TaMDAR6 acts as a negative regulator of plant cell death andparticipates indirectly in stomatal regulation during thewheat stripe rust–fungus interactionMohamed Awaad Abou-Attiaa,b, Xiaojie Wanga,*, Mohamed Nashaat Al-Attalaa, Qiang Xua,Gangming Zhana and Zhensheng Kanga,*
aState Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Shaanxi, People’s Republic ofChinabIdentification of Microorganisms and Biological Control Unit, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt
We identified a new monodehydroascorbate reductase (MDAR) genefrom wheat, designated TaMDAR6, which is differentially affected bywheat–Puccinia striiformis f. sp. tritici (Pst) interactions. TaMDAR6 is a neg-ative regulator of plant cell death (PCD) triggered by the Bax gene and Pst.Transcript levels of TaMDAR6 are significantly upregulated during a com-patible wheat–Pst interaction, indicating that TaMDAR6 may contribute toplant susceptibility. In addition, H2O2 production and PCD are significantlyinduced and initial pathogen development is significantly reduced in theTaMDAR6 knocked-down plants upon Pst infection. Thus, the suppression ofTaMDAR6 enhances wheat resistance to Pst. Besides, the suppression of TaM-DAR6 during an incompatible interaction induces a change in the morphologyof stomata, which leads to poor stoma recognition and as a consequence toreduced infection efficiency. The percentage of infection sites that developsubstomatal vesicles decreases in the TaMDAR6 knocked-down plants duringthe incompatible interaction presumably due to the increase in ROS accu-mulation, which is likely to activate other resistance mechanisms that have anegative effect on substomatal vesicle formation. TaMDAR6 can therefore beconsidered a negative regulator of PCD and of wheat defense to Pst.
Introduction
Wheat (Triticum aestivum) is one of the most importantcereal crops in the world, which is the primary sourceof vegetable protein in human food (Chandra et al.2014, Langridge 2012). Therefore, research on wheat has
provided a basic understanding of this food crop toimprove its productivity. By contrast, plant diseases havea negative impact on wheat production, and one of themost important diseases of wheat worldwide is the wheatstripe rust, caused by Puccinia striiformis f. sp. tritici (Pst)(Zheng et al. 2013). Allison and Isenbeck (1930) were
the first to establish the existence of races in Pst based ondifferential susceptibility of wheat cultivars. China is thelargest epidemic region in the world (Stubbs 1988, Asadet al. 2012); the most destructive epidemics of stripe rustoccurred in 1950, 1964 and 1990, caused yield lossesof 29.3, 13.3 and 1.8% of the national total production,respectively (Li and Zeng 2000).
Pst is a biotrophic fungus that infects living cells andtakes up water and nutrients from the living host. Infec-tion can occur from the seedling to maturity stage. Thefungus forms yellow to orange colored pustules (ure-dinia), and each uredinium contains thousands of ure-diospores, which spread from the pustules by wind.Infection requires high humidity for 4–6 h at 10–15∘C.Compared to other rust fungi, Pst prefers a lower tem-perature for development, which limits this fungus as amajor disease in many areas of the world (Stubbs 1985,Wellings and McIntosh 1990).
Reactive oxygen species (ROS) are toxic by-productsof many aerobic metabolic processes. Moreover, vari-ous stress conditions cause increases in ROS generation,leading to oxidative damage of lipids, proteins and DNA(Apel and Hirt 2004). Under biotic stress, ROS act assignaling molecules to activate pathogenesis-related pro-teins and systemic acquired resistance in cells adjacentto the infection site to prevent further pathogen spread(Draper 1997). ROS serve as signaling molecules at lowlevels, but can also induce cell death at high levels(Sharma et al. 2012). The production of ROS is normallycounter-acted by an enzymatic anti-oxidative system[catalase (CAT); ascorbate peroxidase (APX); glutathioneperoxidase (GPX); and superoxide dismutase (SOD)],and by a non-enzymatic anti-oxidative system [ascorbicacid (AA); glutathione (GSH); tocopherols (TOCs); andphenolic compounds] to protect plant cells against ROStoxicity. The monodehydroascorbate reductase (MDAR)enzyme is involved in the ascorbate–glutathione cycle,and plays an important role in directly reducing monode-hydroascorbate (oxidized ascorbate) to ascorbate usingNAD(P)H as an electron donor (Apel and Hirt 2004).
The roles of MDAR have been extensively reportedunder abiotic stress such as ozone, salt, polyethyleneglycol (PEG) (Sharma and Davis 1997, Eltayeb et al.2007) and drought (Sharma and Dubey 2005), whichshowed that MDAR genes are regulated by abioticstresses to reduce oxidative damages through maintain-ing redox status (Eltelib et al. 2011). Therefore, MDARgenes have been used as indicators of plant resistanceto several abiotic stresses (Ali et al. 2005, Sharma andDubey 2005). For example, in Brassica campestris, tran-script levels of BcMdhar were strongly upregulatedin response to oxidative stress (Yoon et al. 2004). InPhyscomitrella patens, PpMDHAR1 and PpMDHAR3
were induced during salt stress and osmotic stress (Lundeet al. 2006). In contrast, few studies have examined theroles of MDAR under biotic stress, particularly duringthe wheat–Pst interactions. In our previous studies, weidentified two members encoding MDAR from wheat(TaMDHAR4 and TaMDHAR), which demonstrated thatthe suppression of TaMDHAR4 and TaMDHAR enhanceswheat resistance to Pst (Feng et al. 2014a). Besides,TaMDHAR is regulated by miRNA PN-2013 during Pstinfection (Feng et al. 2014b). TaMDHAR is a cytoplas-mic protein. In addition, TaMDHAR4 has been consid-ered a peroxisomal protein (Feng et al. 2014a,2014b).By contrast, TaMDAR6 is a chloroplastic protein. Plantchloroplasts are the most significant generators of ROS(Ishikawa and Shigeoka 2008, Maruta et al. 2012), andare important elements in the regulation of programmedcell death (PCD), which is an essential component ofplant defense to pathogen attacks. Thus, the sub-cellularlocalization of TaMDAR6 in plant chloroplasts opens thepossibility of the involvement of TaMDAR6 in the regula-tion of PCD. Therefore, TaMDAR6 was specially investi-gated. Our findings demonstrated that TaMDAR6 acts asa negative regulator of PCD. In addition, we reported forthe first time the involvement of TaMDAR6 gene in stom-atal regulation and substomatal vesicles formation duringthe wheat–Pst interaction. Therefore, this study providesnew insights into the role of TaMDAR in the wheat–Pstinteraction.
