*2.3. Fungal Recovery Assay*

Tomato stems were collected three weeks after inoculation and surface sterilized with 70% ethanol as described [9]. In short, the ethanol was removed by pouring into a collection tube, and stems were rinsed twice with sterile water to remove the ethanol. Subsequently, the extremities of the stem were trimmed and one piece at the cotyledon and one at crown level were cut and placed on PDA plates containing 200 mg/<sup>L</sup> streptomycin and 100 mg/l penicillin to prevent bacterial growth. When transgenic fungal strains were used, the PDA plates also contained 100 mg/<sup>L</sup> zeocin or 100 mg/<sup>L</sup> hygromycin for ensuring selection of the right fungal strain. Plates were incubated for four days at 25◦C in the dark, after which Fo outgrowth was assessed as follows: 0= no fungal outgrowth, 1= fungal outgrowth of stem pieces isolated from either crown or cotyledon level, and 2= fungal outgrowth from stems isolated at both crown and cotyledon level.

### *2.4. Fungal DNA Isolation and Sequencing*

To confirm that the mycelium emerging from tomato stems corresponded to the originally inoculated strain, the mycelium was scraped from the PDA plate and used for gDNA isolation and PCR as described by the authors of [25]. In short, mycelium scrapes were placed in a 2 mL tube containing 400 μL TE bu ffer (1 mM EDTA pH = 8, 10 mM Tris pH= 8), 200-300 μL glass beads, and 300 μL phenol:chloroform (1:1), followed by disruption using a TissueLyser (Qiagen; Venlo, Netherlands) for 2 min at 30 Hz. Afterwards, the samples were centrifuged for 10 min. The upper phase was transferred to a new 2 mL tube and was subjected to another round of phenol:choloroform extraction. PCR amplification of *EF1-alpha* gene (see Table S1 for primers) was performed in 20 μL reaction consisting of of 0.4 μL dNTPs (10 mM), 4 μL SuperTaq bu ffer (10x), 0.1 μL SuperTaq (5 U/μl), 1 μL of DNA template, and 1 μL primers (5 pmol/μL). The cycling program for PCR amplification was 95◦C for 5 min, 35 cycles of 30s at 95◦C, 30s at 55◦C, and 1 min at 72◦C, with a final elongation step of 3 min at 72◦C. The PCR amplicon was sequenced and compared with the *EF1-alpha* sequence of the strain inoculated originally.

### *2.5. Analysis of Fungal Colonization by Quantitative PCR*

Tomato roots were harvested three weeks after inoculation, washed, snap-frozen in liquid nitrogen, and freeze-dried overnight. Samples were ground in a mortar cooled with liquid nitrogen using a pestle and approximately 100 mg of the resulting powder was using for gDNA isolation. gDNA isolation and purification were performed using GeneJET plant Genomic purification Kit (Thermo Scientific; Walthamm MA, USA). DNA concentration was estimated by spectroscopy using Nanodrop (Thermo Scientific, Walthamm MA, USA), and the quality of the gDNA was assessed by agarose gel electrophoresis. The 10 μL qPCR mixture contained 10 ng of gDNA, 10 pM of each primer, and 2 μL of HOT FirePolEvaGreen qPCR Mix Plus (Solis BioDyne; Tartu, Estonia) were performed in QuantStudioTM3 (Thermo Scientific; Walthamm MA, USA). The cycling program was set to 15 min at 95◦C, 40 cycles of 15s at 95◦C, 1 min at 60◦C, and 30s at 72◦C. The melting curve analysis was performed afterwards as follows: 15s at 95◦C, 1 min at 60◦C, and 15s at 95◦C. The sequences of the primers used are summarized in Table S1 [26,27]. For InterGenic Spacer (IGS) primers two standard curves (four- or ten- times dilution) were performed with a starting concentration of 10 ng, resulting with a primer efficiently of 110 and 95%, respectively. Three technical replicates were used per biological sample, and data was normalized to plant tubulin gene level, using qbase+3.1 (Biogazelle; Ghent, Belgium).
