Abstract
Key message
We report plausible disease resistance mechanisms induced by barley resistant genotype CI89831 against Fusarium head blight (FHB) based on metabolo-transcriptomics approach. We identified HvCERK1 as a candidate gene for FHB resistance, which is functional in resistant genotype CI9831 but non-functional in susceptible cultivars H106-371 and Zhedar-2. For the first time, we were able to show a hierarchy of regulatory genes that regulated downstream biosynthetic genes that eventually produced resistance related metabolites that reinforce the cell walls to contain the pathogen progress in plant. The HvCERK1 can be used for replacing in susceptible commercial cultivars, if non-functional, based on genome editing.
Abstract
Fusarium head blight (FHB) management is a great challenge in barley and wheat production worldwide. Though barley genome sequence and advanced omics technologies are available, till date none of the resistance mechanisms has been clearly deciphered. Hence, this study was aimed at identifying candidate gene(s) and elucidating resistance mechanisms induced by barley resistant genotype CI9831 based on integrated metabolomics and transcriptomics approach. Following Fusarium graminearum infection, we identified accumulation of specific set of induced secondary metabolites, belonging to phenylpropanoid, hydroxycinnamic acid (HCAA) and jasmonic acid pathways, and their biosynthetic genes. In association with these, receptor kinases such as chitin elicitor receptor kinase (HvCERK1) and protein kinases such as MAP kinase 3 (HvMPK3) and MAPK substrate 1 (HvMKS1), and transcription factors such as HvERF1/5, HvNAC42, HvWRKY23 and HvWRKY70 were also found upregulated with high fold change. Polymorphism studies across three barley genotypes confirmed the presence of mutations in HvCERK1 gene in two susceptible genotypes, isolating this gene as a potential candidate for FHB resistance. Further, the silencing of functional HvCERK1 gene in the resistant genotype CI9831, followed by gene expression and metabolite analysis revealed its role as an elicitor recognition receptor that triggered downstream regulatory genes, which in turn, regulated downstream metabolic pathway genes to biosynthesize resistance related (RR) metabolites to contain the pathogen to spikelet infection. A putative model on metabolic pathway regulation is proposed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106–R106. doi:10.1186/gb-2010-11-10-r106
Ashkani S, Rafii MY, Shabanimofrad M et al (2014) Molecular progress on the mapping and cloning of functional genes for blast disease in rice (Oryza sativa L.): current status and future considerations. Crit Rev Biotechnol 36:353–367. doi:10.3109/07388551.2014.961403
Ayliffe M, Singh R, Lagudah E (2008) Durable resistance to wheat stem rust needed. Curr Opin Plant Biol 11:187–192. doi:10.1016/j.pbi.2008.02.001
Ballini E, Morel J-B, Droc G et al (2008) A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance. MPMI 21:859–868. doi:10.1094/MPMI-21-7-0859
Boddu J, Cho S, Kruger WM, Muehlbauer GJ (2006) Transcriptome analysis of the barley-fusarium graminearum interaction. MPMI 19:407–417. doi:10.1094/MPMI-19-0407
Boddu J, Cho S, Muehlbauer GJ (2007) Transcriptome analysis of trichothecene-induced gene expression in barley. MPMI 20:1364–1375. doi:10.1094/MPMI-20-11-1364
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. doi:10.1111/j.1364-3703.2010.00643.x
Brotman Y, Landau U, Pnini S, Lisec J et al (2012) The LysM receptor-like kinase LysM RLK1 is required to activate defense and abiotic-stress responses induced by overexpression of fungal chitinases in Arabidopsis plants. Mol Plant 5:1113–1124. doi:10.1093/mp/sss021
Buerstmayr H, Ban T, Anderson JA (2009) QTL mapping and marker-assisted selection for Fusarium head blight resistance in wheat: a review. Plant Breeding 128:1–26. doi:10.1111/j.1439-0523.2008.01550.x
Bushnell W, Hazen B, Pritsch C, Leonard K (2003) Histology and physiology of fusarium head blight. In: Leonard KJ, Bushnell WR (eds) Fusarium head blight of wheat and barley. APS Press, St. Paul, pp 44–83
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. doi:10.1556/CRC.36.2008.Suppl.B.18
