Zr fungal bacterial rna miniprep

Transcription unit comprising pgn_1808 was examined by reverse transcription (RT)-PCR. Total RNA was isolated using ZR Fungal/Bacterial RNA MiniPrep (Zymo Research Corp., Irvine, CA, USA) and treated with DNase I (Zymo Research Corp.) to remove residual DNA strands. No genomic DNA contaminants were detected in the total RNA samples (data not shown). The pure RNA was used to generate cDNA with a PrimeScript RT-PCR Kit and random 6 mers (Takara Bio Inc., Otsu, Japan). Transcription unit was examined by a standard PCR for intergenic regions using the cDNA as a template and primers listed in S1 Table.

Amycolatopsis mediterranei strains were grown in liquid Bennet medium for 48 h at 30 °C, and then cells were inoculated into a fresh liquid Bennet medium with the final OD₆₀₀ diluted to 0.2. To culture vector+ and rifQ+ strains, erythromycin was added into the medium at a final concentration of 200 µg/mL to maintain the plasmids. Cells were harvested at different time points, representing the early- and middle-exponential growth phases, respectively. Total RNA was then extracted with the ZR Fungal/Bacterial RNA MiniPrep (Zymo Research), and further treated with RNase-free DNase I (TaKaRa) to remove trace genomic DNA. Random hexamers were employed for RT, and 3 µg total RNA was reverse-transcribed into cDNA in a 30-µL reaction system, using the Super-Script III reverse transcriptase (Thermo Fisher Scientific). PCR was performed using 20 ng cDNA as the template in a 20-µL reaction system, using either oneTaq DNA polymerase (NEB) or EasyTaq DNA polymerase (Transgen), and rpoB was employed as an internal control. The gene transcriptional level was monitored by agarose gel electrophoresis, followed by ethidium bromide staining. A negative control was performed following the same procedures except the addition of reverse transcriptase was omitted.

Three biological replicates of healthy developing asymptomatic endophyte-infected inflorescences and choke stromata were collected from the same strong creeping red fescue plant (plant 6035-5 A10-484) from Rutgers research farm in Adephia, NJ on 10 May 2016. The choke stromata samples included both the epiphytic fungal tissue and the internal arrested developing inflorescence tissue. Healthy developing inflorescences were collected before anthesis, and choke stromata were collected before the development of perithecia, when the fungal stroma turns yellow to orange color. Samples were immediately frozen on dry ice and then stored at −80 °C before RNA extraction. Total RNA was extracted from both tissue types using ZR Fungal/Bacterial RNA MiniPrep (Zymo Research, Orange, CA, USA) and treated with DNase to remove DNA, following the manufacturer’s recommendations.

Cells taken from each replicate incubation at 48 h were collected in pre-chilled Corning tubes and were centrifuged at 4 °C for 2 min; then, the cell pellets were stored at − 80 °C before analysis. Total DNA was extracted using the Yeast Genomic DNA Extraction kit (Solarbio, China) and total RNA was extracted using the ZR Fungal/Bacterial RNA MiniPrep™ (Zymo Research, CA). The samples were then sent to the Institute of Biochemistry and Cell Biology (Shanghai, China) for quality and quantity evaluation and sequencing. Samples for RNA-seq and DNA-seq investigation were both in biological triplicate.

Strains were grown in liquid LB medium with appropriate antibiotics at 37 °C overnight with shaking at 220 r.p.m., then 50 μl culture was inoculated into 5 ml fresh liquid LB medium and further incubated till the OD₆₀₀ reached 1.2. To derepress the transcription of lac operon, 0.5 mm IPTG was added and cells were cultured for further 1 h before cell harvesting. To shock with nitrogen-limited conditions, cells were first washed with liquid M9 medium, and then resuspended in liquid M9 medium and cultured for another 1 h. Total RNA was extracted using the ZR Fungal/Bacterial RNA MiniPrep (Zymo Research, Irvine, CA, USA), and further treated with RNase-free DNase I (TaKaRa) to prevent contamination of trace genomic DNA.