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Plant Molecular Biology

, Volume 90, Issue 1–2, pp 19–31 | Cite as

Functional analysis of NtMPK2 uncovers its positive role in response to Pseudomonas syringae pv. tomato DC3000 in tobacco

  • Xingtan Zhang
  • Genhong Wang
  • Junping Gao
  • Mengyun Nie
  • Wenshan Liu
  • Qingyou XiaEmail author
Article

Abstract

Mitogen-activated protein kinase cascades are highly conserved signaling modules downstream of receptors/sensors and play pivotal roles in signaling plant defense against pathogen attack. Extensive studies on Arabidopsis MPK4 have implicated that the MAP kinase is involved in multilayered plant defense pathways. In this study, we identified tobacco NtMPK2 as an ortholog of AtMPK4. Transgenic tobacco overexpressing NtMPK2 markedly enhances resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) virulent and avirulent strains. Transcriptome analysis of NtMPK2-dependent genes shows that possibly the basal resistance system is activated by NtMPK2 overexpression. In addition to NtMPK2-mediated resistance, multiple pathways are involved in response to the avirulent bacteria based on analysis of Pst-responding genes, including SA and ET pathways. Notably, it is possible that biosynthesis of antibacterial compounds is responsible for inhibition of Pst DC3000 avirulent strain when programmed cell death processes in the host. Our results uncover that NtMPK2 positively regulate tobacco defense response to Pst DC3000 and improve our understanding of plant molecular defense mechanism.

Keywords

NtMPK2 Pst DC3000 Plant defense response RNA-seq 

Notes

Acknowledgments

The research was supported by National Basic Research Program of China (No. 2012CB114600) and Fundamental Research Funds for the Central Universities (No. XDJK2013C043).

Author’s contribution

XZ and QX designed the project. XZ, GW, JG, MN and WL performed the experimental analysis. XZ performed the bioinformatics analysis and wrote the manuscript. All of the authors have read the manuscript and approved the submission.

Supplementary material

11103_2015_391_MOESM1_ESM.doc (41 kb)
Supplementary Table 1 The primer sequences used in this study (DOC 41 kb)
11103_2015_391_MOESM2_ESM.doc (33 kb)
Supplementary Table 2 Statistics of sequencing and de novo assembly (DOC 33 kb)
11103_2015_391_MOESM3_ESM.png (197 kb)
Supplementary Fig. 1 Screening of NtMPK2-OX T1 generation transgenic lines using Hygromycin antibiotics (PNG 196 kb)
11103_2015_391_MOESM4_ESM.jp2 (2.7 mb)
Supplementary Fig. 2 H2O2 accumulation in WT and TG19 tobacco (JP2 2758 kb)
11103_2015_391_MOESM5_ESM.jpg (33 kb)
Supplementary Fig. 3 QRT-PCR validation of differentially expressed genes (JPEG 32 kb)
11103_2015_391_MOESM6_ESM.jpg (58 kb)
Supplementary Fig. 4 QRT-PCR analysis of 4 selected genes (JPEG 57 kb)

