Pathogen corruption and site-directed recombination at a plant disease resistance gene cluster

Abstract

The Pc locus of sorghum (Sorghum bicolor) determines dominant sensitivity to a host-selective toxin produced by the fungal pathogen Periconia circinata. The Pc region was cloned by a map-based approach and found to contain three tandemly repeated genes with the structures of nucleotide binding site-leucine-rich repeat (NBS-LRR) disease resistance genes. Thirteen independent Pc-to-pc mutations were analyzed, and each was found to remove all or part of the central gene of the threesome. Hence, this central gene is Pc. Most Pc-to-pc mutations were associated with unequal recombination. Eight recombination events were localized to different sites in a 560-bp region within the approximately 3.7-kb NBS-LRR genes. Because any unequal recombination located within the flanking NBS-LRR genes would have removed Pc, the clustering of cross-over events within a 560-bp segment indicates that a site-directed recombination process exists that specifically targets unequal events to generate LRR diversity in NBS-LRR loci.

Figure 1.

Annotation of the Pc region in sorghum cultivar Colby as shown in an Apollo output diagram. The annotation results are shown in the black fields. Yellow blocks represent the gene homologies identified by BLASTX; pink blocks signify the predicted genes (FGENESH). The names below the identified homologies indicate the proteins that are predicted to be encoded by these candidate genes. The Pc gene is the central member of the NBS–LRR gene triplet.

Figure 2.

Unequal recombination events between paralogs A and C in Pc mutants M1–M7.

Figure 3.

Expression analysis on susceptible Colby (Pc) and nine derived pc isolines using RT-PCR. A primer pair common to all three NBS–LRR paralogs was used. Negative controls (C.), lacking reverse transcriptase in their reaction mixtures, were also included. The PCR products were cloned and sequenced to identify expressed paralogs.

Figure 4.

Distribution of unequal recombination events along the consensus sequence of the NBS–LRR paralogs A and C (3737 bp) and their upstream noncoding regions (870 bp). Similarity between paralogs A and C is shown using a color-coded bar below the distribution graph. Seven of the 11 A–C recombinants and the single B/C recombinant (M1–M7, M13) were localized in a highly variable segment of the LRR region. Recombination frequency in this region was significantly higher (P < 0.0005) than the overall frequency measured along the complete gene. A 468-bp internal deletion in paralog B of M12 overlaps with the A/C recombination hot-spot, suggesting that an imprecisely repaired double-strand break also occurred in the high-recombination region.