SB001 bacterium-Arabidopsis interaction
The Arabidopsis plants were treated with aliquots of 30 mL (4.77  109 cfu/mL) of endophytic bacterium. The bacterium was inoculated in the sterilized substrate around the plants (five-day-old). The colonization of bacterium in roots was verified using the same protocol used in the isolation of this strain from wild plant species on LB plates in different time-points. The endophytic bacterium was well-stablished 72 hrs post-inoculation. Leaves, stems and roots from five plants were collected 72 hrs post-inoculation. Plants treated with water were used as control. Treatments and controls were repeated three times per group. The total RNA was extracted using the Qiagen RNeasy Midi Kit (Hilden, Germany), and the concentration of the total RNA was determined using spectrometry. The treatment and control samples were repeated three times per group. After extracting the total RNA, eukaryotic mRNA was enriched using oligo (dT) beads. The samples were sequenced using Illumina HiSeq 2000 (Personalbio Co. (Shanghai, Peoples Republic of China). High-quality reads were processed using the Perl script, and the differentially expressed genes were identified using the edgeR package (www.r-project.org/). Genes with a fold change  2 were considered significant differentially expressed genes. Gene ontology (GO) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to characterise the differentially expressed genes. GO functional annotations were obtained from the non-redundant annotation results. In addition, the GO annotations were analysed using the Blast2GO software (www.blast2go.com) (Conesa et al. 2005). The RNA sequencing was done following these steps: 1-Total RNA extraction. 2-Total RNA quality test. 3-rRNA removal. 4-RNA fragmentation between 200-300 bp by ion disruption. 5-PCR: the first chain of cDNA was synthesized by random primers and reverse transcriptase; when the second chain of cDNA was synthesized, the library of chain specific components was constructed, and dTTP was replaced by dUTP, which greatly improved the accuracy of the results. 6-PCR enrichment of library fragments: the size of the library ranged from 300 to 400 bp. 7-Library quality inspection: the size of the library was detected by Agilent 2100 Bioanalyzer, and the total concentration of the library was detected by fluorescence quantitative analysis. 8-Illumina sequencing: the best amount of sample was selected for PCR amplification. Annealing of sequencing at the same time of synthesis. The prokaryote genetic analysis process firstly filtered the raw off-machine data to obtain a high-quality sequence, comparing the filtered sequence to the species reference genome. Based on the results of the comparison, the amount of expression of each gene was calculated. On this basis, the expression difference analysis, enrichment analysis and cluster analysis of the sample were further carried out. The transcription structure of the sample was analyzed and the manipulative substructure of the sample gene obtained. UTR, SNP and InDel were obtained. HTSeq 0.6.1p2 (http://www-huber.embl.de/users/anders/HTSeq) was used to compare the read count values from each gene as the original expression of the gene. To make gene expression levels comparable between different genes and samples, we normalized the sequencing depth and gene length using fragments per kilobase of transcript per million (FPKM). FPKM calculates the number of fragments that two reads can compare to the same transcript. In the reference transcription group, we generally taking account the FPKM >1 when the gene was expressed. Pearson correlation coefficients were used to indicate the level of expression correlation between genes in the sample to test the reliability of experiments and whether sample selection was reasonable. The level correlation of gene expression between samples was an important index to test the reliability of experiments and whether sample selection was reasonable. The log2 Fold-Change was calculated by dividing the FPKM values from the bacterium during the interaction with Arabidopsis (SB001) and the bacterium without the interaction with Arabidopsis (B001).