Function and interaction of the coupled genes responsible for Pik-h encoded rice blast resistance

Abstract

Pik-h, an allele of Pik, confers resistance against the rice blast pathogen Magnaporthe oryzae. Its positional cloning has shown that it comprises a pair of NBS-LRR genes, Pikh-1 and Pikh-2. While Pikh-1 appears to be constitutively transcribed, the transcript abundance of Pikh-2 responds to pathogen challenge. The Pikh-1 CC (coiled coil) domain interacts directly with both AvrPik-h and Pikh-2. Transient expression assays demonstrated that Pikh-2 mediates the initiation of the host defence response. Nucleocytoplasmic partitioning of both Pikh-1 and Pikh-2 is required for their functionalities. In a proposed mechanistic model of Pik-h resistance, it is suggested that Pikh-1 acts as an adaptor between AvrPik-h and Pikh-2, while Pikh-2 transduces the signal to trigger Pik-h-specific resistance.

Figure 1. Transcription profiles of the coupled genes Pikh-1 and Pikh-2, as assayed by qRT-PCR.

Data represent mean ± standard deviation (n = 3). Similar results were obtained from two independent experiments. Lower case letter (a, b) used to indicate whether the treatment and control means differed at P<0.01 or P<0.05 level, respectively. The pathogen-inducible gene PBZ1 was used as a reference.

Figure 2. Interactions among Pikh-1, Pikh-2 and AvrPik-h proteins as detected by the yeast two hybrid, pull-down, and co-immunoprecipitation systems.

(A) Pikh-1_FL and Pikh-2_FL vs full length or truncated AvrPik-h proteins. (B) Defining the Pikh-1 domain interacting with AvrPik-h_FL and AvrPik-h_22–113. (C) Full length and truncated Pikh-1 vs Pikh-2. (D) Pikh-1_CC vs AvrPik-h_FL, AvrPik-h_22–113 and Pikh-2_CC using the pull-down assay. (E) Pikh-1_FL vs AvrPik-h_FLand AvrPik-h_22–113 using the co-immunoprecipitation system. Western blots employed anti-FLAG (Pikh-1_CC and Pikh-1_FL), anti-GST (AvrPik-h_FL, AvrPik-h_22–113 and Pikh-2_CC), and anti-Myc (AvrPik-h_FL and AvrPik-h_22–113) antibodies.

Figure 3. Interactions among Pikh-1, Pikh-2, and AvrPik-h proteins as detected by bimolecular fluorescence complementation (BiFC) assay.

The images shown are representative of at least three independent experiments. (A) Pikh-1_FL complexed with AvrPik-h_FL, AvrPik-h_22–113, and Pikh-2_CC in both the nucleus and the cytoplasm, while Pikh-1_CC produces such interactions only in the nucleus. (B) The targeting signals NLS and NES redirected interactions to, respectively, the nucleus and the cytoplasm. ARF19IV-mCherry, nuclear marker.

Figure 4. Functionality upon interactions among Pikh-1, Pikh-2, and AvrPik-h proteins.

(A) Transient expression in N. benthamiana. Pikh-2 on its own triggered an HR-like response (left). The Pik-h domains required for the HR (middle). The response induced by various versions of Pikh-2 (right). The NES or NLS redirected distributions of Pikh-2 resulted in the loss of its functionality. NP: Native promoter. The p35S-INF construct was used as positive control. (B) Transient assay of HR in cv. Nipponbare protoplasts using firefly luciferase as a reporter. The R gene constructs were driven by their NP, and Avr by the maize ubiquitin promoter. The reduction in luminescence was compared with the control, where an empty vector, instead of AvrPik-h_22–113, was uesd for protoplast transformation. Each assay consisted of three technical and three biological replicates. ***P<0.001. Co-expression of the full length Pikh-1 and Pikh-2 was necessary to confer AvrPik-h-dependent HR, and the NES or NLS redirected subcellular distributions of Pikh-1 and Pikh-2 resulted in loss of functionality.

Figure 5. A proposed model for the function of the coupled Pik-h genes.

(A) Pikh-1 perceives the Avr signal and relays it to Pikh-2, whereupon Pikh-2 defence response. (B) Pikh-1 is constitutively associated with Pikh-2. The recognition of AvrPik-h destabilizes this association, thereby lifting the constraints on the conformation of Pikh-2, and restoring its signalling potential.