4.6. Quantitative Analysis of Defense-Related Gene Expression by Real-Time PCR

The plants were distributed in four treatments: control plants treated with distilled water (C), plants inoculated with H. vastatrix (H), plants treated with 0.05% high-density chitosan (Q), and plants treated with 0.05% high-density chitosan and inoculated with the pathogen (QH). Three mL of distilled water were sprayed in treatments C and H, and 3 mL of high-density chitosan in treatments Q and QH. Seven days after the application of water or chitosan, 3 mL were inoculated with the suspension of urediniospores of H. vastatrix. Plants were kept in an incubation room at 26 °C ± 2 °C, and leaf samples were taken at 12, 24, and 48 h after inoculation with the pathogen [9]. On each sampling day, 3 plants were taken for the corresponding evaluation, and each test was performed in duplicate.

Primers coding for the genes peroxidase (POX), catalase (CAT), ß-1,3-glucanase (GLU), and phenylalanine ammonia lyase (PAL) were designed. The genomic sequences were obtained from the Phytozome repository. Subsequently, the OligoAnalyzer Tool from IDT and Primer-BLAST from NCBI were used. Additionally, glyceraldehyde-3-phosphate dehydrogenase (GADPH), actin 8, and 14-3-3 primers were designed as constitutive controls. Table 2 shows the characteristics of each pair of oligonucleotides. The stability of the primers was determined according to the method of Vandesompele et al. [45].

Oligonucleotides.

The TRIzol extraction protocol (Invitrogen, Carlsbad, CA, USA) was used with some modifications) [46]. In total, 200 mg of sample were macerated with liquid nitrogen, and 1 mL of TRIzol was added for 5 min. The samples were centrifuged at 11,000× g for 15 min at 4 °C, the supernatant was removed, and 500 µL chloroform (Sigma-Aldrich, Dorset, UK) were added, shaken for 20 min at −20 °C, then centrifuged at 11,000× g for 15 min at 4 °C. the supernatant was removed, 500 µL chloroform (Sigma-Aldrich, Dorset, UK). Subsequently, the supernatant was recovered, and 500 µL of isopropanol (Sigma-Aldrich, Dorset, UK) were added, left to stand for 2 h at −80 °C, and centrifuged at 11,000× g for 15 min at 4 °C, after which the supernatant was removed. Three washes were performed with 75% ethanol (Sigma-Aldrich, St. Louis, MO, USA), and the pellet was dried and resuspended in 20 µL of RNAse-free water. RNA was quantified at 260 nm and migrated on 0.8% agarose gel. For cDNA synthesis, the Maxima H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) was used. Synthesis was performed according to the manufacturer’s instructions, starting from 100 ng of total RNA.

RT qPCR analyses were performed on a Step-One Real-Time PCR System thermal cycler (Applied Biosystems, Life Technologies, San Francisco, CA, USA) using SYBR Green as the detection system. The reaction conditions were 2 min at 50 °C, 10 min at 60 °C, followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C, ending the cycle with 15 s at 95 °C. The reaction was carried out with 50 ng of cDNA, 0.2 µL of each primer, and 7.5 µL of SYBR green PCR Master Mix (Invitrogen, St. Louis, MO, USA) and RNAse-free water. For the evaluation of each gene, three biological samples with two technical replicates were performed. Relative quantification (RQ) was performed using the ddCT method [47].