Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. Domain structure of the N protein and expression levels of HA-tagged versions of N-derived protein fragments. Left: diagram representing the domain structure of N protein and a selection of N-derived protein fragments. Right: expression levels of HA-tagged versions of a selection of N-derived protein fragments. Constructs were transiently expressed in N.benthamiana leaves. Two days later, protein extracts were subjected to immunoprecipitation experiments using anti-HA agarose beads followed by western blotting. The band present in all lanes corresponds to the IgG. (1: TIR, 2: NBS, 3: NBL, 4: TNBS, 5: TNBL, 6: LRR1, 7: LRR4, 8: FIN)
• Supplemental Figure 2 - Supplemental Figure 2. Effect of the presence of P50 on TIR domain co-immunoprecipitation. HA- and myc-tagged versions of the TIR domain from N were co-expressed into N. benthamiana either alone or together with the P50 elicitor or the PVY CP. myc-tagged TIR expressed alone was used as control. Two days later, protein extracts were subjected to immunoprecipitation experiments using anti-HA (IP: α-HA) agarose beads followed by western blotting. The asterisk indicates the Ig. myc-tagged TIR showed some unspecific binding to the anti-HA beads.
• Supplemental Figure 3 - Supplemental Figure 3. Specificity of the TIR domain co-immunoprecipitation. Combinations of HA- and myc-tagged versions of the TIR domain from N, a HA-tagged version of the TIR from RPS4 and a myc-tagged version of the TIR from Bs4 were co-expressed into N. benthamiana. Two days later, protein extracts were subjected to immunoprecipitation experiments using anti-myc (IP: α-MYC) agarose beads followed by western blotting. The difference in size between NTIR:MYC and BS4TIR:MYC is due to the number of copies of the myc epitope present in each clone (5xmyc in NTIR:MYC and 10xmyc in BS4TIR:MYC).
• Supplemental Figure 4 - Supplemental Figure 4. Loading controls for western blots from Figures 4 and 5. A. Complete picture of western blot corresponding to the second panel in Figure 4C. B. Complete picture of western blot corresponding to the second panel in Figure 5C. The Igs (*) show loading.
• Supplemental Table 1 - Supplemental Table 1. Clones expressing HA-tagged fragments of the N protein.
• Supplemental Table 2 - Supplemental Table 2. Combinations of N domains tested for trans-complementation.
• Supplemental Table 3 - Supplemental Table 3. Combinations of N and N domains assayed for physical interaction by immunoprecipitation.