Quantitative reverse transcription–PCR (RT–qPCR)

Roots were collected from R. solanacearum-infected or water-treated Arabidopsis plants at 0, 24, and 48 hpi. Briefly, roots from ~40 seedlings were cut and pooled. Roots were rapidly washed in water and dried before freezing in liquid nitrogen. Samples were stored at –80 °C. RNA was extracted using the Maxwell 16 LEV Plant RNA Kit (Promega, Australia) according to the manufacturer´s recommendations. RNAs were treated with DNase-free RNase (Promega, Australia) and the concentration measured with an ND-8000 Nanodrop. cDNA was synthesized from 2 µg of RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer’s instructions. According to the SYBR Green PCR mix instructions (Roche, Switzerland), 2.5 µl of cDNA were used in a final reaction volume of 10 µl in the LightCycler 480 System (Roche, Switzerland). Melting curves and relative quantification of target genes were determined using the software LightCycler V1.5 (Roche, Switzerland). The amplification program was set to an initial step of 10 min at 95 °C followed by 45 cycles using 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s. All samples were run in triplicate for each biological replicate, and the target gene was normalized to the endogenous control gene Arabidopsis tubulin β-1 chain (At1g75780). To visualize the data, we calculated the fold change of each biological replicate in 24 h and 48 h samples by normalizing to the ΔCt of time point 0 hpi of the mock and infected samples separately. The statistical analysis of the normalized data was performed using the ‘rstatix’ R package (ver. 0.6.0). To test for differences in gene expression between mock and infected samples, the normalized data were tested for normality and homogeneity of variances. If these two requirements were fulfilled, the parametric t-test was performed for each time point to compare between mock and infected samples. All primer sequences used were obtained from previous publications and are listed in Supplementary Table S6. qPCR analysis conforms to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009).