Analyses of the cis-Regulatory Regions Responsible for the Transcriptional Activation of the N Resistance Gene by Tobacco mosaic virus

Although the tobacco N gene, which confers resistance to Tobacco mosaic virus (TMV), is transcriptionally induced by infection with TMV, the regulatory mechanism has not previously been elucidated. We found that a 5¢-flanking region of the N gene exhibits TMVinducible promoter activity and that the region from )290 to )271 includes a cis-acting element in response to infection. The activity required N protein and signal components of N-mediated resistance. These results suggest that transcription of the N gene is positively regulated by N-mediated resistance signalling. Introduction The tobacco N gene confers resistance to Tobacco mosaic virus (TMV) (Whitham et al. 1994) through recognition of its avirulence factor p50, a 50-kDa fragment of TMV replicase. Alternative splicing of the N mRNA after TMV infection is necessary for complete resistance (Dinesh-Kumar and Baker 2000), and transcription of the N gene is induced by infection (Levy et al. 2004). However, the mechanism of the transcriptional regulation has not yet been elucidated. Here, we show that a 5¢-flanking region of N exhibits p50-inducible promoter activity, and the activity depends on components of N-mediated TMV resistance signalling. Materials and Methods A 1908-bp 5¢-flanking fragment with the first 38-bp coding sequence of N and the Cauliflower mosaic virus (CaMV) 35S promoter region were inserted into pGreen0029 (Hellens et al. 2000) and fused to an intron-containing b-glucuronidase (GUSint), flanked by the nopaline synthase terminator sequence (TNOS), to obtain PN::GUSint and P35S::GUSint, respectively. GUSint of P35S::GUSint was substituted for an introncontaining luciferase (LUCint) to obtain P35S::LUCint. P35S of P35S::GUSint was substituted for the CaMV 35S minimal promoter region to obtain P35Sm::GUSint. Four repeats of the -300 to -251 region of the N promoter were inserted into P35Sm::GUSint to obtain P4xM0::GUSint. The p50 sequence was inserted into pER8 (Zuo et al. 2000) to obtain pER8::p50. A genomic sequence of the N gene fused to the coding sequence for the triple hemagglutinin (HA)-tag was inserted into pEl2W (Ohtsubo et al. 1999) to obtain P35S::N3xHA. Agrobacterium (GV3101) transformed with the constructs was cultured at 28 C, harvested, suspended in 10 mm MES-NaOH, pH 5.6, 10 mm MgCl2, and 150 lm acetosyringone, and incubated for 2 h at 25 C. Leaves of Nicotiana benthamiana were infiltrated with Agrobacterium (OD600 = 0.5) containing the mixture of PN::GUSint or P35S::GUSint, P35S::LUCint, P35S::N3xHA, and pER8::p50 (33 : 33 : 33 : 1). After incubation at 25 C for 36 h, the leaves were infiltrated with 10 lm b-estradiol, incubated for 12 h, and used for the measurement of GUS activity and immunoblotting analysis. The level of GUS activity was normalized using LUC activity. Geldanamycin (GDA, 10 lm) treatment was performed simultaneously b-estradiol treatment. We silenced two N. benthamiana SGT1 genes NbSGT1.1 and NbSGT1.2 (Peart et al. 2002) with a highly homologous region between the two genes using Tobacco rattle virus (TRV) based virus-induced gene silencing system (Ratcliff et al. 2001). Results and Discussion For analysis of N promoter activity, we used an Agrobacterium-mediated transient expression system consisting of a 1908-bp fragment from the 5¢-flanking J Phytopathol 158:826–828 (2010) doi: 10.1111/j.1439-0434.2010.01686.x 2010 Blackwell Verlag GmbH