, we named this gene TaMDHAR4 for T. aestivum monodehydroascorbate reductase 4 (GenBank: JX034702). The deduced TaMDHAR4 protein consisted of 477 amino acid residues. Using the Compute pI/MW software, the predicted TaMDHAR4 protein had a molecular weight of 52.2 kDa with a pI of 8.51. The protein conserved domain analyses using the InterProScan and TMHMM programs indicated that TaMDHAR4 contains a signal peptide (residues 1e19), two transmembrane domains (residues 5e24; 448e470), a FAD/NAD(P)-linked reductase (residues 335e426), and an FAD/NAD(P)-binding domain (residues 7e

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Fig. 1. Sequence information of the TaMDHAR4 gene. The sequences corresponding to the gene-specific primers for RT-PCR are underlined, and an arrow indicates the amplification direction. The triple bases in the box show the start and stop codons. The signal peptide is highlighted in red, the two transmembrane domains are labeled with rectangles, the FAD/ NAD(P)-linked reductases domain is shown in italics, and the FAD/NAD(P)-binding domain is shown in bold italics. The mPTS-like TMDs predicted with the TMHMM program (version 2.0) and the basic (positively charged) amino acid clusters are double-underlined and shown in bold type, respectively. In addition, the sequences highlighted in green and yellow were used for BSMV-g5V and BSMV-g3V, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

212). In addition, TaMDHAR4 possesses a C-terminal extension mPTS-like sequence that comprises a predicted transmembrane domain followed by five arginine residues eRKRRR (Fig. 1). This finding indicates that TaMDHAR4 might be a peroxisomal membrane MDHAR, which is consistent with AtMDHAR4.

3.2. TaMDHAR4 was down-regulated in the incompatible interaction between wheat and Pst It is widely known the plants generate ROS in response to pathogen infection, particularly in incompatible interactions

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between plants and pathogens. Because MDHAR is a key enzyme of the ascorbate-glutathione cycle, we examined the mRNA level of TaMDHAR4 in wheat leaves inoculated with Pst through qRT-PCR analyses (Fig. 2). In the compatible interaction, TaMDHAR4 was stably expressed from 12 to 24 hpi. The transcription of TaMDHAR4 was induced strongly, reached a peak that was approximately 4.5fold higher compared with the control (0 hpi) at 48 hpi, and then decreased from 72 to 120 hpi to the control level. In the incompatible interaction, the relative expression of TaMDHAR4 decreased to approximately 3.3-fold of the control level (0 hpi) from 12 to 18 hpi and recovered at 24 hpi; TaMDHAR4 was also induced at 48 hpi, with an approximately 2.7-fold higher than the control, and decreased to the control level from 72 to 120 hpi. These results indicate that the suppression of TaMDHAR4 may be associated with the early incompatible interaction between wheat and Pst. 3.3. SOD and APX affect the accumulation of TaMDHAR4 in wheat leaves inoculated with Pst To further validate the role of TaMDHAR4 in the incompatible interaction between the wheat cultivar Suwon 11 and the Pst race CYR23, we analyzed the transcriptional level in the wheat leaves injected with SOD and APX. SOD, which is an enzyme that participates in ROS metabolism, reduces O 2 into H2O2, and APX catalyses H2O2 into H2O and O2. As shown in Fig. 3A, the quantity of Pstinduced H2O2 could be enhanced in wheat leaves injected with SOD and decreased in leaves injected with APX, which demonstrates that the treatments were effective. Consequently, we analyzed the expression level of TaMDHAR4 by qRT-PCR (Fig. 3B). After treatment with SOD, the expression of TaMDHAR4 was down-regulated at 12 hpt, which is approximately 24 h post inoculation with Pst. In contrast, the expression of TaMDHAR4 was upregulated at 6 hpt in the leaves injected with APX, which is approximately 18 h post inoculation with Pst. Both changes occurred during the early stage of the interaction between wheat and Pst, which is the key stage of ROS burst. Hence, we deduced that the expression of TaMDHAR4 was affected by the accumulation of H2O2 and that it is involved in the wheat-Pst interaction through the regulation of the ROS metabolism pathway. 3.4. TaMDHAR4 could be induced by various exogenous hormone treatments qRT-PCR analyses were performed to investigate the expression pattern of TaMDHAR4 in wheat leaves treated with exogenous ABA,