Cakir C, Gillespie ME, Scofield SR (2010) Rapid determination of gene function by virus-induced gene silencing in wheat and barley. Crop Sci 50:S–77
Canel C, Moraes RM, Dayan FE, Ferreira D (2000) Podophyllotoxin. Phytochemistry 54:115–120. doi:10.1016/S0031-9422(00)00094-7
Choo TM, Vigier B, Shen QQ et al (2004) Barley traits associated with resistance to fusarium head blight and deoxynivalenol accumulation. Phytopathology 94:1145–1150. doi:10.1094/PHYTO.2004.94.10.1145
Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890. doi:10.1093/nar/16.22.10881
Danan S, Veyrieras J-B, Lefebvre V (2011) Construction of a potato consensus map and QTL meta-analysis offer new insights into the genetic architecture of late blight resistance and plant maturity traits. BMC Plant Biol 11:1–17. doi:10.1186/1471-2229-11-16
Dao TTH, Linthorst HJM, Verpoorte R (2011) Chalcone synthase and its functions in plant resistance. Phytochem Rev 10:397–412. doi:10.1007/s11101-011-9211-7
Dhokane D, Karre S, Kushalappa AC, McCartney C (2016) Integrated metabolo-transcriptomics reveals fusarium head blight candidate resistance genes in wheat QTL-Fhb2. PLoS One 11:e0155851. doi:10.1371/journal.pone.0155851
Dobritzsch M, Lübken T, Eschen-Lippold L et al (2016) MATE transporter-dependent export of hydroxycinnamic acid amides. Plant Cell 28:583–596. doi:10.1105/tpc.15.00706
Facchini PJ, Hagel J, Zulak KG (2002) Hydroxycinnamic acid amide metabolism: physiology and biochemistry. Can J Bot 80:577–589. doi:10.1139/b02-065
Fiehn O (2002) Metabolomics: the link between genotypes and phenotypes. In: Town C (ed) Functional genomics. Springer Netherlands, Dordrecht, pp 155–171
Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev Phytopathol 9:275–296. doi:10.1146/annurev.py.09.090171.001423
Gardiner SA, Boddu J, Berthiller F et al (2010) Transcriptome analysis of the barley–deoxynivalenol interaction: evidence for a role of glutathione in deoxynivalenol detoxification. MPMI 23:962–976. doi:10.1094/MPMI-23-7-0962
Golkari S, Gilbert J, Ban T, Procunier JD (2009) QTL-specific microarray gene expression analysis of wheat resistance to Fusarium head blight in Sumai-3 and two susceptible NILs. Genome 52:409–418. doi:10.1139/G09-018
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 10:3400–3419. doi:10.3390/ijms10083400
Gunnaiah R, Kushalappa AC (2014) Metabolomics deciphers the host resistance mechanisms in wheat cultivar Sumai-3, against trichothecene producing and non-producing isolates of Fusarium graminearum. Plant Physiol Biochem 83:40–50. doi:10.1016/j.plaphy.2014.07.002
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. doi:10.1371/journal.pone.0040695
Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347–369. doi:10.1146/annurev.pp.40.060189.002023
Hamzehzarghani H, Kushalappa A, Dion Y et al (2005) Metabolic profiling and factor analysis to discriminate quantitative resistance in wheat cultivars against fusarium head blight. Physiol Mol Plant Pathol 66:119–133
Huang Y, Li L, Smith KP, Muehlbauer GJ (2016) Differential transcriptomic responses to Fusarium graminearum infection in two barley quantitative trait loci associated with Fusarium head blight resistance. BMC Genom 17:387. doi:10.1186/s12864-016-2716-0
Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG (2012) ZINC: a free tool to discover chemistry for biology. J Chem Inf Model 52:1757–1768. doi:10.1021/ci3001277
Katajamaa M, Orešič M (2005) Processing methods for differential analysis of LC/MS profile data. BMC Bioinform 6:179–179. doi:10.1186/1471-2105-6-179
Katajamaa M, Miettinen J, Orešič M (2006) MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data. Bioinformatics 22:634–636. doi:10.1093/bioinformatics/btk039
Kelley LA, Mezulis S, Yates CM et al (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858
Kohorn BD, Kohorn SL (2012) The cell wall-associated kinases, WAKs, as pectin receptors. Front Plant Sci 3:88. doi:10.3389/fpls.2012.00088
Kou Y, Wang S (2010) Broad-spectrum and durability: understanding of quantitative disease resistance. Curr Opin Plant Biol 13:181–185. doi:10.1016/j.pbi.2009.12.010