Materials and methods
Plant material and inoculation
Compatible and incompatible interactions were estab-lished using the wheat cultivar Suwon 11 and twoChinese races of Pst (CYR23 and CYR31). Suwon 11shows high susceptibility to CYR31 and high resistanceto CYR23 (Wang et al. 2010). Plant cultivation andinoculation with Pst were performed according to Kanget al. (2003). Inoculated leaves were sampled, quicklyfrozen in liquid nitrogen and stored at −80∘C until usedfor RNA isolation.
RNA extraction and first strand cDNA synthesis
Total RNA (3 μg) was extracted using the BIOZOL TotalRNA Extraction Reagent (Bioer Technology CompanyLimited, Binjiang, China) following the manufacturer’sprotocol. Any DNA contamination was removed bytreatment with DNase1 before synthesis of cDNA.Extracted RNA was reversely transcribed into cDNAusing the i-SCRIPT Kit (Bio-Rad, Hercules, CA) accordingto the manufacturer’s instructions.
The full-length sequence was obtained by screen-ing of our cDNA database of wheat–Pst interactions(Ma et al. 2009) and the wheat EST database in theNational Center for Biotechnology Information (NCBI).Homologous sequences were assembled using theCAP3 (http://pbil.univ-lyon1.fr/cap3.php/) and multiplesequence alignments were performed using the DNAMAN
6.0 software (Lynnon BioSoft, Pointe-Claire, Canada).Specific primers were designed according to the assem-bled sequence using the PRIMER 5.0 software (Table S1,Supporting Information). The full length open readingframe (ORF) was amplified from cDNA synthesis usingRNA isolated from wheat cv. Suwon 11 leaves afterinoculation with Pst CYR23 (incompatible interaction)or CYR31 (compatible interaction). Amplification wasconducted in a DNA thermocycler (Bio-Rad) usingthe following program: 95∘C for 3 min; 35 cycles of95∘C for 30 s, 55∘C for 30 s, and 72∘C for 70 s; and72∘C for 10 min. The amplified ORF was ligated intothe pGEM T-easy vector (Promega, Madison, WI) fol-lowing the manufacturer’s protocol and transformedinto competent Escherichia coli (JM109) by the CaCl2procedure (Ausubel et al. 1997). Sequencing was per-formed using an ABI PRISM 3130XL Genetic Analyzer(Applied BioSystems, Foster City, CA). The amino acidsequence and conserved domains were analyzed usingNCBI (http://www.ncbi.nlh.nih.gov/gorf/gorf.html), Inter-ProScan (http://www.ebi.ac.uk/cgi-bin/iprscan/), Com-pute pI/MW (http://web.expasy.org/compute_pi/) andProtParam (http://www.expasy.org/tools/pi_tool.html).The prediction of chromosomal location of the corre-sponding gene was performed using the InternationalWheat Genome Sequencing Consortium database(http://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST).The sequence is deposited at GenBank under accessionnumber KP201873.
Quantitative real-time PCR
TaMDAR6 transcript levels were analyzed by qRT-PCR(7500 Real-Time PCR System, Applied Biosystems) usingsynthesized cDNA, as previously explained, at differenttime points after inoculation (12, 18, 24, 48 and 120hpi) and primer pairs (Table 1) specific to the gene ofinterest. Previous time points were chosen according toour previous studies of the interactions between ChinasePst races (CYR31, CYR23) and wheat cv. Suwon 11(Wang et al. 2007). The RT-PCR (real-time PCR) Systemwas programmed as follows: 95∘C for 1 min; 40 cyclesat 95∘C for 10 s, 60∘C for 20 s, and 72∘C for 40 s; 1cycle at 95∘C for 15 s, 60∘C for 1 min and 95∘C for 15 s;and finally 60∘C for 15 s. The relative expression of the
investigated gene was normalized using the referencegene TaEF-1 (GenBank accession number: Q03033),and relative expression was estimated using the 2–ΔΔCT
method (Livak and Schmittgen 2001). Data are themeans of three independent experiments.
Sub-cellular localization of TaMDAR6in Nicotiana benthamiana
To construct the fusion vector pCaMV35S:TaMDAR-GFP,the complete ORF of TaMDAR6 was amplified using spe-cific primers that have restriction sites for SpeI and AvrII(Table 1). The digested amplicon was ligated into the 5′
end of the green fluorescent protein (GFP) coding regionof pCaMV35S:GFP. The newly constructed plasmid wasintroduced into Agrobacterium tumefaciens GV3101through electroporation (Wise et al. 2006). Transformedcells were infiltrated into Nicotiana benthamiana leaves(Van der Hoorn et al. 2000). GFP signals were detectedusing an Olympus BX-51 fluorescence microscope(Olympus Corp., Tokyo, Japan). The experiment wasperformed twice and with three replicates each time.
