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. 2019 May 20;181(1):289–304. doi: 10.1104/pp.19.00124

Figure 2.

Figure 2.

The interaction between NPR1 and WRKY18 is substantially enhanced by SA. A, NPR1 but not npr1-1 or npr1-2 interacts with WRKY18 in Y2H assays. Yeast strain AH109 was cotransformed with pGBKT7-WRKY6 or WRKY18 and pGADT7-NPR1, npr1-1, npr1-2, or an empty vector (EV). Cotransformed yeast selected from a double dropout (DD) plate (−Leu, −Trp) was recultured, diluted to OD600 nm = 1, 0.1, or 0.01, and then 10 µL of the diluted liquid cultures was placed onto a DD plate and a TD plate (−Leu, −Trp, −His) with or without 200 μm SA. B, Co-IP between HA-NPR1 and GFP, WRKY6-GFP, or WRKY18-GFP. HA-NPR1 and GFP, WRKY6-GFP, or WRKY18-GFP proteins, expressed in N. benthamiana leaves by agroinfiltration, were immunoprecipitated with GFP-Trap for 2 h. The beads were washed, eluted with 2× loading buffer, and immunoblotted with an anti-GFP or an anti-HA antibody. C, In vitro pull-down between 6xHis-MPB-NPR1 and WRKY18-GFP. GFP or WRKY18-GFP was immunoprecipitated from N. benthamiana leaf extracts with GFP-Trap. The beads were washed and incubated with purified 6xHis-MBP-NPR1 with or without 200 μm SA. After overnight incubation, beads were washed three times with washing buffer, then eluted, and detected with anti-GFP or anti-6xHis antibody. The number beneath each blot indicates the relative strength of the band. The experiments were repeated two times with similar results.