Characterization and cloning of TMV resistance gene N homologues from Nicotiana tabacum

Tobacco cultivars Nicotiana tabacum cv. Samsun NN plants carrying the N gene contain a multitude of N-related genes. We cloned a few N homologues and isolated two full-length cDNAs of NL-C26 and NL-B69 genes from N. tabacum cv. Samsun NN. Nucleotide sequence analysis showed that the coding regions of NL-C26 (3,498 bp) and NL-B69 (3,510 bp) had 86 and 83% nucleotide identities with the N gene, respectively. Amino acid sequence analysis revealed that NL-C26 and NL-B69 had the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR/NBS/LRR) structure with 78 and 73% identities to N, respectively. These result indicated that NL-C26 was more similar to N than NL-B69. Tobacco mosaic virus (TMV) infection experiments suggest that NL-C26 and NL-B69 could interact with distinct avirulence (Avr) proteins of yet unidentified pathogens and function as potential HR-inducing resistance proteins similar to N.


INTRODUCTION
Plants suffer many diseases from bacteria, fungi and viruses as well as other organisms.But plants have developed defense mechanisms to protect themselves against invading pathogens by employing a variety of strategies including the pathogen recognition system (Burch-smith et al., 2007), which operates through the interaction between a plant resistance (R) gene and the corresponding pathogen avirulence (Avr) gene (Martin et al., 2003), as described by the gene-for-gene hypothesis (Flor, 1971).The recognition often leads to hypersensitive response (HR) that results in programmed cell death and limits the spread of a pathogen from the site of infection *Corresponding author.E-mail address: gaojsh1221 @hotmail.com.Tel: +86 551 5786232.Fax: +86 551 5786967.
Abbreviations: RACE, Rapid amplification of cDNA ends; RT-PCR, reverse transcription polymerase chain reaction; MP, movement protein; CP, coat protein; NBS, nucleotide-binding site; LRR, leucine-rich repeat; TIR, Toll-interleukin1 receptor; CC, coiled-coil; HR, hypersensitive response; TMV, Tobacco mosaic virus; Avr, avirulence; cDNA, complementary DNA.(Dodds et al., 2006).This defense response is now referred to as the plant innate immunity and known to involve many R genes of diverse plant species for many different pathogens (Chisholm et al., 2006).
In recent years, many R genes conferring specific resistance to viral, bacterial, fungal, oomycete, nematode, and insect pathogens have been cloned from diverse plant species (Baker et al., 1997;Hammond-Kosack and Jones, 1997;Chisholm et al., 2006).Interestingly, the proteins encoded by the R genes are structurally similar and characterized by several conserved domains including centrally located nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains (Ellis et al., 2000;Dangl and Jones, 2001).The NBS/LRR type proteins are further subdivided into two classes based on the N-terminal region having either a Toll-interleukin1 receptor (TIR) or coiled-coil (CC) domain (Dangl and Jones, 2001;Hulbert et al., 2001).Genomic studies have established that TIR/NBS/LRR and CC/NBS/LRR class R genes are present in plants as multigene families.
The interaction between Tobacco mosaic virus (TMV) and the tobacco N gene is a classical model system for the study of plant disease resistance (Holmes, 1938).TMV is a single-stranded, positive-sense RNA virus belonging to the tobamovirus family (Dawson and Lehto, 1990).The genomic RNA consists of 6,395 nucleotides and encodes at least four proteins: two replicase proteins of 126 and 183 kDa, a movement protein (MP) of 30 kDa and a coat protein (CP) of 17.5 kDa (Goelet et al., 1982;Dawson, 1992).The N gene was originally cloned from Nicotiana glutinosa by transposon tagging several years ago (Whitham et al., 1994).Molecular analysis reveals that the N gene confers resistance to TMV and encodes a TIR-NBS-LRR class protein.
In Nicotiana tabacum, the dominant N gene from N. glutinosa was introgressed into TMV-sensitive tobacco cultivars by crossing and repeated backcrossing (Holmes, 1938).The cultivar Samsun NN carrying the N gene exhibits a local HR upon infection with most TMV strains, thereby restricting the viruses to the infection sites.In contrast, the cultivar Samsun nn lacking the N gene allows the viruses to move locally and systemically and develops systemic mosaic.However, Xanthi nn plants infected by TMV-Cg, a tobamovirus isolated from cruciferous plants (Yamanaka et al., 1998), displayed unexpected local and systemic responses, termed a HR-like response (Arce-Johnson et al., 2003).Stange et al. (2004) have cloned the NH gene, an N gene homologue from Xanthi nn plants, and postulated that the gene might be involved in the HR-like response (Stange et al., 2008).Although Nicotiana species are proposed to carry a multitude of N-related gene sequences, which are probably clustered together (Whitham et al., 1994), only limited structural and functional information on these N-related genes is available (Stange et al., 2004).
In this study, we isolated at least 20 cDNA clones closely related to, but distinct from the N gene from N. tabacum cv.Samsun NN.We designated the genes as N homologues and tentatively classified into four groups A, B, C and D based on the N-terminal proximal partial sequence homology.Through sequencing analyses, we obtained two full-length NL-C26 and NL-B69 cDNAs.Amino acid sequences analyses revealed that NL-C26 and NL-B69 proteins show 78 and 73% identities to N, respectively and that their structures are similar to N belonging to the TIR/NBS/LRR class.It is possible that the newly identified tobacco N homology proteins may be involved in HR induction upon interactions with distinct Avr proteins of yet unidentified pathogens and function as resistance proteins similar to N. This study is very helpful for advanced under-standing of the molecular mechanisms underlying the HR induction by the resistance proteins.