Agrobacterium-mediated transientgene expression
PVX:BAX, which was kindly provided by Dr D. Dou(Purdue University, West Lafayette, IN), was digestedwith ClaI and XmaI. The BAX fragment was replacedwith GFP to construct PVX:GFP or with TaMDAR6to construct the PVX:TaMDAR (Fig. S3). Agrobacteriacarrying respective plasmids were cultured in 5 ml ofLB medium, supplemented with kanamycin, rifampicinand gentamycin. Cells were harvested at an OD600of 0.6 to 1.2 and resuspended in 10 mM MgCl2. Bac-terial suspensions were adjusted to a final density of0.2–0.5 at OD600. Transient expression assays wereperformed using 4- to 5-week-old N. benthamianaplants. Suspensions were infiltrated into N. benthamianaleaves (abaxial surface) using a 2-ml disposable syringewithout a needle (Van der Hoorn et al. 2000). At leastsix leaves on different plants were used in infiltrationexperiments; each leaf was divided into four sites theninfiltrated with A. tumefaciens cell suspensions as fol-lows; first and second sites were infiltrated with A.tumefaciens carrying PVX:00 (empty vector), third andfourth sites were infiltrated with A. tumefaciens carrying(PVX:TaMDAR). Then the infiltrated leaf was incubatedat 25∘C. After 2 days, the same areas were infiltratedagain as follows; first and third sites were infiltrated withA. tumefaciens carrying (PVX:00), second and fourthsites were infiltrated with A. tumefaciens carrying(PVX:BAX). A. tumefaciens strains carrying PVX:GFP
Table 1. Effect of TaMDAR6 on programmed cell death induced by the mouse Bax gene in Nicotiana benthamiana. aCo-infiltration was performedby infiltrating cultures in Nicotiana benthamiana leaves. bNecrosis activity at 5, 6, 7 and 8 dpi with PVX:EV+ PVX:EV (empty vector as negative control);PVX:BAX+ PVX:EV (as positive control); PVX:TaMDAR+PVX:EV and PVX:BAX +PVX:TaMDAR (co-infiltration); − = no response; + = very weak necrosis;++ = weak necrosis; +++ moderate necrosis; and ++++ = severe necrosis. cNecrotic area % = percentage of necrotic area of the total infiltratedregion.
No visible No visible No visible No visible - - - - 0 0 0 0
PVX:BAX &PVX:TaMDAR(co-infiltration)a
Very smallnecroticlesions
Small necroticlesions
Large necroticlesions
Necrosis + ++ +++ ++++ 10 30±5 80±5 100
were used to confirm the efficiency of our experiments.Pictures were taken 5–8 days after the last infiltration.The experiment was repeated three times.
Virus-induced gene silencing (VIGS)
VIGS was mediated by the barley stripe mosaic virus(BSMV). BSMV vectors (𝛼, 𝛽, 𝛾 and BSMV:PDS4as) wereprovided by Dr Scofield (Purdue University). A139 bpfragment was chosen and amplified from a plasmid con-taining the TaMDAR6 gene using specific primers withrestriction sites for NotI and PacI (Table 1, Fig. S6). ThePCR product was cloned into the pGEM T-simple vector.Extracted plasmids were digested with NotI and PacI, fol-lowed by replacement of the TaPDS coding sequence inBSMV:TaPDS to construct plasmid BSMV:TaMDARas forgene silencing according to Holzberg et al. (2002). Lin-earized plasmids were transcribed into RNA using themMessage T7 in vitro transcription kit (Ambion, Austin,TX) following the manufacturer’s instructions.
The second leaves of wheat seedlings were gentlyinoculated by rubbing the leaf surfaces from base totip two times using a mixture of each transcriptionalreaction. In total, 30 plants were used for each treatment(5 plants/pot), and another 15 plants were inoculatedwith 1× FES buffer (0.1 M glycine, 0.06 M K2HPO4, 1%w/v tetrasodium pyrophosphate, 1% w/v bentonite and1% w/v celite, pH 8.5) as control. Post 9 days virus inoc-ulation, the fourth leaf was inoculated with urediosporesof Pst CYR23 or CYR31. Infection types of Pst wererecorded 15 dpi. The fourth leaves were also sampledfor RNA isolation and histological observation. Relativetranscript levels of TaMDAR6, pathogenesis-related
protein genes (TaPR2, DQ090946; TaPR5, FG618781)and reactive oxygen species-related genes (TaCAT, cata-lase, X94352; TaPOD, class III peroxidase, TC303653)were assessed using qRT-PCR as described above. Theexperiment was repeated three times.
Histological analysis
Samples were stained as described (Wang et al. 2007).According to Parlevliet (1986), aborted substomatalvesicles were considered aborted penetration attempts.Therefore, only infection sites where substomatal vesi-cles had formed were considered. Fungal developmentand host responses for each treatment (a minimum of 50infection sites for each time point) were observed underan Olympus BX-51 microscope (Olympus Corp., Tokyo,Japan) using bright field and UV light. Auto-fluorescenceof mesophyll cells in infected leaves was measuredas necrotic area using epifluorescence microscopy(excitation filter, 485 nm; dichromic mirror, 510 nmand barrier filter, 520 nm). The percentage of stomatathat remained open was determined from records of atleast 100 stomata observed on four independent leaves.Hyphal length, hyphal branches and necrotic area werecalculated using DP-BSW software. Statistical analyseswere performed using SPSS software.
Results
Cloning and sequence analyses of TaMDAR6
Specific primers were designed to amplify the cDNAfragments of the corresponding gene. The full-length
cDNA sequence was isolated from cDNA of wheatcv. Suwon 11, which is highly susceptible to viru-lent Pst race CYR31 and highly resistant to avirulentPst race CYR23 (Wang et al. 2010). The predictedORF of TaMDAR6 spans 1458 bp and encodes a pro-tein of 485 amino acids with a predicted molecularmass of 52.04 kDa. The prediction of protein domainsusing InterProScan and NCBI databases indicated thatTaMDAR6 protein contains pyridine nucleotide-disulfideoxidoreductase domains (pyr redox 2, pyr redox andNAD(P)-binding Rossmann-like domain) (Fig. 1, Fig. S1).In addition, the corresponding gene is a homolog ofAtMDAR6, which encodes an MDAR protein localizedin chloroplasts of Arabidopsis. Hence, the correspond-ing gene was designated as TaMDAR6. The predictionof chromosomal location using the International WheatGenome Sequencing Consortium database (http://wheat-urgi.versailles.inra.fr/Seq-Repository/BLAST) indi-cated that there were three copies in the wheat genome,located on chromosomes 7AL, 7BL and 7DL, respec-tively, indicating that the TaMDAR6 gene family maybe located in the homologous group 7 chromosomes.In addition, phylogenetic analysis and cDNA multiplesequences alignment both revealed that our correspond-ing gene of this study is located on chromosome 7DL(Fig. 2, Fig. S2). Moreover, phylogenetic analysis (Fig. 2,Table S2) revealed that TaMDAR6 is most similar toTaMDAR6-a, BdMDAR chloroplastic isoforms (X1 andX2) from Brachypodium distachyon, and TaMDAR6-b,respectively, but showed the lowest similarity with otherwheat monodehydroascorbate reductase (TaMDHAR4and TaMDHAR).
