Body of Abstract: A layer of epicuticular wax is present as a coating on the aerial organs of all seed plants. Despite the ubiquitous presence of epicuticular wax, a comprehensive understanding of wax biosynthesis in Arabidopsis, and a confirmed role for this wax layer in preventing water loss, further functions remain undetermined. To address this issue, we generated a population of epicuticular wax biosynthesis mutants in Nicotiana glauca (tree tobacco), a species with unusually abundant surface wax, and examined the responses of these mutants to a variety of interactors under varying experimental conditions, ranging from ultra-regulated assays in the lab to natural outdoor settings. When conditions were selective in natural settings, all components of the epicuticular wax layer contributed to plant fitness. However, strong effects that were seen under controlled conditions in the lab often vanished in the field, and interactions that we did not hypothesize would be affected by the different wax composition were sometimes associated with a specific metabolic shift in this layer. One of the more interesting hypotheses regarding function of plant epicuticular waxes, is that they generate a “self-cleaning” rough surface, which causes water to wash away pathogenic particles from the leaves. To examine this, we performed a microbiome analysis of adaxial and abaxial epidermal tissues of our wax mutant population in the greenhouse and in the field. This showed that, despite field-grown plants being exposed to a rainy winter, the microbial composition was similar between the different mutants and was more strongly affected by the growth location than the surface wax composition. We concluded that wax composition affects the interaction with specific microbes but has a smaller effect on the general microbial community in this setting.