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, 97 (26), 14789-94

Structure-function Analysis of the Tobacco Mosaic Virus Resistance Gene N

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Structure-function Analysis of the Tobacco Mosaic Virus Resistance Gene N

S P Dinesh-Kumar et al. Proc Natl Acad Sci U S A.

Abstract

The tobacco N gene is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant resistance (R) genes and confers resistance to tobacco mosaic virus (TMV). We investigated the importance of specific domains of N in inducing TMV resistance, by examining various N deletion and point mutations that introduce single amino acid substitution mutants in vivo. Our deletion analysis suggests that the TIR, NBS, and LRR domains play an indispensable role in the induction of resistance responses against TMV. We show that amino acids conserved among the Toll/IL-1R/plant R gene TIR domain and NBS-containing proteins play a critical role in N-mediated TMV resistance. Some loss-of-function N alleles such as the TIR deletion and point mutations in the NBS (G216A/E/V/R, G218R, G219D, K222E/N, and T223A/N) interfere with the wild-type N function and behave like dominant negative mutations. These F(1) plants mount a hypersensitive response (HR) that is indistinguishable from that of the wild-type N plants, yet TMV was able to move systemically, causing a systemic hypersensitive response (SHR). Many amino acid substitutions in the TIR, NBS, and LRR domains of N lead to a partial loss-of-function phenotype. These mutant plants mount delayed HR compared with the wild-type N plants and fail to contain the virus to the infection site. In addition, some partial loss-of-function alleles (W82S/A, W141S/A, G218V/S, and G219V) interfere with the wild-type N function, leading to SHR. The partial loss-of-function and dominant negative mutant alleles described in this report will be useful in furthering our understanding of the TIR-NBS-LRR class of R genes.

Figures

Figure 1
Figure 1
Summary of the N gene deletion mutants and their phenotypes. The 13 deletion constructs (B to N) are depicted schematically. The phenotypes of primary transformants (T0), selfed T0 progeny (T1), and progeny derived from the cross between mutant T0 and wild-type NN plant (F1) are shown. TIR, NBS, and LRR are structural domains of the N gene. 6× Myc, six copies of Myc tag (EQKLSEEDLE) are inserted at the C terminus of the N gene. HR, hypersensitive response; SHR, systemic hypersensitive response; M, mosaic.
Figure 2
Figure 2
Comparison of TIR domains of Toll, human IL-1R, and plant resistance proteins. Alignment was performed with the clustal w program (60). Amino acids conserved in all of these proteins are represented by an asterisk (*). Two dots represent conservative changes, and one dot represents semiconservative changes. Amino acids that are subjected to mutagenesis in this study are highlighted.
Figure 3
Figure 3
Response to TMV of plants expressing the N gene TIR domain deletion and various amino acid substitution mutants. Primary TMV-U1-inoculated (1o I.L.) and secondary uninoculated upper leaves (2o U.I.L.) of mutant plants and progeny derived from mutants crossed to wild-type NN plants are shown.
Figure 4
Figure 4
Response of plants expressing various amino acid substitution mutations in the N gene NBS and LRR domains to TMV. Primary TMV-U1-inoculated (1o I.L.) and secondary uninoculated upper leaves (2o U.I.L.) from mutant plants and progeny derived from mutants crossed to wild-type NN plants are shown.

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