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. doi:10.1016/j.jcs.2015.04.005
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. doi:10.1093/jxb/erw187
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. doi:10.1007/s10886-011-9989-1
Kushalappa AC, Gunnaiah R (2013) Metabolo-proteomics to discover plant biotic stress resistance genes. Trends Plant Sci 18:522–531. doi:10.1016/j.tplants.2013.05.002
Kushalappa AC, Yogendra KN, Karre S (2016a) Plant innate immune response: Qualitative and quantitative resistance. Crit Rev Plant Sci 35:38–55. doi:10.1080/07352689.2016.1148980
Kushalappa AC, Yogendra KN, Sarkar K et al (2016b) Gene discovery and genome editing to develop cisgenic crops with improved resistance against pathogen infection. Can J Plant Pathol 1–17. doi:10.1080/07060661.2016.1199597
Landeo JA, Gastelo M, Pinedo H, Flores F (1995) Breeding for horizontal resistance to late blight in potato free of R-genes. In: Dowley LJ, Bannon E, Cooke LR, Keane T, O’Sullivan E (eds) Phytophthora infestans 150. Boole Press, Dublin, pp 268–274
Li G, Yen Y (2008) Jasmonate and ethylene signaling pathway may mediate fusarium head blight resistance in wheat. Crop Sci 48(5):1888–1896. doi:10.2135/cropsci2008.02.0097
Libault M, Wan J, Czechowski T et al (2007) Identification of 118 arabidopsis transcription factor and 30 ubiquitin-ligase genes responding to chitin, a plant-defense elicitor. MPMI 20:900–911. doi:10.1094/MPMI-20-8-0900
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative pcr and the 2–∆∆CT method. Methods 25:402–408. doi:10.1006/meth.2001.1262
Ma M, Yan Y, Huang L, Chen M, Zhao H (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. doi:10.1186/1471-2229-12-141
Macoy DM, Kim W-Y, Lee SY, Kim MG (2015) Biosynthesis, physiology, and functions of hydroxycinnamic acid amides in plants. Plant Biotechnol Rep 9:269–278. doi:10.1007/s11816-015-0368-1
Mao G, Meng X, Liu Y et al (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive mapks drives phytoalexin biosynthesis in arabidopsis. Plant Cell 23:1639–1653. doi:10.1105/tpc.111.084996
Mert-Türk F (2002) Phytoalexins: defence or just a response to stress. J Cell Mol Biol 1:1–6
Miya A, Albert P, Shinya T et al (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA 104:19613–19618. doi:10.1073/pnas.0705147104
Muroi A, Ishihara A, Tanaka C et al (2009) Accumulation of hydroxycinnamic acid amides induced by pathogen infection and identification of agmatine coumaroyltransferase in Arabidopsis thaliana. Planta 230:517–527. doi:10.1007/s00425-009-0960-0
Naoumkina MA, Modolo LV, Huhman DV et al (2010) Genomic and coexpression analyses predict multiple genes involved in triterpene saponin biosynthesis in medicago truncatula. Plant Cell 22:850–866. doi:10.1105/tpc.109.073270
Paranidharan V, Abu-Nada Y, Hamzehzarghani H et al (2008) Resistance-related metabolites in wheat against Fusarium graminearum and the virulence factor deoxynivalenol (DON). Botany 86:1168–1179. doi:10.1139/B08-052
Pecher P, Eschen-Lippold L, Herklotz S et al (2014) The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of “VQ-motif”-containing proteins to regulate immune responses. New Phytol 203:592–606. doi:10.1111/nph.12817
Pirgozliev SR, Edwards SG, Hare MC, Jenkinson P (2003) Strategies for the control of fusarium head blight in cereals. Eur J Plant Pathol 109:731–742. doi:10.1023/A:1026034509247
Poland JA, Balint-Kurti PJ, Wisser RJ et al (2009) Shades of gray: the world of quantitative disease resistance. Trends Plant Sci 14:21–29. doi:10.1016/j.tplants.2008.10.006
Proctor RH, Hohn TM, McCormick SP (1995) Reduced virulence of Gibberella zeae caused by disruption of a trichthecine toxin biosynthetic gene. Mol Plant Microbe Interact 8:593–601
Pushpa D, Yogendra KN, Gunnaiah R et al (2013) Identification of late blight resistance-related metabolites and genes in potato through nontargeted metabolomics. Plant Mol Biol Rep 32:584–595. doi:10.1007/s11105-013-0665-1
Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:1–9. doi:10.1186/gb-2010-11-3-r25
Schweiger W, Steiner B, Ametz C et al (2013) Transcriptomic characterization of two major Fusarium resistance quantitative trait loci (QTLs), Fhb1 and Qfhs.ifa-5A, identifies novel candidate genes. Mol Plant Pathol 14:772–785. doi:10.1111/mpp.12048
