qRT-PCR Primer Design and Optimization

The qRT-PCR primers for the genes of interest (Table S4) were designed by Primer Express, version 3.0 (Life Technologies, Carlsbad, CA, USA) to amplify approximately 100 bp segments from regions with uniform coverage in the RNA-Seq reads as confirmed using Geneious software. For primer optimization (90–110% efficiency) and qRT-PCR (Applied Biosystems 7300 Real-Time PCR System), the primers were all at 0.4 µM concentration, with the exception of CKS_3793 (0.6 µM), as this was optimized from a previous study (Kernell Burke et al., 2015 (link)). RNA for each sample type was harvested using the same methods as for the RNA-Seq following the miRNeasy kit protocol. Each sample had a RIN value of at least 7. Once extracted, the RNA was converted to cDNA using the ABI High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). The Pfaffl method was used to determine the fold change differences between samples from the in planta culture and either the pre-inoculum in vitro liquid culture or in vitro plate culture.