• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Isolation and characterization of a novel wall-associated kinase gene TaWAK5 in wheat(Triticum aestivum)

    2014-03-13 05:50:30KunYangLinQiZengyanZhang
    The Crop Journal 2014年5期

    Kun Yang,Lin Qi,Zengyan Zhang*

    The National Key Facility for Crop Gene Resources and Genetic Improvement,Key Laboratory of Biology and Genetic Improvement of Triticeae Crops,Ministry of Agriculture,Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China

    1.Introduction

    Plants are continuously threatened by a broad range of pathogens,including fungi,oomycetes,viruses,and bacteria.To defend themselves against pathogen attack,plants have evolved an array of response systems,in which external cues are deciphered and translated into effective defense responses [1].Receptor-like kinases (RLKs) play fundamental roles in the perception of external stimuli and activate defense-associated signaling pathways,thereby regulating cellular responses to pathogen infection[1].For example,FLAGELLIN SENSTIVE2 (FLS2) and bacterial translation elongation factor EF-Tu receptor(EFR)act as pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns(PAMPs)and play key roles in PAMP-triggered immunity in Arabidopsis thaliana[2,3].The cell surface receptor chitin elicitor receptor kinase 1 of Arabidopsis (AtCERK1) directly binds chitin through its lysine motif (LysM)-containing ectodomain(AtCERK1-ECD)to activate defense responses[4].

    Wall-associated kinases (WAKs) and WAK-like kinases(WAKLs) are a unique RLK subfamily that contains excellent candidates which may directly link and enable communication between the extracellular matrix (ECM) and the cytoplasm[5,6].WAK proteins possess a typical cytoplasmic Ser/Thr kinase signature,and have an extracellular domain(ectodomain) with similarity to vertebrate epidermal growth factor (EGF)-like domains [7].WAKs have been shown to perceive damage-associated molecular patterns (DAMPs),which are comprised of the pectin and oligogalacturonide(OG)molecules that are released from the plant cell wall following damage caused by pathogen attack.WAKs then function to communicate these damage signals,thereby modulating both plant defense and development[5,8].

    In Arabidopsis,26 WAK/WAKL genes have been identified.Five of these WAK genes(AtWAK1–5)were shown to be clustered on chromosome 1.Certain WAK homologues have been identified in rice(Oryza sativa),tobacco(Nicotiana tabacum),maize(Zea mays),barley (Hordeum vulgare),and wheat(Triticum aestivum)[9].AtWAK1 in Arabidopsis is the most studied WAK receptor kinase.The transcription of AtWAK1 is induced by OG molecules and salicylic acid(SA)[10].AtWAK1 was shown to bind OG molecules and to mediate the perception of OG molecules [5].Transgenic plants overexpressing AtWAK1 showed elevated resistance to the necrotrophic pathogen Botrytis cinerea[5].Both AtWAK1 and AtWAK2 were shown to bind pectin in vitro.AtWAK2 was shown to be required for the pectin-induced activation of numerous genes,many of which were involved in defense responses [8].OsWAK1 transcript was significantly induced after infection with the rice blast fungus (Magnaporthe oryzae) and also induced following treatment with exogenous SA or methyl jasmonate(MeJA).Transgenic rice lines overexpressing OsWAK1 showed enhanced resistance to M.oryzae strain P007[11].Although four WAKs (TaWAK1–4) and two WAKLs (TaWAKL1 and TaWAKL2)have been isolated from wheat[12],their functional roles remain poorly understood.

    Phyto-hormones,including SA,JA,ethylene,and abscisic acid(ABA),are known to play important roles in plant responses to biotic and abiotic stresses[13–19].Upon microbial attack,plants modify the relative abundance of these hormones as a defense mechanism that can then activate efficient defense responses at the molecular genetic level,enabling plant survival [20].SA is involved in recognition of pathogen-derived components and the subsequent establishment of local and systemic acquired resistance [21,22].JA and ethylene signaling act synergistically and regulate induced systemic resistance.ABA also plays an important role in plant defense response,and the ABA signaling pathway interacts with other phyto-hormone signaling pathways in plant defense responses[23–25].

    Wheat is one of the most important staple crops in the world and plays a fundamental role in food security.Sharp eyespot disease,mainly caused by the necrotrophic fungal pathogen R.cerealis,is one of the most devastating diseases in wheat production [26,27].In infected wheat plants,R.cerealis may destroy the stems and sheaths of host plants and can cause lodging and dead spikes[27].Few wheat genes involved in wheat defense responses to R.cerealis have been identified or characterized to date.Moreover,it is not known whether protein kinases participate in wheat responses to the pathogen infection during the developing process of sharp eyespot disease.

    The goal of this research was to understand the roles of WAKs in wheat defense responses to R.cerealis infection.By using the Agilent wheat microarrays,we studied the transcriptomic profiles of WAK/WAKL genes in resistant and susceptible wheat lines following inoculation with R.cerealis.A WAK gene named TaWAK5 was identified to be significantly up-regulated at 21 days post inoculation (dpi) in the resistant wheat line CI12633 as compared with susceptible wheat cultivar Wenmai 6.This paper reports the identification,molecular characterization,and functional analysis of TaWAK5.The transcript abundance of TaWAK5 was markedly induced after R.cerealis infection.Its expression was also induced following exogenous application of SA,ABA,and MeJA.The protein was localized at the sub-cellular level to the plasma membrane in onion cells.We further analyzed the function of TaWAK5 in wheat defense responses to R.cerealis using virus-induced gene silencing(VIGS)technique.

    2.Materials and methods

    2.1.Plant and fungal materials and treatments

    Six wheat (T.aestivum L.) lines/cultivars exhibiting different levels of resistance and susceptibility to R.cerealis were used in this study.They included CI12633 and Shanhongmai(resistant to R.cerealis); Navit 14,and Shannong 0431 (moderately resistant to R.cerealis);Wenmai 6(susceptible to R.cerealis);and Yangmai 158(moderately susceptible to R.cerealis)[28].

    A major Jiangsu virulent isolate strain of pathogen fungus R.cerealis causing the sharp eyespot disease,R0301,was provided by Profs.Huaigu Chen and Shibin Cai from Jiangsu Academy of Agricultural Sciences,China.

    Wheat plants were grown in a 14 h light/10 h dark(22 °C/10 °C) regime.At the tillering stage,the 2nd base sheath of each wheat plant was inoculated with small toothpick fragments harboring well-developed mycelia of the pathogen R.cerealis following Chen [27].Mock treatment (control) plants were inoculated with small toothpick fragments soaked in liquid potato dextrose agar(PDA).Inoculated plants were grown at 90%relative humidity for 4 days.The inoculated stems were sampled at 0,4,7,10,14,and 21 days post inoculation,quickly frozen in liquid nitrogen,and stored at-80 °C prior to total RNA extraction.At 4 dpi,the roots,sheaths,stems,and leaves of the inoculated CI12633 plants were collected,respectively.At 45 dpi,the roots,stems,leaves,and young spikes of the inoculated CI12633 plants were separately sampled and used for RNA extraction and the tissue expression profiles of TaWAK5.

    In additional experiments,the seedlings at the three-leaf stage of the resistant line CI12633 were treated with phyto-hormones,including 1.0 mmol L-1SA,0.1 mmol L-1MeJA (JA analog),ethylene released from 0.2 mmol L-1ethephon,and 0.2 mmol L-1ABA,following Zhang et al.[29].Leaves were collected for RNA extraction at 0,1,3,6,12,and 24 h after treatment with these hormones.

    2.2.RNA extraction and cDNA synthesis

    Total RNA was extracted using TRIzol reagent(Qiagen,China)according to the manufacturer's instructions.DNase I treatment was used to remove genomic DNA.First-strand cDNA was synthesized using 2 μg purified RNA,AMV reverse transcriptase,and oligo (dT15) primers (TaKaRa Inc.,Tokyo,Japan) according to the manufacturer's instructions for the cDNA synthesis kit.

    2.3.Cloning of full-length sequence of TaWAK5 cDNA

    Based on microarray analysis results,a partial cDNA fragment(GenBank accession number CA642360),which was differentially expressed between the resistant wheat genotype CI12633 and the susceptible wheat Wenmai 6,was identified.Based on the sequence of CA642360 and using a 3′-Full RACE Core Set kit v.2.0 from TaKaRa Inc.,the sequence of the 3′ untranslated region (UTR) was amplified from cDNA of CI12633 stems that had been challenged with the pathogen R.cerealis for 21 days.Using CA642360 as a query sequence,the DNA sequence was retrieved from the Cereals Data Base (Cereals DB,http://www.cerealsdb.uk.net/CerealsDB/Documents/ DOC_CerealsDB.php),and the gene structure was analyzed using SoftBerry FGENESH software program in LINUX system (http://linux1.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind).Gene-specific primers TaWAK5-ORF-F/TaWAK5-ORF-R were then designed and used to amplify the full-length open reading frame(ORF)sequence of TaWAK5 from the cDNA of the CI12633.The purified PCR products were cloned to the pMD-18T vector from TaKaRa Inc.and selected to identify the positive clones.Five positive clones were then sequenced with an ABI PRISM 3130XL Genetic analyzer (Applied Biosystems,Foster City,CA).The full-length cDNA sequence of the resulting TaWAK5 gene with 2282 bp length was obtained by analyzing the aligned sequences.

