TaMCA1, a regulator of cell death, is important for the interaction between wheat and Puccinia striiformis

Metacaspase orthologs are conserved in fungi, protozoa and plants, however, their roles in plant disease resistance are largely unknown. In this study, we identified a Triticum aestivum metacaspase gene, TaMCA1, with three copies located on chromosomes 1A, 1B and 1D. The TaMCA1 protein contained typical structural features of type I metacaspases domains, including an N-terminal pro-domain. Transient expression analyses indicated that TaMCA1 was localized in cytosol and mitochondria. TaMCA1 exhibited no caspase-1 activity in vitro, but was able to inhibit cell death in tobacco and wheat leaves induced by the mouse Bax gene. In addition, the expression level of TaMCA1 was up-regulated following challenge with the Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaMCA1 via virus-induced gene silencing (VIGS) enhanced plant disease resistance to Pst, and the accumulation of hydrogen peroxide (H2O2). Further study showed that TaMCA1 decreased yeast cell resistance similar to the function of yeast metacaspase, and there was no interaction between TaMCA1 and TaLSD1. Based on these combined results, we speculate that TaMCA1, a regulator of cell death, is important during the compatible interaction of wheat and Pst.

Metacaspases are present in fungi, protozoa and plants based on predicted structural homologies with the catalytic domains of caspases 1 . In plant systems, metacaspases are subdivided into two types (type I and type II) based on their structures. Specifically, type I metacaspases have an N-terminal pro-domain that is not identified in type II, while type II metacaspases harbor a longer linker region between the putative small (p10) and large (p20) subunits [1][2][3] . In the last decade, several metacaspase genes have been found to be involved in cell death. For example, the yeast metacaspase (YCA1) knock-out (yca1Δ ) survives in the presence of hydrogen peroxide (H 2 O 2 ) 4 . AtMC1, the homologue of YCA1 in Arabidopsis thaliana, was up-regulated in plants challenged with bacterial pathogens 5 . A recent study reported that AtMC1 and AtMC2 antagonistically control hypersensitive response (HR)-associated cell death that is activated by intracellular immune receptors 6 . Similar to the function of AtMC1, AtMC4 plays a positive regulatory role in biotic and abiotic stress-induced cell death 7 . Interestingly, further study showed that AtMC8 might be involved in the cell death induced by UVC or H 2 O 2 , and AtMC8 knockout lines exhibit reduced cell death 8 . TaMCA4 is a novel plant metacaspase gene cloned from wheat (Triticum aestivum). Knockdown of TaMCA4 expression enhances the susceptibility of the host plant to the avirulent P. striiformis f. sp. tritici (Pst) race CYR23 and reduces the necrotic area per infection site. The HR is a rapid plant-initiated cell death [9][10][11] that is associated with the recognition of avirulence products by the corresponding resistance genes. Additionally, HR helps plants defend themselves against pathogens by sacrificing plant cells at the infection sites to limit pathogen growth 12 . Stripe rust caused by Pst is one of the most destructive of the fungal wheat diseases worldwide 13 . However, the physiological roles of type I metacaspase genes in the wheat-Pst interaction have not been well characterized. In the present study, we isolated an AtMC1 homolog TaMCA1 in wheat. The TaMCA1 contained typical structural features of type I metacaspases domains and is located in cytosol and mitochondria. TaMCA1 inhibited cell death in tobacco and wheat cells. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses showed that TaMCA1 was up-regulated in wheat leaves challenged by Pst race CYR23 and CYR31. Furthermore, knockdown of TaMCA1 in wheat using virus-induced gene silencing (VIGS) enhanced plant disease resistance to Pst race CYR31, and TaMCA1 was partly complement the function of the YCA1.

Results
Cloning of the TaMCA1 homologue and sequence analyses. One wheat metacaspase homologue with a complete open reading frame (ORF) was cloned from wheat 'Suwon11' . The predicted ORF encoded a protein of 292 amino acid residues with a molecular weight of 32.1 kDa. Sequence alignment with the T. aestivum cv. Chinese Spring (CS) genome sequence revealed that three copies were located on chromosomes 1A, 1B and 1D. Multi-sequence alignments with other plant metacaspase proteins revealed that the protein contained structural features common to the type I family (Fig. S1), and a phylogenetic tree analysis confirmed its relatedness to AtMC1 and other plant metacaspase proteins (Fig. S2). Therefore, we named the gene TaMCA1 in this study (KU958719).

