Supporting Figures

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Fig. 5. Western analysis of HA-tagged wild-type Rx and Rx mutants. Agrobacterium clones containing HA-tagged Rx constructs under control of the wild-type Rx promoter were diluted to an OD₆₀₀ of 0.25 and infiltrated into the leaves of Nicotiana benthamiana. Leaf discs were collected for Western analysis at 48 h postinfiltration. In each experiment, duplicate samples collected from two different leaves were separated by SDS/PAGE and visualized by immunodetection with an anti-HA antibody. Subsequent to immunodetection, each membrane was stained with Coomassie blue to demonstrate loading. The similar levels of wild-type and mutant Rx constructs shows that none of the mutants is overexpressed.

Fig. 6. Alignment of CP sequences from PVX and PoMV coat protein (CP) sequences of PVX-TK, PoMV (ab strain), and PoMV (II) were aligned in Vector NTI. The underlined residues in the CP-TK sequence correspond to the T121 and K127 positions affecting recognition by Rx¹.

Fig. 7. PVX-KR resistance phenotype of Rx transgenic Nicotiana benthamiana Each bar in the histogram summaries the range of symptoms developing in 12 two-leaf stage transgenic plants (T₁ generation) from a single transgenic line. The nontransgenic T₁ progeny were excluded from this analysis. Different bars represent independent transgenic lines. Each plant was inoculated with PVX-HB-GFP, a derivative of the natural Rx-resistance breaking strain PVX-HB in which the CP is similar to CP-KR (1), and in which there is an inserted GFP marker gene. Plants carrying Rx transgenes exhibited the following range of symptoms: (i) Death, inoculated leaf collapse by 7 dpi (no local lesions) systemic HR and death by 12 dpi. (ii) Systemic HR, spreading local lesions on inoculated leaf and systemic necrosis. (iii) Spreading local lesions on the inoculated leaf with no systemic symptoms. (iv) Local lesions on the inoculated leaf and no systemic symptoms. (v) Extreme resistance, no cell death on inoculated leaf and no systemic symptoms. Plants were monitored for 5 weeks. The results are shown as the proportion of Rx transgenic plants exhibiting each phenotype.

Fig. 8. PoMV resistance phenotype of Rx transgenic N. benthamiana. Twelve two-leaf stage plants (T₁ generation) from each transgenic line were rub-inoculated with sap from PoMV (II) infected N. benthamiana plants. Plants carrying versions of the Rx transgenes exhibited the following range of symptoms classified as: (i) Susceptible, mild chlorotic local lesions on the inoculated leaves and mosaic symptoms by 8 dpi. (ii) Death, systemic HR and death by 12 dpi. (iii) Systemic leaf collapse and leaf curling, no local lesions on inoculated leaf with systemic leaf chlorosis and leaf curling or leaf collapse. (iv) Resistant, no local lesions on inoculated leaf and no mosaic symptoms. Plants were monitored for 18 days. The results are shown as the proportion of Rx transgenic plants exhibiting each phenotype.

Fig. 9. CMV resistance phenotype of Rx transgenic N. benthamiana. Twelve two-leaf-stage plants (T1 generation) from each transgenic line were rub-inoculated with sap from CMV+Ysatellite RNA infected N. benthamiana plants. All inoculated plants developed yellowing and chlorosis by 8 days as shown on representative T₁ plants with mutant or wild-type forms of Rx that were photographed at 21 dpi.

1. Goulden MG, Köhm BA, Santa Cruz S, Kavanagh TA, Baulcombe DC (1993). Virology 197:293-302.