    The TaWAK5 gene was analyzed using several bioinformatics tools.First,the cDNA sequence data was analyzed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and ORF Finder(http://www.ncbi.nlm.nih.gov/gorf/).The deduced protein sequence was then analyzed with the Compute pI/Mw tool(http://web.expasy.org/compute_pi/)which is used for computation of the theoretical iso-electric point and protein molecular weight,InterPro-Scan(http://www.ebi.ac.uk/interpro/)for domain identification and Smart software(http://smart.embl-heidelberg.de/smart/set_mode.cgi? GENOMIC = 1) for prediction of the conserved motifs of domains.DNAMAN software was then used for sequence alignment and MEGA 5.0 software for constructing a phylogenetic tree.The region upstream (1000 bp) of the start codon was analyzed using the plant cis-acting regulatory DNA element(PLACE)database(http://www.dna.affrc.go.jp/PLACE/).

    2.4.Subcellular localization of TaWAK5

    The coding region of TaWAK5 lacking the stop codon was amplified using gene-specific primers TaWAK5-GFP-F/TaWAK5-GFP-R.The amplified fragment was digested with restriction enzymes Pst I and Xba I,then subcloned in-frame into the 5′-terminus of the GFP(green fluorescent protein)coding region in the pCaMV35S:GFP vector (kindly provided by Dr.Daowen Wang,Chinese Academy of Sciences),resulting in the TaWAK5-GFP fusion construct pCaMV35S:TaWAK5-GFP.

    The p35S:TaWAK5-GFP fusion construct or p35S:GFP control construct was separately bombarded into epidermal cells of a white onion according to the protocol described by Zhang et al.[30].To induce the expression of the introduced GFP proteins,the transformed onion cells were incubated at 25 °C for 16 h.The GFP signals were then observed and photographed using a Confocal Laser Scanning Microscope (Zeiss LSM 700,Germany) with a Fluar 10X/0.50M27 objective lens and an SP640 filter.The plasmolysis of the onion cells was undertaken by addition of 0.8 mol L-1sucrose solution for 5 min,as described by Lang-Pauluzzi and Gunning[31].

    2.5.Functional analysis of TaWAK5 through virus-induced gene silencing

    A virus-induced gene silencing (VIGS) technique was previously developed with barley stripe mosaic virus (BSMV) and found to be an effective reverse genetics tool for investigating rapidly the functions of some genes in barley and wheat[32,33].To generate a BSMV:TaWAK5 construct,a 298 bp sequence of TaWAK5 (from nucleotide position 1913 to 2211 in the TaWAK5 cDNA sequence)was amplified from the cDNA sequence for TaWAK5 from the genotype CI12633 with the primers TaWAK5-VIGS-F/TaWAK5-VIGS-R.PCR-amplified cDNA fragments were digested with Pac I and Not I,then ligated into the BSMV:RNAγ vector digested with Pac I-Not I,resulting in the recombinant construct RNAγ:TaWAK5-as.

    Following a previously described protocol [32],the tripartite cDNA chains of BMSV:TaWAK5,or the control virus BMSV:GFP genome,were separately transcribed into the RNAs,then mixed and used to infect CI12633 plants at the 2-leaf stage.At the same time,CI12633 plants were inoculated with only the buffer without virus.Hereafter,these plants treated only with buffer are referred to as mock treatments.The 4th leaves of the inoculated seedlings were collected and analyzed for the virus infection based on the RNA transcript presence of the BSMV coat protein gene using primers BSMV-CP-F/BSMV-CP-R.These tissues were also evaluated for changes in TaWAK5 expression with primers TaWAK5-Q-F/TaWAK5-Q-R at 10 days after BSMV infection.

    For R.cerealis inoculation,the fungus was cultured on potato dextrose agar at 25 °C for 10 days,then 1 cm2plugs from the edge of R.cerealis colonies were placed into liquid PDA medium and cultured at 25 °C for 2 weeks,to develop the mycelia.The 4th base sheath of wheat plants was inoculated with 15 μL of the R.cerealis liquid culture at 20 days after BSMV virus inoculation.

    Inoculated plants were grown at 90%relative humidity for 4 days.Sharp eyespot symptoms were observed respectively at 14 days and 40 days after fungal inoculation.These are the times when sharp eyespot symptoms are normally present at the infected sheaths and stems,respectively,of the susceptible cultivar Wenmai 6.

    2.6.RT-PCR and real-time quantitative RT-PCR (qRT-PCR)analysis

    RT-PCR was performed with 20 μL reaction volumes from the TaKaRa Inc.kit containing 1× PCR buffer,2.0 μL 10× first strand cDNA,150 μmol L-1of each dNTP,and 1 U Taq polymerase,plus 0.25 μmol L-1of each primer.The program used was as follows: initial denaturation at 94 °C for 5 min;followed by 30 cycles of 30 s at 94 °C,30 s at 60 °C,and 30 s at 72 °C;and final extension at 72 °C for 5 min.The PCR products were detected on 2%agarose gels.In all the semi-quantitative RT-PCR experiments,wheat elongation factor 1 alpha-subunit(TaEF-1a) was used to normalize the cDNA contents among various samples.

    qRT-PCR was performed using SYBR Green I Master Mix from TaKaRa Inc.in a volume of 25 μL on an ABI 7300 RT-PCR system (Applied Biosystems Corp.).Reactions were set up with the following thermocycling profile: 95 °C for 5 min,followed by 41 cycles of 95 °C for 15 s and 60 °C for 31 s.The products were continuously examined with a melting curve analysis program.All qRT-PCR reactions were repeated three times.The relative expression of the gene TaWAK5 was calculated with the 2-ΔΔCTmethod [34],where the wheat TaActin gene was used as the internal reference.

    The sequences of primers used are listed in Table S1.

    3.Results

    3.1.The transcription of TaWAK5 is induced by R.cerealis infection

    Microarray analysis is a frequently used molecular genetic technique for the identification of target genes that are expressed differentially between different plant tissue samples or the same samples under different treatments.In this study,we used Agilent wheat microarrays to identify WAK genes that were differentially expressed between the resistant wheat genotype CI12633 and susceptible wheat cultivar Wenmai 6 following infection with R.cerealis.Based on differentially-expressed gene analysis,a wheat cDNA fragment CA642360 had a 30-fold increase in transcript level in the resistant CI12633 as compared with the susceptible Wenmai 6 at 21 dpi.BLAST searching against the GenBank database showed that this gene was homologous to the genes encoding WAKs in plants.As four WAK genes,TaWAK1,TaWAK2,TaWAK3,and TaWAK4,were isolated from wheat in a previous study [12],hereafter,this novel wheat WAK gene induced by R.cerealisis designated as TaWAK5.

    To further investigate the involvement of TaWAK5 in wheat responses against R.cerealis,qRT-PCR was used to analyze the transcript profile of TaWAK5 in wheat infected with the fungal pathogen R.cerealis.The analysis over a 21-day pathogen inoculation time-series showed that TaWAK5 was induced by R.cerealis infection in both the resistant CI12633 and in the susceptible Wenmai 6,whereas the induction degree was higher in CI12633 as compared to Wenmai 6 (Fig.1-A).The expression level of TaWAK5 in CI12633 was about 15 times higher than the level in Wenmai 6 at 21 dpi,consistent with the result of the microarray analysis and with the level of resistance displayed by the genotypes.Following R.cerealis infection,TaWAK5 transcripts in the resistant CI12633 were induced at 4 dpi,reached a first peak at 10 dpi (about 24-fold increase over 0 dpi),decreased at 14 dpi,and reached a second peak at 21 dpi (about 33-fold increase over 0 dpi).

    Meanwhile,the expression of TaWAK5 in different tissues of the R.cerealis-inoculated CI12633 was assessed using qRT-PCR (Fig.1-B).At 4 dpi,the TaWAK5 gene was expressed most highly in the roots(10-fold over in the stems)than in the sheaths and leaves.The lowest expression was found in the stems.The expression level of TaWAK5 in the sheaths was 7 times higher than that in the stems.At 45 dpi,the transcriptional level of TaWAK5 was the highest in the root samples and lowest in the young spike tissue,with 107 times higher expression level in the former root tissue.The expression level of TaWAK5 was elevated 2-fold in stems and 1.99-fold in leaves compared with the young spike.

