Aphid infestation

A M. persicae colony was maintained on pepper plants as described previously [50]. Two independent assays have been performed: one with a low density of aphids (20 per plant) confined to a single leaf with the aid of a clip cage (see below for description). In the second experiment, a high aphid density (200 per plant) was used without restrictions of movement. In both cases, experiment started 5 weeks after sowing and pepper plants were infested with wingless adult aphids.

Twenty aphids were placed on the abaxial surface of a single leaf at the second true leaf pair of leaves and confined into a clip cage (BioQuip Products, Inc. USA), thus preventing them from freely moving throughout the plant. The infestations with aphids were initiated in a staggered manner [97], so that all tissue samples were harvested at the same time, to avoid any bias derived from diurnal cycling and/or changes in environmental conditions. Samples were collected at 3 h post-infestation (hpi), 2 days post-infestation (dpi) and 4 dpi. Appropriate controls consisting on uninfested plants that received empty clip cages for the same time periods as the aphid-infested plants were also included. At the end of the experiment, the leaf area under the cages was cut and aphids brushed off. The resulting leaf discs were then flash frozen in liquid nitrogen and stored at − 80 °C prior to freeze-drying. Tissue was finally grounded and stored at 4 °C into airtight vials until extraction. Four biological replicates were included at each time point, each one consisting on pooled samples from two plants.

Plants were infested with 200 aphids distributed evenly throughout the plant without any additional restriction for their movement. This high density of aphids was used to ensure the infestation of all plant leaves. Infested plants were enclosed into nets to avoid aphid transference among treatments. Plants destined to be controls of the experiment were also enclosed into a net for the same time periods but without aphids. As above, plants were infested sequentially and leaf tissue was harvested at 2 dpi, 4 dpi and 7 dpi. At the end of the experiment aphids were brushed off and all the fully-expanded leaves of the plants were collected. All plant material was collected at the same time, flash frozen in liquid nitrogen, freeze-dried and grounded. Four biological replicates were included at each time point, each one consisting on pooled samples from two plants.