Plant materials
N. tabacum cv.Samsun NN and cv.Samsun nn were used.Plants were grown at 25°C under a 16/8 h light-dark cycle in a virus-free growth chamber.Water with 0.1% Hyponex (Hyponex Japan) was given to the pots twice a week.Gao et al. 7999

TMV strains
Two TMV strains, TMV-OM (a common Japanese strain) and TMV-Cg (the crucifer-and garlic-infecting TMV strain), purified by ultracentrifugal method (Konagaya, 2004) from infected tobacco plants by wild type viruses and resuspended in 10 mM phosphate buffer (pH 7.0), were used.

TMV inoculation
In the experiment, the purified TMV-OM (15 mg/ml) and TMV-Cg (14 mg/ml), which are stored at -80°C, were diluted to a concentration of 5 ng/µl in 10 mM phosphate buffer (pH 7.0) and stored at 4°C.TMV suspensions were mixed with carborundum (Nacalai tesque) of 800 meshes and directly mechanically inoculated in one leaf of 6 to 8 week-old tobacco plants with the fingers equipped in a finger sack or a latex glove.Carborundum on the inoculated leaf was immediately washed with dH2O.Plants were grown in a growth chamber at 25°C under 16 h photoperiod.Infection symptoms were carefully monitored.
For sequence determination, at least 5 -10 independent clones were sequenced by the dideoxy chain termination method using BigDye Terminator Ver.3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an ABI3130 Genetic Analyzer (Applied Biosystems).The overlapping sequences were assembled by using the GENETYX-MAC/ATSQ program (Software Development co., Japan) to obtain the cDNA sequences of NL-C26 and NL-B69.Amino acid alignments and homologies were also determined by using the GENETYX-MAC program.DNA sequence data were edited with the GENETYX-MAC software system (Software Development co., Japan).Sequence contigs were assembled using GENETYX-MAC/ATSQ program (Software Development co., Japan).Homology searches were done using the Basic Local Alignment Search Tool (BLAST) program (Altschul et al., 1990).

Isolating of N homologues from N. tabacum cv. Samsun NN
The N gene from N. glutinosa was introduced into the tobacco cultivar N. tabacum to generate N. tabacum cv.Samsun NN, which contains the N gene and confers resistance to TMV.In our previous study, we isolated the N cDNA (DDBJ accession no.AB120513) from N. tabacum cv.Samsun NN by RT-PCR with mRNA as a template (Konagaya et al., 2004).During isolation of the N cDNA, we cloned a TIR domain homolog with Ntir-F01 and Ntir-R01 primers of the N gene, named TIR2, which have 92.5% nucleotide identity to the TIR domain of the N gene.To isolate N homologues from N. tabacum cv.Samsun NN, we designed the primer sets tir2-F04/Nlrr-R02 and tir2-F04/Nlrr-R00 corresponding to the TIR2 and 3′ region sequences of the N cDNA.About 2.9 and 3.6 kb DNA frag-ments were amplified by RT-PCR, respectively.The DNA fragments were cloned into pCR2.1 vector (Invitrogen) (ligation numbers ASL and ASM for 2.9 and 3.6 kb DNA fragments, respectively) and sequenced.Interestingly, partial sequence analyses of 5′ and 3′ regions revealed that cDNA sequences encoded by these clones were closely related to, but distinct from the N gene.We designated these genes as N homologues and tentatively classified into four groups A, B, C and D based on the N-terminal proximal partial sequence homology (Figure 1).