    A more detailed analysis of the expression patterns of TaWAK5 was carried out in R.cerealis-resistant lines (CI12633,Shanhongmai,Navit14,and Shannong 0431) and susceptible lines (Wenmai 6 and Yangmai 158).As shown in Fig.1-C,transcript abundances of TaWAK5 were higher in the resistant lines than in the susceptible lines at 7 dpi,with highest level found in the highly resistant wheat CI12633,and lowest level found in the moderately-susceptible wheat Yangmai 158.The above results suggested that TaWAK5 may be involved in wheat defense response to R.cerealis infection.

    3.2.TaWAK5 encodes a wall-associated kinase

    The full-length cDNA sequence (2282 bp) of TaWAK5 was obtained from the resistant wheat genotype CI12633 and deposited in the GenBank database (accession number KF710462).The cDNA contained an ORF of 2112 nucleotides(from 21 to 2132 bp),encoding a protein of 703 amino acids with an estimated molecular mass of 77.0 kDa and a predicted pI of 6.7.BLAST searching against the GenBank database indicated that the TaWAK5 gene was homologous to WAK genes from Aegilops tauschii (GenBank entry,EMT17650)with 67% identity,from Triticum urartu (GenBank entry,EMS57881) with 60% identity,from Setaria italic (GenBank entry,XP_004959009) with 55% identity,and from O.sativa(GenBank entry,AAX95007)with 42%identity.

    The deduced amino acid sequence of TaWAK5 was found to contain various signals and protein domains(Fig.2).In the N-terminal region,there was a predicted signal peptide at amino acids 1–37,which may cause membrane targeting.Two EGF-like repeats at amino acids 268–319 and 323–363 were identified in the putative extracellular domain of the sequence.Additionally,the TaWAK5 protein had a putative protein kinase catalytic domain (residues 429–694) that included an ATP binding site and a Ser/Thr kinase active site(ILHGDVKPANILL,residues 549–561).TaWAK5 is non-arginine aspartate (RD)-type protein,as it carries a glycine (G) rather than an arginine (R) residue immediately preceding the conserved aspartate (D)in the catalytically-active subdomain VIb.

    Fig.1-Expression patterns of TaWAK5 in wheat responding to R.cerealis inoculation.A:Transcript levels of the TaWAK5 gene in the R.cerealis-resistant line CI12633 and the susceptible cultivar Wenmai 6 over a 21 day pathogen inoculation time-series.Relative expression of TaWAK5 indicated the changing fold expression level of the gene transcript in CI12633 over Wenmai 6 at 0 dpi.B:Expression patterns of TaWAK5 in different tissues of R.cerealis-inoculated CI12633 plant at 4 dpi and 45 dpi.C:Expression patterns of TaWAK5 in R.cerealis-resistant lines and susceptible lines after R.cerealis inoculation for 7 days.Transcript levels of TaWAK5 in wheat lines were measured by qRT-PCR relative to highly susceptible cultivar Wenmai 6.The mean and standard error(SE) of three replicates are presented.The transcript abundances with different letters are significantly different from each other,based on inferential statistical comparison using SPSS19.

    Phylogenetic analysis was performed to decipher the relationship between TaWAK5 and any related RLKs.Twenty-one available RLK sequences from different plant species were used to construct a rooted phylogenetic tree.These RLK sequences formed four different subgroups of RLKs including WAK,leucine-rich repeat (LRR)-RLK,LysM-RLK,and lectin-RLK.In the first group,the proteins for TaWAK5,TaWAK1,TaWAK2,TaWAK3,TaWAK4,OsWAK,HvWAK,AtWAK1,AtWAK2,AtWAK3,AtWAK4,and AtWAK5 were clustered into a single WAK clade(Fig.3-A).

    We performed a comparison of amino acid sequences of WAK proteins to determine their similarity.TaWAK5 was found to be closely related to HvWAK from H.vulgare (56.6%identity),followed by OsWAK from O.sativa (47.0% identity),suggesting that these are orthologs of each other from different cereals in the Gramineae family.Meanwhile,TaWAK5 shared 31.5–38.6% protein sequence identities with the four reported wheat WAK paralogs,TaWAK1,TaWAK2,TaWAK3,and TaWAK4.The sequence identities between TaWAK5 and Arabidopsis WAKs were only 30.6–32.8%,showing the distance between monocot and dicot homologues of WAK genes.We then carried out a multiple alignment of EGF-like repeats of TaWAK5 and WAKs from wheat,barley,rice,and Arabidopsis,in which each EGF-like repeat contained six conserved cysteine residues (Fig.3-B).The positions of the six cysteine residues are conserved in TaWAK5 and the other tested WAKs,although the amino acid sequences between the cysteine residues varied.

    3.3.TaWAK5 was localized to the plasma membrane in planta

    To study the subcellular localization of TaWAK5,the p35S:TaWAK5-GFP and p35S:GFP constructs were separately introduced into onion epidermal cells.As presented in Fig.4-A,the TaWAK5-GFP fusion proteins were localized on the cell periphery,whereas the fluorescence of GFP alone as a control was distributed throughout the cell.To verify the nature of the subcellular localization of TaWAK5,a plasmolysis experiment was performed.When onion cells expressing TaWAK5-GFP were plasmolyzed in a 0.8 mol L-1sucrose solution,TaWAK5-directed GFP fluorescence signal was observed on the plasma membrane(Fig.4-B).Thus,TaWAK5 may be a plasma membrane-localized protein.

    3.4.TaWAK5 was induced by exogenous SA,ABA,or MeJA application

    Fig.2-Sequenced nucleotide and deduced amino acid sequences of wheat wall-associated kinase TaWAK5.The conserved EGF-like motif is marked by the boxed residues and located between the signal peptide(indicated by dotted line)and the transmembrane domain (underlined by a double line).The kinase domain(underlined by a single line)follows the transmembrane domain.The roman numerals mark the eleven subdomains(shaded residues)conserved in the plant serine/threonine protein kinase family.Arrowheads indicate the three kinase catalytic sites.

    Fig.3-Phylogenetic analysis of the deduced amino acid sequences and comparison of EGF-like repeat sequences.A:Phylogenetic tree constructed by neighbor-joining algorithms with multiple RLK protein sequences.Bootstrapping was performed 1000 times to obtain support values for each branch.Four groups of RLK proteins,WAK,LRR-RLK,LysM-RLK,and Lectin-RLK were found and are represented,respectively,by the letters A,B,C,and D.The GenBank accession numbers of RLK protein sequences are shown in Table S2.The scale bar indicates sequence divergence.B:Amino acid sequence alignment of EGF-like motifs between TaWAK5 and WAK.Boxes in gray represent the six conserved cysteine residues.

    Fig.4-Plasma membrane localization of TaWAK5-GFP fusion protein.A:Subcellular localization of the fused 35S:TaWAK5-GFP in onion epidermal cells.The 35S:TaWAK5-GFP and 35S:GFP constructs,respectively,were introduced into onion epidermal cells by bombardment and expressed under control of the CaMV35S promoter.Bars = 50 μm.B:TaWAK-GFP fusion protein or GFP alone expressed under the control of the CaMV35S promoter in onion cells treated with 0.8 mol L-1 sucrose solution.Bars = 50 μm.

    Fig.5-Expression patterns of TaWAK5 in wheat before(0 h)and after treatment with exogenous phyto-hormones SA(A),ABA(B),MeJA(C),and ET(D) for 0,1,3,6,12,and 24 h.Relative expression of TaWAK5 as fold change of the transcript over the control(0 hpt).Three biological replicates for each time point were averaged,with standard error of mean indicated.Asterisks indicate statistically significant variation calculated using Student's t-test(*P <0.05,**P <0.01).

    To determine if TaWAK5 was responsive to various phytohormone (SA,ABA,ethylene,or MeJA) treatments,we used qRT-PCR to monitor the transcriptional patterns of TaWAK5 in wheat following treatment for 0,1,3,6,12,and 24 h with exogenous SA,ABA,ethylene or MeJA.As shown in Fig.5,the expression of TaWAK5 was significantly induced by SA,ABA,or MeJA treatment.The greatest induction effect was observed with the SA treatment.Upon SA treatment,the expression of TaWAK5 was induced at 1–12 h post-treatment (hpt),reached a peak at 6 hpt(about 33-fold over that of 0 hpt),and then decreased to a normal level by 24 hpt (Fig.5-A).The expression pattern of TaWAK5 after treatment with ABA was similar to that induced by SA;the induction reached a peak(about 17-fold over that of 0 hpt)at 6 hpt (Fig.5-B).Upon MeJA treatment,the transcript of TaWAK5 was induced from 1 to 24 hpt,and peaked at 12 hpt(more than 11-fold over that of 0 hpt) (Fig.5-C).Upon ethylene treatment,the transcriptional level of TaWAK5 decreased from 1 to 24 hpt(Fig.5-D).These results suggested that TaWAK5 may be responsive to the SA,ABA,and MeJA signals.

