Abstract
A novel pathogen-induced gene encoding the RAV (Related to ABI3/VP1) transcription factor, CARAV1, was isolated from pepper leaves infected with Xanthomonas campestris pv. vesicatoria. CARAV1 contains two distinct DNA-binding domains AP2 and B3 uniquely found in higher plants. Transient expression analysis of the smGFP:CARAV1 fusion construct in Arabidopsis protoplasts and pepper epidermal cells revealed the CARAV1 protein to be localized in the nucleus. The N-terminal region of CARAV1 fused to the GAL4 DNA-binding domain was required to activate transcription of reporter genes in yeast. In yeast one-hybrid, the recognition of CAACA and CACCTG motifs also were essential for the CARAV1 protein to bind to a specific target gene and activate the reporter gene. The expression of the CARAV1 gene was strongly induced early in pepper leaves during the pathogen infection, abiotic elicitors and environmental stresses. CARAV1 transcripts were localized in the phloem cells of leaf tissues during pathogen infection and ethylene treatment. Ectopic expression of the CARAV1 gene in transgenic Arabidopsis plants induced some PR genes and enhanced resistance against infection by Pseudomonas syringae pv. tomato DC3000 and osmotic stresses by high salinity and dehydration. The CARAV1 promoter activation was induced by P. syringae pv. tabaci, salicylic acid and abscisic acid. These data suggest that pathogen- and abiotic stress-inducible CARAV1 functions as a transcriptional activator triggering resistance to bacterial infection and tolerance to osmotic stresses.
Similar content being viewed by others
References
Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D, Shinozaki K (1997) Role of Arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression. Plant Cell 9:1859–1868
Bestwick CS, Brown IR, Bennett MHR, Mansfield JW (1997) Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv. phaseolicola. Plant Cell 9:209–221
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254
Chen W, Provart NJ, Glazebrook J, Katagiri F, Chang HS, Eulgem T, Mauch F, Luan S, Zou G, Whitham SA, Budworth PR, Tao Y, Xie Z, Chen X, Lam S, Kreps JA, Harper JF, Si-Ammour A, Mauch-Mani B, Heinlein M, Kobayashi K, Hohn T, Dangle JL, Wang X, Zhu T (2002) Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell 14:559–574
Chomczynski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743
Dangl JL, Dietrich RA, Richberg MH (1996) Death don’t have no mercy: cell death programs in plant-microbe interactions. Plant Cell 8:1793–1807
Do HM, Hong JK, Jung HW, Kim SH, Ham JH, Hwang BK (2003) Expression of peroxidase-like genes, H2O2 production, and peroxidase activity during the hypersensitive response to Xanthomonas campestris pv. vesicatoria in Capsicum annuum. Mol Plant-Microbe Interact 16:196–205
Ezcurra I, Wycliffe P, Nehlin L, Ellerstrom M, Rask L (2000) Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis-elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G-box. Plant J 24:57–66
Finkelstein RR, Lynch TJ (2000) The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell 12:599–609
Giraudat J, Jauge BM, Valon C, Smalle J, Parcy F, Goodman HM (1992) Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4:1251–1261
Glazebrook J (1999) Genes controlling expression of defense responses in Arabidopsis. Curr Opin Plant Biol 2:280–286
Hammond-Kosack KE, Jones DG (1996) Resistance gene-dependent plant defense responses. Plant Cell 8:1773–1791
Hao D, Ohme-Takagi M, Sarai A (1998) Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF domain) in plant. J Biol Chem 273:26857–26861
Heinekamp T, Kuhlmann M, Lenk A, Strathmann A, Droge-Laser W (2002) The tobacco bZIP transcription factor BZI-1 binds to G-box elements in the promoters of phenylpropanoid pathway genes in vitro, but it is not involved in their regulation in vivo. Mol Gen Genomics 267:16–26
Hill A, Nantel A, Rock CD, Quatrano RS (1996) A conserved domain of the viviparous-1 gene product enhances the DNA binding activity of the bZIP protein EmBP-1 and other transcription factors. J Biol Chem 271:3366–3374
Hong JK, Hwang BK (2005) Functional characterization of PR-1 protein, β-1,3-glucanase and chitinase genes during defense response to biotic and abiotic stresses in Capsicum annuum. Plant Pathol J 21:195–206
Iwabuchi K, Li B, Bartel P, Fields S (1993) Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene 8:1693–1696
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol 21:121–131
Jung HW, Hwang BK (2000) Isolation, partial sequencing, and expression of pathogenesis-related cDNA genes from pepper leaves infected by Xanthomonas campestris pv. vesicatoria. Mol Plant-Microbe Interact 13:136–142
Kagaya Y, Ohmiya K, Hattori T (1999) RAV1, a novel DNA-binding protein, binds top bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res 27:470–478
Kang SG, Jin JB, Piao HL, Pih KT, Jang HJ, Lim JH, Hwang I (1998) Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids. Plant Mol Biol 38:437–447
Kim YJ, Hwang BK (2000) Pepper gene encoding a basic pathogenesis-related protein1 is pathogen and ethylene inducible. Physiol Plant 108:51–60
Kim YJ, Martin GB (2004) Molecular mechanisms involved in bacterial speck disease resistance of tomato. Plant Pathol J 20:7–12
Kim SH, Hong JK, Lee SC, Sohn KH, Jung HW, Hwang BK (2004) CAZFP1, Cys2/His2-type zinc-finger transcription factor gene functions as a pathogen-induced early-defense gene in Capsicum annuum. Plant Mol Biol 55:883–904
Lamb C, Dixon R (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275
Lee SC, Hwang BK (2003) Identification of the pepper SAR8.2 gene as a molecular marker for pathogen infection, abiotic elicitors and environmental stresses in Capsicum annuum. Planta 216:387–396