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GAs, SA, MeJA, ETH, and BA (Fig. 4). As shown, compared to control (0 hpt), approximately 2.9-fold and 3.9-fold significant increases in TaMDHAR4 gene expression were observed at 2 hpt. Similarly, the expression of TaMDHAR4 was markedly increased and peaked at 12 hpt in wheat leaves treated with ETH; this peak was approximately 3.7-fold higher compared with the control. In contrast, TaMDHAR4 expression showed no significant induction in response to the SA, GAs, and BA treatments. 3.5. TaMDHAR4 regulates wheat resistance to Pst through the AsAGSH cycle To identify the function of TaMDHAR4 in wheat during Pst infection, we constructed the BMSV-g5V and BMSV-g3V vectors to silence TaMDHAR4. All of the BSMV-inoculated plants displayed mild chlorotic mosaic symptoms at 10 days post inoculation (dpi) but had no obvious defects in further leaf growth. Typical photo bleaching occurred on the leaves of wheat plants pre-inoculated with BSMV:gPDS at 14 dpi. Conspicuous HR and sporulation were elicited on the fourth leaves of the control seedlings (CK and BSMVg) inoculated with Pst races CYR23 and CYR31 at 14 dpi, respectively. In the TaMDHAR4-knocked down seedlings (BMSV-g5V and BMSV-g3V), no fungal sporulation could be found on the fourth leaves, and cell death was more vigorous in the incompatible interaction. In the compatible interactions, the sporulation was reduced in the TaMDHAR4-knocked down seedlings compared with the control (Fig. 5). qRT-PCR was used to calculated the efficiency of VIGS at 24, 48, and 120 hpi. In the incompatible interaction, compared to the BSMV-g inoculated leaves, the expression levels of TaMDHAR4 were decreased to 69%e83% (Fig. 6A), and, in the compatible interaction, the expression leaves of TaMDHAR4 was reduced to 68%e82% (Fig. 6B). Detailed histological changes in the TaMDHAR4-knocked down plants were also observed. The leaf segments inoculated with the Pst races CYR23 and CYR31 were sampled and prepared for histological observation (Table 1 and Fig. 7). The necrotic area generated at the infection site and the Pst hyphal lengths were observed and calculated. In the incompatible interaction, the necrotic area in wheat leaves pre-inoculated with BSMV-g5V and BSMV-g3V was significantly (P < 0.05) greater at 48 and 120 hpi compared with that obtained in wheat leaves pre-inoculated with BSMV-g. The Pst hyphal lengths in wheat leaves pre-inoculated with BSMV-g5V and BSMV-g3V were significantly (P < 0.05) shorter at 120 hpi compared with that observed in wheat leaves pre-inoculated with BSMV-g. In the compatible interaction, the necrotic area in the wheat leaves pre-inoculated with BSMV-g5V and BSMV-g3V was also significantly (P < 0.05) greater at 48 and 120 hpi compared with that obtained in wheat leaves pre-inoculated with BSMV-g. The Pst hyphal lengths in the wheat leaves pre-inoculated with BSMV-g5V was significantly (P < 0.05) shorter at both 48 and 120 hpi compared with that observed in the wheat leaves pre-inoculated with BSMV-g and significantly (P < 0.05) shorter at 120 hpi compared with that observed in the wheat leaves pre-inoculated with BSMV-g3V. 4. Discussion

Fig. 2. Expression profile of TaMDHAR4 in wheat leaves inoculated with Pst races CYR23 and CYR31. The data, which were obtained through qRT-PCR, were normalized to the wheat elongation factor 1 alpha-subunit (EF) expression level. The relative expression of TaMDHAR4 was calculated through the comparative threshold (2DDCT) method. The means and standard deviations were calculated with data from three independent replicates. * represents fold difference values that are significantly different than control (P < 0.05).

Reactive oxygen species (ROS) in plants are accumulated in different cellular compartments during the responses to abiotic and biotic stresses. The accumulation of ROS, particularly O 2 and H2O2, has been successively reported in plants infected with various pathogens and is considered as one of the early signs of plante pathogen interactions (Pitzschke et al., 2006; Shetty et al., 2008).