    3.5.Promoter characterization for TaWAK5

    Transcriptional regulation is important in mediating the responses of plants to external stimuli.To study which stimuli TaWAK5 may respond to,we analyzed cis-acting elements in the TaWAK5 promoter region using the PLACE database.Many important transcriptional motifs were identified in the promoter of TaWAK5,including a TATA box (at position 956),basal transcription,transcription factor binding site,hormone (ABA,SA,gibberellins,cytokinins,and auxin)responsiveness sites,and sites for responsiveness to elicitors and other processes(Table S3).

    3.6.TaWAK5-silencing did not obviously impair wheat resistance to R.cerealis

    To investigate whether TaWAK5 plays a critical role in wheat resistance response to R.cerealis,we used a BSMV-based VIGS technique to down-regulate TaWAK5 transcript levels in resistant wheat CI12633.At 10 days after the virus inoculation,the BSMV CP transcript was detected in plants inoculated with BSMV virus,but not in the mock plants,revealing successful virus infection.As expected,the TaWAK5 transcript levels were considerably reduced in CI12633 plants infected by BSMV:TaWAK5 (Fig.6-A),suggesting that the TaWAK5 transcripts were silenced in these CI12633 plants infected by BSMV:TaWAK5.In disease screening tests of non-infected plants,the 4th sheaths of mock-treated CI12633 and those infected with the BSMV:TaWAK5 virus were inoculated with mycelia of R.cerealis.The 4th sheaths of the susceptible cultivar Wenmai 6 were used as a positive control to show successful R.cerealis inoculation.At 2 weeks post R.cerealis inoculation,lesions with dark-brown margins (an early symptom of sharp eyespot disease) were observed on the 4th sheaths of the susceptible Wenmai 6,but not on the BSMV:TaWAK5-inoculated,BSMV:GFP-inoculated,or mock-treated CI12633 plants (Fig.6-B).The resistance continued to be present through more mature stages.No sharp eyespot symptoms were observed at 4th sheaths and stems of BSMV:TaWAK5-inoculated,BSMV:GFP-inoculated,and mock CI12633 plants,but obvious symptoms were present on the 4th sheaths and stems of Wenmai 6 plants.These results suggested that the silencing of TaWAK5 did not directly compromise wheat resistance to R.cerealis in CI12633.

    4.Discussion

    Fig.6-Effect of silencing TaWAK5 on the resistance response of CI12633 to the necrotrophic fungal pathogen R.cerealis,causal agent for sharp eyespot disease.A:Relative transcript levels of TaWAK5 and BSMV CP genes in the 4th leaves of the mock-treated plants,or those infected by BSMV:GFP and BSMV:TaWAK5 as evaluated by semi-quantitative RT-PCR using gene-specific primers.Amplification of the wheat TaEF-1a gene served as the internal control.B:Response of the 4th sheaths of the mock-treated,BSMV virus-inoculated CI12633 genotype,and positive control Wenmai 6 reactions to R.cerealis.The photographs were taken 2 weeks after R.cerealis inoculation.M:Mock plant;G-1:BSMV:GFP-1 plant;G-2:BSMV:-GFP-2 plant;G-3:BSMV:GFP-3 plant;K5-1:BSMV:TaWAK5-1 plant;K5-2:BSMV:TaWAK5-2 plant;K5-3:BSMV:TaWAK5-3 plant.

    In this study,we isolated a novel wheat WAK gene,TaWAK5,from R.cerealis-resistant wheat CI12633,based on a cDNA transcript that was differentially expressed between resistant wheat genotype CI12633 and susceptible wheat cultivar Wenmai 6.qRT-PCR analysis revealed that the transcript abundance of TaWAK5 in wheat was rapidly increased by R.cerealis infection.Additionally,TaWAK5 in the R.cerealis-resistant lines was induced to higher levels than in R.cerealis-susceptible lines at 7 dpi with R.cerealis.These results suggested that TaWAK5 may be involved in wheat defense responses to R.cerealis infection.

    Sequence analysis and phylogenetic analysis revealed that TaWAK5 was a member of the WAK sub-group of the RLK family in wheat.Several WAK genes have been shown to play important roles in regulating plant defense responses.WAK1 from Arabidopsis and OsWAK1 from rice were shown to enhance resistance to the pathogens B.cinerea and M.oryzae,respectively[5,11].TaWAK5 is a non-RD-type protein,as it has an HGD motif in its subdomain VIb.Out of 38 receptor kinases tested in plants,the six which fall into the non-RD class all function in disease resistance and act as PRRs,while the remaining 32 kinases are RD or alternative catalytic function(ACF) kinases and are involved in developmental processes[35],suggesting that all the non-RD RLKs seemed to participate in innate immunity.The TaWAK5 protein was localized to the plasma membrane in onion epidermal cells,consistent with the signal sequence and receptor responses.

    Many WAKs have been shown to be involved in hormonal signals.Arabidopsis WAK1 is induced by both SA and the SA analog 2,2-dichloroisonicotinic acid(INA),and ectopic expression of the entire WAK1 or the kinase domain alone was shown to provide resistance to lethal SA levels[36].According to cDNA microarray analysis in Arabidopsis,AtWAK1 is induced by MeJA and ethylene [37].In this study,qRT-PCR analyses revealed that TaWAK5 could be induced by application of exogenous SA,ABA,and MeJA.Although an antagonistic interaction between SA-and JA-dependent signaling has been suggested[38–40],in some cases,SA does not inhibit JA biosynthesis and may even contribute to JA-mediated signaling pathway function [41].In Arabidopsis,concentrations of both SA and JA and the timing of initiation of SA and JA signaling are important for the outcome of the complex SA-JA signal interaction [42,43].ABA has been shown to interact with the SA-JA network.ABA has been suggested to affect JA biosynthesis and resistance against the JA-inducing,necrotrophic pathogen Pythium irregular[23,24],and to suppress SA-dependent disease resistance [44].Related to the role of phyto-hormones in WAK expression,the region upstream of the start codon(1000 bp)of TaWAK5 was analyzed in this study.The promoter region contained one ABRE-like motif (ACGTG),but no SA-,or JA-responsive elements (shown in Table S3).Several studies have suggested that modulation of gene expression is accomplished through the interaction of induced regulatory proteins and specific DNA regions [45–47].For instance,the induction of a dehydration-responsive gene,rd22,is mediated by ABA.MYC and MYB recognition sites in the rd22 promoter region function as cis-acting elements that interact specifically with AtMYC2 and AtMYB2; transgenic plants overexpressing AtMYC2 and/or AtMYB2 cDNAs have higher sensitivity to ABA[47].In this study,TaWAK5 promoter had five binding sites of an ABA-regulated protein,two of a SA-regulated protein,and one of a JA-regulated protein(Fig.S1),suggesting that TaWAK5 was also regulated possibly through SA-,ABA-,and MeJA-hormones.

    In this study,VIGS,which has been an efficient tool for rapidly analyzing the functions of plant genes [48–51],was also used to evaluate the disease resistance role of TaWAK5.In wheat,infection with barley stripe mosaic virus (BSMV)constructs carrying a fragment of the resistance gene Lr21 caused conversion of incompatible interactions of wheat and leaf rust pathogen to compatible reactions after the gene silencing,whereas infection with a control construct or one that silences phytoene desaturase gene had no effect on resistance or susceptibility[33].Knocking down the transcript levels of three wheat RLK genes TaRLK-R1,TaRLK-R2,or TaRLKR3 individually or all together by VIGS and the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the wheat hypersensitive reaction to stripe rust fungus,suggesting that TaRLK-R1,TaRLK-R2,TaRLK-R3,and TaHsp90.2 and TaHsp90.3 were positive contributors in the wheat hypersensitive reaction to stripe rust fungus[50,51].These studies suggested that VIGS is an effective reverse genetic tool for investigating the functions of genes in wheat by knocking down the transcripts of target genes during the development of disease resistance.Conventional methods for gene functional analysis of plant genes,including transformation are not easily accomplished given wheat's large genome [52].Transformation is also time-consuming because the function of a target gene should be tested over multiple generations [53].In contrast to the conventional methods,the main advantage of VIGS is the generation of a rapid phenotype without the need for plant transformation[54].Moreover,the VIGS method provides a large-scale screening of genes for functional analysis; only a single plant is enough to follow phenotype with targeted silencing [55].In this study,the VIGS approach was utilized to investigate the function of TaWAK5 in wheat defense response to R.cerealis.Although the TaWAK5 transcript level was reduced in CI12633 plants infected by BSMV:TaWAK5,down-regulation of TaWAK5 in resistant CI12633 did not result in an obvious impairment of wheat resistance to R.cerealis.Plant defense is a complicated network in which some components and network sectors interact with each other in complex ways.The function of an individual component of a network can be compensated for by some other component.Therefore,functional characterization of disease resistance components by knockout of an individual component is difficult and multi-gene knock outs or gene × gene interactions need to be considered [56].In Arabidopsis,it has been suggested that there is functional redundancy between the WAKs,as induced silencing of individual AtWAK1or AtWAK2 using gene-specific antisense transcripts did not cause any phenotypic alteration[57].In this study,knocking down TaWAK5 expression did not cause the compromised resistance phenotype of the host CI12633 to R.cerealis.The reason might be that TaWAK5 is not the major gene controlling wheat defense response to R.cerealis,or that TaWAK5 is functionally redundant with other wheat WAK genes that help replace its functionalities when it is knocked out by VIGS experiments.