TaMDAR6 is localized in chloroplastsof N. benthamiana
Agrobacteria carrying pCaMV35S:TaMDAR-GFP wereinfiltrated into N. benthamiana leaves to determinethe sub-cellular localization of TaMDAR6. Fluorescencemicroscopic analysis revealed that green fluorescence ofTaMDAR-GFP was only detected in chloroplast, totallymatched with the red autofluorescence of chlorophyll.This result indicated that TaMDAR-GFP is localized inthe chloroplast of N. benthamiana (Fig. 2B).
TaMDAR6 has a negative effect on programmedcell death (PCD) induced by the Bax gene
To improve our understanding concerning TaMDAR6function in PCD at the molecular level, TaMDAR6 tran-sient expression was performed through the infiltration ofA. tumefaciens into N. benthamiana leaves. To evaluatethe efficiency of our approach, the transient expression
of the GFP gene was investigated. N. benthamiana leaveswere infiltrated with A. tumefaciens carrying PVX:GFP.Fluorescence microscopic analysis revealed that greenfluorescence of PVX:GFP was detected in N. benthami-ana cells. These results indicated that GFP was success-fully expressed, which demonstrated that our transientexpression system was functional (Fig. S4).
N. benthamiana leaves were infiltrated withA. tumefaciens carrying PVX:00 empty vector+ PVX:00empty vector, or PVX:BAX+PVX:00 empty vector, orPVX:TaMDAR+ PVX:00 empty vector, or PVX:Bax+PVX:TaMDAR. As shown in Fig. 3 and in Table 1, nonecrotic area occurred at areas infiltrated with PVX:00+PVX:00, or PVX:TaMDAR+ PVX:00. By contrast, areasinfiltrated with A. tumefaciens carrying PVX:Bax+PVX:00 became completely necrotic within 5 dpi. Todetermine whether TaMDAR6 enhances or suppressesPCD triggered by Bax, A. tumefaciens cultures carryingPVX:Bax and PVX:TaMDAR vectors were infiltrated intoN. benthamiana leaves at the same site. The percentageof infiltrated leaf area that had become necrotic at 5, 6,7 and 8 dpi increased over time, however, this increasewas much slower than observed for the positive control(PVX:Bax+ PVX:00) (Table 1). Only extremely smallnecrotic lesions were observed 6 dpi at the infiltrationarea with PVX:Bax+PVX:TaMDAR, and these lesionsconstantly evolved to include all of the infiltrated areaafter 8 dpi. This result indicates that the area infiltratedwith PVX:Bax+ PVX:TaMDAR became completelynecrotic after 8 dpi, which was much slower than thenecrosis that occurred with PVX:Bax+PVX:00 (Fig. 3).
Transcriptional responses of TaMDAR6to Pst infection
In the incompatible interaction, transcript levels of TaM-DAR6 remained at the control level from 12 to 18 hpi, atranscript peak was observed at 24 hpi, with an approx-imately 2.2-fold increase, and subsequently a return tocontrol levels at 120 hpi (Fig. 4). In the compatible inter-action, transcript levels of TaMDAR6 were changed alittle at 12 to 18 hpi. However, t ranscript levels werestrongly upregulated at 24 and 48 hpi, by approximately3.9- and 4.7-fold, respectively. At 120 hpi, the controllevel was reached again (Fig. 4). Therefore, the relativetranscript level of TaMDAR6 in the compatible interac-tion was much higher than that in the incompatible inter-action, at least at 24 and 48 hpi (Fig. 4).
Suppression of TaMDAR6 enhances wheatresistance to Pst
A virus-induced gene silencing (VIGS) system estab-lished in cv. Suwon 11 was used to evaluate the function
Fig. 1. Alignment of TaMDAR6 (7DL) with monodehydroascorbate reductase proteins from Brachypodium distachyon (BdMDAR chloroplastic isoformsX1 and X2), Triticum aestivum (TaMDAR6-a (7AL), TaMDAR6-b (7BL), TaMDHAR, TaMDHAR4) and Arabidopsis thaliana (AtMDAR6). Black shadow:amino acids are conserved in all sequences, pink shadow: amino acids are conserved among at least six sequences, light blue shadow: amino acidsare conserved between at least five sequences, yellow shadow: amino acids are conserved between at least three sequences. Red line: Pyr_redox2=pyridine nucleotide-disulfide oxidoreductase 2 domains, blue line: NAD(P)-binding Rossmann-like domain, and green line: Pyr_redox=pyridinenucleotide-disulfide oxidoreductase domains. Sequence alignment was performed using DNAMAN6 software.
of TaMDAR6 gene during the interaction between wheatand Pst (Fig. S5). To evaluate the efficiency of our VIGSsystem, we tested the silencing of the wheat phytoenedesaturase gene (PDS). The second leaves of a two-leafwheat seedling was inoculated with BSMV:TaPDSas orBSMV:𝛾 (empty vector) as positive control, or FAS bufferas negative control. As shown in Fig. 5A, mild virussymptoms occurred on the new leaves at 15 dpi. Mean-while, BSMV:TaPDSas inoculated plants showed largephoto-bleached areas on the fourth leaf, while there wereno such symptoms on plants treated with FAS buffer,which showed that our VIGS system was functional.
