Abstract
The tobacco resistance gene N is known to be upregulated transcriptionally upon infection with Tobacco mosaic virus or the transient expression of the virus elicitor (termed p50), which is the helicase domain of the virus replicase, leading to cell death-associated defense responses. To investigate the roles of the encoded protein and introns of the N gene in its elicitor-triggered upregulation, we developed an Agrobacterium-mediated transient gene transfer system to express the N transgene controlled by the upstream region of the N gene itself in N-lacking tobacco. The transcript level of the N transgene with the introns was enhanced sharply when the p50 transgene was coexpressed. Introduction of a frameshift mutation in the intron-containing N transgene abolished its response to the elicitor, but an additional expression of the functional N protein supplied in trans complemented the upregulation of the mutant transgene. In contrast, an intron-less N transgene failed to be upregulated by the p50 coexpression, resulting in delayed and attenuated cell death and slow induction of defense-related gene expression. In addition, introduction of a frameshift mutation in the intron-less N transgene resulted in no response to the elicitor even with coexpression of the functional N protein. Our data provide the first evidence for important roles of introns of a resistance gene in elicitor-responsive gene regulation and efficient defense induction.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abbink TEM, Tjernberg PA, Bol JF, Linthorst HJM (1998) Tobacco mosaic virus helicase domain induces necrosis in N gene-carrying tobacco in the absence of virus replication. Mol Plant Microbe Interact 12:1242–1246
Brederode FT, Linthorst HJM, Bol JF (1991) Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding. Plant Mol Biol 17:1117–1125
Cao Y, Ding X, Cai M, Zhao J, Lin Y, Li X, Xu C, Wang S (2007) The expression pattern of a rice disease resistance gene Xa3/Xa26 is differentially regulated by the genetic backgrounds and developmental stages that influence its function. Genetics 177:523–533
Christie M, Croft LJ, Carroll BJ (2011) Intron splicing suppresses RNA silencing in Arabidopsis. Plant J 68:159–167
Dinesh-Kumar SP, Baker BJ (2000) Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance. Proc Natl Acad Sci USA 97:1908–1913
Eulgem T, Tsuchiya T, Wang XJ, Beasley B, Cuzick A, Tör M, Zhu T, McDowell JM, Holub E, Dangl JL (2007) EDM2 is required for RPP7-dependent disease resistance in Arabidopsis and affects RPP7 transcript levels. Plant J 49:829–839
Friedrich L, Vernooij B, Gaffney T, Morse A, Ryals J (1995) Characterization of tobacco plants expressing a bacterial salicylate hydroxylase gene. Plant Mol Biol 29:959–968
Gao J-S, Sasaki N, Kanegae H, Konagaya K, Takizawa K, Hayashi N, Okano Y, Kasahara M, Matsushita Y, Nyunoya H (2007) The TIR–NBS but not LRR domains of two novel N-like proteins are functionally competent to induce the elicitor p50-dependent hypersensitive response. Physiol Mol Plant Pathol 71:78–87
Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207
Gu K, Yang B, Tian D, Wu L, Wang D, Sreekala C, Yang F, Chu Z, Wang GL, White FF, Yin Z (2005) R gene expression induced by a type-III effector triggers disease resistance in rice. Nature 435:1122–1125
Halterman DA, Wei F, Wise RP (2003) Powdery mildew-induced Mla mRNAs are alternatively spliced and contain multiple upstream open reading frames. Plant Physiol 131:558–567
Haque MdA, Sasaki N, Kanegae H, Mimori S, Gao J-S, Nyunoya H (2009) Identification of a Tobacco mosaic virus elicitor-responsive sequence in the resistance gene N. Physiol. Mol Plant Pathol 73:101–108
Heil M, Baldwin IT (2002) Fitness costs of induced resistance: emerging experimental support for a slippery concept. Trends Plant Sci 7:61–67
Kathiria P, Sidler C, Woycicki R, Yao Y, Kovalchuk I (2013) Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter. Plant Signal Behav 8:e24760
Kiba A, Tomiyama H, Takahashi H, Hamada H, Ohnishi K, Okuno T, Hikichi Y (2003) Induction of resistance and expression of defense-related genes in tobacco leaves infiltrated with Ralstonia solanacearum. Plant Cell Physiol 44:287–295
Kobayashi M, Ishihama N, Yoshioka H, Takabatake R, Tsuda S, Seo S, Ohashi Y, Mitsuhara I (2010) Analyses of the cis-regulatory regions responsible for the transcriptional activation of the N resistance gene by Tobacco mosaic virus. J Phytopathol 158:826–828