    5.Conclusions

    A wheat WAK gene,TaWAK5,was identified by microarray analysis of differentially expressed genes between R.cerealisresistant line CI12633 and susceptible cv.Wenmai 6 and characterized.TaWAK5 was rapidly induced by R.cerealis infection,and by exogenous SA,MeJA,or ABA application.The deduced TaWAK5 protein shares the structural characteristic of a wall-associated kinase,possessing two EGF-like repeats and a kinase catalytic domain.The TaWAK5 protein was localized to the plasma membranes in onion epidermal cells.Our results provide new insights into the WAK sub-group of the RLK family and will provide the foundation for further research into functions of WAKs in wheat.

    This work was funded by the National Natural Science Foundation of China (31271799),and the National “Key Sci-Tech”program,China (2013ZX08002-001-004),and the China–Czech Government Science and Technology Cooperation Project (40–3 and LH12196).Editing assistance from Chinese Academy of Agricultural Sciences (CAAS) and from M.Blair is gratefully acknowledged.

    Supplementary material

    Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.cj.2014.04.010.

    [1] M.Antolín-Llovera,M.K.Ried,A.Binder,M.Parniske,Receptor kinase signaling pathways in plant-microbe interactions,Annu.Rev.Phytopathol.50(2012) 451–473.

    [2] D.Chinchilla,C.Zipfel,S.Robatzek,B.Kemmerling,T.Nurnberger,J.D.Jones,G.Felix,T.Boller,A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence,Nature 448 (2007) 497–500.

    [3] L.Gómez-Gómez,T.Boller,FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis,Mol.Cell 5(2000) 1003–1011.

    [4] T.Liu,Z.Liu,C.Song,Y.Hu,Z.Han,J.She,F.Fan,J.Wang,C.Jin,J.Chang,J.M.Zhou,J.Chai,Chitin-induced dimerization activates a plant immune receptor,Science 336 (2012)1160–1164.

    [5] A.Brutus,F.Sicilia,A.Macone,F.Cervone,G.De Lorenzo,A domain swap approach reveals a role of the plant wall-associated kinase 1(WAK1)as a receptor of oligogalacturonides,Proc.Natl.Acad.Sci.U.S.A.107 (2010)9452–9457.

    [6] B.D.Kohorn,Plasma membrane-cell wall contacts,Plant Physiol.124 (2000) 31–38.

    [7] Z.H.He,M.Fujiki,B.D.Kohorn,A cell wall-associated,receptor-like protein kinase,J.Biol.Chem.271 (1996)19789–19793.

    [8] B.D.Kohorn,S.Johansen,A.Shishido,T.Todorova,R.Martinez,E.Defeo,P.Obregon,Pectin activation of MAP kinase and gene expression is WAK2 dependent,Plant J.60(2009)97–982.

    [9] V.Kanneganti,A.K.Gupta,Wall associated kinases from plants–an overview,Physiol.Mol.Biol.Plant 14(2008)109–118.

    [10] C.Denoux,R.Galletti,N.Mammarella,S.Gopalan,D.Werck,G.De Lorenzo,S.Ferrari,F.M.Ausubel,J.Dewdney,Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings,Mol.Plant 1(2008) 423–445.

    [11] H.Li,S.Y.Zhou,W.S.Zhao,S.C.Su,Y.L.Peng,A novel wall-associated receptor-like protein kinase gene,OsWAK1,plays important roles in rice blast disease resistance,Plant Mol.Biol.69 (2009) 337–346.

    [12] Y.Liu,D.Liu,H.Zhang,H.Gao,X.Guo,X.Fu,A.Zhang,Isolation and characterisation of six putative wheat cell wall-associated kinases,Funct.Plant Biol.33(2006) 811–821.

    [13] B.Mauch-Mani,F.Mauch,The role of abscisic acid in plant–pathogen interactions,Curr.Opin.Plant Biol.8(2005)409–414.

    [14] L.C.Van Loon,B.P.Geraats,H.J.Linthorst,Ethylene as a modulator of disease resistance in plants,Trends Plant Sci.11(2006) 184–191.

    [15] M.Pozo,L.C.Loon,C.J.Pieterse,Jasmonates–signals in plant-microbe interactions,J.Plant Growth Regul.23(2004)211–222.

    [16] G.Loake,M.Grant,Salicylic acid in plant defence–the players and protagonists,Curr.Opin.Plant Biol.10(2007)466–472.

    [17] B.Vernooij,S.Uknes,E.Ward,J.Ryals,Salicylic acid as a signal molecule in plant–pathogen interactions,Curr.Opin.Cell Biol.6(1994) 275–279.

    [18] R.A.Creelman,J.E.Mullet,Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress,Proc.Natl.Acad.Sci.U.S.A.92 (1995) 4114–4119.

    [19] J.R.Ecker,The ethylene signal transduction pathway in plants,Science 268 (1995) 667–675.

    [20] M.A.Lopez,G.Bannenberg,C.Castresana,Controlling hormone signaling is a plant and pathogen challenge for growth and survival,Curr.Opin.Plant Biol.11 (2008) 420–427.

    [21] J.A.Ryals,U.H.Neuenschwander,M.G.Willits,A.Molina,H.Y.Steiner,M.D.Hunt,Systemic acquired resistance,Plant Cell 8(1996) 1809–1819.

    [22] K.E.Hammond-Kosack,J.D.Jones,Resistance genedependent plant defense responses,Plant Cell 8 (1996)1773–1791.

    [23] V.Flors,J.Ton,R.van Doorn,G.Jakab,P.Garcia-Agustin,B.Mauch-Mani,Interplay between JA,SA and ABA signalling during basal and induced resistance against Pseudomonas syringae and Alternaria brassicicola,Plant J.54(2008) 81–92.

    [24] B.A.Adie,J.Pérez-Pérez,M.M.Pérez-Pérez,M.Godoy,J.J.Sánchez-Serrano,E.A.Schmelz,R.Solano,ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis,Plant Cell 19 (2007) 1665–1681.

    [25] J.P.Anderson,E.Badruzsaufari,P.M.Schenk,J.M.Manners,O.J.Desmond,C.Ehlert,D.J.Maclean,P.R.Ebert,K.Kazan,Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis,Plant Cell 16(2004) 3460–3479.

    [26] M.S.Hamada,Y.Yin,H.Chen,Z.Ma,The escalating threat of Rhizoctonia cerealis,the causal agent of sharp eyespot in wheat,Pest Manag.Sci.67(2011) 1411–1419.

    [27] L.Chen,Z.Zhang,H.Liang,H.Liu,L.Du,H.Xu,Z.Xin,Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat,J.Exp.Bot.59(2008) 4195–4204.

    [28] K.Yang,W.Rong,L.Qi,J.Li,X.Wei,Z.Zhang,Isolation and characterization of a novel wheat cysteine-rich receptor-like kinase gene induced by Rhizoctonia cerealis,Sci.Rep.3(2013)3021,http://dx.doi.org/10.1038/srep03021.

    [29] Z.Zhang,X.Liu,X.Wang,M.Zhou,X.Zhou,X.Ye,X.Wei,An R2R3 MYB transcription factor in wheat,TaPIMP1,mediates host resistance to Bipolaris sorokiniana and drought stresses through regulation of defense-and stress-related genes,New Phytol.196 (2012) 1155–1170.

    [30] Z.Zhang,W.Yao,N.Dong,H.Liang,H.Liu,R.Huang,A novel ERF transcription activator in wheat and its induction kinetics after pathogen and hormone treatments,J.Exp.Bot.58 (2007) 2993–3003.

    [31] I.Lang-Pauluzzi,B.Gunning,A plasmolytic cycle:the fate of cytoskeletal elements,Protoplasma 212 (2000) 174–185.

    [32] S.Holzberg,P.Brosio,C.Gross,G.P.Pogue,Barley stripe mosaic virus-induced gene silencing in a monocot plant,Plant J.30(2002) 315–327.

    [33] S.R.Scofield,L.Huang,A.S.Brandt,B.S.Gill,Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway,Plant Physiol.138 (2005) 2165–2173.

    [34] K.J.Livak,T.D.Schmittgen,Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod,Methods 25(2001) 402–408.

    [35] C.Dardick,P.Ronald,Plant and animal pathogen recognition receptors signal through non-RD kinases,PLoS Pathog.2(2006) e2.

    [36] Z.H.He,D.He,B.D.Kohorn,Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response,Plant J.14(1998)55–63.

