PRJNA730797: The RNA-seq analysis of rice roots colonized by the endophytic bacterium Azoarcus olearius BH72 after one-, three- and seven-days post inoculation and after three days of boosted colonization
概要: Purpose: Transcriptional profiling of Oryza sativa japonica Nipponbare roots after one, three and seven days post inoculation with Azoarcus olearius BH72 (vs. non-inoculated controls) to understand the changes in transcriptomic response of rice roots to colonization by bacterial endophyte at initial stages of interaction; Additional set-up was included in which bacterial growth was boosted (through increasing 20-times carbon source - malic acid in the plant's hydroponic medium) to study rice roots transcriptome during enhanced colonization by the endophyte after three days post inoculation. Methods: Rice root mRNA profiles after one day, three days (including additional set-ups for boosted colonization), and seven days post inoculation with Azoarcus olearius BH72 and corresponding non-inoculated controls were generated by RNA sequencing, in triplicates, using Illumina NextSeq 500. Raw reads were then filtered, trimmed (PHRED > 33) and mapped onto IRGSP-1 version of Oryza sativa ssp. japonica cv. Nipponbare genome using CLC Genomics Workbench 8.5.1 (Qiagen, Germany). Expression of 17 selected genes was confirmed via RT-qPCR. Results: Using the RNA-Seq technology we obtained transcriptomic data from 24 sequencing libraries, with an average 46,181,160 clean reads per library, of which 87% or more were mapped onto the Oryza sativa ssp. japonica cv. Nipponbare IRGSP-1.0 genome (Fig. S3). We considered genes as differentially regulated (DEG) that exhibited at least 1.5-fold-change in expression level between Azo-colonized and non-colonized roots and FDR<0.05. Conclusions: Bacteria appeared to short-circuit the initial root defense responses for a compatible interaction during endophytic establishment, involving previously unknown putative rice candidate genes.
项目整体设计: Experiments included generating Oryza sativa japonica Nipponbare roots mRNA profiles at three time points (one, three and seven days post inoculation with Azoarcus olearius BH72), which were either subjected or not subjected to treatment (inoculated with endophytic bacterium Azoarcus olearius BH72 or non-inoculated), in three independent biological replicates (18 samples in total). Additional root mRNA profiles were obtained from plants subjected to boosted colonization by the endophyte or non-inoculated plants at one time point (three days post inoculation) also in three independent biological replicates (6 samples in total).
Surface sterilization of dehusked rice caryopsis was carried out as described by Hurek et al., 1994. For germination, seeds were then transferred into GA7 Magenta boxes (Sigma-Aldrich, USA) filled with 100 ml of solid plant medium (Egener et al., 1999), supplemented with 8 g per l Bacto Agar, 20 mg of neutralized DL-malic acid per l and a potassium phosphate buffer (0,439 g KH2PO4, 0,561 g K2HPO4 at pH 6.8), and incubated at 300C in the dark for three days, and in light in the phytotron (at 30°C, at 80% humidity and with a 14-h-day and 10-h-night cycle at approximately 100 μmol photons m–2 s–1) for one day. Then, one day before inoculation with bacteria, seedlings were transferred into hydroponic cultures: they were placed on top of metal mash adaptors in 200 x 30,00 x 1,1-1,2 mm glass tubes containing 50 ml of liquid plant medium (as described above but without addition of Bacto agar) and transferred back to the phytotron for an additional 24 h incubation. Certain modifications in the composition of the liquid plant medium were made according to the experimental set-up: for the boosted-colonization, liquid plant medium was supplemented with 400 mg of neutralized DL-malic acid per l instead of 20 mg, and for ammonia-containing controls 68 mg per l (NH4)2SO4 was added.
处理方案:
Parallel to seed preparation, bacterial cells of Azoarcus olearius BH72 or mutant strains were grown until the late exponential growth phase (optical density at 578 nm [OD578nm] = 1.0 to 1.2), washed in an N-free medium (Egener et al., 1999), and directly added to hydroponic cultures to yield an optical density of 0.067. Inoculated plants were then incubated in the phytotron for one, three or seven days depending on the experimental set-up.
提取方案:
In order to extract total RNA, six rice roots were pooled together and the protocol of RNeasy Plant Mini Kit (Qiagen, Germany) was followed. Afterward, RNA quality and purity were evaluated via NanoDrop 2000 (Thermo Fisher Scientific, USA) and Agilent Bioanalyzer 2100 system (Agilent Technologies, USA), and only RNA samples with an RNA integrity number >8 were chosen for further analysis.
建库方案:
mRNA fraction was purified from 1 μg of the total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, USA) and libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s recommendations. Quality of prepared libraries was assessed using Agilent Bioanalyzer 2100 system and quantity via NEBNext Library Quant Kit for Illumina (NEB, USA).
