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, 181 (4), 1627-38

Rice Pi5-mediated Resistance to Magnaporthe Oryzae Requires the Presence of Two Coiled-Coil-Nucleotide-Binding-Leucine-Rich Repeat Genes

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Rice Pi5-mediated Resistance to Magnaporthe Oryzae Requires the Presence of Two Coiled-Coil-Nucleotide-Binding-Leucine-Rich Repeat Genes

Sang-Kyu Lee et al. Genetics.

Abstract

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice. To understand the molecular basis of Pi5-mediated resistance to M. oryzae, we cloned the resistance (R) gene at this locus using a map-based cloning strategy. Genetic and phenotypic analyses of 2014 F2 progeny from a mapping population derived from a cross between IR50, a susceptible rice cultivar, and the RIL260 line carrying Pi5 enabled us to narrow down the Pi5 locus to a 130-kb interval. Sequence analysis of this genomic region identified two candidate genes, Pi5-1 and Pi5-2, which encode proteins carrying three motifs characteristic of R genes: an N-terminal coiled-coil (CC) motif, a nucleotide-binding (NB) domain, and a leucine-rich repeat (LRR) motif. In genetic transformation experiments of a susceptible rice cultivar, neither the Pi5-1 nor the Pi5-2 gene was found to confer resistance to M. oryzae. In contrast, transgenic rice plants expressing both of these genes, generated by crossing transgenic lines carrying each gene individually, conferred Pi5-mediated resistance to M. oryzae. Gene expression analysis revealed that Pi5-1 transcripts accumulate after pathogen challenge, whereas the Pi5-2 gene is constitutively expressed. These results indicate that the presence of these two genes is required for rice Pi5-mediated resistance to M. oryzae.

Figures

F<sc>igure</sc> 1.—
Figure 1.—
Chromosomal location of the Pi5 locus in the RIL260/IR50 population. (Top) The 170-kb Pi5 resistance genomic region is shown between the markers C1454 and S04G03 in RIL260/CO39 and RIL260/M202 (Jeon et al. 2003). (Bottom) A schematic of the eight rare recombinants in the Pi5 region identified in the RIL260/IR50 population. Breakage points are indicated between the relevant molecular markers. Open bars indicate the presumed RIL260 genome, solid bars indicate the IR50 genome, and shaded bars indicate that the region is heterozygous between the two genomes. The arrow indicates the 130-kb minimal interval carrying the Pi5 locus, delimited by analysis of the mapping population. Resistance to M. oryzae PO6-6 were determined in the F3 progeny of each line. R, resistant; S, susceptible; R/S, segregating line.
F<sc>igure</sc> 2.—
Figure 2.—
Genomic sequence comparison at the Pi5 loci in the RIL260 and Nipponbare cultivars. Predicted ORFs determined by RiceGAAS are shown for both genomes. NB–LRR genes, Pi5-1 alleles, and the Pi5-2 and Pi5-3 genes are indicated by black arrows. The N-terminal region of the Pi5-1 Nipponbare allele that is absent in RIL260 is shown in green. Putative transposons and hypothetical genes are indicated by blue and gray arrows, respectively. Open arrows with numbers are predicted to encode the following proteins: 1, putative eukaryotic translation initiation factor; 2, putative GTP-binding protein; 3, putative tetrahydrofolate synthase; 4, putative aldose 1-epimerase; 5, putative histone H5; 6, putative cold-shock DEAD-box protein A; 7 and 10, ankyrin-like proteins; 8 and 9, HGWP-repeat containing proteins. The red line indicates high similarity (>90%) between the RIL260 and Nipponbare ORFs. The chromosomal region that shows little or no homology is indicated by a thin line. The arrows indicate the direction of transcription. A gap in the DNA sequence in RIL260 is indicated by the dotted box.
F<sc>igure</sc> 3.—
Figure 3.—
Analysis of transgenic rice plants. (A) RT–PCR analysis of Pi5-1, Pi5-2, and Pi5-1/Pi5-2 F1 transgenic rice plants 2 days after inoculation with M. oryzae PO6-6. The rice Actin1 gene was used as an internal control in these reactions. (B) Disease symptoms in Pi5-1, Pi5-2, and Pi5-1/Pi5-2 F1 transgenic plants 7 days after inoculation with M. oryzae PO6-6. (C) Genomic DNA PCR analysis and disease reaction of F2 progeny derived from Pi5-1-63/Pi5-2-74 F1 transgenic plants in response to M. oryzae PO6-6 infection. A resistant cultivar, RIL260 carrying Pi5, and a susceptible cultivar, Dongjin (DJ) lacking the Pi5 gene, were used as controls.
F<sc>igure</sc> 4.—
Figure 4.—
Genomic structure of the Pi5-1 and Pi5-2 genes and their gene products. (A) Gene structure of Pi5-1 and Pi5-2. Exons are indicated by lightly shaded boxes and introns are indicated by thick lines. The 5′- and 3′-untranslated regions are indicated by darkly shaded boxes. ATG and TGA denote the translation initiation and stop codons, respectively, and the numbers indicate the amino acid positions. (B) Pi5-1 protein. (C) Pi5-2 protein. Both resistance proteins contain a CC, NB, LRR, and C-terminal region (CT). Amino acids 31–67 of Pi5-1 and 26–87 of Pi5-2, shown in underlined italics, contain CC motifs. The conserved internal motifs characteristic of NB proteins, namely the P-loop, kinase-2, RNBS-B, GLPL, RNBS-D, and MHDV domains, are underlined and in boldface type. A conserved xLDL motif found in the LRR of many NB–LRR proteins is also underlined.
F<sc>igure</sc> 5.—
Figure 5.—
RT–PCR analysis of the Pi5 genes and the PBZ1 gene in the RIL260 cultivar inoculated with M. oryzae PO6-6. cDNAs prepared from the leaf tissue of RIL260 at 0, 4, 12, 24, 48, and 72 hr after pathogen inoculation were used in the experiment. The rice Actin1 gene was used as an internal control.

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