    [37] P.M.Schenk,K.Kazan,I.Wilson,J.P.Anderson,T.Richmond,S.C.Somerville,J.M.Manners,Coordinated plant defense responses in Arabidopsis revealed by microarray analysis,Proc.Natl.Acad.Sci.U.S.A.97(2000) 11655–11660.

    [38] C.A.Preston,C.Lewandowski,A.J.Enyedi,I.T.Baldwin,Tobacco mosaic virus inoculation inhibits wound-induced jasmonic acid-mediated responses within but not between plants,Planta 209 (1999) 87–95.

    [39] J.Cui,A.K.Bahrami,E.G.Pringle,G.Hernandez-Guzman,C.L.Bender,N.E.Pierce,F.M.Ausubel,Pseudomonas syringae manipulates systemic plant defenses against pathogens and herbivores,Proc.Natl.Acad.Sci.U.S.A.102(2005)1791–1796.

    [40] A.P.Kloek,M.L.Verbsky,S.B.Sharma,J.E.Schoelz,J.Vogel,D.F.Klessig,B.N.Kunkel,Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive(coi1) mutation occurs through two distinct mechanisms,Plant J.26 (2001) 509–522.

    [41] J.?a?niewska,V.K.Macioszek,C.B.Lawrence,A.K.Kononowicz,Fight to the death: Arabidopsis thaliana defense response to fungal necrotrophic pathogens,Acta Physiol.Plant.32(2010) 1–10.

    [42] L.A.Mur,P.Kenton,R.Atzorn,O.Miersch,C.Wasternack,The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy,antagonism,and oxidative stress leading to cell death,Plant Physiol.140 (2006) 249–262.

    [43] A.Koornneef,A.Leon-Reyes,T.Ritsema,A.Verhage,F.C.Den Otter,L.C.Van Loon,C.M.Pieterse,Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation,Plant Physiol.147 (2008)1358–1368.

    [44] M.Yasuda,A.Ishikawa,Y.Jikumaru,M.Seki,T.Umezawa,T.Asami,A.Maruyama-Nakashita,T.Kudo,K.Shinozaki,S.Yoshida,H.Nakashita,Antagonistic interaction between systemic acquired resistance and the abscisic acid-mediated abiotic stress response in Arabidopsis,Plant Cell 20(2008)1678–1692.

    [45] S.H.Spoel,A.Koornneef,S.M.Claessens,J.P.Korzelius,J.A.Van Pelt,M.J.Mueller,A.J.Buchala,J.P.Metraux,R.Brown,K.Kazan,L.C.Van Loon,X.Dong,C.M.Pieterse,NPR1 modulates cross-talk between salicylate-and jasmonate-dependent defense pathways through a novel function in the cytosol,Plant Cell 15(2003) 760–770.

    [46] S.Nakamura,T.J.Lynch,R.R.Finkelstein,Physical interactions between ABA response loci of Arabidopsis,Plant J.26(2001) 627–635.

    [47] H.Abe,T.Urao,T.Ito,M.Seki,K.Shinozaki,K.Yamaguchi-Shinozaki,Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling,Plant Cell 15 (2003) 63–78.

    [48] S.H.Gabriels,J.H.Vossen,S.K.Ekengren,G.van Ooijen,A.M.Abd-El-Haliem,G.C.van den Berg,D.Y.Rainey,G.B.Martin,F.L.Takken,P.J.de Wit,M.H.Joosten,An NB-LRR protein required for HR signalling mediated by both extra-and intracellular resistance proteins,Plant J.50(2007) 14–28.

    [49] I.Hein,M.Barciszewska-Pacak,K.Hrubikova,S.Williamson,M.Dinesen,I.E.Soenderby,S.Sundar,A.Jarmolowski,K.Shirasu,C.Lacomme,Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley,Plant Physiol.138(2005)2155–2164.

    [50] G.F.Wang,X.Wei,R.Fan,H.Zhou,X.Wang,C.Yu,L.Dong,Z.Dong,Z.Kang,H.Ling,Q.H.Shen,D.Wang,X.Zhang,Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90(Hsp90):functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance,New Phytol.191(2011)418–431.

    [51] H.Zhou,S.Li,Z.Deng,X.Wang,T.Chen,J.Zhang,S.Chen,H.Ling,A.Zhang,D.Wang,X.Zhang,Molecular analysis of three new receptor-like kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive response to stripe rust fungus infection,Plant J.52(2007) 420–434.

    [52] H.D.Jones,Wheat transformation: current technology and applications to grain development and composition,J.Cereal Sci.41(2005) 137–147.

    [53] C.A.Newell,Plant transformation technology,Mol.Biotechnol.16(2000) 53–65.

    [54] T.M.Burch-Smith,J.C.Anderson,G.B.Martin,S.P.Dinesh-Kumar,Applications and advantages of virus-induced gene silencing for gene function studies in plants,Plant J.39(2004) 734–746.

    [55] A.Becker,M.Lange,VIGS–genomics goes functional,Trends Plant Sci.15(2010) 1–4.

    [56] M.Sato,K.Tsuda,L.Wang,J.Coller,Y.Watanabe,J.Glazebrook,F.Katagiri,Network modeling reveals prevalent negative regulatory relationships between signaling sectors in Arabidopsis immune signaling,PLoS Pathog.6(2010)e1001011.

    [57] T.A.Wagner,B.D.Kohorn,Wall-associated kinases are expressed throughout plant development and are required for cell expansion,Plant Cell 13(2001) 303–318.