To make the silencing specific to TaMDAR6 (7DL), a139 bp fragment was chosen and amplified using specificprimers (Table 1, Fig. S6). The fragment located at the 5′
untranslated region (UTR) and the origin of the ORF. Rig-orous efforts were made to choose the VIGS fragment toget a specific silencing construct. Nucleotide sequencesof wheat MDAR genes were downloaded from NCBI andalignment analysis was conducted. Alignment analysisindicated no homology between the TaMDAR6 gene andother wheat MDAR genes in the VIGS fragment (Fig. S6).Al-Attala et al. (2014) and Holzberg et al. (2002) sup-ported the view that a sequence with 81% identity or less
Fig. 2. Phylogenetic and sub-cellular localization of TaMDAR6. (A) Phy-logenetic tree of TaMDAR6 and other monodehydroascorbate reduc-tase proteins: Brachypodium distachyon (BdMDAR chloroplastic iso-forms X1 and X2); Triticum aestivum (TaMDAR6, TaMDAR6-a (7AL),TaMDAR6-b (7BL), TaMDAR6-d (7DL), TaMDHAR, TaMDHAR4); Vitisvinifera (VvMDAR chloroplastic-like); Solanum lycopersicum (SlMDARchloroplastic-like); Nicotiana tabacum (NtMDAR); Cucumis sativus(CsMDAR chloroplastic-like) and Arabidopsis thaliana (AtMDAR6). TaM-DAR6 is our sequence, the sequences of TaMDAR-d (7DL), TaMDAR6-a(7AL) and TaMDAR6-b (7BL) were obtained from the InternationalWheat Genome Sequencing Consortium. The phylogenetic tree wasconstructed using DNAMAN6 software based on the maximum likelihoodmethods (JTT model); bootstrap values (>50%) of 100 replicates areindicated at each branch. (B) Sub-cellular localization of TaMDAR6 inNicotiana benthamiana. Red color is the autofluorescence of chlorophyll(B1, B3); green fluorescence shows the localization of TaMDAR-GFP (B2);yellow indicates co-localization of fusion protein and chlorophyll contain-ing chloroplasts (B3); (B3) Merger of (B1) and (B2). Scale bars=20 μm.
does not cause targeted gene silencing. Therefore, thesecond leaves of a two-leaf tested plant were inoculatedwith BSMV:TaMDARas or with BSMV:𝛾 (empty vector ascontrol). All BSMV-infected plants displayed mild virussymptoms. The fourth leaves were inoculated with aviru-lent race CYR23 for the incompatible interaction or withvirulent race CYR31 for the compatible interaction.
To clarify whether TaMDAR6 was successfullysilenced, transcript levels of all three TaMDAR6 copies(7AL, 7BL, 7DL) were examined using quantitative RT-PCR (qRT-PCR). In comparison to BSMV:𝛾-infectedplants, the expression level of TaMDAR6 (7DL) wasdecreased in the fourth leaves of BSMV:TaMDARas-infected plants at 0, 24, 48 and 120 hpi with Pst by
Fig. 3. Transient overexpression of TaMDAR6 in Nicotiana benthami-ana leaves. Transient expression of TaMDAR6 delayed programmedcell death induced by the Bax gene. A–D comparison betweenphenotypes of localized HR induced by (PVX:BAX+ PVX:EV) and by(PVX:Bax+ PVX:TaMDAR) at different time points (A) 5 dpi, (B) 6 dpi, (C)7 dpi and (D) 8 dpi. Black circles mark the positions of infiltrated areas.
approximately 80, 88, 90.5 and 80% for the incom-patible interaction and by 80, 89.7, 92.2 and 80%for the compatible interaction, respectively (Fig. S7).These results indicated that TaMDAR6 was successfullysilenced. By contrast, transcript levels of other twocopies TaMDAR6-a (7AL) and TaMDAR6-b (7BL) werenot affected (Fig. S7), indicating that the VIGS constructwas very specific to TaMDAR6 (7DL).
Disease development was scored at 15 dpi with Pstraces to evaluate the effect of gene silencing on thewheat–Pst interaction. BSMV:𝛾-infected plants (emptyvector) had no changes in their resistance or susceptibil-ity against CYR31 or CYR23 (Fig. 5B). In the incompatibleinteraction, both treatments (BSMV:TaMDARas andBSMV:𝛾) had no changes in their resistance (infec-tion type 1) according to the 0–9 scale of McNealet al. (1971), HR areas were elicited in both treat-ments with BSMV:TaMDARas and BSMV:𝛾, and nouredosori formed (Table 2, Fig. 5B). In the compatibleinteraction, although heavy sporulation was observedin both treatments (BSMV:TaMDARas and BSMV:𝛾),
Physiol. Plant. 2015
Fig. 4. Relative transcript levels of TaMDAR6 in response to Pst infection in compatible and incompatible interactions at 12, 18, 24, 48 and 120 hpi.Transcripts were quantified using the comparative threshold (2–ΔΔCT) method. Means and standard deviations were calculated using data from threeindependent biological replicates.
BSMV:TaMDARas-infected plants showed a higherdegree of resistance (infection type 8) than control plants(infection type 9) according to the 0–9 scale of McNealet al. (1971). Moreover, PCD areas were elicited aroundthe infection sites in the BSMV:TaMDARas-infectedplants, but no such cell death areas were observed inthe control (Table 3, Fig. 5B).
To demonstrate that the increases of PCD was involvedin the resistance response; the transcriptional responsesof some defense-related genes were examined, becausethose genes have been used as indicators of HR and arenecessary for plant resistance (Schaffrath et al. 1997; VanLoon and Van Strien 2002). The transcriptional responsesof defense-related genes (TaPR2, TaPR5, TaCAT andTaPOD) to TaMDAR6 suppression were determined byqRT-PCR during incompatible and compatible interac-tions at different time points 0, 24 and 48 hpi. Transcriptlevels of PR genes were significantly upregulated dur-ing compatible and incompatible interactions at all timepoints (Fig. 6). By contrast, transcript levels of catalase(TaCAT) and class III peroxidase (TaPOD) were signifi-cantly reduced in the compatible interaction (Fig. 6). Inaddition, there was no significant change in the tran-script levels of TaCAT and TaPOD (reduced a little) inthe incompatible interaction (Fig. 6). These results indi-cated that the hypersensitive and resistance responses ofwheat to Pst were enhanced. Therefore, the increasesof PCD may be involved in the resistance responsesof wheat to Pst.
