Trizon reagent

The WT strain was cultured on PDA for 10 days at 25°C in the dark and spread with 100-μl aliquots of a suspension of 10⁷ conidia/ml. Total RNAs were extracted from samples that had been separately collected at time points of 36 h (hyphal growth), 72 h (conidiophore development), and 240 h (conidium stage) after inoculation using TRIzon reagent (Cwbio, Hefei, China). Then, RNA was reverse transcribed into cDNA using a ReverTra Ace qPCR RT master mix with genomic DNA (gDNA) remover kit (Toyobo, Japan). Three of the cDNA samples were used to assess the transcript levels of Mr-abaA via qRT-PCR with the CFX96 RT-PCR system (Bio-Rad, USA). The fungal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as a standard gene. The 2^−ΔΔCT method was used to calculate the relative gene transcript levels (38 (link)).
To construct the AbaA::EGFP fusion protein, the full sequence of Mr-abaA with an upstream ∼1,000-bp fragment was amplified and cloned with the full sequence of EGFP into the pDHt-SK-bar vector, with which WT cells were transformed. Each transgenic strain was cultured on PDA for initial and full conidiation at 25°C in the dark. Mature conidia and hyphal cells were stained with the nucleus-specific dye DAPI and were then observed for subcellular localization under LSCM.

Total RNA was isolated from the head of adult flies using TRIzon reagent (CW bio, Beijing, China, Catalog No. CW0580). For each extraction, a total of 30 adult heads were dissected in 500 μL of TRIzon reagent. Duplicate or triplicate samples were used for each genotype. After RNA isolation, cDNA was synthesized from 1 μg of total RNA using the cDNA prepared using HiScript III RT SuperMix (Vazyme, Nanjing, China, Catalog No. R323). qPCR was performed using Eastep qPCR Master Mix Kit (Promega, Beijing, China, Catalog No. LS2062) on a LightCycler 96 (Roche, Rotkreuz, Switzerland). All protocols were performed according to the manufacturer’s instructions. The following primers were used (actin5C was used as an internal control): mtt-f: 5′-GCAATCCCTGGTTTGTGGAAT-3′, mtt-r: 5′-GAAAGTCGCTCCTTTGTGGTG-3′, mGluR-f: 5′-AACGGAACAATTTTAGTCGTCGT-3′, mGluR-f: 5′-GCAGAGAAACAGACACTGAATCC-3′, actin5C-f:5′-CAGAGCAAGCGTGGTATCCT-3′, actin5C-r: 5′-CTCATTGTAGAAGGTGTGGTGC-3′.

Total RNA from collected yeast samples was isolated using TRIzon Reagent (Cwbiotech, China) following the manufacturer’s instruction. RNA concentration and integrity were measured using NanoDrop 2000 (Thermo Fisher Scientific Inc., Waltham, United States) and Agilent 2100 Bioanalyzer system (Agilent Technologies Inc., Santa Clara, United States), respectively. RNA-sequencing (RNA-seq) libraries were prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs^® Inc.) according to the manufacturer’s guide, and the libraries were sequenced on an Illumina HiSeq X 10 platform (Illumina Inc., San Diego, CA). The generated reads were trimmed to eliminate adaptors and enhance quality. The filtrated clean reads were mapped to K. apiculata 34-9 genome using Hisat2 (version 2.1.0) and aligned to the generated transcriptome assembly using Bowtie2 (version 2.2.2), respectively. The output results were estimated using RSEM (version 1.3.0) to obtain normalized counts of gene expression level. The values of expression genes were calculated as fragments per kilobase per million mapped reads (FPKM).

HPLCs were pretreated 1 h with Rb3 (0, 25, 50 and 100 μM) and stimulated with 1 μg/mL P. gingivalis LPS for 24 h or 4 d. Culture media were collected and detected with ELISA kit (NeoBioscience, Shenzhen, China) following manufacturer’s instructions. Cells were collected with TRIZON reagent (CWBio, Beijing, China) at the same time and stored at −80 °C for the subsequent mRNA examination.

Using TRIzon Reagent, total RNA was isolated from cells and EVs (CWBio, China). SYBR Green Pro Taq HS Kit was used to evaluate the expression of microRNA and mRNA (Accurate Biology, Jinan, China). The reverse transcription of RNA to cDNA was followed by Evo M-MLV RT Premix for qPCR (Accurate Biology, Jinan, China). The GAPDH gene was utilized as a reference. As internal control and external reference for miRNA expression in cells and EVs, respectively, U6 and cel-miR-39-3p (GenePharma, Shanghai, China) were selected. Using the 2Ct technique, the relative expression of miR-2467-5p and other genes was determined. The sequences of mRNA primers are provided in Additional file 17: Table S5.