Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation

Abstract

The Xa1 gene in rice confers resistance to Japanese race 1 of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial blight (BB). We isolated the Xa1 gene by a map-based cloning strategy. The deduced amino acid sequence of the Xa1 gene product contains nucleotide binding sites (NBS) and a new type of leucine-rich repeats (LRR); thus, Xa1 is a member of the NBS-LRR class of plant disease-resistance genes, but quite different from Xa21, another BB-resistance gene isolated from rice. Interestingly, Xa1 gene expression was induced on inoculation with a bacterial pathogen and wound, unlike other isolated resistance genes in plants, which show constitutive expression. The induced expression may be involved in enhancement of resistance against the pathogen.

Figure 1

(A) Genetic map of the Xa1 region on chromosome 4. Numbers above the vertical lines indicate recombinations between Xa1 and cDNAs derived from the Y5212 region. Y5212L and Y5212R are YAC end clones (27). Seven cDNAs in the open rectangle linked to Xa1 without recombination. (B) Partial restriction enzyme map of the cosmid 3-2 and CLD04541XA: B, BamHI; E, EcoRI. The 13.5-kb EcoRI-BamHI fragment was subcloned into binary vector CLD04541 (kindly provided by J. D. G. Jones) to generate CLD04541XA. Arrows indicate the positions and lengths of the cDNAs c42-1 and c42-2. (C) Physical structure of the Xa1 gene. The initiation (ATG) and termination (TGA) codons are indicated. Exons are indicated by horizontal lines and open squares, and introns are indicated by lines angled downward. Deduced motifs of the Xa1 gene product are indicated: red, NBS; blue, conserved domains (15); and green, LRR.

Figure 2

(A) Race-specific resistance to Xoo conferred by the cloned Xa1 gene. #295 is a transformant made by introduction of a cosmid-containing Xa1 gene into a susceptible line, Nipponbare. The leaves were inoculated with the Japanese Xoo race 1 strain T7174, race 2 strain T7147, and race 3 strain T7133 by the clipping method (19). Photograph taken 14 days after inoculation. (B) Growth of Xoo strain T7174R, a spontaneous, rifampicin-resistant mutant from T7174 (Japanese race 1), in leaves of transgenic rice containing Xa1 (#295), IR-BB1 with Xa1, and the control line Nipponbare. The bacterial populations were determined from two to three leaf samples at each time point. Leaf tips were ground, suspended in sterilized water, and plated on peptone-sucrose media containing 20 mg/ml rifampicin and 100 mg/ml cycloheximide. Data points are the means. The standard deviations of the means are shown by vertical bars (not shown if smaller than the symbols).

Figure 3

Deduced amino acid sequence of the Xa1 gene product. (A) NBS domains with significant homology to other resistance genes (15) are bold and underlined; “conserved domains” indicated in (15) are underlined; LRR are in italics; asparagines (potential N-linked glycosylation sites) are in bold. (B) LRR with consensus sequences listed at bottom. Leucines, asparagines, and cysteines matching with the consensus sequences are in bold; the NBS domains are bold and underlined. Numbers on the left indicate the positions of residues in the Xa1 amino acid sequence. The GenBank accession number for Xa1 genomic and amino acid sequences is AB002266.

Figure 4

Expression of the Xa1 gene. (A) Northern blot analysis of Xa1 mRNA. Four microgram aliquots of poly(A)⁺ RNA were extracted from leaf tip tissue of IR-BB1 and IR24 at 0 (IAI), 1, 3, and 5 DAI, and 0 (IAI) and 5 DAI, respectively. The blot was hybridized with ³²P-labeled c42-1 and actin probes, and positive signals were analyzed by using a BS2000 Bio-imaging Analyzer (Fuji). Size markers (RNA ladder, GIBCO/BRL) are shown in kilobases. (B) Xa1 cDNA detection by RT-PCR. Total RNA was extracted from leaf tip tissue of IR-BB1 and IR24 inoculated by incompatible strain T7174 at 0 (IAI), 1, 2, 3, 5, 7, 10, and 15 (lanes 1–8), and 0 (IAI), 3, 5, and 10 DAI (lanes 12–15), respectively. Total RNA from IR-BB1 of 5 DAI with water (lane 9) and compatible strain T7133 (lane 10) was also analyzed. Genomic DNA (lane 11) was extracted from IR-BB1 (26). To prevent amplification of contaminant genomic DNA in total RNA samples, Xa1-specific primers were designed for both sides of the second intron (105 bp). Thus, the expected lengths of amplified products from total RNA and genomic DNA were 447 and 552 bp, respectively.