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Fig. 3. Histological observation and transcriptional changes of TaMDHAR4 in wheat leaves injected with SOD and APX during Pst infection. The wheat leaves inoculated with stripe rust CYR23 (incompatible reaction) were injected with MES buffer (CK), 500 mg/ml SOD (SOD), and 300 mg/ml APX (APX) at 11 hpi (0 hpt). A, The histological changes of the wheat leaves were observed at 24 hpi. H: haustoria; HMC: haustoria mother cell; IH: initial hypha; SV: substomatal vesicle. The H2O2 was stained with DAB. Bar ¼ 20 mm. B, The data were normalized to the wheat elongation factor 1 alpha-subunit (EF) expression level. The relative expression of TaMDHAR4 was calculated through the comparative threshold (2DDCT) method. The means and standard deviations were calculated with data from three independent replicates. * represents fold difference values that are significantly different than control (P < 0.05).

The ascorbate-glutathione (AsA-GSH) cycle, as the main antioxidant pathway in plant cells, regulates ROS to stimulate the redoxregulated plant defense (Noctor and Foyer, 1998). AsA, which is a reaction substrate of the AsA-GSH cycle, is important for its antioxidative and metabolic functions. As the key enzyme for AsA regeneration, MDHAR plays a crucial role in plantepathogen interactions. In this study, one cDNA sequence was isolated from

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Fig. 4. Expression profile of TaMDHAR4 in wheat leaves treated with exogenous hormones. The data were normalized to the wheat elongation factor 1 alpha-subunit (EF) expression level. The relative expression of TaMDHAR4 was calculated through the comparative threshold (2DDCT) method. The means and standard deviations were calculated with data from three independent replicates. * represents fold difference values that are significantly different than control (P < 0.05). ABA: abscisic acid; BA: benzyladenine; ETH: ethylene; GAs: gibberellins; MeJA: methyl jasmonate; SA: salicylic acid.

wheat leaves, and its amino acid sequence showed high similarity to MDHAR genes, which resulted in it being designated TaMDHAR4. Bioinformatics studies predict that TaMDHAR4 is located in the peroxisome. Interestingly, the most homologous MDHAR gene in Arabidopsis, AtMDHAR4, is located in the peroxisome (Lisenbee et al., 2005). Plant peroxisomes perform various functions, including photorespiration detoxification reactions, the synthesis of plant hormones, and the metabolism of ROS (Hayashi and Nishimura, 2003; Hu et al., 2012). Moreover, plant peroxisomes play essential roles in plantepathogen interactions (McCartney et al., 2005; Bednarek et al., 2008). Thus, TaMDHAR4 has a potential function in plantepathogen interactions through the AsA-GSH cycle. To determine whether TaMDHAR4 is involved in the interactions between wheat and Pst, we first examined the mRNA levels of TaMDHAR4 in wheat responding to Pst infection through qRT-PCR analyses. The expression of TaMDHAR4 was significantly decreased at 12 and 18 hpi in the incompatible interaction, but no significant change was observed in the compatible interaction at the early stage of infection. Previous histological studies showed that the early recognition of Pst occurs at 12 hpi, and a strong active oxygen burst occurred at 24 hpi. Meanwhile, the accumulation of necrotic area caused by the hypersensitive response (HR) was also observed as early as 24 hpi (Wang et al., 2007; Kang et al., 2002). So, TaMDHAR4 may function at the early recognization stage of wheatPst interactions. In 2005, Ku zniak et al. reported that the activity of MDHAR in peroxisomes was decreased when tobacco leaves were

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Fig. 5. Phenotype observation of the wheat leaves of TaMDHAR4-knocked down plants. Phenotypes changes on the fourth leaves of wheat plants whose second leaves were preinoculated with FES buffer (A), empty BSMV vectors (B), and positive control combination BSMV:PDS vectors (C) were observed at 14 days post treatment. The phenotypes of the fourth leaves inoculated with CYR23 (DeG)/CYR31 (HeK) of wheat plants whose second leaves were pre-injected with FES buffer (D, H), empty BSMV vectors (E, I), BSMV-g5V (F, J), and BSMV-g3V (G, K) were observed at 14 days post inoculation of Pst.

