eviously (59). In vitro development and microsclerotia production of the VdAMP3 deletion mutant were tested and quantified as described previously (18). In Vitro Microbial Growth Assays. Bacterial isolates were grown on lysogeny broth agar at 28 . Single colonies had been selected and grown overnight in low-salt lysogeny broth (LB) (ten g/L tryptone, 5 g/L yeast extract, and 0.5 g/L sodium chloride) at 28 while shaking at 200 rpm. Overnight cultures had been resuspended to optical density (OD)600 = 0.025 in fresh low-salt LB supplemented with 20 M VdAMP3 or ultrapure water (Milli-Q). In vitro growth was quantified using a CLARIOstar plate reader (BMG Labtech) as described previously (18). Fungal isolates had been grown on potato dextrose agar (PDA) at 22 . For yeasts, single colonies had been selected and grown overnight in 0.05potato dextrose broth (PDB) at 28 whilst shaking at 200 rpm. Overnight cultures have been resuspended to OD600 = 0.01 in fresh five PDB supplemented with ultrapure water (Milli-Q), 5 M VdAMP3, or five M VdAMP3 that was incubated within a PCR thermocycler at 95 for 16 h. Alternatively, for filamentous fungi, spores were harvested from PDA and suspended in five PDB supplemented with 5 M VdAMP3 or ultrapure water (Milli-Q) to a final concentration of 104 spores/mL. Subsequent, 200 L of your fungal suspensions was aliquoted in clear 96-well flatbottom polystyrene tissue-culture plates. Plates had been incubated at 28 , and fungal growth was imaged utilizing an SZX10 stereo microscope (Olympus) with EP50 camera (Olympus). Microbiome Analysis. Inoculation and incubation of N. benthamiana plants had been performed as described above. Subsequent genomic DNA isolation and V. dahliae biomass quantification had been performed as previously described working with the primers listed in SI Appendix, Table 3 (60). Immediately after four wk of incubation in plastic bags at area temperature inside the dark, the decaying N. benthamiana phyllosphere samples colonized by V. dahliae WT and the VdAMP3 deletion mutant were collected. The phyllospheres of fresh 3-wk-old N. benthamiana plants have been incorporated as controls. All samples have been flash-frozen in liquid nitrogen and ground using mortar and pestle, and genomic DNA was isolatedPNAS j 9 of 11 doi.org/10.1073/pnas.PLANT BIOLOGYusing the DNeasy PowerSoil Kit (Qiagen). Sequencing libraries were ready applying the TruSeq DNA Nano kit (Illumina), and paired-end 150-bp sequencing was performed on the Illumina NextSeq500 platform in the Utrecht Sequencing Facility. The sequencing data have been processed as follows. Excellent control in the reads, adapter trimming, and removal of N. benthamiana reads had been performed using the ATLAS metagenomic workflow employing the default parameters from the configuration file (61). Reads of the various samples have been combined and assembled using metaSPAdes (used k-mer sizes: 21, 33, and 55) to acquire a single metagenome cross-assembly (62). Subsequently, the cross-assembled contigs had been taxonomically classified employing Contig Annotation Tool and binned per genus (63). The reads in the person samples were mapped to the binned contigs working with Burrows-Wheeler Aligner Maximal Exact Match (64). Next, the mapping files have been converted to bam format making use of Brd web SAMtools (65) HDAC10 custom synthesis version 1.10, along with the variety of reads mapped to the contigs of a single genus had been converted to “reads per million” for the individual samples. The generated taxonomy table and abundance table were subsequently transformed into a phyloseq (66) object (version 1.30.0) in R (version three.six.1) to