TaMCA1 exhibited no caspase-1 activity in vitro.
Purified TaMCA1 was assayed with western blotting experiments (Fig. S3a). To measure the activity of TaMCA1 in vitro, the fluorogenic substrate Ac-YVAD-AMC (a substrate of caspase-1) was utilized as previously described 14,15 . The total protein extracted from the wheat leaves was used as a control. The caspase-1 cleavage product was present in the protein extracted from the wheat leaves, whereas no fragments were detected in the TaMCA1 expressed in E. coli strain (Fig. S3b).
Transcriptional changes of TaMCA1 induced by P. striiformis f. sp. tritici infection. After inoculating seedling 'Suwon11' wheat plants with the avirulent race CYR23 or the virulent race CYR31, qRT-PCR was performed to determine the transcript profiles of TaMCA1 during Pst infection. In the compatible interaction (CYR31), the expression of TaMCA1 was induced at 24 hours post inoculation (hpi), subsequently peaked at 48 hpi, and gradually reduced at 72 and 120 hpi. In contrast, TaMCA1 was only minimally up-regulated at 72 hpi in the incompatible interaction (CYR23) (Fig. 1). Subcellular localization of TaMCA1. In plants, several compartments including vacuole, cytosol, chloroplasta and mitochondrion display caspase activity. AtMC1 was cytosolic enzymes, and it was predicted to be localized at mitochondria 16 . To determine the subcellular localization of TaMCA1, the TaMCA1-GFP fusion protein was expressed in wheat seedlings protoplasts, and existed in the form of dots in cytosol. To further confirm its subcellular localization, TaMCA1-GFP was co-expressed with SLO2-DsRed, a well-known mitochondria marker protein (At2g13600) 17 . As shown in Fig. 2, the green fluorescence was distributed in cytosol, and a few colocalized with SLO2-DsRed at mitochondria.
TaMCA1 suppresses cell death in N. enthamiana and T. aestivum. In the compatible interaction (CYR31), the expression level of TaMCA1 exhibited a significant up-regulation at 48 hpi. Therefore, we speculate that TaMCA1 may play an important role in the cell death to Pst in wheat. To prove this idea, TaMCA1 was transiently overexpressed in N. benthamiana using potato virus X (PVX) delivery in combination with the Bax system either alone or followed 24 h later with an A. tumefaciens strain carrying the mouse Bax gene. The results indicated that the tobacco leaves infiltrated with Bax ( Fig. 3a: circle 1), infiltration buffer (BF) + Bax (Fig. 3a: circle 2), empty vector (EV) + Bax (Fig. 3a: circle 7) or eGFP + Bax (Fig. 3a: circle 8) exhibited a cell death phenotype, and green fluorescence could be detected at 3-7 days in the eGFP treatment (Fig. S4), which indicated that the    (Fig. 3a: circle 4) suppressed cell death, which indicated that TaMCA1 is related to cell death via the suppression of the cell death induced by the mouse Bax gene in tobacco, and this supposition was proven again by the Bio-Rad Gene Gun for co-bombardment assays in N. benthamiana as described 18 . Blue spots observed on leaves represented the quantity of living cells, when the tobacco leaves were bombarded with EV + Bax + Gus (β -glucuronidase, Gus), a 79.8% reduction in the number of blue spots was observed compared to leaves that were shot with TaMCA1 + Bax + Gus (Fig. 3b,c). To determine whether TaMCA1 was able to suppress cell death in wheat, we used the attachment of the Bio-Rad Gene Gun for bombardment assays with wheat leaves as described 19,20 . Numerous blue spots were observed on wheat leaves bombarded with EV + Gus, EV + Bax + Gus, TaMCA1 + Gus or TaMCA1 + Bax + Gus (Fig. 4a). As shown in Fig. 4b, the number of blue spots on the leaves bombarded with EV + Gus, TaMCA1 + Gus or TaMCA1 + Bax + Gus showed no significant change. However, when the wheat leaves were bombarded with EV + Bax + Gus, a 60% reduction in the number of blue spots was observed compared to leaves that were shot with TaMCA1 + Bax + Gus. Thus, our results showed that TaMCA1 suppressed the cell