Plant Material
Wild-type Nicotiana benthamiana, N-transgenic N. benthamiana line 310A (Bendahmane et al., 1999
), tobacco (Nicotiana tabacum) cv Samsun (NN), and tobacco cv Petite Havana (nn) plants were grown in glasshouses under controlled light and temperature.
Plasmid Construction
Two pBIN61-derived vectors (Bendahmane et al., 2002) were made for the transient expression of epitope-tagged versions of N protein and fragments of it. A triple HA tag was amplified by PCR from pACTAG2 (Charest et al., 1995) using a forward primer carrying a SpeI site plus extra restriction sites and a reverse primer with an XmaI restriction site. The PCR product was digested with SpeI and XmaI and cloned into pBIN61 digested with XbaI and XmaI, resulting in the vector we refer to as pHAN, which comprises a linker (XhoI-PmlI-AvrII) between the 35S promoter and the triple HA tag. Vector pMYCN was obtained using the same procedure, but the myc tag (five copies) was amplified from plasmid pCS2+MT (Rupp et al., 1994).
To clone N fragments into pHAN and pMYCN, we amplified the different fragments from an N genomic clone (see below) using forward and reverse primers carrying XhoI and AvrII restriction sites, respectively. The amplified products were cloned directionally into pHAN and pMYCN digested with XhoI and AvrII. A list of the different clones obtained, the N sequences they comprise, and the primers used for amplification is shown in Supplemental Table 1 online. Primer sequences are available on request. All PCRs were performed using Pfu polymerase, and the identities of all clones obtained were confirmed by sequencing.
Full-length HA-tagged N (35S:N:HA) consists of the complete N genomic sequence (from start to stop codons) plus the HA tag under the control of the CaMV 35S promoter and terminator. It was built from several N fragments as follows. pHAN:LRR1 and pHAN:LRR2 were digested with BamHI. The insert derived from pHAN:LRR1 and the backbone derived from pHAN:LRR2 were gel-purified and ligated to obtain pHAN:LRR4. Next, this construct and pHAN-TNBL were digested with SalI and DraIII. The insert derived from pHAN:TNBL and the backbone derived from pHAN:LRR4 were gel-purified and ligated to obtain 35S:N:HA. Exactly the same procedure, but with pMYCN-based constructs, was used to create 35S:N:MYC.
To place MYC-tagged N under the control of the N native promoter (gNMYC), a 12.3-kb DNA fragment containing N coding sequence, introns, 4.3 kb of 5' flanking sequence, and 1.3 kb of 3' flanking sequence was transferred from plasmid pTG34 (Whitham et al., 1994) to pBIN19 (Bevan, 1984) using XhoI, creating pBIN19:N. A 5xMYC tag was amplified from pSC2+MT using a reverse primer with an AvrII site and a forward primer with an overhang containing the last 20 nucleotides of N coding sequence, which contains a unique SacI site. The amplified product was digested with AvrII. Next, the 1.3 kb of 3' flanking sequence was amplified using forward and reverse primers carrying AvrII and XhoI sites, respectively, and the PCR product was digested with AvrII, ligated to the AvrII-digested MYC tag, gel purified, and then digested with SacI and XhoI. Finally, pBIN19:N was digested with XhoI and SacI and the released fragment was used in a three-way ligation with the 5xMYC 3'-untranslated region SacI-XhoI fragment and XhoI-digested pBIN19. To obtain gNHA, the SacI-AvrII fragment of gNMYC was replaced with its HA counterpart.
All mutants of N described in this work were obtained by PCR methods. Forward N primers starting at ATG and including an XhoI site (primer Nup) were used with reverse primers including the mutation, whereas forward primers including the mutation were used together with a downstream reverse primer (NBSdw; see Supplemental Table 1 online). Both PCR fragments were gel-purified, subjected to five cycles of PCR without primers, and then amplified with primers Nup and NBSdw. The final amplified fragment contained a StuI site that was always located downstream of the mutation. The PCR product was digested with XhoI and StuI and replaced into 35S:N:HA and 35S:N:MYC. The sequences of primers used for mutagenesis are available on request. All PCRs were performed using Pfu polymerase, and the identities of all clones obtained were confirmed by sequencing.
Mutants in the context of the TIR domain were obtained by PCR methods. Forward N primers starting at ATG and including an XhoI site (primer Nup) were used with reverse primers including the mutation, whereas forward primers including the mutation were used together with a downstream reverse primer (TIRdw; see Supplemental Table 1 online). Both PCR fragments were gel-purified, subjected to five cycles of PCR without primers, and then amplified with primers Nup and TIRdw. The PCR product was digested with XhoI and AvrII and cloned directionally into pHAN and pMYCN.