Histological observation of interactions betweenPst and TaMDAR6 knocked-down plants
To improve our understanding concerning the wheat–Pstinteraction, TaMDAR6 knocked-down plants were
infected by CYR31 (virulent Pst race) or CYR23 (avir-ulent Pst race), then microscopically examined. Inthe incompatible interaction, H2O2 production at theinteraction sites gradually increased in the TaMDAR6knocked-down plants at 24 and 48 hpi. In addition,hypersensitive cell death seemed to be enhanced inthe TaMDAR6 knocked-down plants at 48 hpi (Table 2,Fig. S8). Growth of hyphae was significantly reduced atboth time points (Table 2). Numbers of hyphal branchesand of haustorial mother cells were also significantlyreduced in the TaMDAR6 knocked-down plants at 24and 48 hpi (Table 2, Fig. S8).
In the compatible interaction, hyphal growth wasalso significantly shorter than that observed in theBSMV:𝛾-infected plants at 24 and 48 hpi (Table 3).Numbers of hyphal branches, haustorial mother andhaustoria cells were significantly reduced in the TaM-DAR6 knocked-down plants at 48 hpi (Table 3, Fig. S8).Moreover, H2O2 production at the site of interactionwas induced in the TaMDAR6 knocked-down plantsat 48 hpi (Table 3, Fig. S8). PCD was induced in theTaMDAR6 knocked-down plants at 120 hpi. However, at120 hpi, H2O2 production areas were too large, leadingto difficulties in taking other measurements (hyphalgrowth, numbers of hyphal branches and haustorialmother cells).
In the incompatible interaction, urediospores germi-nated well in BSMV:𝛾 (empty vector as control) andBSMV:TaMDARas plants at all time points. In controlplants, Pst germ tubes reached the stomata, with suc-cessful penetration at 24 and 48 hpi. In the TaMDAR6knocked-down plants, Pst germ tubes also reachedstomata, however, with successful penetration onlyat 24 hpi. At 48 hpi, there were two observations:
Fig. 5. Analysis of TaMDAR6 using VIGS. (A) Phenotypes of wheatleaves after inoculation with FAS buffer (Mock) or BSMV:𝛾 orBSMV:TaMDARas or BSMV:TaPDSas. Mild virus symptoms and largephoto-bleached areas appeared on the fourth leaves of infected plantsat 15 dpi. (B) Phenotypes of the fourth leaves of inoculated plants withFAS buffer or BSMV:𝛾 or BSMV:TaMDAR followed by challenge with viru-lent (CYR31) or avirulent (CYR23) races of stripe rust. BSMV:𝛾: constructcarrying only the BSMV genome (empty vector); BSMV:PDS: silencingconstruct for PDS; BSMV:TaMDAR: silencing construct for TaMDAR6. Typ-ical leaves were photographed at 15 dpi.
(1) germ tubes did not end over stomata, and (2) germtubes grew over stomata, in both cases not penetratingthe stoma (Fig. S9). Moreover, in comparison with controlplants, the percentage of stomata that remained openin BSMV:TaMDARas-infected plants decreased at 24and 48 hpi by approximately 10 and 53%, respectively(Table 2). Meanwhile, a significant change in stomatalshape (length×width) of BSMV:TaMDARas-infectedplants could be observed at 48 hpi (Table 2, Fig. 7).However, no significant difference in stomatal shape Ta
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Physiol. Plant. 2015
Fig. 6. Expression of resistance-related genes in the TaMDAR6knocked-down wheat in response to Pst infection during (A) anincompatible interaction and (B) a compatible interaction. Tran-script levels were assayed using qRT-PCR. TaPR2, 𝛽-1,3-glucanase(DQ090946); TaPR5, Thaumatin-like protein (FG618781); TaCAT , cata-lase (X94352) and TaPOD, class III peroxidase (TC303653). BSMV:𝛾- andBSMV:TaMDARas-infected leaves were sampled 0, 24 and 48 hpi withPst. BSMV:𝛾 construct carrying only the BSMV genome (empty vector).Relative transcript levels were quantified using the comparative thresh-old (2–ΔΔCT) method. Asterisks indicate a significant difference (P <0.05)from the non-silenced plants using t-test.
was observed between BSMV:TaMDARas-infected andBSMV:𝛾-infected leaves at 24 hpi (Table 2, Fig. 7).Besides, the percentage of the infection sites where sub-stomatal vesicles had formed underneath the stomata(successful penetration) in the TaMDAR6 silenced plantswas lower than that in control plants at 48 hpi by approx-imately 56% (Table 2). In the compatible interaction,there was no significant change in stomatal behavior orin percentage of infection sites (Table 3). Ta
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Physiol. Plant. 2015
Fig. 7. Stomatal shape on wheat leaves inoculated with BSMV:𝛾 (empty vector) or BSMV:TaMDARas during an incompatible interaction. Suppressionof TaMDAR6 induced changes in stomatal morphology. (A) Stoma on control plants at 24 hpi. (B) Stoma on the TaMDAR6 knocked-down plants at 24hpi. (C) Stoma on control plants at 48 hpi. (D) Induced changes in stomatal shape on the TaMDAR6 knocked-down plants at 48 hpi. Scale bars=20 μm.
Suppression of TaMDAR6 induces H2O2accumulation without Pst attack
To demonstrate that the suppression of TaMDAR6induces ROS accumulation, wheat leaves were infectedwith BSMV:TaMDARas or with BSMV:𝛾 without Pstinoculation. In comparison to BSMV:𝛾-infected leaves,H2O2 production was induced in the fourth leaves ofBSMV:TaMDARas-infected plants (Table S3, Fig. 8). Inaddition, there also was no significant change in stomatalshape in the fourth leaves of BSMV:TaMDARas-infectedplants (Table S3).
Discussion
TaMDAR6 functions as a negative regulator of PCD
PCD is an important element for plant immunity againstbiotic and abiotic stress as well as for plant developmentand proliferation (Gadjev et al. 2008), including vac-uolar cell death, necrosis and hypersensitive cell death(Van Doorn et al. 2011). In fact, plant hypersensitive celldeath occurs during plant infection by hemibiotrophicand biotrophic pathogens, and displays the same micro-scopic features of both necrosis and vacuolar cell death
Fig. 8. Microscopic observation of H2O2 accumulation in the TaMDAR6knocked-down wheat without pathogen infection. The fourth leaves ofthe wheat seedlings were infected with (A) BSMV:𝛾 (empty vector) or(B) BSMV:TaMDARas, and stained with 3,3-diaminobenzidine. Areas ofH2O2 production were measured by DP-BSW software and calculated fromat least 10 fields for each segment. Scale bars=20 μm.
