The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

Abstract

Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.

Keywords: Magnaporthe oryzae; pathogen recognition; plant immunity; resistance protein; rice.

Figure 1. RGA4 auto-activates HR-like cell death that is repressed by RGA5

1. RGA4:HA, YFP:RGA5, and AVR-Pia constructs were transiently expressed in Nicotiana benthamiana leaves by A. tumefaciens infiltration. Proteins were extracted 24 h after infiltration, and protein extracts were analyzed by immunoblotting with anti-GFP (α-GFP) and anti-HA (α-HA) antibodies. Ponceau staining of Rubisco small subunit was used to verify equal protein loading.

2. The indicated combinations of fusion proteins were transiently expressed in N. benthamiana. The RGA4:HA strain was infiltrated at OD₆₀₀ of 0.2, while strains carrying all other constructs were infiltrated at OD₆₀₀ of 0.4. A bacterial strain carrying the GFP construct was used to equilibrate the total OD₆₀₀ to 1 before infiltration. Cell death induced by the different combinations was visualized 3 days after infiltration. Equivalent results were obtained in at least three independent experiments.

3. Leaf protoplasts of the susceptible rice cultivar Himenomochi (pia) were transfected with a plasmid for constitutive LUC expression in combination with constructs allowing expression of RGA4, RGA5, RGA4+RGA5, or RGA4+RGA5+AVR-Pia. LUC activity was determined in protein extracts of protoplast samples harvested 40 h after transfection. Average values and standard deviations were calculated from three replicate samples, and values were normalized with respect to the average values of the empty vector samples. The experiment was repeated three times with equivalent results. Different letters indicate groups that are significantly different (P < 0.05 in pairwise t-test).

4. Rice protoplast of the resistant rice variety Sasanishiki or of the rga4 mutant line Sas1493 was co-transfected with RNAi constructs directed against RGA4 or RGA5 and the luciferase reporter gene. Luc activity was determined and analyzed as in (C). Average values and standard deviations were calculated from the indicated number of replicate samples (n). The experiment was repeated three times with equivalent results.

Source data are available online for this figure.

Figure 2. An intact nucleotide-binding pocket is required for RGA4 but not for RGA5 activity

A, B Constructs were transiently expressed in N. benthamiana, and protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP (α-GFP) antibodies. C The rga4_K/R:HA strain carrying a construct for the rga4_K209R P-loop mutant was infiltrated alone and in combination with YFP:RGA5 or YFP:RGA5 and AVR-Pia strains in N. benthamiana leaves. The RGA4:HA strain was infiltrated alone as a positive control. Cell death was visualized 3 days after infiltration. Equivalent results were obtained in at least three independent experiments. D, E Rice protoplasts from Oc suspension culture were transfected with plasmids for constitutive LUC expression and different combinations of constructs for expression of: Venus, RGA4, rga4_K209R, RGA5, rga5_K210R, and AVR-Pia. LUC activity was determined in protoplast protein extract samples 40 h after transfection. Average values and standard deviations were calculated from three replicate samples, and values were normalized with respect to the average values of the corresponding Venus samples. The experiments were repeated three times with equivalent results. Average values significantly different from the average value of the corresponding control (Venus) samples are indicated (P < 0.05 in Student's t-test). F The YFP:rga5_K/R strain carrying a construct for the rga5_K210R P-loop mutant was infiltrated alone and in combination with RGA4:HA or RGA4:HA and AVR-Pia strains. The RGA4:HA strain was infiltrated alone as a positive control. Cell death was visualized 3 days after infiltration. Equivalent results were obtained in at least 3 independent experiments. Source data are available online for this figure.

Figure 3. A disturbed MHD motif in RGA4 is crucial for its function

1. Alignment of a portion of the ARC2 domain sequences of RGA4, RGA5, Rx, and MLA10 containing the MHD motif (red box).

2. Proteins were extracted 24 h after infiltration of the indicated RGA4:HA or YFP:RGA5 constructs in N. benthamiana and analyzed by immunoblotting with anti-HA or anti-GFP antibodies (α-HA and α-GFP). Ponceau staining of Rubisco small subunit was used to verify equal protein loading.

3. The rga4_MHD:HA strain harboring a construct for expression of an RGA4 variant with the MHD consensus motif was infiltrated alone and in combination with YFP:RGA5 or YFP:RGA5 and AVR-Pia strains in N. benthamiana. Cell death was visualized 3 days after infiltration. The same results were obtained in at least three independent experiments.

4. The RGA4 MHD mutant constructs rga4_MHD:HA, rga4_MYG:HA, rga4_THG:HA, rga4_TYD:HA and rga4_AAA:HA were expressed in N. benthamiana and analyzed for cell death induction.