death triggered by the mouse Bax gene in both N. benthamiana and T. aestivum. Knocking down TaMCA1 increased the resistance of wheat to Pst. Based on the expression profile of TaMCA1 during Pst infection, the Barley stripe mosaic virus (BSMV)-based VIGS system was applied to further characterize TaMCA1's function in the interaction of wheat and Pst [21][22][23] . Two pairs of primers were designed specifically to knockdown TaMCA1. Moreover, the silencing of the wheat phytoene desaturase gene (PDS) was used as the positive control for the gene silencing system to confirm whether our VIGS conditions were functioning correctly, and this system generated photobleaching symptoms by 9 days post inoculation (dpi). The result showed that the plants treated with the BSMV:γ , BSMV:TaMCA1-1 or BSMV:TaMCA1-2 displayed mild chlorotic mosaic symptoms by 9 dpi but exhibited no obvious defects in further leaf growth (Fig. 5a). The fourth leaves of the wheat plants that were pre-treated with 1 × Fes buffer, BSMV:γ , BSMV:TaMCA1-1 or BSMV:TaMCA1-2 were then inoculated with urediospores of the Pst virulent race CYR31. On average, the knockdown of TaMCA1 expression limited the number of uredium developments, which was equivalent to the development of an increased resistance type to the wheat stripe rust fungus (Fig. 5b). Additionally, the fungal biomass in both TaMCA1-knockdown plants was significantly reduced at 120 hpi compared with the control plants (pre-infected with 1 × Fes buffer or BSMV:γ ) (Table S1), which suggested that the wheat stripe rust fungus growth or development were restricted to a certain extent in both TaMCA1-knockdown plants. The transcripts level of TaMCA1 was significantly suppressed to different extents (71-78%) compared with the BSMV:γ -treated plants (Fig. 5c), which indicated that TaMCA1 was silenced in both TaMCA1-knockdown plants. Histological changes of Pst growth and host response. A microscopic examination revealed that there was no obvious difference in the hyphal branches between the control plants and the plants pre-treated with BSMV:TaMCA1-1 or BSMV:TaMCA1-2 at 24 or 48 hpi (Table 1). Moreover, the hyphal length of the wheat pre-treated with BSMV:TaMCA1-1 or BSMV:TaMCA1-2 were significantly (P < 0.05) shorter than those observed in the control plants at 48 hpi (Table 1), and the colony size in both TaMCA1-knockdown plants was significantly reduced compared with the sizes observed in the control plants (P < 0.05) at 120 hpi (Table 1).
To further understand the correlationship between Pst-induced TaMCA1 and Pst-induced cell death, we assayed the expression levels of a few selected genes in TaMCA1 knockdown plant after infection with the stripe rust fungus, including catalase (TaCAT), class III peroxidase (TaPOD) and superoxide dismutase (TaSOD), Triticum aestivum metacaspase 4 (TaMCA4) and Triticum aestivum defender against cell death (TaDAD2). As shown in Fig. 5d, the levels of TaMCA4 and TaDAD2 showed no change in comparison with BSMV:γ -treated plant. But, the transcript levels of TaCAT, TaPOD and TaSOD were down-regulated in TaMCA1-knockdown plant, particularly at 24 hpi. Together, these results suggested that TaMCA1 may be involved in plant ROS accumulation to influence plant resistance.
Enhanced reactive oxygen species accumulation in TaMCA1-knockdown plant. To further confirm the supposition that TaMCA1 may be involved in plant ROS accumulation to influence plant resistance, we assayed the production of hydrogen peroxide (H 2 O 2 ), the most important component of ROS. The results showed that H 2 O 2 accumulation in both TaMCA1-knockdown plants with Pst race CYR31 was significantly (P < 0.05) greater than that in the control plants with Pst race CYR31 at 24 hpi (Table 1), which was consistent with the result in Fig. 5d. These findings suggest that TaMCA1 may be involved in mediating the plant accumulation of ROS to influence plant resistance during the compatible interaction of wheat and Pst.