infected by the fungus B. cinerea (Elzbieta and Maria, 2005). Plants need sufficient H2O2 to induce HR and activate the series of genes involved in disease resistance. Based on these results, we speculated that the pathogen-induced downregulation of TaMDHAR4 in the incompatible interaction might be relevant for the HR of wheat induced by Pst. In addition, an induction was presented at 48 hpi in both the incompatible and the compatible interactions. In the

interactions between wheat and stripe rust, there are two stages that exhibit an active oxygen burst: a stronger one at 24 hpi and a weaker one at 48 hpi (Wang et al., 2007). In fact, the resistance or susceptibility of wheat to Pst is determined before 48 hpi, the second active oxygen burst hardly affects the response of wheat to Pst. Actually, many studies have reported that the expression of MDHAR was induced by various biotic stresses, the induction of MDHAR genes was explained as regenerating sufficient AsA to reduce the damage caused by H2O2 (Leterrier et al., 2005; Grantz et al., 1995; Eltelib et al., 2011). So, the induction of TaMDHAR4 at 48 hpi mainly functions as an indirect enzyme for ROS scavenging. This may be the function diversity of MDHAR in host responding to biotic and abiotic stresses. AsA is the reaction substrate of APX in the conversion of H2O2 to H2O. Sufficient AsA accumulation can trigger the reduction of cell death. The SOD and APX injection assays indicated that a high concentration of H2O2 down-regulates the expression of TaMDHAR4 and that a low concentration up-regulates the expression of this gene. These results also demonstrate that TaMDHAR4 may

Table 1 Histological analyses during the incompatible/compatible interaction between Suwon 11 and the stripe rust fungus CYR23/31 when the TaMDHAR4 was silenced. Treatmenta

Fig. 6. Relative transcript levels of TaMDHAR4 in knockdown plants. The silencing efficiency of TaMDHAR4 was calculated through qRT-PCR. BSMV-g was used as the control. The data were normalized to the wheat elongation factor 1 alpha-subunit (EF) expression level. The relative expression of TaMDHAR4 was calculated through the comparative threshold (2DDCT) method. The vertical bars represent the standard deviations. The means and standard deviations were calculated with data from three independent replicates.

Necrotic areab

Hyphal lengthc

48 hpi

120 hpi

24 hpi

48 hpi

120 hpi

A BSMV-g BSMV-g5V BSMV-g3V

2.12a 2.52b 2.61b

7.13a 8.47b 9.01b

0.48a 0.51a 0.43a

0.89a 0.82a 0.81a

1.35a 0.94b 1.03b

B BSMV-g BSMV-g5V BSMV-g3V

0.03a 0.06b 0.11b

0.08a 0.22b 0.18b

0.82a 0.79a 0.85a

1.93a 1.65b 2.03a

4.15a 2.77b 3.63b

The analyses of significance was performed using the paired sample t-test method with the SPSS software (P < 0.05). a The second leaves were pre-infected with recombinant BSMV. BSMV-g5V and BSMV-g3V are the cDNA fragments derived from the upstream and downstream sequences of TaMDHAR4, respectively, and BSMV-g was used as the control. The fourth leaves were inoculated with Pst races CYR23 (A) and CYR31 (B); hpi: hours post-inoculation. b Average area of necrotic area calculated from 50 infection sites (unit in 1000 mm2，measured with the DP-BSW software). c Average distance from the base of the substomatal vesicles to the hyphal tips calculated from 50 infection sites (unit in 100 mm, measured with the DP-BSW software).

Fig. 7. Histological observation of wheat leaves infected with Pst. The fourth leaves of wheat plants were challenged with Pst races CYR23 (incompatible reaction; AeF) and CYR31 (compatible reaction; aef) and sampled at 24, 48, and 120 hpi. AeC and aec show the histological features from the control (BSMV-g). DeF and def show the histological features from the TaMDHAR4-knocked down leaves (BSMV-g5V). H: haustoria; HMC: haustoria mother cell; IH: initial hypha; NC: necrotic area; SH: second hypha; SV: substomatal vesicle. Bar ¼ 20 mm.

