Abstract
Crop plant resistance against pathogens is governed by dynamic molecular and biochemical responses driven by complex transcriptional networks. However, the underlying mechanisms are largely unclear. Here we report an interesting role of HvWRKY23 transcription factor (TF) in modulating defense response against Fusarium head blight (FHB) in barley. The combined approach of gene silencing, metabolomics, real time expression analysis and ab initio bioinformatics tools led to the identification of the HvWRKY23 role in FHB resistance. The knock-down of HvWRKY23 gene in the FHB resistant barley genotype CI9831, followed by inoculation with Fusarium graminearum, led to the down regulation of key flavonoid and hydroxycinnamic acid amide biosynthetic genes resulting in reduced accumulation of resistant related (RR) secondary metabolites such as pelargonidin 3-rutinoside, peonidin 3-rhamnoside-5-glucoside, kaempferol 3-O-arabinoside and other flavonoid glycosides. Reduced abundances of RR metabolites in TF silenced plants were also associated with an increased proportion of spikelets diseased and amount of fungal biomass in spikelets, depicting the role of HvWRKY23 in disease resistance. The luciferase reporter assay demonstrated binding of HvWRKY23 protein to promoters of key flavonoid and hydroxycinnamic acid amides (HCAA) biosynthetic genes, such as HvPAL2, HvCHS1, HvHCT, HvLAC15 and HvUDPGT. The accumulation of high abundances of HCAAs and flavonoid glycosides reinforce cell walls to contain the pathogen to initial infection area. This gene in commercial cultivars can be edited, if non-functional, to enhance resistance against FHB.
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References
Agarwal P, Reddy MP, Chikara J (2011) WRKY: its structure, evolutionary relationship, DNA-binding selectivity, role in stress tolerance and development of plants. Mol Biol Rep 38:3883–3896
Ahuja I, Kissen R, Bones AM (2012) Phytoalexins in defense against pathogens. Trends Plant Sci 17:73–90
Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60:379–406
Bollina V, Kumaraswamy GK, Kushalappa AC et al (2010) Mass spectrometry-based metabolomics application to identify quantitative resistance-related metabolites in barley against Fusarium head blight. Mol Plant Pathol 11:769–782
Bollina V, Kushalappa AC, Choo TM et al (2011) Identification of metabolites related to mechanisms of resistance in barley against Fusarium graminearum, based on mass spectrometry. Plant Mol Biol 77:355–370
Cakir C, Scofield S (2008) Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes. Cereal Res Commun 36:217–222
Castro ED, Sigrist CJA, Gattiker A (2006) ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res 34:W362–W365
Chow CN, Zheng HQ, Wu NY et al (2016) PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants. Nucleic Acids Res 44(D1):D1154–D1160
Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16(22):10881–10890
Dang FF, Wang YN, Yu L et al (2013) CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant, Cell Environ 36:757–774
Didi V, Jackson P, Hejátko J (2015) Hormonal regulation of secondary cell wall formation. J Exp Bot 66:5015–5027
Ding L, Xu H, Yi H et al (2011) Resistance to hemi-biotrophic F. graminearum infection is associated with coordinated and ordered expression of diverse defense signaling pathways. PLoS ONE 6:e19008
Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206
Foster-Hartnett D, Danesh D, Peñuela S et al (2007) Molecular and cytological responses of Medicago truncatula to Erysiphe pisi. Mol Plant Pathol 8:307–319
Gao X, Cox LK Jr, He P (2014) Functions of calcium-dependent protein kinases in plant innate immunity. Plants 3(1):160–176
Gil-Humanes J, Wang Y, Liang Z et al (2017) High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J 89:1251–1262
González-Lamothe R, Mitchell G, Gattuso M et al (2009) Plant antimicrobial agents and their effects on plant and human pathogens. Int J Mol Sci 10(8):3400–3419
Grunewald W, De Smet I, Lewis DR et al (2012) Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis. Proc Natl Acad Sci USA 109:1554–1559
Gunnaiah R, Kushalappa AC, Duggavathi R et al (2012) Integrated metabolo-proteomic approach to decipher the mechanisms by which wheat qtl (fhb1) contributes to resistance against Fusarium graminearum. PLoS ONE 7:e40695
Ishihara A, Hashimoto Y, Tanaka C et al (2008) The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production. Plant J 54:481–495
