Hiseq 2000

The whole genome of the progenitor isolate (11–281) sequenced, assembled and annotated as previously reported [49 (link)], was used as a reference genome in this study. The assembled genome sequence of P. striiformis isolate 11–281 is available on GenBank under the accession number SBIN00000000. The version described in this article is version SBIN01000000. The raw data of isolate 11–281 used for this experiment has been submitted to NCBI with SRA study SRR8446446. Urediniospores of the 30 mutant isolates were collected from infected wheat plants grown in growth chambers, dried in a desiccator at 4 °C for about a week, and used for extracting genomic DNA using the cetyl trimethylammonium bromide (CTAB) method with modifications as previously described [73 (link)]. The genomic DNA samples of the 30 mutant isolates were sequenced using the Illumina Hiseq 2000 technology by Novogene Corporation (Sacramento, CA, U.S.A.). The raw data of the 30 mutant isolates have been deposited in the NCBI under BioProject accession PRJNA587768 (containing SRA accessions SRR10413520 to SRR10413549) with the title “Puccinia striiformis f. sp. tritici strain:PST-mutants | isolate:PST-mutants_M11 Raw sequence reads”.

Vegetative hyphae of PH-1 and Fgssn3 mutant M9 were harvested from 36 h liquid CM cultures. For each strain, two biological replicates were used. Total RNAs were extracted with the Qiagen RNeasy Micro kit and treated with RNase-free DNase I. Complementary DNA libraries with the average insert size of 330 bp were constructed with the Illumina TruSeq RNA Sample Preparation Kit and sequenced with Illumina HiSeq 2000 at the Novogene Bioinformatics Institute (Beijing, China). For each sample, at least 18 Mb paired-end reads were obtained. The resulting RNA-seq reads were mapped onto the reference genome of F. graminearum strain PH-1 with Tophat 2.0.1259 (link). The number of reads (counts) aligned to each predicted transcript was calculated by FeatureCounts60 (link). Differential expression analysis of genes was performed with the edgeRun package61 (link) using the UCexactTest function with the Benjamini and Hochberg’s algorithm to control the false discovery rate (FDR). To filter out weakly expressed genes, only genes with a minimum expression level of 1 count per million in at least two samples were included in the analysis. Genes with a FDR of below 0.05 were considered differentially expressed between Fgssn3 mutant and PH-1. The RNA-Seq data have been deposited in the NCBI Sequence Read Archive database with accession code PRJNA289285.

The total RNA of three biological replicates for ΔAfRafA, ΔAfStuA, and WT grown in YES and YEP was sequenced. Libraries were prepared according to standard protocols from Illumina Inc. (San Diego, CA, United States) and sequenced on a HiSeq 2000 platform (Novogene, Beijing, China). Low-quality reads (Phred ≤ 20) and adaptor sequences were filtered out, and the Q20, Q30, and GC content of the clean data were calculated (Table 2). Sequenced clean reads were mapped against predicted transcripts of the A. flavus NRRL 3357 genome¹ using Tophat v2.0.4 (Trapnell et al., 2009 (link)), and only unique matches were allowed. Transcript abundance (i.e., FPKM) were estimated using the HTSeq package. Differentially expressed genes were analyzed with the DESeq package (Anders and Huber, 2010 (link)), and both a twofold change cut-off and an adjusted p-value of ≤0.05 were established as thresholds. An enrichment analysis of differential expression was performed using the GOSeq R package (Young et al., 2010 (link)). GO terms (including cellular component, molecular function, and biological process) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were classified as significantly enriched among differentially expressed genes only when their Benjamini adjusted p-values were ≤0.05. All the RNA-seq data had been stored in GEO database with an ID of GSE107025.

The purified DNA was sent to Novogene Co. Ltd. (Beijing, China) for Hi-seq 2000 Next Generation Sequencing (NGS). After sequencing, clean data were obtained, assembled by SOAP denovo (version 2.04) [46 (link),47 (link)], SPAdes (version 3.6.2) [48 (link)], and AbySS (version 2.0) [49 (link)], and finally integrated by CISA (version 1.3) [50 (link)]. The protein sequences of the predicted genes were compared to the GO, KEGG, and COG databases as well as the classic protein function database using Diamond (version 2.1.8) [51 ]. The amino acid sequences were aligned to VFDB (version 2022) and ARDB (version 1.1) [52 (link)] databases using Diamond. The amino acid sequences of target species were compared with the CARD database (version 3.2.2) using Resistance Gene Identifier (RGI) software provided by the CARD database, which obtained annotated results [53 (link)]. The raw data were uploaded to NCBI database with the accession number PRJNA970803.

A specificity profile of the modified crRNAs was created as previously described^{11 (link)}. Briefly, 200 nM of the pre-selection library was incubated with 1000 nM gRNA and 1000 nM Cas9 in NEB Buffer 3.1 for 1 h at 37 °C to create the post-selection library. In addition, 200 nM of the library was incubated with 2U of BspMI using the same reaction conditions as above, to create the final pre-selection library. Both digestion reactions were purified using a QiaQuick PCR Purification Kit (Qiagen) and ligated to 10 pmol of barcoded adaptor S50X-F/R (post-selection) or lib_adapter1 with ABO/HLA_lib_adapter2 (pre-selection) using 1000U of T4 DNA Ligase (NEB) for 16 hrs at room temperature. Ligation reactions were purified using the MinElute PCR Purification Kit (Qiagen) then amplified using primer PE2_short with barcoded primer HLA/ABO-N70X (post-selection) or primer lib_PCR_F with barcoded ABO/HLA_PCR_R (pre-selection) using Q5 Hot Start High-Fidelity Master Mix (NEB). Products were gel extracted and purified using MinElute Gel Extraction Kit (Qiagen) and quantified with a Qubit 2.0 Fluorometer. Finished libraries were run on a HiSeq 2000 (Novogene), demultiplexed, and analyzed as previously described^{45 (link)}. The sequences used for this protocol are listed in Supplementary Table 2.