TaMCA1 decreases the yeast resistance to H 2 O 2 . Firstly, we examined the effects of TaMCA1 on the survival of yeast cells subjected to H 2 O 2 . As shown in Fig. 6a, the viability was severely reduced in the TaMCA1-transformed yeast cells grown on inducing medium with H 2 O 2 compared with the cells grown on repressing medium with H 2 O 2 . Similar results were also obtained in the complementation experiment, the yca1Δ and yca1Δ expressing the empty vector (yca1Δ + EV) survived the H 2 O 2 stimuli, and by contrast, both the wild type (W) and the yca1Δ expressing TaMCA1 (yca1Δ + TaMCA1) were shown to have a reduction of survival, respectively (Fig. 6b). Therefore, TaMCA1 could decrease yeast cell resistance to H 2 O 2 , and was able to partly complement the function of the YCA1.

Discussion
In plant cell, cell death is often accompanied by biochemical and morphological hallmarks similar to those observed in animal apoptosis 25,26 . However, the orthologs of animal caspases, i.e., cysteinyl aspartate-specific proteases, which are highly conserved in animal cells, have not yet been identified in plants 27 . In the last decade, a family of genes encoding cysteine-type C14 proteases that is more structurally similar to mammalian caspases than any other caspase-like proteases in plants were named metacaspases 1,28 . Within plant genomes, the Arabidopsis thaliana genome encodes three type I and six type II metacaspases, and the Oryza sativa sp. japonica genome encodes four type I and four type II metacaspases 29  that regulate wheat plant resistance against Pst. In the present study, we obtained a novel Triticum aestivum metacaspase gene, TaMCA1, from Triticum aestivum cv. Suwon11. TaMCA1 was predicted to be a member of the wheat type I metacaspase family, and contained a variable-sized N-terminal extension upstream of the p20 caspase-like domain and a short linker between the p20 and p10 domains. Caspase (clan CD, family C14) is a member of the cysteine protease family and specifically cleaves after aspartate 32 . Caspases are synthesized inside the cells as inactive zymogens, but their activation can be specifically measured with synthetic substrates 3 . Our results showed that the synthetic substrate (Ac-YVAD-AMC) was not degraded by the recombinant TaMCA1 in vitro, although the substrate was degraded by the extract from wheat leaves (Fig. S3). A large amount of evidence demonstrate that recombinant metacaspases do not degrade synthetic substrates in vitro 2,8,33 , but caspase-like activity is found to be present in extracts from mosaic virus-infected tobacco leaves 34 and barley (Hordeum vulgare) embryonic suspension cells 35 . Therefore, we speculate that TaMCA1 may be activated through the proteolytic cleavage of zymogens or conformational changes inside plant cells induced by some signals.
Metacaspase has been identified as a major player in cell death 7,8,19,29,31,36,37 . In the present study, our work showed that TaMCA1 could suppresse cell death induced by the Bax gene in N. benthamiana and wheat leaves (Figs 3 and 4). TaMCA4 has also been shown to be involved in the cell death triggered by the Bax gene in N. benthamiana and wheat leaves 19 . Hence, we speculate that TaMCA1 may be involved in wheat-Pst interaction as a negative cell death regulator. Furthermore, the expression level of TaMCA1 was up-regulated (approximately 4-fold) at 72 hpi following challenge with Pst race CYR23 and remarkably up-regulated (approximately 38-fold) at 48 hpi following challenge with Pst race CYR31 (Fig. 1), which indicated that TaMCA1 may be play an important  (Table 1), and the Pst biomass in both TaMCA1-knockdown plants was significantly reduced at 120 hpi compared with the control plants (Table S1).