    精品一区二区三区四区五区乱码| 国产激情偷乱视频一区二区| 免费看十八禁软件| 他把我摸到了高潮在线观看| 亚洲av片天天在线观看| 欧美丝袜亚洲另类 | 淫秽高清视频在线观看| 床上黄色一级片| 国产精品九九99| 天堂√8在线中文| 亚洲精品美女久久av网站| 丝袜人妻中文字幕| 国产美女午夜福利| 精品一区二区三区av网在线观看| av福利片在线观看| av欧美777| 男女之事视频高清在线观看| av片东京热男人的天堂| 黑人操中国人逼视频| 每晚都被弄得嗷嗷叫到高潮| 一二三四社区在线视频社区8| 啪啪无遮挡十八禁网站| 99久久国产精品久久久| 国产成人福利小说| 欧美精品啪啪一区二区三区| 国产亚洲欧美98| 三级毛片av免费| 高清毛片免费观看视频网站| 国产精品日韩av在线免费观看| 色吧在线观看| 草草在线视频免费看| www日本黄色视频网| 国产乱人视频| 一二三四在线观看免费中文在| 一级毛片女人18水好多| 亚洲欧美精品综合久久99| 亚洲成a人片在线一区二区| 久久精品国产99精品国产亚洲性色| 校园春色视频在线观看| 欧洲精品卡2卡3卡4卡5卡区| 亚洲第一电影网av| 免费观看精品视频网站| 国产伦一二天堂av在线观看| www日本在线高清视频| 成年女人看的毛片在线观看| 久久精品人妻少妇| 最新中文字幕久久久久 | 好男人在线观看高清免费视频| 亚洲精品456在线播放app | 亚洲av熟女| 少妇的丰满在线观看| 久久午夜综合久久蜜桃| 亚洲欧美日韩高清专用| 亚洲欧美日韩卡通动漫| 天堂动漫精品| 国产一区二区三区在线臀色熟女| 国产精品九九99| 18禁黄网站禁片免费观看直播| 草草在线视频免费看| 波多野结衣高清作品| 久久天堂一区二区三区四区| 美女免费视频网站| 国产精品影院久久| 丁香欧美五月| 日本成人三级电影网站| 欧美日本视频| 国产黄片美女视频| 12—13女人毛片做爰片一| 丁香欧美五月| 18禁国产床啪视频网站| 久久久水蜜桃国产精品网| 精品国产乱码久久久久久男人| 亚洲国产欧美一区二区综合| tocl精华| 老司机午夜十八禁免费视频| 欧美中文综合在线视频| 亚洲午夜精品一区,二区,三区| 天天躁狠狠躁夜夜躁狠狠躁| 啪啪无遮挡十八禁网站| 国产 一区 欧美 日韩| 国语自产精品视频在线第100页| 国产主播在线观看一区二区| 综合色av麻豆| 精品日产1卡2卡| 9191精品国产免费久久| 一二三四在线观看免费中文在| 人人妻人人澡欧美一区二区| av国产免费在线观看| 国产爱豆传媒在线观看| 少妇熟女aⅴ在线视频| 成人精品一区二区免费| 亚洲第一电影网av| xxx96com| 美女扒开内裤让男人捅视频| 桃色一区二区三区在线观看| 婷婷丁香在线五月| 成熟少妇高潮喷水视频| 欧美极品一区二区三区四区| 老司机午夜十八禁免费视频| 精品无人区乱码1区二区| 久久中文字幕人妻熟女| 岛国视频午夜一区免费看| 俄罗斯特黄特色一大片| 精品久久久久久久末码| 免费在线观看成人毛片| 久久人妻av系列| 国产免费男女视频| 成年女人看的毛片在线观看| 亚洲成人精品中文字幕电影| 亚洲熟妇中文字幕五十中出| 一个人免费在线观看电影 | 国产精品99久久99久久久不卡| 一区二区三区高清视频在线| 亚洲精品在线观看二区| 一本精品99久久精品77| 国产单亲对白刺激| bbb黄色大片| 欧美日韩福利视频一区二区| 男插女下体视频免费在线播放| 99久久精品国产亚洲精品| 两人在一起打扑克的视频| 国产成人欧美在线观看| 成熟少妇高潮喷水视频| 国产av一区在线观看免费| 最近最新中文字幕大全电影3| 成熟少妇高潮喷水视频| 欧美丝袜亚洲另类 | 韩国av一区二区三区四区| 99视频精品全部免费 在线 | 成人鲁丝片一二三区免费| 久久久精品大字幕| 亚洲av五月六月丁香网| 亚洲真实伦在线观看| 亚洲,欧美精品.| 欧美在线一区亚洲| 亚洲成a人片在线一区二区| 精品国产乱子伦一区二区三区| 两个人的视频大全免费| 一个人免费在线观看的高清视频| 精品久久蜜臀av无| 在线观看免费视频日本深夜| 久久中文看片网| 久久久色成人| 日本在线视频免费播放| 在线免费观看不下载黄p国产 | 中亚洲国语对白在线视频| 精品免费久久久久久久清纯| 成人性生交大片免费视频hd| 两个人看的免费小视频| 老熟妇乱子伦视频在线观看| 啦啦啦免费观看视频1| 午夜福利欧美成人| av天堂在线播放| 成人特级av手机在线观看| 男人和女人高潮做爰伦理| 一本一本综合久久| 国产又黄又爽又无遮挡在线| 久久久久免费精品人妻一区二区| 久久久久久九九精品二区国产| 日本精品一区二区三区蜜桃| 免费人成视频x8x8入口观看| 久久久国产成人免费| 午夜成年电影在线免费观看| 亚洲成av人片在线播放无| 亚洲熟女毛片儿| 国内揄拍国产精品人妻在线| 制服丝袜大香蕉在线| 国产成人aa在线观看| 亚洲国产精品久久男人天堂| 成人一区二区视频在线观看| 色综合婷婷激情| 99久久精品一区二区三区| 小蜜桃在线观看免费完整版高清| 757午夜福利合集在线观看| 亚洲美女黄片视频| 欧美日韩中文字幕国产精品一区二区三区| 麻豆国产97在线/欧美| 母亲3免费完整高清在线观看| 欧美日韩福利视频一区二区| 最新美女视频免费是黄的| netflix在线观看网站| 国产视频一区二区在线看| 国产一区二区在线观看日韩 | 黑人操中国人逼视频| 国产精品1区2区在线观看.| 成年女人看的毛片在线观看| 美女午夜性视频免费| 亚洲美女视频黄频| 国产高清视频在线观看网站| 深夜精品福利| 精品电影一区二区在线| 又紧又爽又黄一区二区| 国产成人av激情在线播放| 国内久久婷婷六月综合欲色啪| 日韩大尺度精品在线看网址| 两性午夜刺激爽爽歪歪视频在线观看| 亚洲在线观看片| 香蕉久久夜色| 日韩欧美国产一区二区入口| 精品国产三级普通话版| 亚洲中文av在线| 日韩欧美免费精品| 亚洲aⅴ乱码一区二区在线播放| 母亲3免费完整高清在线观看| 国产精华一区二区三区| 欧美日韩一级在线毛片| 亚洲欧美精品综合久久99| 国产激情久久老熟女| 亚洲在线自拍视频| 成年版毛片免费区| 欧美在线黄色| 深夜精品福利| 国产精品女同一区二区软件 | 精品无人区乱码1区二区| 精品一区二区三区av网在线观看| 女人高潮潮喷娇喘18禁视频| 国产精品电影一区二区三区| 国产97色在线日韩免费| 男插女下体视频免费在线播放| 后天国语完整版免费观看| 欧美丝袜亚洲另类 | 成人av在线播放网站| 女警被强在线播放| 亚洲欧美精品综合久久99| 成熟少妇高潮喷水视频| 九色国产91popny在线| 亚洲在线自拍视频| 精品久久久久久久人妻蜜臀av| 国产亚洲精品综合一区在线观看| 好看av亚洲va欧美ⅴa在| 99热精品在线国产| 在线国产一区二区在线| 国产成人精品久久二区二区免费| 免费看光身美女| 国产乱人伦免费视频| 99久久久亚洲精品蜜臀av| 日本五十路高清| avwww免费| 琪琪午夜伦伦电影理论片6080| 亚洲精品中文字幕一二三四区| 午夜福利视频1000在线观看| 午夜精品在线福利| 国产精品 国内视频| 亚洲色图 男人天堂 中文字幕| 在线看三级毛片| 亚洲av电影不卡..在线观看| 成人一区二区视频在线观看| 欧美绝顶高潮抽搐喷水| 99视频精品全部免费 在线 | 国模一区二区三区四区视频 | 夜夜看夜夜爽夜夜摸| 亚洲成人精品中文字幕电影| 亚洲av美国av| 久久久久久久精品吃奶| 精品熟女少妇八av免费久了| 99久久综合精品五月天人人| 免费观看精品视频网站| 手机成人av网站| 亚洲专区中文字幕在线| 1024香蕉在线观看| 免费大片18禁| 别揉我奶头~嗯~啊~动态视频| 亚洲成a人片在线一区二区| 色尼玛亚洲综合影院| 国产成人精品久久二区二区免费| 一级黄色大片毛片| 在线视频色国产色| 亚洲精品国产精品久久久不卡| 亚洲五月天丁香| 在线免费观看不下载黄p国产 | 一本精品99久久精品77| 黑人操中国人逼视频| 在线a可以看的网站| 97碰自拍视频| 亚洲激情在线av| 日本免费a在线| 操出白浆在线播放| 国产av一区在线观看免费| 国产欧美日韩精品一区二区| 香蕉久久夜色| 变态另类成人亚洲欧美熟女| 日韩精品青青久久久久久| 久久精品夜夜夜夜夜久久蜜豆| 国产私拍福利视频在线观看| 九九热线精品视视频播放| 一个人观看的视频www高清免费观看 | 亚洲成人中文字幕在线播放| 亚洲熟妇熟女久久| 麻豆av在线久日| 老司机福利观看| 波多野结衣巨乳人妻| 激情在线观看视频在线高清| 此物有八面人人有两片| 亚洲片人在线观看| 国产一区二区在线av高清观看| 亚洲黑人精品在线| 桃色一区二区三区在线观看| 国内精品久久久久精免费| 国产精品一区二区三区四区免费观看 | av欧美777| 久久久精品欧美日韩精品| 亚洲欧美日韩高清专用| 丰满人妻熟妇乱又伦精品不卡| 亚洲一区二区三区不卡视频| 悠悠久久av| 色综合亚洲欧美另类图片| 91在线精品国自产拍蜜月 | 三级毛片av免费| 九色国产91popny在线| 小说图片视频综合网站| www日本黄色视频网| 观看美女的网站| 日韩有码中文字幕| 精品久久久久久久久久免费视频| 国产黄片美女视频| 亚洲一区二区三区色噜噜| 在线永久观看黄色视频| 亚洲国产欧美人成| 99在线视频只有这里精品首页| 国产精品永久免费网站| 欧美激情久久久久久爽电影| 午夜福利18| 淫秽高清视频在线观看| 午夜精品久久久久久毛片777| 中文在线观看免费www的网站| 一个人看的www免费观看视频| 搡老岳熟女国产| 91av网站免费观看| 热99re8久久精品国产| 啦啦啦观看免费观看视频高清| 精品福利观看| 精华霜和精华液先用哪个| 听说在线观看完整版免费高清| 很黄的视频免费| 美女被艹到高潮喷水动态| 色哟哟哟哟哟哟| 美女黄网站色视频| a在线观看视频网站| 丰满人妻熟妇乱又伦精品不卡| 一个人看的www免费观看视频| 丁香欧美五月| 少妇裸体淫交视频免费看高清| 国产日本99.