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

Abstract

Background: Arabidopsis NPR1 is a master regulator of systemic acquired resistance. NPR1 binds to TGA transcription factors and functions as a transcriptional co-activator. In rice, NH1/OsNPR1 functions to enhance innate immunity. NRR disrupts NH1 function, when over-expressed.

Results: We have established a rice transient protoplast assay to demonstrate that NH1 is a transcriptional co-activator and that NRR represses NH1-mediated activation. We identified three NRR homologues (RH1, RH2, and RH3). RH1 and RH3, but not RH2, also effectively repress NH1-mediated transcriptional activation. NRR, RH1, RH2, and RH3 share sequence similarity in a region beyond the previously identified NPR1-interacting domain. This region is required for strong interaction with NH1. A double point mutation, W66A/F70A, in this novel NH1-interacting domain severely reduces interaction with NH1. Mutation W66A/F70A also greatly reduces the ability of NRR to repress NH1-mediated activation. RH2 carries a deviation (amino acids AV) in this region as compared to consensus sequences (amino acids ED) among NRR, RH1, and RH3. A substitution (AV to ED) in RH2 results in strong binding of mutant RH2ED to NH1 and effective repression of NH1-mediated activation.

Conclusions: The protoplast-based transient system can be used to dissect protein domains associated with their functions. Our results demonstrate that the ability of NRR and its homologues to repress NH1-mediated transcriptional activation is tightly correlated with their ability to bind to NH1. Furthermore, a sequence is identified as a novel NH1-interacting domain. Importantly, this novel sequence is widely present in plant species, from cereals to castor bean plants, to poplar trees, to Arabidopsis, indicating its significance in plants.

Figure 1

Transient cell assay on transcriptional activation activity of rTGA2.1, rLG2, and NH1. Protoplast cells were prepared from 10 days old green, transgenic rice seedlings, containing a UAS-Luc reporter. Protoplasts were transfected with combinations of plasmid constructs and Luc and Gus enzyme activities assayed after 20 h incubation at 28°C in growth chamber. The Ubi-Gus plasmid was included in all transfections and the Gus activity assayed for reference. In blanks, a Ubi-pUC plasmid was included to compensate for the amount of input DNA. rTGA2.1 and rLG2 are fused to the Gal4 DNA binding domain respectively, generating Gal4:rTGA2.1 and Gal4:rLG2. NH1 was expressed from the Ubi-NH1 construct. The UAS-Luc reporter activity is expressed as Luc/Gus. Each bar represents the average and standard deviation of three independent transfections.

Figure 2

Transient assay for effects of NRR, RH1, RH2, and RH3 on NH1-mediated activation. (A) Protoplasts preparation and transfection were done as described in Figure 1. The Gal4:rLG2 and Ubi-NH1 constructs are as described above. NRR, RH1, RH2, and RH3 were expressed from the Ubi-1 promoter. Each bar represents the average and standard deviation of three replicates. (B) Sequence lineup of the NPR1-interacting domain and the NH1-interacting domain. Sequences within the NPR1 interacting and NH1-interacting domains of rice NRR, RH1, RH2, RH3, and wheat NRR ortholog (wNRR) are lined up. A consensus sequence is deduced. (C) Yeast two-hybrid assay. The NH1 protein is fused to the LexA protein (bait) and NRR, RH1, RH2, and RH3 are fused to the B42AD protein (prey). A positive interaction between the bait and the prey results in blue colors.

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).

Figure 4

Effects of the point mutations on the ability of NRR to repress NH1-mediated activation. (A) Protoplast transient assay. The experiment was done as in Figure 1. (B) Western blot analysis of total protein extracted from protoplast cells transfected with the different combinations of plasmids. Four replicates of transfection for each combination of plasmids were carried out and the cells combined at the end of transfection. Cells were incubated for 20 h before harvest. The amount of protein loaded for each sample was normalized to the Gus activity expressed from the Ubi-Gus construct included as a reference. Protein on the blot was probed with an anti-His antibody. The wild type NRR protein and mutants W66, WF, and DL are tagged with 6× histidine at the C-terminus.

Figure 5

Effects of the RH2ED mutation on interaction with NH1 and NH1-mediated transcriptional activation. (A) Protein-protein interaction in yeast two-hybrid. Yeast two-hybrid tests were done as described in Figure 3. (B) Western analysis of yeast expressed fusion proteins. Protein was extracted from yeast cells containing constructs expressing LexA:NH1 plus B42AD (labeled B42), B42AD:NRR (NRR), B42AD:RH1 (RH1), B42AD:RH2 (RH2), B42AD:RH3 (RH3), or B42AD:RH2ED (RH2ED). 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 and anti-LexA antibodies sequentially. Protein loading was normalized to amount of input yeast cells. (C) Effects of the RH2ED mutation on NH1-mediated transcriptional activation. The protoplast transient assay was done as described in Figure 1. (D) Western analyses of transiently expressed proteins in protoplasts. Protoplast transfection and protein preparation were done as described in Figure 4. Protein on a nitrocellulose membrane was probed with an anti-HA antibody. A duplicate membrane was probed with anti-NH1 and anti-Gal4DB antibodies sequentially. The amount of protein loaded was normalized to the Gus activity.