5. YFP:RGA5, YFP:rga5_LHV, YFP:rga5_LAH, and RGA4:HA were expressed in N. benthamiana, and cell death was visualized 3 days after infiltration. Similar results were obtained in at least three independent experiments.

Source data are available online for this figure.

Figure 4. RGA4 and RGA5 form hetero- and homo-complexes in planta

A RGA4:T7, 10×Myc:RGA5, RGA5, and 10×Myc:GFP were transiently expressed in rice protoplasts from Oc cells. Proteins were extracted 16 h after transfection and analyzed by immunoblotting with anti-c-Myc (α-c-Myc) and anti-T7 antibodies (α-T7) (Input). In addition, immunoprecipitations were performed with anti-c-Myc beads (IP c-Myc) and analyzed by immunoblotting with anti-c-Myc antibodies for the detection of immunoprecipitated 10×Myc:RGA5 and 10×Myc:GFP and with anti-T7 antibodies for the detection of co-precipitated RGA4:T7. B, C Different combinations of RGA4:CFP, RGA4:HA, YFP:RGA5, HA:RGA5, GFP, and AVR-Pia constructs were transiently expressed in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). In addition, immunoprecipitations were performed with anti-GFP beads (IP GFP) and analyzed by immunoblotting with anti-GFP antibodies for the detection of immunoprecipitated RGA4:CFP, YFP:RGA5, and GFP proteins and with anti-HA antibodies for the detection of co-precipitated RGA4:HA and HA:RGA5 proteins. Source data are available online for this figure.

Figure 5. The CC domains of RGA4 and RGA5 form homo- and hetero-complexes and the RATX1 domain of RGA5 self-interacts in yeast

1. Pairwise interaction of CC, NB-ARC, and LRR domains from RGA4 and RGA5, as well as the RGA5 RATX1 domain (see Supplementary Fig S9A for domain borders) was evaluated by blue coloration and growth of yeast cells on basal media lacking Trp, Leu, Ade, and His and supplemented with 5-bromo-4-chloro-3-indolyl α-D-galactopyranoside (X-a-gal). Pictures were taken after 3 days of growth.

2. RGA4 and RGA5 CC domain homo- and hetero-complexes. Dilution series of yeast cells expressing GAL4-AD and GAL4-BD fusions of RGA5 1–228 and/or RGA4 1–229 CC domains on non-selective synthetic media lacking Trp and Leu (-TL) and selective media additionally lacking His (-HTL). The wheat Sr33 (1–225) CC domain was used as a negative control. Pictures were taken after 3 days of growth. Growth on -HTL media is indicative of interaction.

3. GAL4-AD and -BD fusions of CC domains of RGA4, RGA5, and Sr33 were detected by immunoblotting of yeast protein extracts with, respectively, anti-HA and anti-Myc antibodies. Equal loading was verified by red Ponceau staining.

Source data are available online for this figure.

Figure 6. RGA4 and RGA5 CC domains form homo- and hetero-complexes in planta

RGA4_1–229:CFP, RGA4_1–229:HA, YFP:RGA5_1–228, HA:RGA5_1–228, and GFP constructs were transiently expressed in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitations were performed with anti-GFP beads (IP GFP) and analyzed by immunoblotting with anti-GFP antibodies for the detection of immunoprecipitated RGA4_1–229:CFP, YFP:RGA5_1–228, and GFP proteins and with anti-HA antibodies for the detection of co-precipitated RGA4_1–229:HA and HA:RGA5_1–228 proteins. Source data are available online for this figure.

Figure 7. The RGA5 CC domain is required but not sufficient for repression of RGA4-mediated cell death, while the RATX1 domain is dispensable

The indicated RGA5 deletion constructs were co-expressed with RGA4 in N. benthamiana, and development of cell death was visualized 4 days after infiltration.

Figure 8. RGA4 and RGA5 are mainly localized in the cytosol of rice protoplasts

A, B Rice protoplasts derived from Oc cells (Baba et al, 1986) were transiently transformed with combinations of constructs allowing the expression of RGA4:GFP, Venus:RGA5, NLS:2×mOrange, NLS:Cerulean, mCherry, and Cerulean. Confocal images of protoplasts were taken 16–22 h after transfection. Scale bars, 5 μm.

Figure 9. Localization patterns of RGA4 and RGA5 are not affected by the presence of AVR-Pia or RGA4-RGA5 co-expression

Rice protoplasts derived from Oc cells (Baba et al, 1986) were transfected with combinations of constructs for the expression of RGA4:GFP, Venus:RGA5, AVR-Pia, mCherry and Cerulean, and un-labeled RGA4 and RGA5. Confocal images of protoplasts were taken 16–22 h after transfection. Scale bars, 5 μm.