Jin J, Zhang H, Kong L et al (2014) PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Res 42:D1182–D1187
Kage U, Yogendra KN, Kushalappa AC (2017) TaWRKY70 transcription factor in wheat QTL-2DL regulates downstream metabolite biosynthetic genes to resist Fusarium graminearum infection spread within spike. Nat Sci Rep 7:42596
Karre S, Kumar A, Dhokane D, Kushalappa AC (2017) Metabolo-transcriptome profiling of barley reveals induction of chitin elicitor receptor kinase gene (HvCERK1) conferring resistance against Fusarium graminearum. Plant Mol Biol 93(3):247–267
Katajamaa M, Orešič M (2005) Processing methods for differential analysis of LC/MS profile data. BMC Bioinform 6:1–12
Katajamaa M, Miettinen J, Orešič M (2006) MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data. Bioinformatics 22:634–636
Kelley LA, Mezulis S, Yates CM et al (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protocols 10:845–858
Kosugi S, Hasebe M, Matsumura N et al (2008) Six classes of nuclear localization signals specific to different binding grooves of importin α. J Biol Chem 284(1):478–485
Kumar A, Karre S, Dhokane D et al (2015) Real-time quantitative PCR based method for the quantification of fungal biomass to discriminate quantitative resistance in barley and wheat genotypes to Fusarium head blight. J Cereal Sci 64:16–22
Kumar A, Yogendra KN, Karre S et al (2016) WAX INDUCER1 (HvWIN1) transcription factor regulates free fatty acid biosynthetic genes to reinforce cuticle to resist Fusarium head blight in barley spikelets. J Exp Bot 67(14):4127–4139
Kumaraswamy KG, Kushalappa AC, Choo TM et al (2011) Mass spectrometry-based metabolomics to identify potential biomarkers for resistance in barley against Fusarium head blight (Fusarium graminearum). J Chem Ecol 37:846–856
Kushalappa AC, Gunnaiah R (2013) Metabolo-proteomics to discover plant biotic stress resistance genes. Trends Plant Sci 18:522–531
Kushalappa AC, Yogendra KN, Karre S (2016a) Plant innate immune response: qualitative and quantitative resistance. Crit Rev Plant Sci 35:38–55
Kushalappa AC, Yogendra KN, Sarkar K et al (2016b) Gene discovery and genome editing to develop cisgenic crops with improved resistance against pathogen stress. Can J Plant Pathol. https://doi.org/10.1080/07060661.2016.1199597
Lai Z, Vinod K, Zheng Z et al (2008) Roles of arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens. BMC Plant Biol 8:1–13
Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275
Letunic I, Doerks T, Bork P (2015) SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43:D257–D260
Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16:319–331
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2−ΔΔCT method. Methods 25:402–408
Lorenc-Kukuła K, Jafra S, Oszmiański J, Szopa J (2005) Ectopic expression of anthocyanin 5-o-glucosyltransferase in potato tuber causes increased resistance to bacteria. J Agric Food Chem 53:272–281
Ma M, Yan Y, Huang L et al (2012) Virus-induced gene-silencing in wheat spikes and grains and its application in functional analysis of HMW-GS-encoding genes. BMC Plant Biol 12:141
Martin JT (1964) Role of cuticle in the defense against plant disease. Annu Rev Phytopathol 2:81–100
Masada S, Terasaka K, Oguchi Y et al (2009) Functional and structural characterization of a flavonoid glucoside 1,6-glucosyltransferase from Catharanthus roseus. Plant Cell Physiol 50:1401–1415
Mierziak J, Kostyn K, Kulma A (2014) Flavonoids as important molecules of plant interactions with the environment. Molecules 19(10):16240–16265
Mukhtar MS, Deslandes L, Auriac MC et al (2008) The Arabidopsis transcription factor WRKY27 influences wilt disease symptom development caused by Ralstonia solanacearum. Plant J 56:935–947
Munteanu MG, Vlahovicek K, Parthasarathy S et al (1998) Rod models of DNA: sequence-dependent anisotropic elastic modelling of local bending phenomena. Trends Biochem Sci 23:341–347
Onkokesung N, Reichelt M, Doorn AV et al (2014) Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae. J Exp Bot. https://doi.org/10.1093/jxb/eru096
Padmavati M, Sakthivel N, Thara KV, Reddy AR (1997) Differential sensitivity of rice pathogens to growth inhibition by flavonoids. Phytochemistry 46:499–502
Pandey SP, Somssich IE (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol 150:1648–1655
Parvez MM, Tomita-Yokotani K, Fujii Y et al (2004) Effects of quercetin and its seven derivatives on the growth of Arabidopsis thaliana and Neurospora crassa. Biochem Syst Ecol 32:631–635
