Activation of plant immune responses by a gain-of-function mutation in an atypical receptor-like kinase

Abstract

Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases.

Figure 1.

Characterization of the snc4-1D mutant. A, Morphology of wild type (WT) and snc4-1D. Plants were grown on soil and photographed 3 weeks after planting. B, Growth of H. a. Noco2 on wild-type and snc4-1D mutant plants. Two-week-old seedlings were sprayed with H. a. Noco2 spores (5 × 10⁴ spores/mL). Infection was scored 7 d later by counting the number of conidia spores with a hemocytometer. The values presented are averages of three replicates ± sds. C to E, Expression of PR1 (C), PR2 (D), and PDF1.2 (E) in wild-type and snc4-1D mutant seedlings. Total RNA was extracted from 2-week-old seedlings grown on one-half-strength Murashige and Skoog medium at 23°C. Relative levels of PR1, PR2, and PDF1.2 were determined by real-time PCR. Values were normalized to the expression of ACTIN1. Error bars represent sd from three measurements. F, DAB staining of true leaves of wild-type and mutant plants. G, Trypan blue staining of true leaves of wild-type and mutant plants.

Figure 2.

Map-based cloning of snc4-1D. A, Map of the snc4-1 locus. B, Exon/intron structure of SNC4. The coding regions are indicated with boxes. snc4-2, Salk_122292; snc4-3, WiscDsLox444C5. C, Predicted protein structure of SNC4. TM, Transmembrane motif; PTK, protein Tyr kinase. The locations of the mutations in SNC4 are as indicated. aa, Amino acids.

Figure 3.

Complementation analysis of snc4-1D. A, Morphology of wild-type (WT), snc4-1D, WT::SNC4 (wild type transformed with a SNC4 genomic clone), and WT::snc4-1D (wild type transformed with a snc4-1D genomic clone) transgenic lines. All plants were grown on soil and photographed when they were 3 weeks old. B to D, Expression of PR1 (B), PR2 (C), and PDF1.2 (D) in wild-type, snc4-1D, WT::SNC4, and WT::snc4-1D transgenic lines. Values were normalized to the expression of ACTIN1. Error bars represent sd from averages of three measurements. E, Growth of H. a. Noco2 on wild-type, snc4-1D, WT::SNC4, and WT::snc4-1D transgenic lines. Infection was performed and scored as in Figure 1B. The values presented are averages of three replicates ± sd. [See online article for color version of this figure.]

Figure 4.

Characterization of the intragenic suppressors of snc4-1D. A, Morphology of wild-type (WT), snc4-1D, and the intragenic suppressor mutant plants. Plants were grown on soil at 23°C and photographed 3 weeks after planting. B, Mutations identified in the intragenic suppressor mutant alleles. C to E, Expression of PR1 (C), PR2 (D), and PDF1.2 (E) in wild type, snc4-1D, and the suppressor mutants of snc4-1D. Values were normalized to the expression of ACTIN1. Error bars represent sd from averages of three measurements. aa, Amino acid. [See online article for color version of this figure.]

Figure 5.

Analysis of the snc4-1D ndr1-1 and snc4-1D eds1-2 double mutants. A, Morphology of 3-week-old plants of the indicated genotypes. B to D, Expression of PR1 (B), PR2 (C), and PDF1.2 (D) in the indicated genotypes. Values were normalized to the expression of ACTIN1. Error bars represent sd from averages of three measurements. E, Growth of H. a. Noco2 on the indicated genotypes. Infection was performed and scored as in Figure 1B. The values presented are averages of three replicates ± sd. [See online article for color version of this figure.]

Figure 6.

Analysis of snc4-1D mks1-1 double mutant. A, Morphology of 3-week-old wild type (WT), mks1-1, snc4-1D, and two representative lines for the snc4-1D mks1-1 double mutant. B to D, Expression of PR1 (B), PR2 (C), and PDF1.2 (D) in wild-type, mks1-1, snc4-1D, and snc4-1D mks1-1 seedlings. Values were normalized to the expression of ACTIN1. The values presented are averages of three replicates ± sd. E, Growth of H. a. Noco2 on wild-type, snc4-1D, and snc4-1D mks1-1 plants. Infection was performed and scored as in Figure 1B. The values presented are averages of three replicates ± sd. [See online article for color version of this figure.]

Figure 7.

Characterization of snc4-1D opr3-2 double mutants. A, Morphology of wild-type (WT), snc4-1D, opr3-2, and snc4-1D opr3-2 mutant plants. Plants were grown on soil and photographed when they were 3 weeks old. B to D, Expression of PDF1.2 (B), PR1 (C), and PR2 (D) in wild-type, snc4-1D, opr3-2, and snc4-1D opr3-2 mutant seedlings. Error bars represent sd from averages of three measurements. Values were normalized to the expression of ACTIN1. E, Growth of H. a. Noco2 on wild-type, snc4-1D, opr3-2, and snc4-1D opr3-2 double mutant plants. Infection was performed and scored as in Figure 1B. The values presented are averages of three replicates ± sd. [See online article for color version of this figure.]