Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance

Abstract

The rice blast resistance gene Pikm was cloned by a map-based cloning strategy. High-resolution genetic mapping and sequencing of the gene region in the Pikm-containing cultivar Tsuyuake narrowed down the candidate region to a 131-kb genomic interval. Sequence analysis predicted two adjacently arranged resistance-like genes, Pikm1-TS and Pikm2-TS, within this candidate region. These genes encoded proteins with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) and were considered the most probable candidates for Pikm. However, genetic complementation analysis of transgenic lines individually carrying these two genes negated the possibility that either Pikm1-TS or Pikm2-TS alone was Pikm. Instead, it was revealed that transgenic lines carrying both of these genes expressed blast resistance. The results of the complementation analysis and an evaluation of the resistance specificity of the transgenic lines to blast isolates demonstrated that Pikm-specific resistance is conferred by cooperation of Pikm1-TS and Pikm2-TS. Although these two genes are not homologous with each other, they both contain all the conserved motifs necessary for an NBS-LRR class gene to function independently as a resistance gene.

Figure 1.—

Physical maps of the Pikm gene region and the structures of the two genes constituting Pikm. (A) Physical maps of the Pikm locus generated by using the Nipponbare (Pikm⁻) genome sequence (top) and Tsuyuake (Pikm⁺) sequence (bottom). The candidate gene region is delimited by the marker 85H07S5 from the proximal side and the marker k3951 from the distal side. The numbers above each of the maps represent distances in kilobases. The arrows below each map represent putative genes predicted by the annotation tools in RiceGAAS. The six boldface arrows on the Nipponbare map and the two boldface arrows on the Tsuyuake map represent putative R genes. (B) Structures of Pikm1-TS and Pikm2-TS. Dark-shaded boxes are exons, light-shaded boxes are 5′- or 3′-UTRs, and the lines are introns.

Figure 2.—

Complementation testing and molecular analysis of the transgenic lines. (A) Reaction to inoculation with the blast isolate Ina 86-137 in Tsuyuake, Nipponbare, and a set of F₂ progeny (numbers 1–7) derived from a cross between a T₁ plant harboring Pikm1-TS and another T₁ plant harboring Pikm2-TS. The isolate Ina 86-137 is incompatible with the Pikm-donor cultivar Tsuyuake and compatible with the transformation-recipient cultivar Nipponbare. (B) Cosegregation of the resistance phenotype with the presence of both the Pikm1-TS gene and the Pikm2-TS gene. The presence of Pikm1-TS and Pikm2-TS in the transgenic lines is shown by the presence of a product from PCR amplification with the primers Gene2TY1 (top) and Gene1TY1 (bottom), respectively. The amplified products were separated through a 2.0% agarose gel. The transgenic lines numbered 3 and 6 contain both Pikm1-TS and Pikm2-TS, those numbered 2 and 4 contain only Pikm1-TS, those numbered 1 and 5 contain only Pikm2-TS, and that numbered 7 contains neither of the transgenes.

Figure 3.—

Deduced amino acid sequence of the Pikm1-TS protein. The three conserved motifs, i.e., kinase 1a, kinase 2, and kinase 3a, forming the NBS domain and the nT motif upstream of the NBS domain are in boldface type. The LRR repeat region and non-LRR region are shown detached from the rest of the sequence. The LRR region contains six repeat units that show a good match to the consensus sequence Lxx Lxx Lx LxxTxLxxLPxxLxx. In this LRR region, leucines and other residues matching the consensus sequence are shown in boldface type.

Figure 4.—

Deduced amino acid sequence of the Pikm2-TS protein. The three conserved motifs (i.e., kinase 1a, kinase 2, and kinase 3a) forming the NBS domain and the nT motif upstream of the NBS domain are in boldface type. The C-terminal LRR is shown detached from the rest of sequence. In the LRR region, leucines and other residues matching the consensus sequence LxxLxxLxLxxTxLxxLPxxLxx are shown in boldface type.

Figure 5.—

Expression profiles of Pikm1-TS and Pikm2-TS in the cultivar Tsuyuake inoculated with the blast isolate Ina 86-137. Expression of these genes was examined 0, 0.5 (i.e., 12 hr), 1, 3, and 5 days after inoculation, together with 5 days after inoculation without blast spores (negative control inoculation).