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. doi:10.1104/pp.105.061861
Senthil-Kumar M, Rame Gowda HV, Hema R et al (2008) Virus-induced gene silencing and its application in characterizing genes involved in water-deficit-stress tolerance. J Plant Physiol 165:1404–1421. doi:10.1016/j.jplph.2008.04.007
Shimizu T, Nakano T, Takamizawa D et al (2010) Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice. Plant J 64:204–214. doi:10.1111/j.1365-313X.2010.04324.x
Shin S, Torres-Acosta JA, Heinen SJ et al (2012) Transgenic arabidopsis thaliana expressing a barley udp-glucosyltransferase exhibit resistance to the mycotoxin deoxynivalenol. J Exp Bot 63:4731–4740. doi:10.1093/jxb/ers141
St.Clair DA (2010) Quantitative disease resistance and quantitative resistance loci in breeding. Annu Rev Phytopathol 48:247–268. doi:10.1146/annurev-phyto-080508-081904
Starkey DE, Ward TJ, Aoki T et al (2007) Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity. Fungal Genet Biol 44:1191–1204. doi:10.1016/j.fgb.2007.03.001
Thakur RP (2007) Host plant resistance to diseases: potential and limitations. Indian J Plant Protect 35(1):17–21
Tovchigrechko A, Vakser IA (2006) GRAMM-X public web server for protein–protein docking. Nucleic Acids Res 34:W310–W314. doi:10.1093/nar/gkl206
Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20. doi:10.1093/mp/ssp106
Wan J, Zhang S, Stacey G (2004) Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin. Mol Plant Pathol 5:125–135. doi:10.1111/j.1364-3703.2004.00215.x
Wan J, Zhang X-C, Neece D et al (2008) A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in arabidopsis. Plant Cell 20:471–481. doi:10.1105/tpc.107.056754
Wang Y, Kwon SJ, Wu J et al (2014) Transcriptome analysis of early responsive genes in rice during magnaporthe oryzae infection. Plant Pathol J 30:343–354. doi:10.5423/PPJ.OA.06.2014.0055
Wen L (2013) Cell death in plant immune response to necrotrophs. J Plant Biochem Physiol. doi:10.4172/2329-9029.1000e103
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. doi:10.1105/tpc.105.037523
Yamaguchi K, Yamada K, Ishikawa K et al (2013) A receptor-like cytoplasmic kinase targeted by a plant pathogen effector is directly phosphorylated by the chitin receptor and mediates rice immunity. Cell Host Microbe 13:347–357. doi:10.1016/j.chom.2013.02.007
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. doi:10.1186/1471-2105-13-134
Yogendra KN, Pushpa D, Mosa KA et al (2014) Quantitative resistance in potato leaves to late blight associated with induced hydroxycinnamic acid amides. Funct Integr Genom 14:285–298. doi:10.1007/s10142-013-0358-8
Yogendra KN, Kumar A, Sarkar K et al (2015a) Transcription factor StWRKY1 regulates phenylpropanoid metabolites conferring late blight resistance in potato. J Exp Bot 66:7377–7389. doi:10.1093/jxb/erv434
Yogendra KN, Kushalappa AC, Sarmiento F et al (2015b) Metabolomics deciphers quantitative resistance mechanisms in diploid potato clones against late blight. Funct Plant Biol 42:284–298
Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of cereals. Weed Res 14:415–421. doi:10.1111/j.1365-3180.1974.tb01084.x
Zheng Z, Qamar SA, Chen Z, Mengiste T (2006) Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J 48:592–605. doi:10.1111/j.1365-313X.2006.02901.x
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 thank Dr. TM Choo, AAFC, for providing the seeds of barley genotypes used here.
Author contributions
SK, conducted the experiment, analyzed the data, wrote the manuscript; AK, DD helped in analyzing metabolite and transcriptome data, and developing figures; ACK*, conceived the idea, aided in designing the experiment and edited the manuscript.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Karre, S., Kumar, A., Dhokane, D. et al. Metabolo-transcriptome profiling of barley reveals induction of chitin elicitor receptor kinase gene (HvCERK1) conferring resistance against Fusarium graminearum . Plant Mol Biol 93, 247–267 (2017). https://doi.org/10.1007/s11103-016-0559-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-016-0559-3