Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector

Abstract

Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50 kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a prerecognition complex containing the p50 effector and NRIP1.

Figure 1. NRIP1 interacts with the TIR domain of N and is required for N-mediated resistance to TMV

(A) NRIP1 interacts with N's TIR domain in a yeast two-hybrid assay (row 2) but not with other N domains or with full length N (row 3–6). NRIP1 does not interact with the TIR domain from the R protein, BS4 (row 1), or LexA alone (row 7). Interactions were determined by the ability to turn on the LEU2 reporter gene and subsequent growth on plates lacking leucine (Column 3). (B) Coimmunoprecipitation of NRIP1-Cerulean with either gN-TAP or N(TIR)-TAP in the presence or absence of p50. All proteins were expressed transiently in N. benthamiana. The top panel shows the input of NRIP1-Cerulean detected with α-GFP. Middle panel shows gN-TAP, N(TIR)-TAP or TAP control immunoprecipitated with IgG beads and detected with α-Myc to the 9xMyc in the TAP tag. The bottom panel shows the NRIP1-Cerulean co-immunoprecipitated with gN-TAP, N(TIR)-TAP or TAP and detected with α-GFP. NRIP1-Cerulean co-immunoprecipitated with gN-TAP and N(TIR)-TAP in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of p50. The TAP alone control was not pulled down (lane 5). (C) N-containing N. benthamiana plants were infiltrated with VIGS vector control, VIGS-STR14, VIGS-N, VIGS-NRIP1(3') or VIGS-NRIP1(FL). Silenced plants were infected with TMV-GFP nine days after infiltration of silencing constructs. TMV-GFP appears green on a background of reddish-brown chlorophyll autofluorescence under UV light. TMV-GFP overcomes N-mediated resistance and moves from the inoculated leaves to upper, uninfected leaves in NRIP1(3')- and NRIP1(FL)-silenced plants (row 1 and 2 respectively) while in STR14-silenced plants (row 4) and the vector control plants (row 5) the virus does not spread to the upper uninfected parts of the plants. TMV-GFP moved robustly in N-silenced plants (Row 3).

Figure 2. NRIP1 localization is p50-dependent

(A) In NRIP1-Cerulean transgenic plants, NRIP1-Cerulean colocalized with the red autofluorescence of chloroplasts. NRIP1-Cerulean was found in stromules (arrows). Scale bar is 20 μm. (B) NRIP1-Cerulean localized to the chloroplasts in wild-type NRIP1-Cerulean (column 1) and N-containing NRIP1-Cerulean (Column 2) transgenic plants expressing TAP alone. NRIP1-Cerulean redistributed to chloroplasts, cytoplasm, and nucleus in the presence of p50-TAP in NRIP1-Cerulean transgenic plants without N (column 3) and with N (column 4). Red structures are chloroplasts. Yellow arrows mark stromules. Scale bars equal 20 μm.

Figure 3. NRIP1 associates with p50

(A) NRIP1 interacts with LexA-p50 (top row) but not LexA alone (bottom row) in a yeast two-hybrid assay. The interaction was determined by the ability to turn on the LEU2 reporter gene and to grow on plates lacking leucine (column 3). (B) TAP alone or p50-TAP was expressed in non-N-containing (lane 1 and 3) or N-containing (lane 2 and 4) NRIP1-Cerulean transgenic plants. Transient expression of NRIP1-Cerulean was coexpressed with p50-TAP in non-N-containing (lane 1) or N-containing (lane 2) N. benthamiana plants. The level of TAP-containing proteins was detected with α-Myc antibodies to the 9xMyc in the TAP tag (top panel). The level of NRIP1-Cerulean was detected with α-GFP antibodies (Middle panel). α-PEPC was used to detect largest PEPC band as a loading control (Bottom panel). (C) Coimmunoprecipitation of NRIP1-Cerulean with p50. Top panel shows input of NRIP1-Cerulean detected with α-GFP. Middle panel shows p50-TAP, p50-U1-Ob-TAP or TAP control immunoprecipitated with IgG beads. α-Myc was used to detect the 9xMyc in the TAP tag. The bottom panel is NRIP1-Cerulean coimmunoprecipitated with p50-TAP, p50-U1-Ob-TAP, or TAP control and detected with α-GFP (TAP proteins detected by α-GFP are not shown). NRIP1-Cerulean was pulled down by both p50-TAP (lane 1) and p50-U1-Ob-TAP (lane 2) but not the TAP alone control (lane 3). (D) BiFC assays to confirm the interaction of NRIP1 with p50. Coexpression of NRIP1-YC with p50-YN reconstituted BiFC (panel 2) but not with GUS-YN (panel 1). Scale bars equal 20 μm.

