Ortholog alleles at Xa3/Xa26 locus confer conserved race-specific resistance against Xanthomonas oryzae in rice

Abstract

The rice disease resistance (R) gene Xa3/Xa26 (having also been named Xa3 and Xa26) against Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight disease, belongs to a multiple gene family clustered in chromosome 11 and is from an AA genome rice cultivar (Oryza sativa L.). This family encodes leucine-rich repeat (LRR) receptor kinase-type proteins. Here, we show that the orthologs (alleles) of Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3, from wild Oryza species O. officinalis (CC genome) and O. minuta (BBCC genome), respectively, were also R genes against Xoo. Xa3/Xa26-2 and Xa3/Xa26-3 conferred resistance to 16 of the 18 Xoo strains examined. Comparative sequence analysis of the Xa3/Xa26 families in the two wild Oryza species showed that Xa3/Xa26-3 appeared to have originated from the CC genome of O. minuta. The predicted proteins encoded by Xa3/Xa26, Xa3/Xa26-2, and Xa3/Xa26-3 share 91-99% sequence identity and 94-99% sequence similarity. Transgenic plants carrying a single copy of Xa3/Xa26, Xa3/Xa26-2, or Xa3/Xa26-3, in the same genetic background, showed a similar resistance spectrum to a set of Xoo strains, although plants carrying Xa3/Xa26-2 or Xa3/Xa26-3 showed lower resistance levels than the plants carrying Xa3/Xa26. These results suggest that the Xa3/Xa26 locus predates the speciation of A and C genome, which is approximately 7.5 million years ago. Thus, the resistance specificity of this locus has been conserved for a long time.

Figure 1.

Comparison of Organization of the Xa3/Xa26 Gene Family in Two Wild Oryza Species, O. officinalis and O. minuta, with that in Cultivated Rice Varieties, Minghui 63 and Teqing.Members of the Xa3/Xa26 family and their transcription orientation are indicated by arrows. Regions showing extreme sequence identity (95% DNA identity overall) between the two wild species are shadowed. Organization of the Xa3/Xa26 family in Minghui 63 and Teqing is drawn according to published data (Sun et al., 2004, 2006); the dotted slant indicates the border between two sequence contigs; the question mark indicates that the neighborhood of two adjacent contigs is deduced according to sequence similarity and the Xa3/Xa26 family organization in other cultivated rice varieties.

Figure 2.

Enhanced Resistance to Xoo Strain PXO61 Associated with the Presence of a GUS Marker Gene that Was Tightly Linked to OoRKb1 or OmRKb1 in T₁ Families at Booting Stage.Bars represent mean (three to five replicates) ± standard deviation. The ‘a’ or ‘b’ indicates that a significant difference between transgenic and wild-type (WT) Mudanjiang 8 was detected at P < 0.01 or P < 0.05, respectively.

Figure 3.

Growth of Xoo Strains PXO341 and PXO61 in Leaves of OoRKb1-Carrying (D101OM) and OmRKb1-Carrying (D103OM) Plants at Adult (Booting) Stage (T₁ Plants) and Seedling (Six-Leaf) Stage (T₃ Homozygous Lines).Bacterial populations were determined from three leaves at each time point by counting colony-forming units (cfu). Mudanjiang 8 was wild-type. Rb49 was a transgenic line carrying Xa3/Xa26 regulated by its native promoter in Mudanjiang 8 background.

Figure 4.

Resistance Spectrum of Xa3/Xa26-2 and Xa3/Xa26-3 to Xoo at Adult (Booting) and Seedling (Six-Leaf) Stages.All the transgenic plants carried a single copy of transgene. D101OM43, D103OM26, and D103OM42 are resistance T₁ transgenic plants. D101OM1-4 is resistance T₂ transgenic plants. D101OM1-20 and M103OM26-9 are homozygous resistance transgenic lines (T₃ generation). Rb49 is a resistance transgenic line carrying Xa3/Xa26 regulated by its native promoter with the genetic background of Mudanjiang 8 (Cao et al. 2007a). Bars represent mean (6–10 replicates for D101OM and D103OM and 3–5 replicates for Rb49 and wild-type at booting stage and 3–6 replicates at six-leaf stage) ± standard deviation. The ‘a’ or ‘b’ indicates that a significant difference between transgenic and wild-type was detected at P < 0.01 or P < 0.05, respectively. The ‘c’ or ‘d’ indicates that a significant difference between the Xa3/Xa26-2-carrying or Xa3/Xa26-3-carrying plants and Xa3/Xa26-carrying plants was detected at P < 0.01 or P < 0.05, respectively.

Figure 5.

Comparison of the Structures of Xa3/Xa26-2 and Xa3/Xa26-3 with Xa3/Xa26.The coding regions (black boxes) of the genes are interrupted by one intron (line). The positions of 5' and 3' UTR (white boxes), translation start codon (ATG), and translation stop codon (TGA) are also indicated. The numbers indicate the nucleotides of each substructure.

Figure 6.

Alignment of Xa3/Xa26-2, Xa3/Xa26-3, and Xa3/Xa26 Proteins.All sequences are compared with the reference Xa3/Xa26. The predicted signal peptide sequence is underlined. The predicted transmembrane region is double underlined. The arrows above the amino-acid residues indicate the LRR repeats and the xxLxLxx motifs of the LRR domain are boxed. Dots represent identical amino acids of Xa3/Xa26-2 and Xa3/Xa26-3 to Xa3/Xa26. The dash represents the single amino acid absent in Xa3/Xa26-2 and Xa3/Xa26-3.