(Van Doorn et al. 2011). In addition, ROS have been con-sidered as key factors of the induction and modulationof the PCD during plant–pathogen interaction (Desikan
et al. 1998, Gadjev et al. 2008). The lifetime of ROSwithin the cellular environment is determined by theantioxidative system, which provides crucial protectionagainst oxidative damage (Apel and Hirt 2004). As men-tioned, MDAR proteins also play important roles in ROSaccumulation. It is worth mentioning that plant chloro-plasts are considered as the most important sources ofROS (Ishikawa and Shigeoka 2008, Maruta et al. 2012).In addition, the role of TaMDAR6 encoding chloroplas-tic TaMDAR protein in the wheat–Pst interaction andin PCD has not been reported. Therefore, it is temptingto speculate that the chloroplastic TaMDAR protein mayparticipate in PCD regulation. To study this hypothesis,we isolated the TaMDAR6 gene encoding chloroplasticTaMDAR from wheat, then studied its role in PCD trig-gered by a mammalian Bax gene and by Pst.
The mammalian Bax gene is an animal pro-apoptoticprotein, absent in plants, which induces PCD (HR-likecell death) primarily through the accumulation ofROS similar to plant hypersensitive cell death (Caiand Jones 1998, Fujita et al. 1998, Aravind et al.1999, Kawai-Yamada et al. 2001). The co-infiltrationof TaMDAR6 together with Bax resulted in a slowercell death than that induced by Bax alone. Thisfinding is consistent with most reports that haveindicated plant antioxidants as a negative regulatorof Bax-induced PCD (Kampranis et al. 2000, Moonet al. 2002 and Chen et al. 2004). In these studies,the over-expression of plant antioxidants (ascorbateperoxidase; glutathione-S-transferase/peroxidase, andphospholipid hydroperoxide glutathione peroxidase)reduces ROS accumulation and suppresses Bax expres-sion in N. benthamiana and in yeast. The authorsconclude that plant antioxidants suppress Bax-inducedPCD through decreasing ROS generation. Takentogether, we suggest that the differences observed in thenecrotic development are caused by TaMDAR6 expres-sion, which in turn functions as a negative regulator ofPCD triggered by Bax.
In this study, a VIGS approach was used to deter-mine the role of TaMDAR6 (located on chromosome7DL) in pathogen-induced cell death. Macroscopic andmicroscopic analyses showed that the average necroticarea was significantly increased in the TaMDAR6knocked-down plants upon Pst infection. El-Zahabyet al. (1995) stated that plant antioxidants suppress theformation of necrotic symptoms during barley–Bghinteractions. Therefore, we conclude that silencing ofTaMDAR6 is able to increase PCD during the wheat–Pstinteractions. Meanwhile, H2O2 accumulation was sig-nificantly increased in the TaMDAR6 knocked-downplants over time, suggesting that increase in necrotic
area may be linked to an increase in H2O2 accumu-lation, indicating that TaMDAR6 silencing increasesPCD through increased H2O2 accumulation. Thus,TaMDAR6 has a negative effect on PCD through reduc-ing H2O2 accumulation. Therefore, we concludethat TaMDAR6 is involved in PCD during wheat–Pstinteractions.
TaMDAR6 has a negative role in Pst recognition
Our previous histological studies stated that an oxida-tive burst was triggered following the recognition of elic-itor(s), released from haustoria of an avirulent race bya receptor of the host cell (Wang et al. 2007, 2010).From this study, we detected H2O2 accumulation atthe infection sites after TaMDAR6 was knocked-downin the compatible interaction, indicating that the wheatcells became able to recognize the virulent race of Pst.Thus, the interaction between pathogen elicitors andhost receptors probably activates a signal transductioncascade that involves H2O2 accumulation. ThereforeTaMDAR6 has a negative role in pathogen recognitionduring the wheat–Pst interaction. Furthermore, hyphalgrowth, the number of hyphal branches, and the num-ber of haustoria and haustorial mother cells were signif-icantly reduced in the TaMDAR6 knocked-down plantsfor both interactions. These results open the possibilitythat increased H2O2 production reduces initial growthof Pst, consistent with the findings of Feng et al. (2014a,2014b) in wheat against Pst during suppression of TaMD-HAR4 and TaMDHAR genes. Ellingboe (1972) reportedthat development of hyphae is a good indicator for estab-lishment of a compatible interaction. Therefore, we con-clude that the knocking down of TaMDAR6 enhancesplant resistance to Pst.
TaMDAR6 negatively regulates ROS accumulation
Transcript levels of TaMDAR6 were upregulated uponPst infection in compatible and incompatible interac-tions. Therefore, we suggest that TaMDAR6 is involvedin wheat–Pst interactions. Moreover, transcript levelsof TaMDAR6 were higher in a compatible interactionthan that in incompatible interaction. Burhenne andGregersen (2000) and Vanacker et al. (1998), stated thatthe inoculation of barley leaves with Blumeria graminis(Bgh) causes significant increases in the expression levelsand activities of antioxidant enzymes such as ascorbateperoxidase in compatible interactions more than incom-patible interactions, suggesting that antioxidant enzymesmay contribute to plant sensitivity against Bgh infection.Feng et al. (2014a) described that transcript levels ofTaMDHAR4 gene were downregulated at 12–18 hpi
Physiol. Plant. 2015
during incompatible interaction then upregulated at48 hpi in both interactions. The authors suggested thatsuppression of TaMDHAR4 might be important to theincompatible interaction. In contrast, transcript levelsof TaMDAR6 remained at the control level from 12to 18 hpi then upregulated at 24 and 48 hpi in bothinteractions. Therefore, it is tempting to speculate thatthere may be a required level of TaMDAR6 expression inwheat plants to show incompatible interaction againstPst, and that an increase in that level may contribute toplant sensitivity against Pst.