Figure 4. NRIP1 associates with N only in the presence of p50

BiFC assays were used to study the association of NRIP1 and N in the context of subcellular compartmentalization. Coexpression of NRIP1-YC and N-YN did not result in Citrine BiFC in the presence of Cerulean alone (column 1). However, in the presence of p50-Cerulean, NRIP1-YC and N-YN resulted in Citrine BiFC (column 3). NRIP1-YC and the control GUS-YN did not reconstitute Citrine BiFC in the presence of p50-Cerulean (column 2). Scale bars equal 20 μm.

Figure 5. Constitutively localized cytoplasmic (-SP)NIP interacts with N only in the presence of p50

(A) (-SP)NRIP1-Cerulean coinfiltrated with p50-TAP (top right) induces HR cell death in N-containing N. benthamiana plants. (-SP)NRIP1-Cerulean coinfiltrated with TAP alone (top left) does not induce cell death. HR occurs normally when Cerulean alone is coinfiltrated with p50-TAP (bottom right). Coinfiltration of Cerulean alone and TAP alone does not induce HR (bottom left). The black outlines mark the site of infiltration. (B) BiFC assays were implemented to study the association of (-SP)NRIP1 with N. Coexpression of (-SP)NRIP1 with N-YN in the presence of p50-Cerulean (not shown) reconstituted Citrine BiFC (panel 3). However, coexpression of (-SP)NRIP1 and GUSYN with p50-Cerulean (not shown) did not reconstitute Citrine BiFC (panel 1). Also, coexpression of (-SP)NRIP1-YC and N-YN without p50 did not result in Citrine BiFC (panel 2) Scale bars equal 20 μm. (C) BiFC assays were implemented to study the association of (-SP)NRIP1 with p50. Coexpression of (-SP)NRIP1-YC with p50-YN reconstituted BiFC (panel 2) but not with GUS-YN (panel 1). Scale bars equal 20 μm.

Figure 6. NRIP1's sulfurtransferase activity is not required for NRIP1 to associate with p50

(A) NRIP1 exhibits thiosulfate sulfurtransferase activity in in vitro assays. GST-NRIP1 generates Fe(SCN)₃ with sodium thiosulfate as a substrate (line 1) but not with 3-mercaptopyruvate (line 2). An inactive mutant (C145S) of NRIP1 was unable to use sodium thiosulfate as a substrate (line 3). Production of Fe(SCN)₃ was quantified in a spectrophotometer at OD₄₆₀. (B) NRIP1(C145S)-Cerulean localized to the chloroplasts of N. benthamiana epidermal cells (Column 1). NRIP1(C145S)-Cerulean changed localization to the cytoplasm and nucleus in the presence of p50-Citrine (column 2). Scale bars equal 20 μm. (C) Coexpression of NRIP1(C145S)-YC and N-YN in the presence of p50-Cerulean (not shown) produced BiFC (column 2) but not with GUS-YN (column 1). Scale bars equal 20 μm. (D) Coexpression of NRIP1(C145S)-YC with p50-YN produced Citrine BiFC fluorescence (column 2) but not with GUS-YN (column 1). Scale bars equal 20 μm.

Figure 7. Model of recognition by an N-NRIP1-p50 complex

(A) In uninfected plants, NRIP1 localizes to the stroma of chloroplasts and N localizes to the cytoplasm and nucleus (not shown). (B) When TMV infects the plant, NRIP1 is first retained in or recruited to the cytoplasm by TMV's p50 domain via protein-protein interactions. (C) NRIP1 mediates the association of p50 and N's TIR domain to form an active immune complex in the cytoplasm. This complex may contain unknown (X) host components required for the activation that are only recruited by a NRIP1-p50 complex. Activated cytoplasmic N either enters the nucleus or sends a signal to nuclear-localized N to initiate a defense response.