Cloning of full-length cDNAs of NL-C26 and NL-B69
We selected two representative clones ASL26 and ASL69 from C and B groups (named NL-C26 and NL-B69 genes, respectively) for further analysis.To determine the full-length coding sequences of the NL-C26 and NL-B69 cDNAs, their 5′ and 3′ distal cDNAs were amplified by RACE method from the total RNA of Samsun NN, and assembled with the overlapping internal cDNA sequences.The reconstructed NL-C26 and NL-B69 cDNAs were 3 585 and 3 631 base pairs (bp) long, respectively (DDBJ accession nos.AB333780 and AB333781).

Characterization and structure of NL-C26 and NL-B69 proteins
The deduced NL-C26 and NL-B69 proteins consisted of According to the amino acid sequences, the NL-C26 and NL-B69 proteins contain an N-terminal TIR domain, a central NBS domain including Kinase1a (P-loop), Kinase2 and Kinase3a motifs, and a C-terminal LRR domain structurally similar to the N protein, shown for comparison.These domains are defined essentially according to Whitham et al. (1994).The amino acid numbers (aa) under the diagrams indicate the borders of each domain.These diagrams are not shown to scale.B. Comparison of amino acid identities.Amino acid sequence identities of the full-length, TIR, NBS, LRR domains of NL-C26 and NL-B69 compared to the corresponding domains of N are shown.and 1,169 amino acid residues, structurally similar to the N protein which belongs to TIR-NBS-LRR class of R protein (Figure 3A).Sequence analyses revealed that the NL-C26 and NL-B69 proteins showed 78 and 73% overall identities to the N protein of the same cultivar, respectively.Their TIR, NBS and LRR domains were defined essentially according to Whitham et al. (1994) and shown to give higher scores: the TIR, NBS and LRR domains of NL-C26 were 91, 88 and 81% identical, whereas those of NL-B69 were 71, 73 and 78% identical, respectively, to the corresponding domains of N (Figure 3B).These results indicated that NL-C26 is more similar to N than NL-B69.Taken together, these data showed that NL-C26 and NL-B69 were novel N homology proteins with TIR-NBS-LRR domains and might function as N.

HR-like induction
Normally, N. tabacum cv.Samsun NN plants carrying the N gene induce HR at the local leaf when inoculated with TMV-OM or TMV-Cg (Figures 5A and B) or most other tobamoviruses (e.g.TMV-U1), whereas N. tabacum cv.Samsun nn plants lacking the N gene are unable to develop HR.Upon infection with TMV-OM, no necrotic lesions or other symptoms of resistance were observed on Samsun nn plants.Instead, the virus spreads systemically through the overall plant, resulting in the appearance of a systemic mosaic with apical leaf deformation (Figure 5C).Unexpectedly, we found that Samsun nn plants developed local lesions (HR-like) in inoculated leaf and systemic symptoms with necrotic lesions and deformations in apical leaves when infected with TMV-Cg (Figure 5D).These results demonstrate that the susceptible Samsun nn plants have the transduction machinery to display an incomplete resistance response.It is a very intriguing possibility that NL-C26 or NL-B69 existing in Samsun nn plants might be a receptor protein that could recognize a TMV-Cg elicitor but not TMV-OM.