In this study, we studied for the first time the relation-ship between TaMDAR6 gene and substomatal vesiclesformation and stomata regulation during the Pst–wheatinteraction. The knocking down of TaMDAR6 duringwheat–Pst interactions reduced disease symptoms andinduced HR, suggesting that the suppression of TaM-DAR6 increases wheat resistance against Pst. Besides,histological observations of the Pst inoculated plantsindicate that urediospores germinated successfully inboth wheat–Pst interactions. However, the percentageof the infection sites where substomatal vesicles formed(successful penetration) was influenced by the knockingdown of TaMDAR6 in the incompatible interaction.Meanwhile, H2O2 accumulation and the expressionlevel of genes encoding pathogenesis-related proteinssignificantly increased over time in the TaMDAR6knocked-down plants. Dangl and Jones (2001) statedthat H2O2 accumulation could activate many plantdefenses to eliminate the pathogen. In addition, Broersand López-Atilano (1996) during a study of the effectsof wheat resistance on the development of Pst, sug-gested that some of the formed substomatal vesiclesdisintegrated later by PR proteins that were activatedafter pathogen colonization. Therefore, we concludethat the decrease in substomatal vesicle formationmight be linked to an increase in H2O2 accumulation,which in turn may trigger other resistance mechanismsthat have a negative effect on the formed substomatalvesicles.
Furthermore, some of the germ tubes of Pst in theincompatible interaction did not reach the stomata orgrew over them without penetration, suggesting thatthe germ tubes became unable to recognize stomataon the TaMDAR6 knocked-down plants. In addition,non-recognition of stomata by the germ tubes of Pstwas associated with changes in the shape of the stom-ata and with decreases in the percentage of stomatathat remained open in the incompatible interaction.This suggests that the suppression of TaMDAR6 inducedchanges in stomatal morphology and enhanced stom-atal closure on the TaMDAR6 knocked-down plants inthe incompatible interaction. In addition, the inability of
Pst germ tubes to recognize stomata may be triggeredby changes in morphological features of the stomata.Moreover, changes of stomatal shape in the TaMDAR6knocked-down plants might be triggered by an increasein H2O2 accumulation in the incompatible interaction.Allen et al. (2000) and McAinsh et al. (1996) statedthat H2O2 has a marked effect on stomatal behavior,induces changes in guard cells and promotes stomatalclosure. In addition, TaMDAR6 localizes to the chloro-plast of N. benthamiana. Maruta et al. (2012) statedthat plant chloroplasts are a major source of H2O2.Thus, TaMDAR6 plays important role in H2O2 regula-tion. Therefore, we conclude that TaMDAR6 participatesindirectly in stomatal regulation through its effects onH2O2 accumulation during the wheat–Pst incompati-ble interaction. According to these results, the knock-ing down of TaMDAR6 enhances plant responses toavoid further penetration, reducing Pst infection effi-ciency through reducing the probability of Pst germ tubesto locate or recognize the stomata during incompatibleinteraction.
In contrast, there was no significant change in stomatalbehavior in silenced plants for either compatible inter-action or non-inoculated plants even though there wasan increase of the H2O2 accumulation in both cases.Bhattacharjee (2012) and Melillo et al. (2006), statedthat ROS may induce different types of responses dur-ing plant infection, depending on their level. Therefore,these results open the possibility that H2O2 levels in bothcases were not sufficient to induce significant changesin stomatal behavior or to reduce the percentage of theinfection sites in the compatible interaction. Therefore,we conclude that change in stomatal behavior dependson the level of ROS.
Collectively, the results of this study provide strong evi-dence that TaMDAR6 functions as a negative regulatorof PCD. In addition, the knocking down of TaMDAR6induces ROS accumulation, which triggers other resis-tance mechanisms, depending on their level, such aschanges in stomatal behavior. Thus, the knocking downof the TaMDAR6 gene enhances the resistance of wheatto Pst.
Author contributions
M. A. A. A., X. W. and Z. K. performed conception,design and interpretation of data; M. A. A. A., M. N. A.A., Q. X.; G. Z. performed the experiments; M. A. A. A.and X. W. analyzed the data; M. A. A. A., X. W. draftedthe article or revised; Z. K. contributed the final approvalof the version to be published.
Physiol. Plant. 2015
Acknowledgements – This study was supported bythe National Basic Research Program of China (No.2013CB127700), the Key Grant Project of the ChineseMinistry of Education (313048), the National NaturalScience Foundation of China (No. 31271990), the Youngstar of science and technology, Shaan’xi (2012KJXX-15)and by the 111 Project from the Ministry of Education ofChina (B07049). We thank Nagi Abou-zeid of the PlantPathology Research Institute, ARC, Egypt for his adviceand continuous guidance; S. R. Scofield of the Departmentof Agronomy, Purdue University, West Lafayette, USA forproviding BSMV vectors; and D. Dou for providing transientexpression vectors.
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Supporting Information
Additional Supporting Information may be found in theonline version of this article:
Table S1. Primers used in this study.
Table S2. Homology matrix of TaMDAR6 and othermonodehydroascorbate reductase proteins from differentplants.
Table S3. Histological observations of the TaMDAR6knocked-down plants without pathogen infection.
Fig. S1. Prediction of conserved domains using the NCBIdatabase.
Fig. S2. cDNA multiple sequence alignment of all TaM-DAR6 copies.
Fig. S3. Maps of the binary PVX-based expression vec-tors.
Fig. S4. Transient overexpression GFP in Nicotiana ben-thamiana.
Fig. S5. BSMV-mediated VIGS system.
Fig. S6. Alignment of the nucleotide sequences of TaM-DAR6 with other MDAR genes from wheat.
Fig. S7. Relative transcript levels of all three TaMDAR6copies in silenced and non-silenced plants.
Fig. S8. Wheat–Pst interactions in response to suppres-sion of TaMDAR6 gene.
Fig. S9. Micrographs of germlings of Pst on the TaM-DAR6 knocked-down wheat leaves in the incompatibleinteraction.