References

  1. Ahuja I, Kissen R, Bones AM (2012) Phytoalexins in defense against pathogens. Trends Plant Sci 17:73–90. doi: 10.1016/j.tplants.2011.11.002 PubMedCrossRefGoogle Scholar
  2. Asai T et al (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983. doi: 10.1038/415977a PubMedCrossRefGoogle Scholar
  3. Bednarek P, Pislewska-Bednarek M, Loren V, van Themaat E, Maddula RK, Svatos A, Schulze-Lefert P (2011) Conservation and clade-specific diversification of pathogen-inducible tryptophan and indole glucosinolate metabolism in Arabidopsis thaliana relatives. New Phytol 192:713–726. doi: 10.1111/j.1469-8137.2011.03824.x PubMedCrossRefGoogle Scholar
  4. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170 PubMedPubMedCentralCrossRefGoogle Scholar
  5. Broekaert WF, Delaure SL, De Bolle MF, Cammue BP (2006) The role of ethylene in host-pathogen interactions. Annu Rev Phytopathol 44:393–416. doi: 10.1146/annurev.phyto.44.070505.143440 PubMedCrossRefGoogle Scholar
  6. Browse J (2009) Jasmonate passes muster: a receptor and targets for the defense hormone. Annu Rev Plant Biol 60:183–205. doi: 10.1146/annurev.arplant.043008.092007 PubMedCrossRefGoogle Scholar
  7. Burow MD, Chlan CA, Sen P, Lisca A, Murai N (1990) High-frequency generation of transgenic tobacco plants after modified leaf disk cocultivation with Agrobacterium tumefaciens. Plant Mol Biol Rep 8:124–139CrossRefGoogle Scholar
  8. Cameron RK, Dixon RA, Lamb CJ (1994) Biologically induced systemic acquired resistance in Arabidopsis thaliana. Plant J 5:715–725CrossRefGoogle Scholar
  9. Chen RE, Thorner J (2007) Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1773:1311–1340. doi: 10.1016/j.bbamcr.2007.05.003 PubMedPubMedCentralCrossRefGoogle Scholar
  10. Chen S, Songkumarn P, Liu J, Wang GL (2009) A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiol 150:1111–1121. doi: 10.1104/pp.109.137125 PubMedPubMedCentralCrossRefGoogle Scholar
  11. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676. doi: 10.1093/bioinformatics/bti610 PubMedCrossRefGoogle Scholar
  12. Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16:10881–10890PubMedPubMedCentralCrossRefGoogle Scholar
  13. Frei dit Frey N et al (2014) Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences. Genome Biol 15:R87. doi: 10.1186/gb-2014-15-6-r87 PubMedPubMedCentralCrossRefGoogle Scholar
  14. Haas BJ et al (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512. doi: 10.1038/nprot.2013.084 PubMedCrossRefGoogle Scholar
  15. Han L, Li GJ, Yang KY, Mao G, Wang R, Liu Y, Zhang S (2010) Mitogen-activated protein kinase 3 and 6 regulate Botrytis cinerea-induced ethylene production in Arabidopsis. Plant J 64:114–127. doi: 10.1111/j.1365-313X.2010.04318.x PubMedGoogle Scholar
  16. Ichimura K, Shinozaki K, Tena G, Sheen J, Henry Y (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7:301–308CrossRefGoogle Scholar
  17. Ishihama N, Yamada R, Yoshioka M, Katou S, Yoshioka H (2011) Phosphorylation of the Nicotiana benthamiana WRKY8 transcription factor by MAPK functions in the defense response. Plant Cell 23:1153–1170. doi: 10.1105/tpc.110.081794 PubMedPubMedCentralCrossRefGoogle Scholar
  18. Jin H, Axtell MJ, Dahlbeck D, Ekwenna O, Zhang S, Staskawicz B, Baker B (2002) NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase, regulates innate immunity and development in plants. Dev Cell 3:291–297PubMedCrossRefGoogle Scholar
  19. Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329. doi: 10.1038/nature05286 PubMedCrossRefGoogle Scholar
  20. Jones P et al (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30:1236–1240. doi: 10.1093/bioinformatics/btu031 PubMedPubMedCentralCrossRefGoogle Scholar
