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Figure 3 | Plant Methods

Figure 3

From: A rice transient assay system identifies a novel domain in NRR required for interaction with NH1/OsNPR1 and inhibition of NH1-mediated transcriptional activation

Figure 3

Effects of point mutations on interaction of NRR with NH1. (A) Yeast two-hybrid assay. The NH1 bait is fused to the LexA protein. NRR and mutants W66A and W66A/F70A are fused to the B42AD protein as prey. Blue colors indicate positive interactions. Protein was extracted from yeast cells containing constructs expressing LexA:NH1 plus B42AD (labeled vector), B42AD:NRR (NRR), B42AD:W66 (W66), or B42AD:WF (WF). Extracted protein samples were run on a 5-20% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was probed with anti-HA antibody. Protein loading was normalized to the amount of input yeast cells. (B) Bimolecular Fluorescence Complementation (BiFC) or split YFP assay. The NH1 protein is fused to the YFP N-terminal half (YN). NRR, W66A (W66), W66A/F70A (WF), and D111A/L112A (DL) are fused to the YFP C-terminal half (YC). When NH1 interacts with NRR, the two halves of YFP are brought together and re-constitute a functional YFP protein, leading to fluorescence. The YFP fluorescence was detected under a fluorescence microscope with a filter set for YFP (excitation: 500 nm; emission: 535 nm).

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Plant Methods

ISSN: 1746-4811