Fig 1.Early blight resistance maps to chromosome 12 of potato.A. 2-3 genotypes of 13 different accessions of S. commersonii and S. malmeanum were inoculated with A. solani altNL03003. 3 plants of each genotype were tested and 3 leaves per plants were inoculated with 6 10 µl droplets with spore suspension. Lesion diameters were measured 5 days post inoculation and visualised using boxplots, with horizonal lines indicating median values and individual measurements plotted on top. Non-expanding lesions (<3 mm) indicate resistance and expanding lesions indicate susceptibility. Some accessions segregate for resistance. B. Accession CGN18024 is an example of an accession that segregates for resistance to A. solani, with CGN18024_1 displaying resistance and CGN18024_3 displaying susceptibility at 5 days after spray-inoculation. C. Progeny from CGN18024_1 x CGN18024_3 was inoculated with A. solani. 3 plants of each genotype were tested and 3 leaves per plants were inoculated with 6 10 µl droplets with spore suspension each. Lesion diameters were measured 5 days post inoculation. 16 progeny genotypes are resistant (with lesion diameters < 2-3 mm) and 14 are susceptible (with expanding lesions). This corresponds to a 1:1 segregation ratio (Χ2 (1, N = 30) = 0.133, ρ= 0.72). D. SNPs derived from a BSRseq analysis using bulks of susceptible and resistant progeny were plotted in 1 Mb windows over the 12 chromosomes of the potato DMv4.03 genome (68) They are almost exclusively located on chromosome 12.Table 1.Genome assembly metrics of S. commersonii cmm1t (70) and CGN18024_1Fig 2.Identification of two glycosyltransferase resistance genes.A. Comparison of the susceptible and resistant haplotype of the Solanum commersonii CGN18024_1 resistance region (delimited by markers 817K and 797K) in a comparative dot plot shows a rearrangement. Locations of markers used to map the resistance region are indicated in grey along the x-and y-axis. The duplicated region of the resistant haplotype contains marker 807K (white asterisk) and two predicted glycosyltransferase genes (ScGTR1 and ScGTR2). Several short ORFs with homology to glycosyltransferase genes that were predicted in the resistance region are indicated by white boxes, but ScGTR1 and ScGTR2 are the only full-length genes. As a result of the rearrangement, the resistance region of the resistant haplotype (27 kb) is 7 kb larger than the corresponding region of the susceptible haplotype (20 kb). B. Alignment of RNAseq reads from the BSRSeq analysis shows that ScGTR1 and ScGTR2 are expressed in bulks of resistant progeny, but not in bulks of susceptible progeny. C. S. tuberosumcv. ‘Atlantic’, S. commersonii CGN18024_1 and CGN18024_3 were agroinfiltrated with expression constructs for ScGTR1 and ScGTR2, ScGTS and empty vector (-). A. solani is inoculated 2 days after agroinfiltration and lesion diameters are measured 5 days after inoculation. Lesion sizes were visualised with boxplots, with horizonal lines indicating median values and individual measurements plotted on top. Agroinfiltration with expression constructs for ScGTR1 and ScGTR2 results in a significant (Welch’s Two Sample t-test, **P < 0.01, ***P < 0.001) reduction of lesion sizes produced by Alternaria solani altNL03003 in S. commersoni iCGN18024_3, but not in S. tuberosum cv. ‘Atlantic’.Fig 3.Leaf compounds from resistant S. commersonii inhibit growth of diverse fungi, including pathogens of potato.A. Crude leaf extract from CGN18024_1/CGN18024_3 was added to PDA plates (5% w/v) and autoclaved (left) or semi-sterilised for 15 min at 60 °C (right). Growth of Alternaria solani altNL03003 was strongly inhibited on PDA plates with autoclaved leaf extract from CGN18024_1 compared to plates with CGN18024_3, as shown on the left two pictures taken at 7 days after placing an agar plug with mycelium of A. solani at the centre of each plate. Abundant fungal contamination appeared after 4 days on plates containing semi-sterilised leaf from CGN18024_3, but not on plates containing material from CGN18024_1 (right two pictures). B. Growth of potato pathogenic fungi A. solani (altNL03003), B. cinerea (B05.10) and F. solani (1992 vr) was followed by measuring the colony diameter on PDA plates containing autoclaved leaf material from CGN18024_1/CGN18024_3. Growth of all three fungi was measured on PDA plates containing CGN18024_1 (red squares), CGN18024_3 (green circles) or plates with PDA and no leaf material (blue triangles). Significant differences in growth on PDA plates containing plant extract compared to PDA plates without leaf extract are indicated with asterisks (Welch’s Two Sample t-test, **P < 0.01, ***P < 0.001).Fig 4.Tetraose steroidal glycoalkaloids from Solanum commersonii provide resistance to Alternaria solani and Colorado potato beetle.Data are visualised with boxplots, with horizonal lines indicating median values and individual measurements plotted on top. A. Tetraose steroidal glycoalkaloids (SGAs) were detected in resistant CGN18024_1 and in CGN18024_3 transformed with ScGTR1/ScGTR2. Susceptible S. tuberosum cv. ‘Atlantic’ and wildtype (WT) CGN18024_3 contain only triose SGAs. Overexpression of ScGTR1 resulted in the addition of a hexose to the triose SGAs from CGN18024_3, resulting in a commertetraose (Gal-Glu-Glu-Glu), while overexpression of ScGTR2 caused the addition of a pentose, resulting in a lycotetraose (Gal-Glu-Glu-Xyl). B. WT CGN18024_1/CGN18024_3 and CGN18024_3 transformants were inoculated with Alternaria solani altNL03003. 3 plants of each genotype were tested and 3 leaves per plants were inoculated with 6 10 µl droplets with spore suspension each. Lesions diameters were measured 5 days post inoculation. ScGTR1 and ScGTR2 can both complement resistance to A. solani in CGN18024_3, as the lesion sizes produced on CGN18024_3 transformants are comparable to resistant CGN18024_1. C. 3 plants per genotype were challenged with 5 Colorado potato beetle larvae each. The tetraose SGAs produced by ScGTR1 and ScGTR2 can provide resistance to Colorado potato beetle, as indicated by reduced larvae survival and total larvae weight. Significant differences with WT CGN18024_3 are indicated with asterisks (Welch’s Two Sample t-test, *P < 0.05, ***P < 0.001). D. Putative structures of SGAs detected in CGN18024_1 and CGN18024_3, based on previous studies (85, 87–89). CGN18024_3 produces triose SGAs and is susceptible to Colorado potato beetle and A. solani. ScGTR1 and ScGTR2 from CGN18024_1 convert these triose SGAs from susceptible S. commersonii to tetraose SGAs, through the addition of a glucose or xylose moiety respectively. Both sugar additions can provide resistance to Colorado potato beetle and A. solani.