The in silico map-based cloning of Pi36, a rice coiled-coil nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast fungus

Abstract

The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silico prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The Pi36-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast R genes Pita, Pib, Pi9, and Piz-t. An RT-PCR analysis showed that Pi36 is constitutively expressed in Kasalath.

Figure 1.—

In silico map-based cloning of Pi36. (A) Physical and genetic map surrounding the Pi36 locus. The numbers below the map represent distances in kilobases, as estimated from the Nipponbare genome sequence. The numbers in parentheses represent the number of recombinants/gametes in the mapping population previously reported (Liu et al. 2005). (B) Pi36 candidate genes. Pi36-1 and Pi36-2 were predicted by RiceGAAS and Pi36-3 by both FGENESH and Gramene. The shaded box represents the coding region, and the hatched boxes represent predicted 5′ promoter and 3′ poly(A) regions, respectively. The numbers above the map refer to location on the reference Nipponbare genomic sequence. The targets for the LR–PCR primers are indicated.

Figure 2.—

Southern blot analysis of blast resistant transgenic plants. Genomic DNA was isolated from resistant transformed and susceptible non-transformed plants. A fragment of hptII was used as a probe. Lane 1, molecular weight marker λHindIII; lane 2, Kasalath (resistant); lane 3, Q1063 (susceptible); lanes 4–12, transgenic T₁ progeny.

Figure 3.—

Pi36 gene complementation test and molecular analysis of the transgenic lines. (A) Resistance phenotypes of the Pi36 gene donor cv. Kasalath and its receptor cv. Q1063 as well as transgenic plants of two T₂ lines segregated into 3R:1S against isolate CHL39. R, resistant; S, susceptible. (B) Cosegregation analysis of the resistance phenotype with the transgenes. The amplified fragment with the primer pair CRG4F and CRG4R was subsequently digested with HaeIII, and the digested product was subjected to 1.5% agarose gel electrophoresis. M represents standard molecular weight marker DL2000.

Figure 4.—

The structure of Pi36, as deduced from its genomic and cDNA sequences. (A) The target gene region. The numbers above the map show genomic positions in the Nipponbare genomic sequence. (B) Gene structure as deduced from the genomic DNA sequence. (C) Gene structure as deduced from the cDNA sequence. The shaded box indicates an exon, and the line an intron. The positions of 5′ and 3′ UTR (hatched boxes), the translation start codon (ATG), and the translation stop codon (TGA or TAG) are also shown. The annealing targets of the RACE and RT-PCR primers are indicated. (D) Structure of the Pi36-encoded protein, in which three tandem conserved domains are shown.

Figure 5.—

Deduced amino acid sequence of the Pi36 encoded protein. The seven conserved motifs forming the NBS region are underlined. Residue 590, the single amino acid substitution distinguishing the blast-resistant from the blast-susceptible form of the protein, is double underlined. The C-terminal LRR region is shown separately from the rest of the sequence.

Figure 6.—

Phylogenetic analysis of Pi36 with other 10 R genes. Multiple amino acid alignments were conducted using ProbCons and a neighbor-joining phylogenetic tree was generated using MEGA. Numbers on branches indicate the percentage of 1000 bootstrap replicates which support the adjacent node. The unit branch length is 0.2 nucleotide substitutions per site, as indicated by the bar.

Figure 7.—

Expression patterns of Pi36 assayed by semiquantitative RT–PCR. Two-week-old resistant Kasalath and susceptible LTH seedlings were inoculated with blast (isolate CHL39). The expression of Pi36 was assayed at various time points postinoculation. Genomic DNA (gDNA) serves as a control to distinguish PCR products from cDNA and gDNA. The rice Actin1 gene acted as a positive control.