Kramer LC, Choudoir MJ, Wielgus SM, Bhaskar PB, Jiang J (2009) Correlation between transcript abundance of the RB gene and the level of the RB-mediated late blight resistance in potato. Mol Plant Microbe Interact 22:447–455
Krizek BA (2015) Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers. BMC Res Notes 8:556
Levy M, Edelbaum O, Sela I (2004) Tobacco mosaic virus regulates the expression of its own resistance gene N. Plant Physiol 135:2392–2397
Li F, Pignatta D, Bendix C, Brunkard JO, Cohn MM, Tung J, Sun H, Kumar P, Baker B (2012) MicroRNA regulation of plant innate immune receptors. Proc Natl Acad Sci USA 109:1790–1795
Liu Y, Burch-Smith T, Schiff M, Feng S, Dinesh-Kumar SP (2004) Molecular chaperone Hsp90 associates with resistance protein N and its signaling proteins SGT1 and Rar1 to modulate an innate immune response in plants. J Biol Chem 279:2101–2108
Marathe R, Anandalakshmi R, Liu Y, Dinesh-Kumar SP (2002) The tobacco mosaic virus resistance gene, N. Mol Plant Pathol 3:167–172
Mohr TJ, Mammarella ND, Hoff T, Woffenden BJ, Jelesko JG, McDowell JM (2010) The Arabidopsis downy mildew resistance gene RPP8 is induced by pathogens and salicylic acid and is regulated by W box cis elements. Mol Plant Microbe Interact 23:1303–1315
Narsai R, Howell KA, Millar AH, O’Toole N, Small I, Whelan J (2007) Genome-wide analysis of mRNA decay rates and their determinants in Arabidopsis thaliana. Plant Cell 19:3418–3436
Ogata T, Kida Y, Arai T, Kishi Y, Manago Y, Murai M, Matsushita Y (2012) Overexpression of tobacco ethylene response factor NtERF3 gene and its homologues from tobacco and rice induces hypersensitive response-like cell death in tobacco. J Gen Plant Pathol 78:8–17
Ogata T, Okada H, Kawaide H, Takahashi H, Seo S, Mitsuhara I, Matsushita Y (2015) Involvement of NtERF3 in the cell death signalling pathway mediated by SIPK/WIPK and WRKY1 in tobacco plants. Plant Biol 17:962–972
Sasaki N, Takaoka M, Sasaki S, Hirai K, Meshi T, Nyunoya H (2013) The splice variant Ntr encoded by the tobacco resistance gene N has a role for negative regulation of antiviral defense responses. Physiol Mol Plant Pathol 84:92–98
Sato Y, Ando S, Takahashi H (2014) Role of intron-mediated enhancement on accumulation of an Arabidopsis NB-LRR class R-protein that confers resistance to Cucumber mosaic virus. PLoS One 9:e99041
Takabatake R, Seo S, Mitsuhara I, Tsuda S, Ohashi Y (2006) Accumulation of the two transcripts of the N gene, conferring resistance to Tobacco mosaic virus, is probably important for N gene-dependent hypersensitive cell death. Plant Cell Physiol 47:254–261
Takano M, Haque MdA, Odaira S, Nakata K, Sasaki N, Nyunoya H (2013) Overexpression of a tobacco Dof transcription factor BBF1 stimulates the transcription of the tobacco mosaic virus resistance gene N and defense-related responses including ROS production. Plant Biotechnol 30:37–46
Wang Z-X, Yamanouchi U, Katayose Y, Sasaki T, Yano M (2001) Expression of the Pib rice-blast-resistance gene family is up-regulated by environmental conditions favouring infection and by chemical signals that trigger secondary plant defences. Plant Mol Biol 47:653–661
Yamamoto S, Nakano T, Suzuki K, Shinshi H (2004) Elicitor-induced activation of transcription via W box-related cis-acting elements from a basic chitinase gene by WRKY transcription factors in tobacco. Biochim Biophys Acta 1679:279–287
Yoshimura S, Yamanouchi U, Katayose Y, Toki S, Wang Z-X, Kono I, Kurata N, Yano M, Iwata N, Sasaki T (1998) Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA 95:1663–1668
Zhang X-C, Gassmann W (2003) RPS4-mediated disease resistance requires the combined presence of RPS4 transcripts with full-length and truncated open reading frames. Plant Cell 15:2333–2342
Zhang X-C, Gassmann W (2007) Alternative splicing and mRNA levels of the disease resistance gene RPS4 are induced during defense responses. Plant Physiol 145:1577–1587
Acknowledgements
This work was supported partly by Japan Society for Promotion of Science (JSPS) KAKENHI Grant number 26450052 (to N.S.).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Taku, K., Sasaki, N., Matsuzawa, K. et al. Introns of the tobacco resistance gene N play important roles in elicitor-responsive upregulation and efficient induction of defense responses. J Gen Plant Pathol 84, 73–84 (2018). https://doi.org/10.1007/s10327-018-0762-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10327-018-0762-y