# Distinct Signatures of Host Defense Suppression by Plant-Feeding Mites
## 4 Material and Methods
### Tomato Infestation and Sampling  
In order to measure phytohormone concentrations and plant defense gene expression upon the various mite infestations, mites were transferred onto 21-day-old, intact tomato plants and infested leaflets were harvested seven days later, following previously described procedures [23; 33], with minor modifications. In short, plants were infested either with one species of mite (_T. urticae, T. evansi_, or _A. lycopersici_), referred to as \"single infestations\", or simultaneously with a combination of two species (_T. urticae_ plus _T. evansi_, or _T. urticae_ plus _A. lycopersici_), referred to as \"dual infestations\". Spider mite infestations were performed by individually transferring adult females, randomly collected from the rearing colony, with a fine brush onto each of three leaflets per plant. For single infestations, each leaflet received 15 spider mites so that in total each plant was infested with 45 spider mites. Russet mite infestations were performed by transferring mites on small pieces (ca. 0.5 cm\({}^{2}\)) of leaflet, cut out from well-infested rearing-plants, onto each of three leaflets per plant. These leaflet pieces were cut out while using a stereo microscope (Leica MZ6; Leica Microsystems, Wetzlar, Germany) to ensure each of them contained ca. 250 mobile stages of russet mites. For single infestations, each plant was thus infested with ca. 750 russet mites. For dual infestations, each of three leaflets received either 15 _T. urticae + 15 T. evansi_ (in total 90 spider mites per plant), or 15 _T. urticae_ + ca. 250 _A. lycopersici_ (in total 45 spider mites and ca. 750 russet mites per plant). A lanolin (Sigma-Aldrich, St. Louis, MO, USA) barrier was made around the petiolule to prevent the mites from escaping. Leaflets from non-infested control plants also got a lanolin barrier. Seven days after the infestations, infested leaflets and corresponding leaflets of non-infested control plants were excised, flash-frozen in liquid nitrogen, and stored at \(-80\)\({}^{\circ}\)C until we extracted their phytohormones and isolated the RNA. Mites were not counted, nor removed, prior to the harvest of leaf material. Likewise, we did not quantify mite-inflicted feeding damage, because russet mite feeding activity cannot be quantified yet. The three leaflets obtained from the same plant were pooled to form one biological replicate. This experiment was replicated four times in consecutive weeks, each time using six plants per treatment, resulting in a total of 144 samples (6 treatments \(\times\) 6 plants \(\times\) 4 experimental replicates). The mite densities and sampling moment used in this study were carefully selected based on previous studies [23; 33; 37; 38] to maximize the opportunity to capture plant defense suppression by mites. That is, although the magnitude of defense responses induced by _T. urticae_ increases as the infestation progresses, tomato leaflets infested with 15 of these mites usually enter senescence after eight to nine days and die shortly after [33]. Leaflets infested with 15 _T. evansi_ do not senesece prematurely [33] and stay green (except for the emptied cells) and turgid for at least 14 days. Similarly, leaflets infested with 250 _A. lycopersici_ do show clear signs of infestation (i.e., \"silvering\") after seven days, but it takes roughly another week before they senesce [23]. The _T. urticae_-induced senescence was not dramatically accelerated in dual-infested leaves, therefore the sampling moment was set at seven days after introduction of the mites, i.e., when _T. urticae_-induced defense responses peak. Russet mitedensities were higher than those of spider mites, because russet mites are much smaller and their per capita consumption rate is thus smaller as well [135; 136]. Yet, we aimed to normalize mite densities on the basis of their estimated egg biomass production (since food is mostly converted into eggs).