Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight

Abstract

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating potato disease in the world. Control of late blight in the United States and other developed countries relies extensively on fungicide application. We previously demonstrated that the wild diploid potato species Solanum bulbocastanum is highly resistant to all known races of P. infestans. Potato germplasm derived from S. bulbocastanum has shown durable and effective resistance in the field. Here we report the cloning of the major resistance gene RB in S. bulbocastanum by using a map-based approach in combination with a long-range (LR)-PCR strategy. A cluster of four resistance genes of the CC-NBS-LRR (coiled coil-nucleotide binding site-Leu-rich repeat) class was found within the genetically mapped RB region. Transgenic plants containing a LR-PCR product of one of these four genes displayed broad spectrum late blight resistance. The cloned RB gene provides a new resource for developing late blight-resistant potato varieties. Our results also demonstrate that LR-PCR is a valuable approach to isolate genes that cannot be maintained in the bacterial artificial chromosome system.

Fig. 1.

Genetic and physical maps of the genomic region containing RB. The RB locus is closely linked to markers CT88, TG495, 273C, and 274A (8, 9). BAC clones from the RB haplotype are represented as filled boxes, and BAC clones from the rb haplotype are represented as open boxes. Both 177O13 and CB3A14 contain one truncated and four complete RGAs. The direction of transcription of each gene is indicated by an arrow. The 3.6-kb deletion region between RGA2 and RGA-tr ismarked. This 3.6-kb fragment is not present in BAC CB3A14 but is drawn for the purpose of comparison with BAC 177O13.

Fig. 2.

Complementation analysis of putative RB genes. Transgenic Katahdin and control plants were inoculated with the US930287 isolate of P. infestans. Disease symptoms were recorded 7 days after inoculation. (A–C) Transgenic Katahdin plants containing constructs RGA1-PCR, RGA2-PCR, and RGA4-PCR, respectively. (D) Control Katahdin plant. (E) Katahdin plant that was not inoculated. (F–I) Transgenic Katahdin plants containing constructs RGA1-BAC, RGA2-BAC, RGA3-BAC, and RGA4-BAC, respectively.

Fig. 3.

Stability analysis of CB3A14. Culture from the original well of BAC CB3A14 was streaked, and plasmid DNA from 12 independent clones (lanes 1–12) was isolated and used as a template for PCR analysis with primers GAP-1a and GAP-1b (Table 3) that span the 3.6-kb deletion (Fig. 1). In the experiment represented by lane 13, genomic DNA from S. bulbocastanum clone PT29 was used as a template. In the experiment represented by lane 14, DNA from BAC 177O13 was used as a template.

Fig. 4.

Structure of the RB gene and the deduced RB protein. (A) Physical structure of the RB gene. Two exons are indicated by open rectangles, and one intron is indicated by lines angled downward. (B) Predicted RB protein sequences. The potential leucine zipper motif and a heptad repeat motif are underlined. The three predicted kinase motifs of the NBS domain are shown in blue. Conserved motifs for plant resistance genes are shown in green. The two amino acid changes (E⁴²⁰-K and K⁶⁶²-M) caused by LR-PCR misincorporation are indicated in bold and underlined. The start point of the 3.6-kb deletion in RGA2-BAC is double underlined. The LRRs are aligned according to the consensus sequence LXXLXXLXXLXLXXN/CXXLXXLXX, where X represents any amino acid. The first L and the last two Ls are not highly conserved in different LRRs. Aliphatic residues L, I, M, V, and F are red; conversed N, C, and T residues are brown.

Fig. 5.

The posterior probability of diversifying selection at each site in RB homologs. The x axis denotes codon position in the alignment of RB homologs. A site with posterior probability >0.95 is considered to be under significant diversifying selection.