Defense Gene Expression
To determine whether salt stress and insect herbivory induced tomato
defense responses, the expression of defense-related genes was measured
(Supplementary Figure S4 ).
A two-factorial assay with salt (0 mM, 100 mM, and 200 mM) and herbivory
(no herbivory, herbivory) was conducted. Following insect herbivory for
3 hours, plants were treated with salt. Treated leaves were flash frozen
in liquid nitrogen and stored at -80°C until further analysis.
For quantitative real-time polymerase chain reaction (qRT-PCR), leaves
were homogenized in a Geno Grinder 2000 (OPS Diagnostics, USA), and
total RNA was purified using TRIZOl. 1 μg of purified RNA was
transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, USA). qRT-PCR primers used are shown inSupplementary Table S2 . qRT-PCR reactions using Power-Track
SYBR Green PCR Master Mix (Applied Biosystems, USA) were run on a 7500
Fast Real-Time PCR System (Applied Biosystems, USA) using a previous
protocol (Acevedo et al. , 2017).
Relative quantification of genes were calculated using the
2−ΔΔC T method (Livak and
Schmittgen, 2001), with 0 mM x no herbivory-treated plants as the
reference group and ubiquitin as a housekeeping gene to normalize
CT values (Rotenberg et al. , 2006).