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》

Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis.

Resistance responses that plants deploy in defence against pathogens are often triggered following a recognition event mediated by resistance (R) genes. The encoded R proteins usually contain a nucleotide-binding site (NB) and a leucine-rich repeat (LRR) domain. They are further classified into those that contain an N-terminal coiled coil (CC) motif or a Toll interleukin receptor (TIR) domain. Such R genes, when transferred into a susceptible plant of the same or closely related species, usually impart full resistance capability. We have used map-based cloning and mutation analysis to study the recognition of Peronospora parasitica (RPP)2 (At) locus in Arabidopsis accession Columbia (Col-0), which is a determinant of specific recognition of P. parasitica (At) isolate Cala2. Genetic mapping located RPP2 to a 200-kb interval on chromosome 4, which contained four adjacent TIR:NB:LRR genes. Mutational analysis revealed three classes of genes involved in specifying resistance to Cala2. One class, which resulted in pleiotropic effects on resistance to other P. parasitica (At) isolates, was unlinked to the RPP2 locus; this class included AtSGT1b. The other two classes were mapped within the interval and were specific to Cala2 resistance. Representatives of each of these classes were sequenced, and mutations were found in one or the other of two (RPP2A and RPP2B) of the four TIR:NB:LRR genes. RPP2A and RPP2B complemented their specific mutations, but failed to impart resistance when present alone, and it is concluded that both genes are essential determinants for isolate-specific recognition of Cala2. RPP2A has an unusual structure with a short LRR domain at the C-terminus, preceded by two potential but incomplete TIR:NB domains. In addition, the RPP2A LRR domain lacks conserved motifs found in all but three other TIR:NB:LRR class proteins. In contrast, RPP2B has a complete TIR:NB:LRR structure. It is concluded that RPP2A and RPP2B cooperate to specify Cala2 resistance by providing recognition or signalling functions lacked by either partner protein.

Sinapidou E ，Williams K ，Nott L ，Bahkt S ，Tör M ，Crute I ，Bittner-Eddy P ，Beynon J ... -  《PLANT JOURNAL》

Virus resistance induced by NB-LRR proteins involves Argonaute4-dependent translational control.

Active resistance to viruses is afforded by plant disease resistance (R) genes, which encode proteins with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains. Upon recognition of pathogen-derived elicitors, these NB-LRR proteins are thought to initiate a number of signaling pathways that lead to pathogen restriction. However, little is known about the molecular mechanisms that ultimately curtail virus accumulation. Here, we show that the co-expression of a plant NB-LRR protein with its cognate elicitor results in an antiviral response that inhibits the translation of virus-encoded proteins in Nicotiana benthamiana. This antiviral response is dependent on viral cis elements, and, upon activation of the NB-LRR protein, viral transcripts accumulate but do not associate with ribosomes. The induced inhibition of viral transcript translation and NB-LRR-mediated virus resistance were compromised by the downregulation of Argonaute4-like genes. Argonaute proteins have been implicated in small RNA-mediated RNA degradation, and in degradation-independent translational control. Our results suggest that the engagement of Argonaute proteins in the specific translational control of viral transcripts is a key factor in virus resistance mediated by NB-LRR proteins.

Bhattacharjee S ，Zamora A ，Azhar MT ，Sacco MA ，Lambert LH ，Moffett P ... -  《-》

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》

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.

The N gene is a Toll/interleukin-1 receptor (TIR)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR)-type resistance (R) gene that generates two alternative transcripts, N(S) and N(L). N(S) encodes the full-length N protein while N(L) is predicted to encode a truncated form of the protein lacking most of the LRR region. We found that the two transcripts were accumulated at 20 degrees C, a permissive temperature, but not at 30 degrees C, a non-permissive temperature for the N gene, in tobacco mosaic virus (TMV)-inoculated leaves. When N gene-dependent cell death was triggered by transient 20 degrees C treatment for 2-6 h, considerable levels of the transcripts were accumulated just before cell death, although the levels of N(S) were always higher. The accumulation was induced by transient expression of the 50 kDa helicase domain (p50) of TMV replicase which is the Avr component of N, but not by transient expression of NtMEK2 (DD)-mediated cell death or N gene-independent hypersensitive cell death. These results suggest that the accumulation of N(S) and N(L) is associated with the function of N and, above a certain threshold, triggers N-mediated hypersensitive cell death.

Takabatake R ，Seo S ，Mitsuhara I ，Tsuda S ，Ohashi Y ... -  《PLANT AND CELL PHYSIOLOGY》