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Soybean rust is an economically significant disease caused by the fungus Phakopsora pachyrhizi that negatively impacts soybean ( Glycine max [L.] Merr.) production throughout the world. Susceptible plants infected by P. pachyrhizi develop tan-colored lesions on the leaf surface that give rise to funnel-shaped uredinia as the disease progresses. While most soybean germplasm is susceptible, seven genetic loci ( Rpp1 to Rpp7 ) that provide race-specific resistance to P. pachyrhizi ( Rpp ) have been identified. Rpp3 was first discovered and characterized in the soybean accession PI 462312 (Ankur), and it was also determined to be one of two Rpp genes present in PI 506764 (Hyuuga). Genetic crosses with PI 506764 were later used to fine-map the Rpp3 locus to a 371-kb region on chromosome 6. The corresponding region in the susceptible Williams 82 (Wm82) reference genome contains several homologous nucleotide binding site-leucine rich repeat (NBS-LRR) genes. To identify Rpp3 , we designed oligonucleotide primers to amplify Rpp3 candidate ( Rpp3C ) NBS-LRR genes at this locus from PI 462312, PI 506764, and Wm82 using polymerase chain reaction (PCR). Five Rpp3C genes were identified in both Rpp3 -resistant soybean lines, and co-silencing these genes compromised resistance to P. pachyrhizi . Gene expression analysis and sequence comparisons of the Rpp3C genes in PI 462312 and PI 506764 suggest that a single candidate gene, Rpp3C3 , is responsible for Rpp3 -mediated resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024., Competing Interests: The author(s) declare no conflict of interest.