Phukan UJ, Jeena GS, Shukla RK (2016) WRKY transcription factors: molecular regulation and stress responses in plants. Front Plant Sci 7(760):1–14
Proctor RH, Hohn TM, McCormick SP (1995) Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol Plant Microbe Interact 8:593–601
Qiu JL, Fiil BK, Petersen K et al (2008) Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. EMBO J 27:2214–2221
Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15:247–258
Sarowar S, Alam ST, Makandar R, Lee H, Trick HN, Dong Y, Shah J (2018) Targeting the pattern-triggered immunity pathway for enhancing resistance to Fusarium graminearum. Mol Plant Pathol. https://doi.org/10.1111/mpp.12781
Schön M, Töller A, Diezel C et al (2013) Analyses of wrky18 wrky40 Plants Reveal Critical Roles of SA/EDS1 Signaling and Indole-Glucosinolate Biosynthesis for Golovinomyces orontii Resistance and a Loss-of Resistance Towards Pseudomonas syringae pv. tomato AvrRPS4. MPMI 26:758–767
Scofield SR, Huang L, Brandt AS, Gill BS (2005) 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 138:2165–2173
Sudisha J, Sharathchandra RG, Amruthesh KN et al (2012) Pathogenesis related proteins in plant defense response. In: Mérillon MJ, Ramawat GK (eds) Plant defence: biological control. Springer, Dordrecht, pp 379–403
Tena G, Asai T, Chiu WL, Sheen J (2001) Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol 4:392–400
Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31:455–461
Tuszynska I, Magnus M, Jonak K et al (2015) NPDock: a web server for protein–nucleic acid docking. Nucleic Acids Res 43:W245–W430
Vandromme M, Gauthier-Rouvière C, Lamb N, Fernandez A (1996) Regulation of transcription factor localization: fine-tuning of gene expression. Trends Biochem Sci 21:59–64
Velasco P, Lema M, Francisco M et al (2013) In vivo and in vitro effects of secondary metabolites against Xanthomonas campestris pv. campestris. Molecules 18(9):11131
Von Arnim AG, Deng XW, Stacey MG (1998) Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene 221(1):35–43
Wang X, Fan C, Zhang X et al (2013) BioVector, a flexible system for gene specific-expression in plants. BMC Plant Biol 13:198
Watanabe N, Lam E (2006) Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell death. Plant J 45:884–894
Xing DH, Lai ZB, Zheng ZY et al (2008) Stress- and pathogen-induced Arabidopsis WRKY48 is a transcriptional activator that represses plant basal defense. Mol Plant 1:459–470
Xu X, Chen C, Fan B, Chen Z (2006) Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. Plant Cell 18:1310–1326
Yamaguchi K, Yamada K, Kawasaki T (2013) Receptor-like cytoplasmic kinases are pivotal components in pattern recognition receptor-mediated signaling in plant immunity. Plant Signal Behav 8:e25662
Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 13:1–11
Yogendra KN, Kumar A, Sarkar K et al (2015) Transcription factor StWRKY1 regulates phenylpropanoid metabolites conferring late blight resistance in potato. J Exp Bot 66:7377–7389
Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: aversatile cell system for transient gene expression analysis. Nat Protoc 2(7):1565–1572
Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of cereals. Weed Res 14:415–421
Acknowledgements
We acknowledge the financial support of Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (MAPAQ), Québec, Canada, and Natural Sciences and Engineering Research Council of Canada (NSERC). We also acknowledge Mr. Sripad Joshi, Plant Science Department, McGill University for assisting in protein–ligand docking.
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SK, conducted the experiment, analyzed the data, wrote the manuscript; YK, helped in luciferase experiment; AK, UK helped in analyzing metabolite data and developing figures; JB, provided guidance and helped to design the molecular work carried in this paper; Ajjamada Kushalappa, conceived the idea, aided in designing the experiment and edited the manuscript.
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Karre, S., Kumar, A., Yogendra, K. et al. HvWRKY23 regulates flavonoid glycoside and hydroxycinnamic acid amide biosynthetic genes in barley to combat Fusarium head blight. Plant Mol Biol 100, 591–605 (2019). https://doi.org/10.1007/s11103-019-00882-2
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DOI: https://doi.org/10.1007/s11103-019-00882-2