  21. King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44:301–307PubMedGoogle Scholar
  22. Kloek AP, Brooks DM, Kunkel BN (2000) A dsbA mutant of Pseudomonas syringae exhibits reduced virulence and partial impairment of type III secretion. Mol Plant Pathol 1:139–150. doi: 10.1046/j.1364-3703.2000.00016.x PubMedCrossRefGoogle Scholar
  23. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome. BMC Bioinformatics 12:323. doi: 10.1186/1471-2105-12-323 PubMedPubMedCentralCrossRefGoogle Scholar
  24. Li G, Meng X, Wang R, Mao G, Han L, Liu Y, Zhang S (2012) Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis. PLoS Genet 8:e1002767. doi: 10.1371/journal.pgen.1002767 PubMedPubMedCentralCrossRefGoogle Scholar
  25. Liu Y, Zhang S (2004) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16:3386–3399. doi: 10.1105/tpc.104.026609 PubMedPubMedCentralCrossRefGoogle Scholar
  26. Liu Y, Wang L, Cai G, Jiang S, Sun L, Li D (2013) Response of tobacco to the Pseudomonas syringae pv. tomato DC3000 is mainly dependent on salicylic acid signaling pathway. FEMS Microbiol Lett 344:77–85. doi: 10.1111/1574-6968.12157 PubMedCrossRefGoogle Scholar
  27. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. doi: 10.1006/meth.2001.1262 PubMedCrossRefGoogle Scholar
  28. Mao G, Meng X, Liu Y, Zheng Z, Chen Z, Zhang S (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 PubMedPubMedCentralCrossRefGoogle Scholar
  29. Meng X, Zhang S (2013) MAPK cascades in plant disease resistance signaling. Annu Rev Phytopathol 51:245–266. doi: 10.1146/annurev-phyto-082712-102314 PubMedCrossRefGoogle Scholar
  30. Mudgett MB, Staskawicz BJ (1999) Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis. Mol Microbiol 32:927–941PubMedCrossRefGoogle Scholar
  31. Muthamilarasan M, Prasad M (2013) Plant innate immunity: an updated insight into defense mechanism. J Biosci 38:433–449. doi: 10.1007/s12038-013-9302-2 PubMedCrossRefGoogle Scholar
  32. Nishihama R, Ishikawa M, Araki S, Soyano T, Asada T, Machida Y (2001) The NPK1 mitogen-activated protein kinase kinase kinase is a regulator of cell-plate formation in plant cytokinesis. Genes Dev 15:352–363. doi: 10.1101/gad.863701 PubMedPubMedCentralCrossRefGoogle Scholar
  33. Petersen M et al (2000) Arabidopsis map kinase 4 negatively regulates systemic acquired resistance. Cell 103:1111–1120PubMedCrossRefGoogle Scholar
  34. Pitzschke A, Schikora A, Hirt H (2009) MAPK cascade signalling networks in plant defence. Curr Opin Plant Biol 12:421–426. doi: 10.1016/j.pbi.2009.06.008 PubMedCrossRefGoogle Scholar
  35. Remm M, Storm CE, Sonnhammer EL (2001) Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. J Mol Biol 314:1041–1052. doi: 10.1006/jmbi.2000.5197 PubMedCrossRefGoogle Scholar
  36. Ren D, Liu Y, Yang KY, Han L, Mao G, Glazebrook J, Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis. Proc Natl Acad Sci USA 105:5638–5643. doi: 10.1073/pnas.0711301105 PubMedPubMedCentralCrossRefGoogle Scholar
  37. Rogers EE, Glazebrook J, Ausubel FM (1996) Mode of action of the Arabidopsis thaliana phytoalexin camalexin and its role in Arabidopsis-pathogen interactions. Mol Plant Microbe Interact: MPMI 9:748–757PubMedCrossRefGoogle Scholar
  38. Sasaki K, Mitsuhara I, Seo S, Ito H, Matsui H, Ohashi Y (2007) Two novel AP2/ERF domain proteins interact with cis-element VWRE for wound-induced expression of the Tobacco tpoxN1 gene. Plant J 50:1079–1092. doi: 10.1111/j.1365-313X.2007.03111.x PubMedCrossRefGoogle Scholar