DISCUSSION
Plant genomes have been known to contain multiple pathogen-resistant genes, whose cDNAs have been isolated in many plant species.The RPP5 gene in Arabidopsis resides at a locus carrying nine RPP5 homologues, while the orthologous Col-0 locus contains eight homologues (Parker et al., 1997).The RPP1 locus in Ws-0 contains three TIR/NBS/LRR R genes that confer resistance to distinct races of Hyaloperonospora parasitica (Botella et al., 1998).TMV-resistant gene N was isolated several years ago from N. glutinosa plants (Whitham et al. 1994).It is the first R gene against the plant virus and is determined to participate in resistance response.Currently, several N gene homologues have been cloned from different plant species (Parker et al., 1997;Hehl et al., 1999;Di Gaspero and Cipriani, 2002;Vidal et al., 2002).However, no N gene homologue was reported in tobacco plants.For up to three years, a novel NH gene was cloned and characterized in N. tabacum cv.xanthi nn plants (Stange et al., 2004).It was, however, the first N gene homologue from tobacco plants.
It has been proposed that Nicotiana species contain multiple gene homologues of the N gene (Whitham et al., 1994).Consistent with this, we cloned many partial cDNAs containing the N-related sequences from N. tabacum cv.Samsun NN (Figure 1).Moreover, we obtained two cDNAs encoding novel N-like TIR/NBS/LRR proteins, NL-C26 and NL-B69 (Figure 2).Sequence analyses have demonstrated that NL-C26 was closer to N than NL-B69 in the overall and domain sequences (Figure 3).In the TIR/NBS/LRR class proteins from diverse plant species, the NL-C26 and NL-B69 proteins have the highest amino acid identities to the N or NH protein (Figure 4).It is not surprising since these proteins are all from N. tabacum species.We have also showed that mRNAs of NL-C26 and NL-B69 were expressed in N. tabacum cv.Samsun nn plants (Gao et al., 2007).Unlike N, NL-C26 and NL-B69, when coexpressed with the elicitor of TMV, failed to induce HR on the sites of agroinfiltration in Samsun nn leaves (Gao et al., 2007).
Most tobamoviruses can infect N. tabacum cv.Xanthi nn as well as Samsun nn and cause systemic symptoms without HR (Abbink et al., 1998;Stange et al., 2004).Exceptionally, TMV-Cg strain (Yamanaka et al., 1998) induces a partial HR-like response locally and systemically in Xanthi nn (Stange et al., 2004) and also in Samsun nn (Figure 5).Recently, an N gene homolog NH has been cloned from Xanthi nn and its expression was analyzed (Stange et al., 2004).The transcript level of the NH gene was shown to increase after inoculation of TMV-Cg, which was implicated in possible involvement of the gene in the HR-like response, although no functional evidence was provided.Phylogenetical analysis indicated that the amino acid identities of NH to N, NL-C26 and NL-B69 were 80, 81 and 69%, respectively.It is an intriguing possibility that NL-C26 and/or NL-B69 may also be involved in the HR-like responses in Samsun nn induced by TMV-Cg or yet other pathogens.

Figure 1 .
Figure 1.A phylogenetic tree was generated based on the 5′end nucleotide sequences of these N homolog cDNAs.It shows that the N homolog cDNAs can be divided into four groups (NL-A, -B, -C and -D).

Figure 2 .
Figure 2. Comparison of deduced amino acid sequences of the NL-C26, NL-B69 and N proteins.Sequences analyses indicated that the NL-C26 and NL-B69 proteins consisted of 1,165 and 1,169 amino acid residues, respectively and had the TIR/NBS/LRR structure similar to the N protein.Dots represent residues in NL-C26 and NL-B69 that are conserved with respect to N. Gaps inserted to maintain the alignment are shown as dashes.Three structural regions TIR, NBS, LRR defined according to Dinesh-Kumar et al. (2000) are indicated in bold and identified by the vertical lines.

Figure 3 .
Figure 3. Structure features of the NL-C26 and NL-B69 proteins.A. Schematic representation of the NL-C26 and NL-B69 proteins.Rectangle boxes represent Open Reading Frames (ORFs).According to the amino acid sequences, the NL-C26 and NL-B69 proteins contain an N-terminal TIR domain, a central NBS domain including Kinase1a (P-loop), Kinase2 and Kinase3a motifs, and a C-terminal LRR domain structurally similar to the N protein, shown for comparison.These domains are defined essentially according toWhitham et al. (1994).The amino acid numbers (aa) under the diagrams indicate the borders of each domain.These diagrams are not shown to scale.B. Comparison of amino acid identities.Amino acid sequence identities of the full-length, TIR, NBS, LRR domains of NL-C26 and NL-B69 compared to the corresponding domains of N are shown.

Figure 5 .
Figure 5. Symptoms of Samsun NN and Samsun nn plants infected with TMV-OM or TMV-Cg.Tobacco plants were inoculated in a basal leaf with 5 ng/ul of TMV-OM or TMV-Cg.Local symptoms were evaluated at 5 days post inoculation (dpi) and systemic symptoms were evaluated at 17 dpi.A. Local lesions (HR) were induced in inoculated leaf of Samsun NN infected with TMV-OM.B. Local lesions (HR) were induced in inoculated leaf of Samsun NN infected with TMV-Cg.C. Systemic mosaic symptoms displayed in apical leaves of Samsun nn infected with TMV-OM.D. Systemic symptoms, necrotic lesions and deformations displayed in apical leaves of Samsun nn infected with TMV-Cg.