Lee SC, Hong JK, Kim YJ, Hwang BK (2000a) Pepper gene encoding thionin is differentially induced by pathogens, ethylene and methyl jasmonate. Physiol Mol Plant Pathol 56:207–216
Lee SC, Lee YK, Kim KD, Hwang BK (2000b) In situ hybridization study of organ- and pathogen-dependent expression of a novel thionin gene in pepper (Capsicum annuum). Physiol Plant 110:384–392
Lee SJ, Lee MY, Yi SY, Oh SK, Choi SH, Her NH, Choi D, Min BW, Yang SG, Harn CH (2002) PPI1: a novel pathogen-induced basic region-leucine zipper (bZIP) transcription factor from pepper. Mol Plant-Microbe Interact 15:540–548
Lee J-H, Kim S-H, Jung Y-H, Kim J-A, Lee M-O, Choi P-G, Choi W, Kim K-N, Jwa N-S (2005) Molecular cloning and functional analysis of rice (Oryza sativa L.) OsNDR1 on defense signaling pathway. Plant Pathol J 21:149–157
Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–595
Liu L, White MJ, MacRae TH (1999) Transcription factors and their genes in higher plants. Eur J Biochem 262:247–257
Luerssen K, Kirik V, Herrmann P, Misera S (1998) FUSCA3 encodes a protein with a conserved VP1/ABI3-like B3 domain which is of functional importance for the regulation of seed maturation in Arabidopsis thaliana. Plant J 15:755–764
McCarty DR, Hattori T, Carson CB, Vasil V, Lazar M, Vasil IK (1991) The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator. Cell 66:895–905
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497
Ohme-Takagi M, Shinshi H (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell 7:173–182
Ohta M, Ohme-Takagi M, Shinshi H (2000) Three ethylene-responsive transcriptional factors in tobacco with distinct transactivation functions. Plant J 22:29–38
Okamuro JK, Caster B, Villarroel R, van Montagu M, Jofuku KD (1997) The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci USA 94:7076–7081
Park JM (2005) The hypersensitive response. A cell death during disease resistance. Plant Pathol J 21:99–101
Penninckx IAMA, Thomma BPHJ, Buchala A, Métraux J, Broekaert WF (1998) Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10:2103–2114
Reidt W, Wohlfarth T, Ellerstrom M, Czihal A, Tewes A, Ezcurra I, Rask L, Baumlein H (2000) Gene regulation during late embryogenesis: the RY motif of maturation-specific gene promoters is a direct target of the FUS3 gene product. Plant J 21:401–408
Riechmann JL, Meyerowitz EM (1998) The AP2/EREBP family of plant transcription factors. Biol Chem 379:633–646
Riechmann JL, Heard J, Martin G, Reuber L, Jiang CZ, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu GL (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110
Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY, Hunt MD (1996) Systemic acquired resistance. Plant Cell 8:1809–1819
Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002) DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem Biophys Res Commun 290:998–1009
Samac DA, Shah DM (1991) Developmental and pathogen- induced activation of the Arabidopsis acidic chitinase promoter. Plant Cell 3:1063–1072
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press
Schwechheimer C, Bevan M (1998) The regulation of transcription factor activity in plants. Trends Plant Sci 3:378–383
Singh KB, Foley RC, Onate-Sanchez L (2002) Transcription factors in plant defense and stress responses. Curr Opin Plant Biol 5:430–436
Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ (2001) LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development. Proc Natl Acad Sci USA 98:11806–11811
Suzuki M, Kao CY, McCarty DR (1997) The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity. Plant Cell 9:799–807
Takeuchi Y, Dotson M, Keen NT (1992) Plant transformation: a simple particle bombardment device based on flowing helium. Plant Mol Biol 18:835–839
Thomma BPHJ, Eggermont K, Penninckx IAMA, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA 95:15107–15111
Thomma BPHJ, Eggermont K, Tierens KFMJ, Broekaert WF (1999) Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiol 121:1093–1101
Triezenberg SJ (1995) Structure and function of transcriptional activation domains. Curr Opin Gen Dev 5:190–196
Ukness S, Mauch-Mani B, Moyer M, Potter S, Williams S, Dincher S, Chandler D, Slusarenko A, Ward E, Ryals J (1992) Acquired resistance in Arabidopsis. Plant Cell 4:645–656
Ülker B, Somssich I (2004) WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 7:491–498
Ulmasov T, Hagen G, Guifoyle TJ (1997) ARF1, a transcription factor that binds to auxin response elements. Science 276:1865–1868
Ward ER, Uknes SJ, Williams SC, Dincher SS, Wiederhold DL, Alexander DC, Ahl-Goy P, Metraux J, Ryals JA (1991) Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3:1085–1094
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
Wubben JP, Lawrence CB, de Wit PJGM (1996) Differential induction of chitinase and 1, 3-β-glucanase gene expression in tomato by Cladosporium fulvum and its race-specific elicitors. Physiol Mol Plant Pathol 48:105–116
Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264
Yang Y, Shah J, Klessig DF (1997) Signal perception and transduction in plant defense responses. Genes Dev 11:1621–1639
Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551
Author information
Authors and Affiliations
Corresponding author
Additional information
Kee Hoon Sohn and Sung Chul Lee contributed equally to the paper
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Sohn, K.H., Lee, S.C., Jung, H.W. et al. Expression and functional roles of the pepper pathogen-induced transcription factor RAV1 in bacterial disease resistance, and drought and salt stress tolerance. Plant Mol Biol 61, 897–915 (2006). https://doi.org/10.1007/s11103-006-0057-0
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11103-006-0057-0