  39. Seo S, Sano H, Ohashi Y (1999) Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase. Plant Cell 11:289–298PubMedPubMedCentralCrossRefGoogle Scholar
  40. Soyano T, Nishihama R, Morikiyo K, Ishikawa M, Machida Y (2003) NQK1/NtMEK1 is a MAPKK that acts in the NPK1 MAPKKK-mediated MAPK cascade and is required for plant cytokinesis. Genes Dev 17:1055–1067. doi: 10.1101/gad.1071103 PubMedPubMedCentralCrossRefGoogle Scholar
  41. Su T et al (2011) Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana. Plant Cell 23:364–380. doi: 10.1105/tpc.110.079145 PubMedPubMedCentralCrossRefGoogle Scholar
  42. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi: 10.1093/molbev/mst197 PubMedPubMedCentralCrossRefGoogle Scholar
  43. Toshio M, Folke S (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  44. Viana RA, Pinar M, Soto T, Coll PM, Cansado J, Perez P (2013) Negative functional interaction between cell integrity MAPK pathway and Rho1 GTPase in fission yeast. Genetics 195:421–432. doi: 10.1534/genetics.113.154807 PubMedPubMedCentralCrossRefGoogle Scholar
  45. Vlot AC, Dempsey DA, Klessig DF (2009) Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol 47:177–206. doi: 10.1146/annurev.phyto.050908.135202 PubMedCrossRefGoogle Scholar
  46. Wang Z, Mao H, Dong C, Ji R, Cai L, Hao F, Liu S (2009) Overexpression of Brassica napus MPK4 enhances resistance to Sclerotinia sclerotiorum in oilseed rape. MPMI 22:235–244. doi: 10.1094/mpmi PubMedCrossRefGoogle Scholar
  47. Wei CF et al (2007) A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana. Plant J 51:32–46. doi: 10.1111/j.1365-313X.2007.03126.x PubMedCrossRefGoogle Scholar
  48. Whalen MC, Innes RW, Bent AF, Staskawicz BJ (1991) Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell 3:49–59. doi: 10.1105/tpc.3.1.49 PubMedPubMedCentralCrossRefGoogle Scholar
  49. Wink M (1988) Plant breeding: importance of plant secondary metabolites for protection against pathogens and herbivores. Theor Appl Genet: TAG 75:225–233CrossRefGoogle Scholar
  50. Xin XF, He SY (2013) Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol 51:473–498. doi: 10.1146/annurev-phyto-082712-102321 PubMedCrossRefGoogle Scholar
  51. Xu J, Zhang S (2015) Mitogen-activated protein kinase cascades in signaling plant growth and development. Trends Plant Sci 20:56–64. doi: 10.1016/j.tplants.2014.10.001 PubMedCrossRefGoogle Scholar
  52. Ye J et al (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293–W297. doi: 10.1093/nar/gkl031 PubMedPubMedCentralCrossRefGoogle Scholar
  53. Zeng Q, Chen JG, Ellis BE (2011) AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis. Plant J 67:895–906. doi: 10.1111/j.1365-313X.2011.04642.x PubMedCrossRefGoogle Scholar
  54. Zhang S, Klessig DF (2001) MAPK cascades in plant defense signaling. Trends Plant Sci 6:520–527PubMedCrossRefGoogle Scholar
  55. Zhang Z et al (2012) Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2. Cell Host Microbe 11:253–263. doi: 10.1016/j.chom.2012.01.015 PubMedCrossRefGoogle Scholar
  56. Zhang X, Cheng T, Wang G, Yan Y, Xia Q (2013) Cloning and evolutionary analysis of tobacco MAPK gene family. Mol Biol Rep 40:1407–1415. doi: 10.1007/s11033-012-2184-9 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Xingtan Zhang
    • 1
    • 2
  • Genhong Wang
    • 1
  • Junping Gao
    • 1
  • Mengyun Nie
    • 1
  • Wenshan Liu
    • 1
  • Qingyou Xia
    • 1
    Email author
  1. 1.State Key Laboratory of Silkworm Genome BiologySouthwest UniversityChongqingChina
  2. 2.FAFU and UIUC-SIB Joint Center for Genomics and BiotechnologyFujian Agriculture and Forestry UniversityFuzhouChina

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