During plant-pathogen interactions, plants have developed a more sophisticated and efficient mechanism to counteract the spread of pathogen invasion, such as ROS bursts, protease activation. ROS accumulation in host cells is a plant defense response that is important for resistance against rust fungi in wheat [38][39][40] . In this study, the transcript levels of TaCAT, TaPOD and TaSOD were down-regulated in TaMCA1-knockdown plant after infection with Pst, especially at 24 hpi. Interestingly, the DAB staining of the TaMCA1-knockdown plants at the infection sites became more extensive at 24 hpi compared with those observed in the control plants (Table 1). Similar observations have been reported in previous studies, AtMC1 mutant was hypersensitive to the salicylic acid agonist benzo(1,2,3)thiadiazole-7-carbothioic acid S-methy lester, and accompanied by ROS accumulation 41 . A pepper (Capsicum annuum L.) metacaspase 9 (Camc9) was reported to be involved in the production of ROS during pathogen-induced cell death 36 . The yca1Δ survived in the presence of H 2 O 2 4 , and AtMC1, AtMC6 and AtMC8 were also able to complement the cell death functions of YCA1 8,33 . In the present study, TaMCA1 in fission yeast decreased the resistance to H 2 O 2 stimuli, and was also able to partly complement the function of the YCA1 (Fig. 6). These data strongly support the notion that TaMCA1 mediated the plant resistance to Pst by regulating ROS accumulation.
Previous reports have demonstrated that AtMC1 interacts with LSD1 in yeast and transgenic Arabidopsis 6 . However, there was no interaction between TaMCA1 and TaLSD1 based on the yeast two-hybrid assays (Fig. 7). These results suggested that the difference between TaMCA1 and AtMC1 is larger because of the wheat genome is much larger than those of species.
In summary, our study indicated that TaMCA1 would participate in the regulation of cell death only after the generation of sufficient signals, including mammalian Bax, Pst or H 2 O 2 . Furthermore, TaMCA1 was able to decrease plant resistance via the management of ROS accumulation during the compatible interaction of wheat and Pst.

Methods
Plant materials and Pst isolate and treatments. Two plants (Triticum aestivum cv. Suwon11 and N. benthamiana) and two Chinese Pst races (CYR23 and CYR31) were used in this study. Suwon11 exhibits a typical HR to CYR23 but is highly susceptible to CYR31 42 . The wheat seedlings were grown, inoculated and maintained following previously described procedures 43 . N. benthamiana was grown in the growth chamber at 22 °C under constant light and used for transient expression.

RNA extraction and qRT-PCR.
For RNA isolation, the leaves were collected at 0, 12, 24, 48, 72 and 120 hpi with Pst. The time points were selected based on microscopic studies 38,44 . RNA was isolated from the wheat leaves using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. First-strand cDNA was synthesized using the GoScript Reverse-Transcription System (Promega Corp., Madison, WI, USA). The reverse transcription reactions were incubated with oligo(dT) primers at 42 °C for 1 h in total volumes of 20 μ l. The primer design and qRT-PCR reactions were conducted as described previously 45 . To standardize the data, the wheat translation elongation factor TaEF-1a gene (Q03033) was used as an internal reference for the qRT-PCR analysis. Dissociation curves were generated for each reaction to ensure specific amplification. The threshold values (CTs) generated with the ABI PRISM 7500 Software Tool (Applied Biosystems) were employed to quantify the relative gene expressions using the comparative 2 −ΔΔCT method 46 . Three independent biological replicates were performed for each experiment.
Sequence analysis, alignments and polymorphism analysis. PROSITE Scan (http://prosite.expasy. org/scanprosite/) and Pfam (http://pfam.sanger.ac.uk/) were used to predict the conserved domains and motifs. CLUSTALW and DNAMAN6.0 were used to perform the multi-sequence alignments. The MEGA5.1 software was used to create the phylogenetic trees of the TaMCA1 members.