H. Feng et al. / Plant Physiology and Biochemistry 76 (2014) 7e16

function in cell death through the down-regulation of its expression to improve the H2O2 content in cells. In the interaction between plant and pathogens, a series of plant genes will be activated, which need lots of molecules to perform signal transduction. SA, ABA, JA, and ET are three key signaling mediators during plant defenses against pathogens (Glazebrook, 1999). Plant peroxisomes function in hormone synthesis (Hu et al., 2012), which may play key roles in the regulation of the induced defense response in plants (van Loon et al., 2006). According to Koo et al. (2006), peroxisomes are involved in the production of lipid-based signaling molecules (jasmonic acid). ABA may function synergistically with JA and displays a complex antagonistic interaction with SA (Fan et al., 2009). In addition, ET was also demonstrated to participate in the disease response in Arabidopsis (Pieterse et al., 1998). Our qRT-PCR assay found that the expression of TaMDHAR4 is induced by some types of exogenous hormones, such as MeJA, ABA, and ETH. Different expression profiles of TaMDHAR4 in exogenous hormones treated plants indicate that TaMDHAR4 may function through some types of signaling pathways. To investigate the function of TaMDHAR4 in wheat during the response to Pst infection, virus-induced gene silencing (VIGS) was employed. The knockdown of TaMDHAR4 resulted in enhanced HR and limited hypha growth. To ensure the reliability of our results, we used two fragments from different parts of the target gene for the construction of the BSMV vectors. The TaPDS gene was selected as the positive control, and qRT-PCR was used to measure the efficiency of the VIGS system. Histological observation was combined with phenotype analysis to detect the subtle changes generated by the knockdown of TaMDHAR4. Based on the phenotype changes, we found that the necrotic area was more intense in the wheat leaf surface of the TaMDHAR4-knocked down plants, and this finding is consistent with the results of the histological observations, which indicate that the level of cell death induced by Pst was much stronger in the TaMDHAR4-knocked down leaves. In fact, the necrotic area of the host could be reflected by the content of H2O2 in the host cells (Wang et al., 2007). Moreover, the Pst hyphae elongation was restricted in the TaMDHAR4-knocked down plants. Combining with the results of histological observation, we speculate that the knockdown of TaMDHAR4 results in an enhancement of the reactive oxygen species and restricted the initial hyphae. Thus, combining the phenotype and microscopic observations, we confirm that TaMDHAR4 plays an important role in the hypersensitive response of wheat to Pst. In our study, we speculated the TaMDHAR4 regulates the resistance of plants through the pathogen-induced ROS metabolism. However, MDHAR and DHAR activity were reported that cannot keep pace with AsA content consumption, even they could have an important role in the removal of the products of AsA oxidation, in some cell compartments (De Tullio et al., 1998). Further studies are needed to identify the molecular mechanisms of how and when TaMDHAR4 function in the interaction between wheat and Pst. MDHAR proteins may function in hypersensitive respond is a complex network; a series of events should be programmed after TaMDHAR gene expression. The proteins and their induced biochemical pathways will be studied in future. 5. Conclusion In the present research, we first cloned a wheat monodehydroascorbate reductase gene TaMDHAR4, a 1431-bp nucleotide sequences which contains typical structural features, such as mPTS-like sequences in the C-terminal extension and transmembrane domain followed by five basic arginine residues (-RKRRR). qRT-PCR analyses demonstrated that TaMDHAR4 could

