Supplemental Data. Tameling et al. (2010). Plant Cell 10.1105/tpc.110.077461 Supplemental Table 1. Primers used in plasmid construction Primer Code Sequence eo1 5’-GCCATGGCTCATGCAAGTGTGGCTTCTC-3’ eo2 5’-CGGTACCAACCATATTGTTCTTAACCTTC-3’ wo88 5’-CTAGAGGTCGACTCTCGAGTGCACGGATCCT-3’ wo89 5’-TCGAAGGATCCGTGCACTCGAGAGTCGACCT-3’ wo93 5’-AACTCGAGTCTAGATCGTCATCTTCTCTCACTGG-3’ wo111 5’-GTCGACATGGATTCTGCAGGATTCTCTCTGA-3’ wo114 5’-GGATCCTATTACACCTTCTGCAGATTCCTTTG-3’ wo125 5’-AGTCGACGGATCCATGGTGAGCAAGGGCGAGG-3’ wo129 5’-TCCCGGGTTACTCGAGCTTGTACAGCTCGTCCATGC-3’ wo155 5’-GATTTGCGGAACAATATGTTTG-3’ wo156 5’-CAAACATATTGTTCCGCAAATC-3’ wo157 5’-GATTTGCGGGCTAATATGTTTGG-3’ wo158 5’-CCAAACATATTAGCCCGCAAATC-3’ wo169 5’-CGAGGGGCCTAAAAAGAAGCGTAAGGTTGAGGACCCTGGATAATCTAGAC-3’ wo170 5’-CCGGGTCTAGATTATCCAGGGTCCTCAACCTTACGCTTCTTTTTAGGCCCC-3’ wo171 5’-TCGAGGGGCCTAAAAACAAGCGTAAGGTTGAGGACCCTGGATAATCTAGAC-3’ wo172 5’-CCGGGTCTAGATTATCCAGGGTCCTCAACCTTACGCTTGTTTTTAGGCCCC-3’ wo177 5’-ACTCGAGGGAGGTGGAATGTTACGTCCTGTAGAAAC-3’ wo178 5’-TCCCGGGTCATTGTTTGCCTCCCTGC-3’ wo190 5’-AGAATTCATGGCTTATGCTGCTGTTAC-3’ wo191 5’-TGGATCCTACATGAGGCGCATGTGATGAG-3’ wo192 5’-AGGATCCGGGGGAGGCAGCATGGCTTATGCTGCTGTTACTT-3’ wo193 5’-TGTCGACTACATGAGGCGCATGTGATGAG-3’ wo198 5’-AGAATTCATGGCTGAGAATATAATGGTTGGCCG-3’ wo199 5’-TGGATCCTCACATGAGGCGCATGTGATGA-3’ wo200 5’-AATTCGTCGACATCGATGGATCCTGAC-3’ wo201 5’-TCGAGTCAGGATCCATCGATGTCGACG-3’ wo227 5’-TCTCGAGTTACTAGTCAATAGCTTCTTGTATAGCTGC-3’ wo228 5’-ACTCGAGCATTGGAGGATGGTCACGG-3’ wo229 5’-AACTAGTCCTTGAGCCTTGTCTCCAACTCG-3’

Supplemental Methods

Plasmid Construction

A fragment of the 3’-coding sequence of RanGAP2 was amplified with the oligonucleotides wo227 and wo93, introducing 5’ XhoI and SpeI sites and 3’ XhoI and XbaI sites, subcloned in the PCR4 Blunt TOPO vector (Invitrogen) resulting in the plasmid SOL195. The RanGAP2 fragment was excised with XhoI and XbaI and ligated in the XhoI/XbaI-digested TRV2 vector (Liu et al., 2002), resulting in pTRV:Rg2-b (SOL199). Subsequently, a fragment of the 3’-coding sequence of RanGAP1 was amplified with the oligonucleotides wo228 and wo229, introducing a 5’ XhoI site and a 3’ SpeI site, subcloned in the PCR4 Blunt TOPO vector (Invitrogen), excised with XhoI and SpeI and ligated in XhoI/SpeI-digested SOL199, resulting in TRV:Rg1+2 (SOL202).

For the domain swap construct pRX1:R1G23R45 (under control of the Rx1 regulatory sequences), encoding the CC and LRR domains of Rx and the NB-ARC domain of GPA2, the internal Nsi and ApaLI sites were used for exchanging corresponding fragments (Slootweg, 2009). For the domain swap construct pGAPII:G13R45 (under control of the GPA2 regulatory sequences) encoding the CC- and NB-ARC domain of GPA2 and the LRR domain of Rx, the internal ApaLI site was used for exchanging corresponding fragments (Slootweg, 2009).

The binary plasmids pBIN61-Rx-NB-ARC-Myc, pBIN61-Rx-CC-HA and the pBIN61-GFP-CP32TK (Moffett et al., 2002), and the binary plasmid pB1-Rx(E400K) (Bendahmane et al., 2002), were described previously.

Virus-Induced Gene Silencing and PVX-Inoculation

VIGS was performed as described in Liu et al. (2002) and TRV:GUS, TRV:Rg2 and TRV:SGT were described in Tameling et al. (2007). Four weeks after TRV inoculation wild type N. benthamiana plants were challenge-inoculated by agroinfiltration in one leaf with an Agrobacterium suspension (OD600 of 0.001) carrying the binary PVX-GFP plasmid pGR208 (Peart et al., 2002). Viral spread was monitored under UV light to detect GFP fluorescence.

Supplemental References

Bendahmane, A., Farnham, G., Moffett, P., and Baulcombe, D.C. (2002). Constitutive gain-of-function mutants in a nucleotide binding site- leucine rich repeat protein encoded at the Rx locus of potato. Plant J. 32, 195-204.

Hillig, R.C., Renault, L., Vetter, I.R., Drell, T.t., Wittinghofer, A., and Becker, J. (1999). The crystal structure of rna1p: a new fold for a GTPase-activating protein. Mol. Cell 3, 781-791.

Kelley, L.A., and Sternberg, M.J. (2009). Protein structure prediction on the Web: a case study using the Phyre server. Nature Protoc. 4, 363-371.

Liu, Y., Schiff, M., Marathe, R., and Dinesh-Kumar, S.P. (2002). Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 30, 415-429.

Moffett, P., Farnham, G., Peart, J., and Baulcombe, D.C. (2002). Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death. EMBO J. 21, 4511-4519.

Peart, J.R., Cook, G., Feys, B.J., Parker, J.E., and Baulcombe, D.C. (2002). An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus. Plant J. 29, 569-579.

Slootweg, E. (2009). Structure, function and subcellular localization of the potato Resistance protein Rx1, PhD thesis.

Tameling, W.I.L., and Baulcombe, D.C. (2007). Physical association of the NB-LRR resistance protein Rx with a Ran GTPase–activating protein is required for extreme resistance to potato virus X. Plant Cell 19, 1682-1694.