Protein expression, purification and Activity assay. Recombinant of TaMCA1 in pET-28a (+ ) was expressed in the E. coli strain BL21 (DE3) (Invitrogen). The cells were cultured in LB medium at 37 °C with ampicillin (50 μ g/ml) to an OD600 of 0.4-0.6, and expression was subsequently induced by 0.5 mM isopropyl-β -d-thiogalactoside (IPTG) at 25 °C 200 rpm for 5 h. The protein was extracted and purified as described previously 33 . Purified TaMCA1 was assayed with western blotting experiments. The fluorogenic substrate Ac-YVAD-AMC (PharMingen; AMC7-amino-4-methylcoumarin, i.e., a substrate of caspase-1) was used to measure the activity of TaMCA1 as described previously 14,47 .

Subcellular localization of TaMCA1 in protoplasts.
In order to block out the chloroplast auto-fluorescence, the wheat seedlings for protoplast transformation were kept in the growth chamber at 16 °C without light for 7 days before using. Protoplast preparation and transformation were performed as described previously 48 . The green channel shows the localization of TaMCA1-GFP; the red channel shows the localization of SLO2-DsRed, a mitochondrial marker protein (At2g13600). Bar = 20 μ m.

Overexpression of TaMCA1 in N. benthamiana and T. aestivum leaves. The reconstructed vectors
PVX-eGFP, PVX-Avr1b, PVX-Bax and PVX-TaMCA1 were transformed individually into the A. tumefaciens strain GV3101 by electroporation. The transformants were grown on LB media plates with 30 μ g/ml rifampicin, 30 μ g/ml chloramphenicol and 30 μ g/ml kanamycin. For the infiltration of the leaves, the A. tumefaciens strains carrying PVX-EV or PVX-Avr1b, PVX-eGFP, PVX-TaMCA1 or PVX-Bax were cultured in LB medium with rifampicin (30 μ g/ml), chloramphenicol (30 μ g/ml) and kanamycin (30 μ g/ml) at 28 °C for 24-48 h. During the logarithmic phase, the cells were collected by centrifugation, washed twice with 10 mM MgCl 2 , and finally suspended to an OD600 of 0.8 with an infiltration media (10 mM MgCl 2 , 10 mM MES, pH 5.6, and 200 mM acetosyringone). Next, the cells were incubated at room temperature for 1-3 h before infiltration. A. tumefaciens strains carrying PVX-EV, PVX-eGFP, PVX-Avr1b or PVX-TaMCA1 were infiltrated into tobacco leaves using a syringe without a needle. The same infiltration site was challenged with a strain carrying PVX-Bax 24 h after the initial infiltration. The green fluorescence was detected in the PVX-eGFP treated leaves 72 h after infiltration and directly imaged on an Olympus BX-51 microscope (Olympus Corporation, Japan; excitation filter, 485 nm; dichromic mirror, 510 nm; barrier filter, 520 nm). Symptom development was monitored visually 3 to 8 days after infiltration 49 .
For the particle bombardment assays, a great quantity of reconstructed plasmid (pUC-EV, pUC-Bax, pUC-Gus or pUC-TaMCA1) was prepared. Leaves from 4-to 6-week-old N. benthamiana plants were bombarded using the Bio-Rad He/1000 particle delivery system with a double-barreled extension attached, and leaves from 2-to 3-week-old T. aestivum plants were bombarded with single-barreled particle delivery as described previously 18,19 . The DNA samples were prepared according to the shooting protocol described previously 19 . After bombardment, the leaves were incubated at 28 °C for 2-3 days in darkness and then stained for 16-24 h at 28 °C using 5-bromo-4-chloro-3-indolyl-D-glucuronic acid (X-α -gluc) at 0.8 mg/ml, 80 mM Na phosphate (pH 7.0), 8 mM Na 2 EDTA, 0.4 mM K 3 Fe(CN) 6 , 0.4 mM K 4 Fe(CN) 6 , 0.06% (vol/vol) Triton X-100 and 20% methanol. After bleaching the leaves using 100% methanol for many days until the blue spots could be observed clearly by microscopy. In total, 14 shots were performed for each treatment, the number of spots for each shot were counted. Analysis of variance (ANOVA) was used to analyze the significant differences between the different treatments using SPSS software 18.0. BSMV-mediated TaMCA1 gene silencing. Capped in vitro transcripts were prepared from linearized plasmids that contained the tripartite BSMV genome 50 using the mMessage mMachine T7 in vitro transcription kit (Ambion, Austin, TX, USA) following the manufacturer's instructions. Suwon11 wheat seedlings at the two-leaf stage were prepared; the second leaf was treated with BSMV virus (BSM:γ , BSMV:PDS, BSMV:TaMCA1-1 or BSMV:TaMCA1-2) as previously described 21,51 and then maintained in a growth chamber at 23 ± 2 °C. The seedlings were mock inoculated with BSMV:PDS as a positive control, and 1 × Fes buffer was used a negative control 52 . The fourth leaves then inoculated with fresh urediniospores of CYR31 at 10 dpi and subsequently sampled at 0, 24, 48 and 120 hpi for histological observation and RNA isolation. The infection types of stripe rust were examined at 15 dpi.
