# Signaling in the Tomato Immunity against _Fusarium oxysporum_
## 4 Materials and Methods
### HS-SPME Extraction and GC-MS Analysis of Volatile Compounds  
The volatile organic compounds (VOCs) were extracted from 0.1 g of ground tomato leaves (6 biological replicates) weighed in a 10 mL headspace screw-cap vial according to [PERSON] et al. 2015 [58]. One milliliter of saturated CaCl\({}_{2}\) at pH 6 and 100 \(\upmu\)L EDTA 750 nM at pH 7.5 were added, and the mixture was sonicated for 5 min. Volatile compound extraction was performed by head space solid-phase microextraction (HS-SPME). Samples were incubated at 50 \({}^{\circ}\)C for 10 min, extracted at the same temperature for 20 min, and adsorbed in a 65 \(\upmu\)m PDMS/DVB fiber (Supelco, Bellefonte, PA, USA). Solid phase microextraction of the adhered compounds was carried out for 1 min at 250 \({}^{\circ}\)C in splitless mode using a CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland). Then, the fiber was cleaned by exposing it for 5 min at 250 \({}^{\circ}\)C in an SPME fiber conditioning station (CTC Analytics) to prevent cross-contamination.  
Separation of the compounds was performed by using an Agilent 6890N gas chromatograph (Santa Clara, CA, USA) coupled to an Agilent 5975B mass spectrometer operating in electronic impact (EI) mode with 70 eV of ionization energy and 230 \({}^{\circ}\)C of source temperature. Chromatographic separation was carried out on a DB-5 ms fused silica capillary column (60 m long, 0.25 mm i.d., 1 \(\upmu\)m film thickness) using helium as the carrier gas at a constant flow of 1.2 mL/min. Temperature conditions established in the oven were 40 \({}^{\circ}\)C for 2 min, a ramp from 5 \({}^{\circ}\)C/min to 250 \({}^{\circ}\)C, and an isothermal at 250 \({}^{\circ}\)C for 5 min. Data acquisition was performed at 6 scans per second in an \(m/z\) range of 35-250. Chromatograms and mass spectra were acquired and processed using the Enhanced ChemStation F.01.03.2357 software (Agilent).  
Unequivocal identification of the VOCs was performed by using commercial compounds served as standards. The coelution and the spectrum equivalence between the commercial compound and the one to determine confirmed unequivocally its identity. Other compounds were tentatively identified by comparing their mass spectra with those listed in the NIST 05 Mass Spectral library.  
\begin{table}
\begin{tabular}{c c c} \hline
**Gene** & **Forward Primer (5’-3’)** & **Reverse Primer (5’-3’)** \\ \hline _PR1_ & ACTCAAGTAGTGCGCGAACTCA & AGTAAGAGAGCTTGTCCGATCGAGT \\ _ACS2_ & GATGGATTTGCGCGTCCACTTT & GATCCAGGAGGAGGTTAG \\ _LEA_ & AGCAGATGTTGGAAAAGG & ATGCCTATGGGGATTGT \\ _TomloxA_ & AAGAAAGCTGAGTTGAATGAA & TTGAAACTTTTCAGCTGGAATTA \\ _TomloxC_ & \(\upalpha\)TAGCCATCATGTGC & GTAATGTCGAAATTCCCTG \\ _TomloxD_ & GGCTTTTACTCTCTG & A AATCAAGGCGCAGTTTT \\ _TomloxF_ & CCGATCAAGGGTGTACTTT & GGTCTGTGTGAATGATG \\ _HPL_ & AGCTACGATTGCGCGTTTAGT & CATTCTCTTTGGTAAGA \\ _AOS_ & CCTCTTCTTCCTTCTCTCACCAAA & GCCGGGTATAGTCCTCGGTAGA \\ _MTS1_ & TGGTGTCACCTTCAAGAGA & GCCTTGTGGTGAAAATAGGA \\ _SAMT1_ & TCCCAGAAAACATTATACATGTGAT & AATGAACCTAACAAGCTTCTGATCCACACATA \\ _EF1\upalpha_ & CCACCTGAGATCCTTAAGG & ACCCTCACGTGTCTCCAG \\ \hline \end{tabular}
\end{table}
Table 1: Primer sequences used for real-time quantitative PCR.