To obtain 35S:NRG1:HA, the NRG1 sequence was amplified from a cDNA clone (Peart et al., 2005) using forward and reverse primers carrying XhoI and AvrII restriction sites, respectively. After digestion, the amplified product was cloned directionally into pHAN digested with XhoI and AvrII.
The sequence of the P50 elicitor (nucleotides 2082 to 3418 from TMV, which correspond to the helicase domain of the viral replicase) (Erickson et al., 1999) was amplified from the U1 strain from TMV using forward and reverse primers with SalI and XmaI restriction sites, respectively. The resulting product was digested with these enzymes and cloned into pBINY53 (Mestre et al., 2000).
To obtain P50:GFP, the same sequence described above was amplified with forward and reverse primers with SalI and XbaI restriction sites, respectively. The resulting product was digested and cloned into a pBINY53-derived binary vector containing the GFP4 sequence (P. Mestre, unpublished data). Primer sequences are available upon request. The other constructs used in this work have been described elsewhere: CP (Mestre et al., 2000); TMV:GFP, TRV:00, and TRV:EDS1 (Peart et al., 2002a); TRV:SGT1 (Peart et al., 2002b); and TRV:NRG1 (Peart et al., 2005).
Agrobacterium tumefaciens–Mediated Transient Expression
Binary constructs were transiently expressed in N. benthamiana and tobacco leaves as described (Mestre et al., 2000). In brief, Agrobacterium cells were inoculated into 5 mL of L medium supplemented with 50 µg/mL kanamycin and 2.5 µg/mL tetracycline and grown at 28°C. After centrifugation, cells were resuspended in 5 mL of a solution containing 10 mM MgCl2 and 150 µM acetosyringone. The cultures were incubated at room temperature for 2 to 3 h before infiltration. N-derived cultures were infiltrated at 0.2 OD600. P50 and CP cultures were infiltrated at 0.1 OD600. TMV:GFP was infiltrated at a 50-fold dilution from 1 OD600.
Protein Extraction, Immunoprecipitation, and Immunoblotting
All procedures were performed exactly as described previously (Moffett et al., 2002). Anti-HA (3F10) agarose beads (Sigma-Aldrich) and anti-myc (9E10) agarose beads (Santa Cruz Biotechnology) were used for immunoprecipitation. Protein gel blot analysis was performed with anti-HA 3F10 antibodies (Roche) and anti-myc A-14 antibodies (Santa Cruz Biotechnology). Coomassie Brilliant Blue R 250 staining of the membranes after protein gel blot analysis was used to confirm equal loading. The intensity of the Ig bands on the protein gel blots of immunoprecipitated samples was used as an additional loading control. These bands are not included in the figures for reasons of clarity. The Ig bands corresponding to Figures 4C and 5C are shown in Supplemental Figure 4 online.
Virus-Induced Gene Silencing
Virus-induced gene silencing experiments were performed using a TRV vector as described elsewhere (Ratcliff et al., 2001). Briefly, N. benthamiana plants were infiltrated with Agrobacterium carrying the different TRV-based constructs, and plants were used for agroinfiltration 3 weeks later.
Sequence Analysis
Alignments were performed using the AlignX application of VectorNTI suite 9. Predictions of TIR secondary structure and solvent accessibility were performed with PHD and PROF (Rost and Sander, 1993).
Accession Numbers
Sequence data for the genes used in this work can be found in the GenBank/EMBL data libraries under the following accession numbers: NbSGT1 (AF516180), NbEDS1 (AF479625), NRG1 (DQ054580), and N (Q40392). The accession numbers for the EMBL/GenBank protein sequences of the resistance proteins shown in the alignments are as follows: Bs4 (AAR21295), RPP1 (AAC72977), RPP5 (AAF08790), L6 (AAA91022), M (AAB47618), RPS4 (CAB50708), and RRS-1 (Q9FH83).
Supplemental Data
The following materials are available in the online version of this article.
- Supplemental Table 1. Clones Expressing HA-Tagged Fragments of the N Protein.
- Supplemental Table 2. Combinations of N Domains Tested for Transcomplementation.
- Supplemental Table 3. Combinations of N and N Domains Assayed for Physical Interaction by Immunoprecipitation.
- Supplemental Figure 1. Domain Structure of the N Protein and Expression Levels of HA-Tagged Versions of N-Derived Protein Fragments.
- Supplemental Figure 2. Effect of the Presence of P50 on TIR Domain Coimmunoprecipitation.
- Supplemental Figure 3. Specificity of the TIR Domain Coimmunoprecipitation.
- Supplemental Figure 4. Loading Controls for Protein Gel Blots from Figures 4 and 5.
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