Dnase treated

Root samples were frozen in liquid nitrogen and total RNA was extracted using TRI REAGENT (MRC), DNase treated (RQ1 Promega), and reverse transcription was achieved with M-MLV reverse transcriptase (RNase H minus, Point Mutant, Promega) using an anchored oligo(dT)₂₀ primer. Accumulation of transcripts was measured by qRT-PCR (LightCycler 480, Roche Diagnostics) using the SYBR^R Premix Ex Taq^TM (TaKaRa). Gene expression was normalized using ACT2 as an internal standard. Sequences of primers used in qPCR for gene expression analysis are listed in Supplementary File 1.

RNA from the FRR2161 type strain (the wild-type strain) was isolated from vegetative hyphal cells grown at 25°C for 2 days in liquid medium, from asexually developing cultures grown on solid medium at 25°C for 7 days, and from yeast cells grown at 37°C for 6 days in liquid medium. RNA was isolated from yeast cells derived either from LPS-activated J774 murine macrophages infected with wild-type conidia at 24 h postinfection or from yeast cells incubated in macrophage growth media for 24 h. Macrophages were infected as described in the "In vivo macrophage assay" section below. RNA was extracted using TRIzol reagent (Invitrogen) and an MP FastPrep-24 bead beater according to the manufacturer’s instructions. RNA was DNase treated (Promega) prior to RT-PCR analysis, and a synthesis control assay lacking cDNA was performed to ensure that no DNA contamination was present. Increasing numbers of cycles were used to ensure that the amplification was in the exponential phase, and the benA gene was used as a loading control. Expression of simA was determined by RT-PCR (Invitrogen Superscript III One-Step RT-PCR with platinum Taq) using primers simA-DD3 (5′-ATCCATCCCCCGTGAAGC-3′) and simA-DD4 (5′-GCCGACACGAAGTGATCC-3′). Band intensity was quantitated in Photoshop, and relative intensity values were calculated using the benA loading controls.