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.

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. Overall design: 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).