Responses of homologous Or67bs from D. melanogaster, S. pallida, and S. flava expressed in the D. melanogaster empty neuron systems to stimulation with natural odor blends. (A) Schematic representation of the single sensillum recording (SSR) using two “empty neuron systems”. Or67b proteins (X_Or67b, where X refers to the fly species) were expressed in a D. melanogaster mutant that lacks its endogenous Or22a in ab3A (antennal basiconic 3A) OSNs (Hallem and Carlson 2006; left), or Or67d in at1 (antennal trichoid 1) OSNs (Kurtovic et al. 2007; right). Note that the at1 empty neuron system was used only for expression of Sfla Or67b2, as this Or was not functional in the ab3A empty neuron system. Although comparison between the responses of this Or with other Or67b proteins should be interpreted cautiously (Syed et al. 2010), Or tuning (but not response intensity) is independent from the empty neuron system expressed for Or expression (supplementary fig. 5, Supplementary Material online). The antennal basiconic sensilla houses the A (which expresses one of the Or67b proteins) and the native intact B neuron; the A neuron has larger amplitude spikes than the B neuron, allowing discrimination of spikes originating from either neuron. The antennal trichoid sensilla houses a single OSN expressing Sfla Or67b2; arrows indicate spikes (see also supplementary fig. 4, Supplementary Material online). Calibration bars (vertical lines to the right of traces): 10 mV throughout all figures unless noted. (B) Representative electrophysiological recordings obtained from the targeted sensilla of flies expressing Or67b in OSNs in response to stimulation with apple cider vinegar, crushed rosette leaves of wild-type Col-0 Arabidopsis thaliana, and phytoalexin deficient 3 (PAD3) and aliphatic and indolic Glucosinolate Knock Out (GKO; myb28 myb29 cyp79b2 cyp79b3) A. thaliana mutants. Although all three A. thaliana genotypes have the same genetic background (Glazebrook and Ausubel 1994; Beekwilder et al. 2008), PAD3 plants are a more appropriate control for this study than GKO or Col-0, because PAD3 is deficient (as is GKO) in the production of camalexin but still produces normal levels of aliphatic or indolic glucosinolates. The bars above records indicate the onset and duration (1 s) of the stimulation throughout all figures unless otherwise noted. (C) Responses (net number of spikes/second, control subtracted, n = 6–9 obtained from 2 to 4 animals) evoked by stimulation with apple cider vinegar, odors from homogenized mustard leaves (arugula and A. thaliana), grated mustard root odors (wasabi, horseradish, turnip, and daikon), homogenized nonmustard leaf odors (tomato), and grated nonmustard root odors (beet, control). The outer edges of the horizontal bars represent the 25% and 75% quartiles, the vertical line inside the bars represents the median, and the whiskers represent 10% and 90% quartiles; each dot represents an individual response. Blue, gray, and green rectangles, respectively, show responses by stimulation with the Drosophila melanogaster attractant apple cider vinegar, non-ITC-bearing plants, and ITC-bearing plants. Asterisks indicate significant differences between the control-subtracted net number of spikes and a threshold value for responsiveness (10 spikes/second), as explained in Materials and Methods (one-sample signed rank tests; *P < 0.05, **P < 0.01). Neurons expressing Dmel Or67b and Spal Or67b, but not those expressing any of the S. flava Or67b paralogs, responded to stimulation with apple cider vinegar. Conversely, only neurons expressing S. flava Or67b paralogs responded to arugula odors (which bear ITCs). None of the S. flava paralogs responded to stimulation with the nonhost (non-ITC bearing) tomato. Dmel Or67b responded to all A. thaliana genotypes, whereas Sfla Or67b1-2 responded only to ITC-bearing A. thaliana, indicating that the presence of ITCs within plants is necessary to evoke responses from these two S. flava paralogs. Stimulation with wasabi root odors evoked responses from all Sfla Or67b paralogs but not from the Dmel or the Spal paralogs.