*2.4. Insects*

Both pyrethroid-susceptible (Py-S) and pyrethroid-resistant (Py-R) *T. infestans* used in this study come from a well-established colony at the insectary of the School of Natural Sciences, National University of Salta, Argentina. Insects were fed on ketamine-anesthetized rat blood once per each development stage. All animal care and laboratory experimental protocols were carried out following the Regulation of the Institutional Committee for Care and Use of Laboratory Animals and Field Studies (CICUALEC), School of Natural Sciences, National University of Salta, Argentina. The colony was periodically renewed by incorporating first generation, field-collected insects.

## *2.5. Mortality Bioassays*

Fungal suspensions were prepared in sunflower oil at a concentration of 1 × 10<sup>12</sup> conidia mL−1, determined with a hemocytometer. Groups of 15 Py-R insects were used for each fungal strain, which included first (NI) and third stage (NIII) nymphs, and adults (A). Each group was placed in Petri dishes (10 cm diameter) containing 1 mL of each fungal formulation homogenously dispersed with a silicone brush (1.3 × 10<sup>10</sup> conidia/cm2). The insects were allowed to be in contact with it for ten minutes, and then transferred individually to acrylic containers and incubated at 27 ◦C with a photoperiod of 12:12 h without feeding. As control treatments, Petri dishes sprayed with sunflower oil were used. Mortality was registered daily and dead insects were put in a humid chamber in order to confirm that death was caused by fungal infection. Medium survival time (MST), Maximum survival reached (S%), and Kaplan and Meier survival curves were performed nine days after fungal infection.

#### *2.6. Gene Expression by qRT-PCR*

We used the dual gene expression approach for studying the molecular interaction between fungi and insects, according to the protocol described by Lobo et al. [13]. For this, two-week-old fourth-instar nymphs, either Py-S or Py-R, were used one week after a blood meal. Individual insects were immersed for 6 s in aqueous (0.01% Tween 80) conidial suspensions of either 0 (control), 1 × 102, or 1 × 10<sup>4</sup> conidia mL−1. Insects were returned and maintained at the rearing conditions described above. At di fferent time periods (three, six, and nine days after treatment), three live insects were separated, and their total RNA was extracted from both fungus-treated and control insects by employing the Tri Reagent ® (Molecular Reagent Centre, Cincinnati, OH, USA) technique, according to manufacturer's instructions. RNA was quantified by a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and its integrity was assessed on a 1% ( *w*/*v*) agarose gel. Two-step real-time polymerase chain reaction (RT-PCR) was carried out with iScript cDNA Synthesis kit and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Amplification was performed on an AriaMx Real-Time PCR (qPCR) Instrument (Agilent Technologies, Santa Clara, CA, USA) employing 40 ng reverse-transcribed total RNA for each sample. Targeted fungal genes were those encoding for enzymes involved in the biosynthesis of some secondary metabolites [13]; i.e., tenellin synthetase (*BbtenS*), beauvericin synthetase (*BbbeaS*) and bassianolide synthetase (*BbbslS*). Insect genes assayed were a limpet transcription factor (*Tilimpet-2*) and three defensin genes (*Tidef-1*, *Tidef-2*, and *Tidef-6*) regulated by limpet [23]. Primers used are listed in Table 1. The following amplification program was used: denaturation at 95 ◦C for 10 min, followed by 40 cycles with three-segment amplification (30 s at 95 ◦C for denaturation, 30 s at 55 ◦C for annealing, and 30 s at 72 ◦C for DNA chain elongation). In order to confirm that only single products were amplified, a temperature-melting step was then performed. Negative controls were performed by using 'cDNA' generated without reverse transcriptase as templates. Reactions containing primer pairs without template were also included as blank controls. The assay was performed in duplicate, and three independent biological replicates were done. To analyze the expression profiles, we applied the NRQ model, consisting of the conversion of quantification cycle values (Cq) into normalized relative quantities (NRQs), the adjustment for differences in PCR efficiency between the amplicons [31], and the normalization with reference genes [32].
