The resistant cherry tomato (Solanum lycopersicum var. cerasiforme) line LC-95, derived from an accession collected in Ecuador, harbors a natural allele (ol-2) that confers broad-spectrum and recessively inherited resistance to powdery mildew (Oidium neolycopersici). As both the genetic and phytopathological characteristics of ol-2-mediated resistance are reminiscent of powdery mildew immunity conferred by loss-of-function mlo alleles in barley and Arabidopsis, we initiated a candidate-gene approach to clone Ol-2. A tomato Mlo gene (SlMlo1) with high sequence-relatedness to barley Mlo and Arabidopsis AtMLO2 mapped to the chromosomal region harboring the Ol-2 locus. Complementation experiments using transgenic tomato lines as well as virus-induced gene silencing assays suggested that loss of SlMlo1 function is responsible for powdery mildew resistance conferred by ol-2. In progeny of a cross between a resistant line bearing ol-2 and the susceptible tomato cultivar Moneymaker, a 19-bp deletion disrupting the SlMlo1 coding region cosegregated with resistance. This polymorphism results in a frameshift and, thus, a truncated nonfunctional SlMlo1 protein. Our findings reveal the second example of a natural mlo mutant that possibly arose post-domestication, suggesting that natural mlo alleles might be evolutionarily short-lived due to fitness costs related to loss of mlo function. Bai, Yuling. Pavan, Stefano. Zheng, Zheng. Zappel, Nana. Reinstädler, Anja. Lotti, Concetta. De, Giovanni. Ricciardi, Luigi. Lindhout, Pim. Visser, Richard. Theres, Klaus. Panstruga, RalphMolecular plant-microbe interactions : MPMI200821(1)30-9