E z n a fungal rna kit

Total RNA was extracted from ~200 mg of deep frozen tissue using the Fungal RNA E.Z.N.A. Kit (Omega Bio-Tek, Norcross, GA) and treated with 1 U of RQ1 DNase (Promega, Madison, WI) per μg of RNA. The RNA integrity was estimated using denaturing electrophoresis on 1% (w/v) agarose gels. The nucleic acid concentrations were measured with a Qubit 2.0 fluorometer (Life Technologies), and the purity of the total RNA was estimated using the 260/280 nm absorbance ratio on a NanoDrop™ 2000 (Thermo Scientific) machine. The total RNA (225 ng) was reverse-transcribed into cDNA in a 20-μl volume using the iScript cDNA Synthesis kit (Bio-Rad, Alcobendas, Spain).

Mycelia were harvested, frozen, and ground in a sterile mortar in the presence of liquid nitrogen. High molecular weight DNA was extracted using the phenol-chloroform protocol described previously [9 ]. DNA sample concentrations were measured using a Qubit® 2.0 Fluorometer (Life Technologies, Madrid, Spain), and DNA purity was measured using a NanoDrop™ 2000 (Thermo-Scientific, Wilmington, DE, USA). DNA quality was verified by electrophoresis in 0.7% agarose gels. Total RNA was extracted from 200 mg of deep-frozen tissue using Fungal RNA E.Z.N.A Kit (Omega Bio-Tek, Norcross, GA, USA), and its integrity was verified using the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA).

RNA was extracted from C. strossmayeri mycelium, harvested from a two-week culture grown in PDB, using the E.Z.N.A^® Fungal RNA Kit (OMEGA bio-tek). Isolated RNA was quality checked using RNA Analysis ScreenTape^® (Agilent). Approximately 500 ng of total RNA was prepared for sequencing using the Illumina TruSeq Total RNA LT Kit (Illumina). The data were processed using RTA version 1.18.64, with default filter and quality settings. The reads were demultiplexed with CASAVA 1.8.4, allowing no mismatches. This was carried out at the Bristol Genomics Facility. RNA-seq data were processed using Galaxy QC and manipulation tools to trim the sequences followed by the TopHat RNA analysis tool to map the RNA-seq reads to the assembled genomes. These data were then viewed in Artemis to evaluate relevant expression levels of genes of interest. Partek^® Genomics Suite was also used to map RNA-seq reads to assembled genomes and to genes of interest. RNA-seq data are available on the NCBI SRA database under the accessions: STUDY: PRJNA604530; SAMPLE: CBS 177.39 (SAMN13973684); EXPERIMENT: C.s (SRX7684385); RUN: AB_C_ACAGTG_L001_R1_001.fastq (SRR11032120).

The wild-type strain A60 and the ΔMohxt2 and ΔMohxt3 mutants were inoculated into liquid CM and cultured at 28 °C with shaking at 180 rpm for 50 h. Total RNA was extracted from harvested mycelia using the E.Z.N.A. Fungal RNA Kit (Omega Bio-Tek, Suite 450, Norcross, GA, USA) and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were constructed using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina NovaSeq 6000 platform with 150 bp paired-end (PE150) sequencing, generating at least 6 Gb of data for each sample. Quality control and filtering of raw data were performed using Trimmomatic (v0.39). Clean reads were aligned to the M. oryzae 70-15 reference genome (MG8, https://www.ncbi.nlm.nih.gov/assembly/GCF_000002495.2/ (accessed on 26 March 2023)) using HISAT2 (v2.1.0). Gene expression levels (FPKM values) were calculated using StringTie (v2.1.3). Differential expression analysis was performed using the DESeq2 (v1.28.1) R package, and genes with log2fold change ≥ 1 and adjusted p-value ≤ 0.05 were identified as differentially expressed genes. KEGG enrichment analysis was conducted using the clusterProfiler (v3.16.1) R package. The experiment was performed with three biological replicates.

All Fo isolates were cultured in PDB at 28 °C for 3 days and the mycelia were collected and finely ground to a powder using a mortar and pestle with liquid nitrogen. The total RNA was extracted using the E.Z.N.A. Fungal RNA Kit (Omega Bio-tek, Inc., Norcross, USA) following the manufacturer’s instructions and used for reverse transcription with the PrimeScript™ RT reagent Kit (TaKaRa).The sequences of FoMyo5 motor domains were amplified from the cDNAs of all Fo isolates using the primer pairs FoMyo5F/FoMyo5R in this study (FoMyo5F:5’-ATGGGAATATCAAGACGC-3’; FoMyo5R: 5’-TTTGATAAAGGCCTTGGT-3’), which were synthesized by Tsingke Biotechnology Co., Ltd. Then these amplicons were gel purified using the OMEGA BIO-TEK (Shanghai, China) gel purification kit, cloned into the PMD18-T vector and sequenced in Tsingke Biotechnology Co., Ltd. And we aligned these FoMyo5 motor domains using Bioedit 7.2 software (Isis Pharmaceuticals).

The conidia of A. flavus or A. parasiticus were harvested from 3-day-old PDA cultures and then spread on a cellophane film placed on PDA in a Petri dish (9 cm in diameter) with 200 μL conidial suspension (1 × 10⁷ conidia/mL). Another Petri dish was loaded with 10 g the 7-day-old AWG culture of strain 3-10 or 10 g fresh AWG (CK). Then, the two dishes were face-to-face sealed by parafilm to form a DDS. After co-culturing at 28°C for 72 h, the mycelia on the film were collected and immediately frozen in liquid nitrogen. Total RNA in the mycelial sample was extracted using E.Z.N.A^® Fungal RNA Kit (Omega Bio-tek, Inc., Norcross, GA, United States) according to the provided manual. Expression of eleven important genes (aflR, AccC, aflCa, aflA, aflS, aflO, aflD, aflF, aflP, aflQ, and aflX) in the aflatoxins biosynthesis pathway in A. flavus and A. parasiticus were determined by quantitative real-time PCR (qRT-PCR) using the method described by Gong et al. (2019) (link). The primers used for the qRT-PCR are listed in Supplementary Table S3.