Late blight caused by Phytophthora infestans greatly constrains potato production. Many Resistance (R) genes were cloned from wild Solanum species and/or introduced into potato cultivars by breeding. However, individual R genes have been overcome by P. infestans evolution; durable resistance remains elusive. We positionally cloned a new R gene, Rpi-amr1, from Solanum americanum , that encodes an NRC helper-dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans isolates tested. Using association genomics and long-read RenSeq, we defined eight additional Rpi-amr1 alleles from different S. americanum and related species.Despite only ~90% identity between Rpi-amr1 proteins, all confer late blight resistance but differentially recognize Avramr1 orthologs and paralogs. We propose that Rpi-amr1 gene family diversity assists detection of diverse paralogs and alleles of the recognized effector, facilitating durable resistance against P. infestans .

1 2 Late blight caused by Phytophthora infestans greatly constrains potato production. 3 Many Resistance (R) genes were cloned from wild Solanum species and/or introduced 4 into potato cultivars by breeding. However, individual R genes have been overcome by 5 P. infestans evolution; durable resistance remains elusive. We positionally cloned a 6 new R gene, Rpi-amr1, from Solanum americanum, that encodes an NRC helper-7 dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans 8 isolates tested. Using association genomics and long-read RenSeq, we defined eight 9 additional Rpi-amr1 alleles from different S. americanum and related species. Despite 10 only ~90% identity between Rpi-amr1 proteins, all confer late blight resistance but 11 differentially recognize Avramr1 orthologs and paralogs. We propose that Rpi-amr1 12 gene family diversity facilitates detection of diverse paralogs and alleles of the 13 recognized effector, enabling broad-spectrum and durable resistance against P. 14 infestans. Pathogen enrichment Sequencing) enable rapid definition of allelic variation 63 and mapping of plant NLRs, or discovery of variation in pathogen effectors 21-23 . 64 Combined with single-molecule real-time (SMRT) sequencing, SMRT RenSeq enabled 65 cloning of Rpi-amr3 from Solanum americanum 24 . Similarly, long read and cDNA 66 PenSeq enabled us to identify Avramr1 from P. infestans 25 . 67 4 68In this study, we further explored the genetic diversity of S. americanum, and by 69 applying sequence capture technologies, we fine-mapped and cloned Rpi-amr1 from S. 70 americanum, (usually) located on the short arm of chromosome 11. Multiple Rpi-amr1 71 homologs were found in different S. americanum accessions and in relatives, including 72Solanum nigrescens and Solanum nigrum. Functional alleles show extensive allelic 73 variation and confer strong, broad-spectrum resistance to all 19 tested diverse P. 74 infestans isolates. Although differential recognition was found between different Rpi-75 amr1 and Avramr1 homologs, all Rpi-amr1 alleles recognize the Avramr1 homologs 76 from Phytophthora parasitica and Phytophthora cactorum. Our study reveals unique 77properties of genetic variation of R genes from "non-host" species. 78 79