Isoplant

Seventeen children (aged 3–9 years) participated in this study (ethics committee approval no. EC20-28; Nihon University School of Dentistry at Matsudo, Chiba, Japan). The decayed, missing, and filled tooth indices of these patients ranged from 4 to 16. Plaque was collected from the tooth surface using a sterile excavator, suspended in 1 mL of 0.9% (w/v) NaCl solution, applied to mitis–salivarius agar, and incubated at 37 °C for 48 h. Rough colonies, characteristic of S. mutans, were isolated. Species in the genus were identified based on the presence or absence of a portion of the htrA gene encoding a surface protease and an intergenic region following polymerase chain reaction (PCR) amplification, as reported by Chen et al. [10 (link)]. Genomic DNA was extracted from the culture medium using ISOPLANT (Nippon Gene Co., Ltd., Tokyo, Japan) following the manufacturer’s protocols. The PCR mixture was prepared by mixing 6 µL PrimeSTAR Max Premix (2×) (Takara Bio Inc., Shiga, Japan), 10 ng genomic DNA, forward (5′-TCGCGAAAAAGATAAACAAACA-3′) and reverse (5′-GCCCCTTCACAGTTGGTTAG-3′) primers (final concentration of 0.2 µM each), and PCR-grade water. The PCR conditions were as follows: 30 cycles at 98 °C for 10 s, 55 °C for 10 s, and 72 °C for 6 s. The PCR products were subjected to MultiNA capillary electrophoresis (MCE-202; Shimadzu Corporation, Kyoto, Japan) to confirm amplification.

DNA was extracted from the isolated MV fraction using ISOPLANT (Nippon Gene) according to the manufacturer’s instructions. A Nextera XT DNA library preparation kit (Illumina) was used for paired-end libraries. DNA-seq was performed using an Illumina MiSeq^TM system.
Low-quality read removal and adapter trimming of the raw FASTQ file obtained were performed using fastp software (version 0.20.1) (Chen et al., 2018 (link)) with the “-q 20 -3” options. The cleaned FASTQ file was mapped to the P. denitrificans Pd1222 genome using Bowtie2 (version 2.4.4). Prior to mapping, a FASTA file of the genome was indexed using the “Bowtie-build” command in Bowtie2. The resulting BAM files were indexed using SAMtools (version 1.12) (Li et al., 2009 (link)). A GTF file of the P. denitrificans Pd1222 genome partitioned into 1,000-bp windows was created using R script, and the number of reads mapped to every 1,000 bp in the genome was counted using featureCounts software (version 2.0.1) (Liao et al., 2014 (link)) with the GTF file and “-p -O -M -C -f -t region -g ID -s 0” options. The number of reads mapped to the gene was also counted with the GTF file of P. denitrificans Pd1222 and the “-p -O -M -C -f -t CDS -g gene_id -s 0” options.

INT3 was excised from the main stem at 07:00–08:00 on each sampling day, immediately frozen in liquid nitrogen, and then stored at -80°C for the following processing. A tissue segment of 2–4 cm from the top of the third internode was removed and crushed into a fine powder with an SK-Mill (Tokken Inc., Chiba, Japan). Total RNA of the internode segment was extracted using RNAs-ici!-S (Rizo Inc., Tsukuba, Japan). The isolated RNA was treated with TURBO DNase (Life Technologies), then precipitated with ethachinmate (NIPPON GENE, Tokyo, Japan) and diluted in DEPC water. Genomic DNA was prepared from young seedlings of each cultivar using ISOPLANT (NIPPON GENE, Tokyo, Japan). The extracted DNA was incubated with RNase (final concentration of 10 μg ml^-1) at 37°C for 30 min, then precipitated with ethachinmate and resuspended in water.