tA single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta

Abstract

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.

Figure 1.

Positional Cloning of Pi-ta. (A) Chromosome walking was initiated with the single-copy RFLP marker p7C3 derived from cosegregating the RAPD marker SP7C3. The long black horizontal line represents the rice chromosome, and markers tested for recombination with p7C3 are listed above this line. The locations of these markers in the BAC contig are indicated by the boldface vertical dotted lines. Black boxes indicate BAC sequences that were mapped physically on this contig by using BAC subclones or end-primer polymerase chain reaction (PCR). Solid vertical lines connecting BAC clones indicate walking steps, and broken vertical lines indicate confirmation of overlaps. One step to the left identified the recombination border delineated by marker SP4B9. DNA markers located to the right (77D8-12, 135SphI, 20E1R, and 113E7R) failed to recombine with p7C3. The dashed horizontal double-headed arrows directly above the listed markers indicate genetic distances between molecular markers SP4B9 and p7C3 and between p7C3 and SP9F3. The solid double-headed arrows at the top denote the approximate physical distance between the markers. The broken double-headed arrow at the top right indicates that the physical distance between 113E7R and SP9F3 is unknown. The broken horizontal bars indicate sequenced BAC clones. (B) Diagrammatic representation of BAC142E8 subclone pCB1641 containing a 7535-bp Pi-ta DNA fragment that includes a 1255-bp native promoter region. The position of the two exons (box and arrow) is shown in base pairs.

Figure 2.

Functional Analysis of Pi-ta by Stable Rice Transformation. Representative leaves are shown from infection assays with hygromycin-resistant primary transgenic Nipponbare rice transformed with the Pi-ta cDNA under the control of 2424 bp of native promoter (pCB1926). Symptoms are shown 7 days after inoculation with M. grisea strain 0-137 containing AVR-Pita. (A) Line 45-1-35-1 without a functional Pi-ta gene. (B) Line 45-6-3-1 transformed to contain a functional Pi-ta gene.

Figure 3.

Deduced Pi-ta Protein Sequence. The three regions that make up the putative NBS are shown in italics and underlined; they are amino acids 236 to 244, which fit the generalized consensus GxGxxG(R/K)V for a phosphate binding (P) loop; amino acids 314 to 323, the probable kinase 2 motif; and amino acids 342 to 354, the probable kinase 3a motif. Potential N-glycosylation sites are double-underlined. Residue 918, the single amino acid that always differs between resistant and susceptible forms of the protein, is in boldface and underlined. All susceptible proteins have a serine at this position. The C-terminal LRD, shown separately from the rest of the sequence, is aligned according to the 10 potential LxxLxxL repeats.

Figure 4.

Pi-ta Transcription in Resistant and Susceptible Rice Varieties in the Absence of Pathogen Challenge. RNA gel blot analysis was performed with poly(A)⁺ mRNA prepared from 2-week-old leaves of either resistant variety YT14 (lane 1) or susceptible variety YT16 (lane 2). Of the two double haploid rice lines, YT14 inherited its Pi-ta gene from Yashiro-mochi, and YT16 inherited its pi-ta^– gene from Tsuyuake. The gel blot was hybridized with a radiolabeled Pi-ta probe, and the single-copy LRD fragment of the Pi-ta gene was cloned in pCB1645. Equal loading of samples was determined by staining gels with ethidium bromide. Numbers at left identify the positions of RNA length markers (0.24 to 9.5 kb RNA ladder from Gibco BRL).

Figure 5.

Transient Expression of the Cloned Pi-ta Gene. Seedlings of two double haploid rice lines were assayed for GUS activity 48 hr after cobombardment of the plasmids indicated with a (+). A (−) indicates that the plasmid was not included in the cobombardment mixture. Histochemical staining identifies localized blue regions resulting from accumulation of active GUS enzyme. GUS activity was high in the absence of the HR and low in cells undergoing HR. Plasmids used in this experiment were pML63, containing 35S::uidA encoding GUS; pCB1947, containing a 35S-Adh1 intron promoter fused with a truncated AVR-Pita cDNA encoding the predicted mature form of AVR-Pita, AVR-Pita₁₇₆; and pCB1920, containing the Pi-ta cDNA under control of a 2.5-kb native promoter sequence. (A) YT16 (pi-ta^–) cobombarded with the GUS reporter and AVR-Pita₁₇₆ genes. (B) YT14 (Pi-ta) cobombarded as in (A). (C) YT16 (pi-ta^–) cobombarded as in (A) except for addition of the cloned Pi-ta gene. (D) YT14 (Pi-ta) cobombarded as in (A) except for addition of the cloned Pi-ta gene.