Histological observations of the fungal growth. A histopathological analysis was performed to characterize the cellular interaction between the wheat and Pst. BSMV-infected wheat leaves with Pst were sampled at 24, 48 and 120 hpi. Leaf segments cut from the inoculated leaves were fixed and decolorized with thanol/trichloromethane (3:1 v/v) containing 0.15% (w/v) trichloroacetic acid for 3-5 days. The segments were soaked in saturated chloral hydrate until translucent and then stained with wheat germ agglutinin (WGA) conjugated to Alexa-488 (Invitrogen) 53 . The hyphal length, colony size and number of hyphal branches of stained tissues were examined under blue light excitation (excitation wavelength 450-480 nm and emission wavelength 515 nm) with an Olympus BX-53 microscope (Olympus Corporation, Japan) and calculated with the cellSens Entry software (Olympus Corporation, Japan) as described 54 . H 2 O 2 was stained in situ using 3,3-diaminobenzidine (DAB; Amresco, Solon, OH, USA) 55 . The infection sites at which appressoria had Scientific RepoRts | 6:26946 | DOI: 10.1038/srep26946 formed over the stomata were considered to have successful penetration, and at least 50 infection sites were examined on each of five randomly selected leaf segments per treatment.

Fungal growth biomass during the wheat-Pst interaction.
To quantify the cDNA of Pst, the standard curves were first created with the Pst translation elongation factor gene via real-time PCR analysis. The threshold cycles (Cq) were plotted against the concentration of cDNA of the Pst race CYR31 (7.215, 3.608, 2.405, 1.804, 0.902 and 0.722 ng/μ l) (Fig. S5). Dissociation curves were generated for each reaction to ensure specific amplification. The quantification of cDNA was performed using a RT-PCR System (Bio-Rad, Hercules, CA, USA). Three independent biological replicates were performed for each experiment.
Expression of TaMCA1 in yeast. The vector pREP3X was used for the overexpression of Schizosaccharomyces pombe 56 . In this system, thiamine was used as a repressor of the pREP3X vector. For the assays of sensitivity to the H 2 O 2 stimuli, the transformed cells were cultured in yeast medium with thiamine at 30 °C with an initial starting optical density at 600 nm (OD600) of 0.2. During the logarithmic phase, the cells were collected by centrifugation, washed thrice with sterile water and finally diluted to densities of 10 6 , 10 5 and 10 4 cell/ml using a blood-counting chamber and then assayed on yeast solid media plates with or without 1. Yeast two-hybrid assay. AtMC1 (At1g02170), TaEIL1 1−650 57 and TaMCA1 were individually inserted into the pGBKT7 vector (pBD); LSD1 (At4g20380) and TaLSD1 (EF553327) were individually inserted into the pGADT7 vector (pAD). The vectors (pBD-AtMC1, pAD-LSD1; pBD-TaEIL1, pAD; pBD-TaMCA1, pAD-TaLSD1) were co-transformed in pairs into the yeast strain AH109, and interactions were tested on selective double dropout/-leucine-tryptophan (SD/-Leu-Trp) media and subsequently on quadruple dropout/-leucine-tryptophan-histidine-adenine (SD/-Leu-Trp-His-Ade) media containing 5-bromo-4-chloro-3-indoxyl-α -D-galactopyranoside (X-α -Gal) as a substrate according to the manufacturer's instructions.