15

be induced by various exogenous hormones, such as ABA, MeJA, and ETH, particularly in the incompatible interaction. TaMDHAR4 is sharply down-regulated at 12 and 18 hpi only in wheat leaves challenged with Puccinia striiformis f. sp. tritici (Pst) race CYR23 and induced at 48 hpi with both Pst races CYR23 and CYR31. SOD and APX injection analyses demonstrated that TaMDHAR4 may be involved in the early stage during the interaction between wheat and Pst, which is the key stage of ROS burst. Moreover, the knockdown of TaMDHAR4 through virus-induced gene silencing (VIGS) enhanced the wheat resistance to Pst by inhibiting sporulation in the compatible interaction. Histological observations also demonstrated that silenced wheat resulted in an increased proportion of necrotic area at the infection sites and suppressed Pst hypha elongation. In a word, TaMDHAR4 plays an important role in the interactions between wheat and Pst. Acknowledgments This study was financially supported by the National Basic Research Program of China (No. 2013CB127700), the National Natural Science Foundation of China (No. 31071651), and the 111 Project from the Ministry of Education of China (No. B07049). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2013.12.015. References Ali, M.B., Hahn, E.J., Paek, K.Y., 2005. Effects of temperature on oxidative stress defense systems, lipid peroxidation and lipoxygenase activity in Phalaenopsis. Plant Physiol. Biochem. 43, 213e223. Arrigoni, O., Dipierro, S., Borraccino, G., 1981. Ascorbate free radical reductase, a key enzyme of the ascorbic acid system. FEBS Lett. 125, 242e245. Asada, K., 1997. The role of ascorbate peroxidase and monodehydroascorbate reductase in H2O2 scavenging in plants. In: Oxidative Stress and the Molecular Biology of Antioxidant Defenses. CSHL Press, New York, pp. 715e735. Bednarek, P., Pislewska-Bednarek, M., Svatos, A., Schneider, B., Doubsky, J., Mansurova, M., Humphry, M., Consonni, C., Panstruga, R., Sanchez-Vallet, A., Molina, A., Schulze-Lefert, P., 2008. A glucosinolate metabolism pathway in living plant cells mediates broad-spectrum antifungal defense. Science 323, 101e106. Dat, J., Vandenabeele, S., Vranová, E., Van Montagu, M., Inzé, D., Van Breusegem, F., 2000. Dual action of the active oxygen species during plant stress responses. Cell. Mol. Life Sci. 57, 779e795. De Tullio, M.C., De Gara, L., Paciolla, C., Arrigoni, O., 1998. Dehydroascorbatereducing proteins in maize are induced by the ascorbate biosynthesis inhibitor lycorine. Plant Physiol Biochem. 36, 433e440. Eltelib, H.A., Badejo, A.A., Fujikawa, Y., Esaka, M., 2011. Gene expression of monodehydroascorbate reductase and dehydroascorbate reductase during fruit ripening and in response to environmental stresses in acerola (Malpighia glabra). J. Plant Physiol. 168, 619e627. Elzbieta, K., Maria, S., 2005. Compartment-specific role of the ascorbate-glutathione cycle in the response of tomato leaf cells to Botrytis cinerea infection. J. Exp. Bot. 56, 921e933. Fan, J., Hill, L., Crooks, C., Doerner, P., Lamb, C., 2009. Abscisic acid has a key role in modulating diverse plantepathogen interactions. Plant Physiol. 150, 1750e1761. Foreman, J., Demidchik, V., Bothwell, J.H.F., Mylona, P., Miedema, H., Torres, M.A., Linstead, P., Costa, S., Brownlee, C., Jones, J.D.G., Davies, J.M., Dolan, L., 2003. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442e446. Glazebrook, J., 1999. Genes controlling expression of defense responses in Arabidopsis. Curr. Opin. Plant Biol. 2, 280e286. Grantz, A.A., Brummell, D.A., Bennett, A.B., 1995. Ascorbate free radical reductase mRNA levels are induced by wounding. Plant Physiol. 108, 411e418. Hayashi, M., Nishimura, M., 2003. Entering a new era of research on plant peroxisomes. Curr. Opin. Plant Biol. 6, 577e582. Holzberg, S., Brosio, P., Gross, C., Pogue, G.P., 2002. Barley stripe mosaic virusinduced gene silencing in a monocot plant. Plant J. 30, 315e327. Hu, J., Baker, A., Bartel, B., Linka, N., Mullen, R.T., Reumann, S., Zolmanh, B.K., 2012. Plant peroxisomes: biogenesis and function. Plant Cell 24, 2279e2303. Kang, Z., Li, Z., 1984. Discovery of a normal T. type new pathogenic strain to Lovrin10. J. Northw. Agric. For. Univ. Nat. Sci. Ed. 4, 18e28.

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