The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice

Abstract

The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9-Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS-LRR gene cluster. Semiquantitative reverse transcriptase (RT)-PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.

Figure 1.

Structure of the 76-kb genomic sequence in the Pi9 region and the rice transformation constructs for complementation analysis of the candidate genes. (A) The six NBS–LRR genes (Nbs1-Pi9–Nbs6-Pi9) were predicted by GENSCAN and BLAST homology search; the Nbs2-Pi9 and Nbs5-Pi9 coding sequences were further confirmed by the Nbs2-Pi9 and Nbs5-Pi9 full-length cDNA sequences. The shaded line represents the whole 76-kb genomic sequence. The exons are indicated by stippled boxes and the introns by open boxes. The arrow above each NBS–LRR gene represents the transcription direction of the gene and the numbers below each gene show the start and stop sites of the coding region. No exon was identified in Nbs4-Pi9 because four stop codons exist in the coding region; the Nbs4-Pi9 region is shown as a box with horizontal lines. The 5′-partial region of Nbs6-Pi9 is shown, and its coding region was disrupted by a solo-LTR retrotransposon. The direct repeats of the solo-LTR terminal sequence (TS) and the inverted repeats of the termini are shown. (B) Rice transformation constructs containing different genomic fragments from the 76-kb NBS–LRR gene cluster. The 45-kb fragment (bp 1–45,348) in pRTAC8-45 kb (I) and pRTAC8-45 kb (II) comprises the candidate genes Nbs1-Pi9, Nbs2-Pi9, and Nbs3-Pi9 and has different cloning orientations. Construct pNBS5 carries a 24.7-kb fragment (49,808–74,581 bp) containing the Nbs5-Pi9 gene. Construct pNBS4 carries a 12.5-kb fragment (45,509–58,068 bp) from the Nbs4-Pi9 region. Constructs pNBS1-1 and pNBS1-2 carry a 10-kb fragment (13,613–23,605 bp) and a 6.9-kb fragment (12,391–19,301 bp), respectively, from the Nbs1-Pi9 region. Construct pNBS2 contains a 13.5-kb fragment (32,363–45,848 bp) spanning the Nbs2-Pi9 region. Construct pNBS3 carries 14.6 kb of the Nbs3-Pi9 region (18,395–33,070 bp).

Figure 2.

Phylogenetic analysis of the six Pi9 candidate genes. ClustalX version 1.83 (Thompson et al. 1997) was used for multiple alignment of the nucleotide sequences of Nbs1-Pi9, Nbs2-Pi9, Nbs3-Pi9, Nbs4-Pi9, Nbs5-Pi9, Nbs6-Pi9, Pib (Wang et al. 1999), and Pi-ta (Bryan et al. 2000). On the basis of the ClustalX analysis results, the phylogenetic tree was further generated using the program TREEVIEW (Page 1996; http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Bootstrap values, corresponding to the match times of branching orders (1000 replicates), are shown at each branch point. The unit of branch length is 0.1 nucleotide substitutions per site, as indicated by a bar at the bottom left corner of the tree.

Figure 3.

PCR analysis of the Pi9 deletion mutants. Genomic DNA from 15 susceptible (S) and five resistant (R) mutant lines was PCR amplified using primers (Table 3) specific to the Nbs1-Pi9 5′ or 3′ region, Nbs2-Pi9, Nbs3-Pi9, Nbs4-Pi9, Nbs5-Pi9, and Nbs6-Pi9. The five resistant plants analyzed in the PCR were obtained from different M₂ populations of the Pi9-susceptible mutants. The positive and negative controls were 75-1-127, the Pi9 parental line, and IR31917, the recipient susceptible cultivar for the introgression of Pi9 from Oryza minuta (Amante-Bordeos et al. 1992), respectively.

Figure 4.

Complementation of susceptible rice cultivar TP309 with the Nbs2-Pi9 gene and cosegregation between the transgene and blast resistance phenotype. (A) 75-1-127 is a Pi9-resistant parent. IR31917 and CO39 are susceptible cultivars. Nos. 12-2R and 12-1S are resistant and susceptible plants, respectively, derived from the no. 12 T₂ line transformed with the Nbs2-Pi9 construct pNBS2. (B) Southern blot analysis of 10 plants from the no. 12 T₂ line. Genomic DNA was digested with EcoRI and probed with a 928-bp Nbs2-Pi9 fragment (Figure 1A, 40,350–41,278 bp). S and R represent susceptible and resistant T₂ plants, respectively. (C) Diagram of the pNBS2 construct and the location of the 928-bp Nbs2-Pi9 hybridization probe.

Figure 5.

Deduced amino acid sequence of the Pi9 protein. The 1032 amino acids of NBS–LRR protein encoded by the Pi9 gene are shown. The NBS domain is between amino acids 193 and 572 and the LRR domain is between amino acids 573 and 975. In the NBS domain, the three underlined sequences, GMGGLGKT (positions 193–200), KRYFVILDDLW (positions 277–287), and GSRIVITTRNVDL (positions 307–319), correspond to kinase 1a (P-loop), kinase 2, and kinase 3a, respectively. The LRR domain is composed of 17 imperfect LRR repeats. The consensus is IXX(L)XX(L)XX(L) in which the L residues are in boldface type. The N-terminal region is the CC domain (positions 1–192). The “nT” sequence motif (WAEQIRDLSYDIEDSLDEF, positions 68–86) is in italics. At the C-terminal region, the last 57 amino acids (positions 976–1032) are a non-LRR sequence.

Figure 6.

The Pi9 gene structure and its expression in the infected rice plants. (A) Structure of the Pi9 gene and positions of the Pi9-specific primers used in semiquantitative RT–PCR analysis. Exons in the Pi9 gene are indicated by horizontal lines and open squares. Introns are indicated by lines angled downward. The initiation (ATG) and termination (TGA) codons are also indicated as are the positions of the RT–PCR primers NBS2-G and NBS2-H. (B) Semiquantitative RT–PCR analysis of the Pi9 expression. Total RNA was isolated from Pi9-resistant plants (75-1-127) at 0, 6, 12, 48, and 72 hr after inoculation (HAI). One microgram of total RNA was pretreated with RNase-free DNase I and subjected to reverse transcription. Semiquantitative PCR with 28 and 30 cycles was performed using Pi9-specific primers NBS2-G and NBS2-H.