免费观看| 国产亚洲精品av在线| 欧美在线黄色| 两性夫妻黄色片| 特级一级黄色大片| 久久久久久久久久黄片| 中文字幕精品亚洲无线码一区| 欧美日韩国产亚洲二区| 日韩欧美一区二区三区在线观看| 在线观看日韩欧美| 亚洲午夜精品一区,二区,三区| 哪里可以看免费的av片| 狂野欧美激情性xxxx| 成人特级黄色片久久久久久久| 国产精品爽爽va在线观看网站| 校园春色视频在线观看| 日韩欧美 国产精品| 亚洲性夜色夜夜综合| 午夜福利成人在线免费观看| 中文亚洲av片在线观看爽| 久久久久久久午夜电影| 久久久国产成人精品二区| 三级男女做爰猛烈吃奶摸视频| 亚洲精品美女久久av网站| e午夜精品久久久久久久| 免费无遮挡裸体视频| 欧美黑人欧美精品刺激| 国产精品美女特级片免费视频播放器 | 观看免费一级毛片| 亚洲va日本ⅴa欧美va伊人久久| 99re在线观看精品视频| 老鸭窝网址在线观看| 亚洲国产中文字幕在线视频| 亚洲欧美精品综合一区二区三区| 久久婷婷人人爽人人干人人爱| 丝袜人妻中文字幕| 淫妇啪啪啪对白视频| 午夜福利高清视频| 精品久久久久久久毛片微露脸| 免费在线观看亚洲国产| 又黄又粗又硬又大视频| 一级a爱片免费观看的视频| 色播亚洲综合网| 精品久久久久久久久久久久久| 九九在线视频观看精品| 亚洲熟妇中文字幕五十中出| 别揉我奶头~嗯~啊~动态视频| 少妇的逼水好多| 午夜影院日韩av| 欧美激情在线99| 日韩欧美在线二视频| 亚洲欧美精品综合一区二区三区| 国产精品,欧美在线| 亚洲国产色片| 亚洲中文日韩欧美视频| 欧美av亚洲av综合av国产av| av视频在线观看入口| 亚洲欧洲精品一区二区精品久久久| 欧美日韩黄片免| 视频区欧美日本亚洲| 欧美丝袜亚洲另类 | 两性午夜刺激爽爽歪歪视频在线观看| 国产真人三级小视频在线观看| 嫩草影院入口| 亚洲在线观看片| 嫩草影视91久久| 成人午夜高清在线视频| 午夜久久久久精精品| 欧美一区二区精品小视频在线| 一个人免费在线观看的高清视频| 成年女人毛片免费观看观看9| 亚洲欧美精品综合一区二区三区| 十八禁网站免费在线| 在线看三级毛片| 色老头精品视频在线观看| 99热只有精品国产| 黄色片一级片一级黄色片| 免费看光身美女| 18禁国产床啪视频网站| 亚洲人成电影免费在线| 日韩国内少妇激情av| 他把我摸到了高潮在线观看| 在线观看日韩欧美| 老司机深夜福利视频在线观看| 国产主播在线观看一区二区| 毛片女人毛片| 在线观看舔阴道视频| 热99在线观看视频| 成人性生交大片免费视频hd| 老司机深夜福利视频在线观看| 国产免费av片在线观看野外av| 麻豆成人午夜福利视频| 国产伦在线观看视频一区| 美女午夜性视频免费| 毛片女人毛片| 香蕉久久夜色| 午夜免费观看网址| 亚洲人成伊人成综合网2020| 老鸭窝网址在线观看| 观看免费一级毛片| av中文乱码字幕在线| 中国美女看黄片| 亚洲av成人一区二区三| 18禁国产床啪视频网站| 狂野欧美激情性xxxx| 老熟妇仑乱视频hdxx| 国产精品久久视频播放| 国产伦在线观看视频一区| 亚洲av成人av| svipshipincom国产片| 国产野战对白在线观看| 成人无遮挡网站| 亚洲成av人片在线播放无| 男女做爰动态图高潮gif福利片| 久久久色成人| www日本在线高清视频| 亚洲av免费在线观看| 美女高潮的动态| 精品国产超薄肉色丝袜足j| 欧美日韩乱码在线| 久久久久久久久免费视频了| 天天躁狠狠躁夜夜躁狠狠躁| 亚洲成av人片免费观看| 亚洲自偷自拍图片 自拍| 亚洲精品美女久久久久99蜜臀| 性欧美人与动物交配| 国产一级毛片七仙女欲春2| 亚洲性夜色夜夜综合| 男女那种视频在线观看| 国产精品久久久av美女十八| 青草久久国产| 欧美精品啪啪一区二区三区| www.999成人在线观看| 精品午夜福利视频在线观看一区| 国产精品亚洲一级av第二区| 午夜亚洲福利在线播放| 国产精品99久久久久久久久| 久久精品影院6| 久久伊人香网站| 99re在线观看精品视频| 欧美3d第一页| 日韩精品青青久久久久久| 最近在线观看免费完整版| 又紧又爽又黄一区二区| 成人午夜高清在线视频| 搡老熟女国产l中国老女人| 国产一区二区激情短视频| 男女那种视频在线观看| 国内揄拍国产精品人妻在线| 久久这里只有精品19| 亚洲一区二区三区色噜噜| 天天躁日日操中文字幕| 又黄又粗又硬又大视频| 色综合站精品国产| 一本精品99久久精品77| 美女午夜性视频免费| 很黄的视频免费| 两个人的视频大全免费| 国模一区二区三区四区视频 | 欧美一区二区国产精品久久精品| 最近最新中文字幕大全电影3| 国产不卡一卡二| 国产69精品久久久久777片 | 久9热在线精品视频| 日本成人三级电影网站| 欧美zozozo另类| 国产伦一二天堂av在线观看| 国产三级黄色录像| 天堂网av新在线| 1024香蕉在线观看| 成熟少妇高潮喷水视频| 午夜影院日韩av| 精品无人区乱码1区二区| 两个人的视频大全免费| 高清毛片免费观看视频网站| 欧美日韩精品网址| а√天堂www在线а√下载| 99久久99久久久精品蜜桃| 国产成人福利小说| 国产aⅴ精品一区二区三区波| 日本 av在线| 又爽又黄无遮挡网站| 国产精品影院久久| 在线观看免费午夜福利视频| 成年女人看的毛片在线观看| 久久天躁狠狠躁夜夜2o2o| 亚洲欧美日韩东京热| 久久中文字幕人妻熟女| 中文字幕av在线有码专区| 亚洲精品美女久久av网站| 亚洲人成网站高清观看| 国产欧美日韩一区二区三| 亚洲男人的天堂狠狠| 美女免费视频网站| 人妻久久中文字幕网| 久久精品aⅴ一区二区三区四区| 色尼玛亚洲综合影院| 岛国视频午夜一区免费看| 亚洲欧美日韩高清在线视频| 国产成年人精品一区二区| 日本在线视频免费播放| 中文字幕人成人乱码亚洲影| 少妇的丰满在线观看| 亚洲自拍偷在线| 非洲黑人性xxxx精品又粗又长| 99久久久亚洲精品蜜臀av| 久久性视频一级片| 99视频精品全部免费 在线 | 久久久久免费精品人妻一区二区| 亚洲中文av在线| 精品国内亚洲2022精品成人| 99热只有精品国产| 国产人伦9x9x在线观看| 国产又色又爽无遮挡免费看| 久久精品国产99精品国产亚洲性色| 亚洲精品在线观看二区| 国产成人aa在线观看| 一区二区三区高清视频在线| 真人一进一出gif抽搐免费| 九九在线视频观看精品| 琪琪午夜伦伦电影理论片6080| 欧美极品一区二区三区四区| 成人三级黄色视频| 亚洲成a人片在线一区二区| 男人舔女人的私密视频| 成人av在线播放网站| 色老头精品视频在线观看| 深夜精品福利| xxx96com| 国产精品久久久人人做人人爽| 可以在线观看的亚洲视频| av福利片在线观看| 国产精品 国内视频| 国产乱人伦免费视频| 99久久无色码亚洲精品果冻| 成人国产综合亚洲| а√天堂www在线а√下载| 欧美激情久久久久久爽电影| 久久亚洲真实| 亚洲专区国产一区二区| 一边摸一边抽搐一进一小说| 在线观看免费视频日本深夜| 男插女下体视频免费在线播放| 国产精品久久久久久久电影 | av视频在线观看入口| 又黄又爽又免费观看的视频| 欧美zozozo另类| 国产乱人伦免费视频| 亚洲精华国产精华精| 日本成人三级电影网站| a在线观看视频网站| 欧美一区二区精品小视频在线| 国产单亲对白刺激| 欧美午夜高清在线| 99精品在免费线老司机午夜| 淫秽高清视频在线观看| 无限看片的www在线观看| 嫩草影院入口| 免费在线观看影片大全网站| 国产视频内射| 婷婷六月久久综合丁香| 亚洲在线观看片| 国内毛片毛片毛片毛片毛片| 免费av不卡在线播放| 别揉我奶头~嗯~啊~动态视频| 色在线成人网| 午夜久久久久精精品| АⅤ资源中文在线天堂| 国产成+人综合+亚洲专区| 全区人妻精品视频| 久久天躁狠狠躁夜夜2o2o| 一区二区三区高清视频在线| 特大巨黑吊av在线直播|