Identification and analyses of candidate genes for rpp4-mediated resistance to Asian soybean rust in soybean

Abstract

Asian soybean rust is a formidable threat to soybean (Glycine max) production in many areas of the world, including the United States. Only five sources of resistance have been identified (Resistance to Phakopsora pachyrhizi1 [Rpp1], Rpp2, Rpp3, Rpp4, and Rpp5). Rpp4 was previously identified in the resistant genotype PI459025B and mapped within 2 centimorgans of Satt288 on soybean chromosome 18 (linkage group G). Using simple sequence repeat markers, we developed a bacterial artificial chromosome contig for the Rpp4 locus in the susceptible cv Williams82 (Wm82). Sequencing within this region identified three Rpp4 candidate disease resistance genes (Rpp4C1-Rpp4C3 [Wm82]) with greatest similarity to the lettuce (Lactuca sativa) RGC2 family of coiled coil-nucleotide binding site-leucine rich repeat disease resistance genes. Constructs containing regions of the Wm82 Rpp4 candidate genes were used for virus-induced gene silencing experiments to silence resistance in PI459025B, confirming that orthologous genes confer resistance. Using primers developed from conserved sequences in the Wm82 Rpp4 candidate genes, we identified five Rpp4 candidate genes (Rpp4C1-Rpp4C5 [PI459025B]) from the resistant genotype. Additional markers developed from the Wm82 Rpp4 bacterial artificial chromosome contig further defined the region containing Rpp4 and eliminated Rpp4C1 (PI459025B) and Rpp4C3 (PI459025B) as candidate genes. Sequencing of reverse transcription-polymerase chain reaction products revealed that Rpp4C4 (PI459025B) was highly expressed in the resistant genotype, while expression of the other candidate genes was nearly undetectable. These data support Rpp4C4 (PI459025B) as the single candidate gene for Rpp4-mediated resistance to Asian soybean rust.

Figure 1.

Motif identification in Rpp4C1 (Wm82), Rpp4C2 (Wm82), Rpp4C3 (Wm82), and RLG (Wm82). The predicted amino acid sequences of the Rpp4 cluster as well as Rpp4L were used for motif prediction. Identified motifs included CC domains (black boxes), NBS domains (brown boxes), and LRR domains (blue vertical lines). Full amino acid length is indicated at right and by the scale at bottom.

Figure 2.

PI459025B response to ASR infection following VIGS. The images at left are the top (adaxial) leaf surfaces and those at right are the bottom (abaxial) leaf surfaces. A to E represent plants subjected to one of five pretreatments. The plant in A was not pretreated. The plant in B was mock inoculated with buffer and carborundum. The plant in C was infected with a BPMV vector lacking an insert (empty vector). The plant in D was inoculated with a BPMV construct carrying the Rpp4 candidate BPMV_NBD_F/R insert (Supplemental Table S4). The plant in E was inoculated with a BPMV construct carrying the Rpp4 candidate BPMV_LRR_F/R insert (Supplemental Table S4). All of these plants were subsequently infected with ASR and phenotyped. Red/brown lesions indicate a resistant interaction, and tan lesions indicate a susceptible interaction. F, Relative expression of P. pachyrhyzi α-tubulin mRNA relative to soybean ubiquitin following BPMV and P. pachyrhyzi inoculation. Each sample includes three biological and three technical replicates. The empty vector (C) sample has significantly lower (2–3-fold) expression of the fungal α-tubulin than either of the Rpp4 candidate gene VIGS constructs. Asterisks designate P < 0.01 from a t test of Rpp4-silenced plants (D and E) compared with empty vector-silenced plants (C).

Figure 3.

Genetic map of the PI459025B and BRS184 F2:3 population with newly developed markers. The genetic map shows chromosome 18 surrounding the Rpp4 locus. New markers added to the map developed by Silva et al. (2008) include sc21_1866, sc21_2024, sc21_2716, sc21_2922, sc21_3420, sc21_3360, and sc21_4808. Recombination events (in relation to Rpp4 phenotype) are indicated in parentheses to the right of each marker. Distance between markers (in cM) is shown on the left side of the map.