The N-homologue LRR domain adopts a folding which explains the TMV-Cg-induced HR-like response in sensitive tobacco plants.

Following leaf infection with the tobacco mosaic virus (TMV), Nicotiana species that carry the disease resistance N gene develop a hypersensitive response (HR) that blocks the systemic movement of the virus. TMV-sensitive tobacco plants that lack the N gene develop classical disease symptoms following infection with most of the tobamoviruses. However, upon infection with TMV-Cg, these plants display a HR-like response that is unable to limit viral spread. We previously identified the NH gene in sensitive plants; this gene is homologous to the resistance N gene and both belong to the TIR/NBS/LRR family. Isolation and analysis of the NH transcript enabled the prediction of the amino acid sequence in which we detected a leucine-rich repeat domain, proposed to be involved in pathogen recognition. This domain is found in four of five classes of pathogen resistant proteins, in which sequence and structural changes may generate different specificities. In order to study the possible functional role of the LRR domain in the HR-like response, we developed a comparative three-dimensional model for the NH and N gene products, by means of functional and structural domains recognition, secondary structure prediction, domain assignment through profile Hidden Markov Models (HMM) and molecular dynamics (MD) simulations. Based on our results we postulate that the NH protein could adopt a LRR fold with a functional role in the HR-like response. Our two reliable LRR three-dimensional models (N-LRR, NH-LRR) can be used as structural frameworks for future experiments in which the structure-function relationships regarding the protein-protein interaction process may be revealed. Evolutionary aspects of the N and NH genes in Nicotiana species are also discussed.

Stange C ，Matus JT ，Domínguez C ，Perez-Acle T ，Arce-Johnson P ... -  《JOURNAL OF MOLECULAR GRAPHICS & MODELLING》

Elicitor-mediated oligomerization of the tobacco N disease resistance protein.

Plant nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins are similar to the nucleotide binding oligomerization domain (NOD) protein family in their domain structure. It has been suggested that most NOD proteins rely on ligand-mediated oligomerization for function, and we have tested this possibility with the N protein of tobacco (Nicotiana tabacum). The N gene for resistance to Tobacco mosaic virus (TMV) is a member of the Toll-interleukin receptor (TIR)-NBS-LRR class of plant disease resistance (R) genes that recognizes the helicase domain from the TMV replicase. Using transient expression followed by immunoprecipitation, we show that the N protein oligomerizes in the presence of the elicitor. The oligomerization was not affected by silencing Nicotiana benthamiana ENHANCED DISEASE SUSCEPTIBILITY1 and N REQUIREMENT GENE1 cofactors of N-mediated resistance, but it was abolished by a mutation in the P-loop motif. However, loss-of-function mutations in the RNBS-A motif and in the TIR domain retain the ability to oligomerize. From these results, we conclude that oligomerization is an early event in the N-mediated resistance to TMV.

Mestre P ，Baulcombe DC 《-》

Isolation and characteristics of the CN gene, a tobacco mosaic virus resistance N gene homolog, from tobacco.

Nicotiana rustica L. HZNH, a native Chinese tobacco germplasm, displays a hypersensitive response (HR) and systemic acquired resistance following infection with tobacco mosaic virus (TMV). A resistance gene, CN, cloned from HZNH plants, was homologous to the N and NH genes identified in other Nicotiana species. The CN coding region (3423 bp) shares 93.63% and 86.50% nucleotide identity with N and NH, respectively. Whereas the five CN exon sequences are highly homologous with those of N and NH, the four introns differ significantly in length and sequence. Sequence analysis revealed that CN belongs to the TIR/NBS/LRR gene class. Expression of CN was up-regulated after TMV infection and was temperature sensitive. Organ-specific expression analysis suggested that CN transcripts accumulated at high levels in leaves, low levels in stems, and minimal levels in roots. When CN was inserted into TMV-susceptible N. tabacum cv. K326 plants by Agrobacterium-mediated transformation, the transgenic plants displayed HR and systemic HR due to uninhibited movement of the virus.

Zhang GY ，Chen M ，Guo JM ，Xu TW ，Li LC ，Xu ZS ，Ma YZ ，Chen XP ... -  《-》

NRG1, a CC-NB-LRR protein, together with N, a TIR-NB-LRR protein, mediates resistance against tobacco mosaic virus.

In animals and plants, innate immunity is regulated by nucleotide binding domain and leucine-rich repeat (NB-LRR) proteins that mediate pathogen recognition and that activate host-cell defense responses. Plant NB-LRR proteins, referred to as R proteins, have amino-terminal domains that contain a coiled coil (CC) or that share similarity with animal Toll and interleukin 1 receptors (TIR). To investigate R protein function, we are using the TIR-NB-LRR protein N that mediates resistance against tobacco mosaic virus (TMV) through recognition of the TMV p50 protein. Here, we describe N requirement gene 1 (NRG1), a novel N-resistance component that was identified by a virus-induced gene silencing (VIGS) screen of a cDNA library. Surprisingly, NRG1 encodes an NB-LRR type R protein that, in contrast to N, contains a CC rather than a TIR domain. Our findings support emerging evidence that many disease-resistance pathways each recruit more than a single NB-LRR protein. The results also indicate that, in addition to the previously recognized role in elicitor recognition, NB-LRR proteins may also function in downstream signaling pathways.

Peart JR ，Mestre P ，Lu R ，Malcuit I ，Baulcombe DC ... -  《CURRENT BIOLOGY》

Over-expression of the triploid white poplar PtDrl01 gene in tobacco enhances resistance to tobacco mosaic virus.

A full-length cDNA, designated as the Populus tomentosa disease resistance-like 01 (PtDrl01) gene, was isolated from triploid white poplar [(Populus tomentosa × P. bolleana) × P. tomentosa]. The protein thought to be produced by the PtDrl01 gene contains a nuclear localisation sequence (NLS), a toll/interleukin-1 receptor (TIR) homologue region, a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) domain. The protein also exhibits a considerable degree of homology to N-like resistance proteins. Real-time quantitative RT-PCR analysis revealed that expression of the PtDrl01 gene in triploid white poplar leaves could be induced by two defence signalling molecules: methyl jasmonate (MeJA) and salicylic acid (SA). Over-expression of the PtDrl01 gene in transgenic tobacco induced enhanced resistance to tobacco mosaic virus (TMV). Long-term resistance from the PtDrl01 gene to TMV infection was also observed in transgenic tobacco plants. Additionally, over-expression of the PtDrl01 gene resulted in transcriptional changes in genes expressing pathogenesis-related proteins in transgenic tobacco under non-stress conditions. These data strongly suggest that the PtDrl01 gene is involved in plant defence responses to pathogen infection.

Zheng HQ ，Zhang Q ，Li HX ，Lin SZ ，An XM ，Zhang ZY ... -  《-》