Project description:Azoarcus olearius strain BH72 is a plant endophyte that can fix atmospheric nitrogen for plant assimilation. Our recent directional RNA-Seq analysis detected widespread presence of reads mapping in the antisense orientation to the annotated genes. Although this has been discovered for several bacteria, the stable expression and functional relevance of such antisense RNAs (asRNAs) are still being debated. In this study we ventured to confirm the single-cell expression and unravel the functionality of antisense transcription in strain BH72. First, from the directional RNA-Seq data we predicted 798 asRNAs that were encoded against 709 genes (18 % of the annotated genome). Using a cassette encoding for two fluorescent reporters (GFP and TdTomato), in convergent orientation with transcriptional terminators in between, we were able to confirm the single-cell co-expression of both the sense and the antisense transcripts within azo1349 in at least 90 % of the bacterial population. By modifying the above experimental setup, we observed the interference exhibited by these overlapping transcripts. Further, decreasing the ribosomal occupancy on one of the overlapping transcripts increased its repression of the complimentary transcript. However, RNA-Seq analysis of a RNase III deficient mutant indicated a minimal effect of the endoribonuclease on the regulation of sense-antisense pairs in strain BH72. Lastly, we elucidated the control mediated by RNase III and asRNA on the expression of genes in the type six secretion system-1 cluster.
Project description:Transcriptional profiling of Oryza sativa japonica Nipponbare roots after 14 days post inoculation with Azoarcus olearius BH72, the goal is to understand the transcriptomic response of rice roots to colonization by bacterial endophyte
Project description:Arabidopsis thaliana associated with Plant Growth Promoting Bacteria (PGPB) Azoarcus olearius (DQS4) innoculated with WS (Wassilewskija) and Col0 (Columbia) ecotypes.
The work (proposal:https://doi.org/10.46936/10.25585/60000448) conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231.
Project description: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.
Project description:Azoarcus olearius BH72 is a diazotrophic endophyte carrying out biological nitrogen fixation (BNF) and supplying nitrogen to its host plant. Our previous microarray approach provided insights in the transcriptome of strain BH72 under N2-fixation in comparison to ammonium-grown conditions, which already indicated induction of genes not related to the BNF process. Due to the known limitations of the technique, we might have missed additional differentially regulated genes (DEGs). Thus we used directional RNA-Seq to better comprehend the transcriptional landscape under these growth conditions. RNA-Seq detected almost 24 % of the annotated genes to be regulated, twice the amount identified by microarray. In addition to confirming entire regulated operons containing known DEGs, the new approach detected induction of genes involved in carbon metabolism and flagellar and twitching motility. On the other hand, genes encoding for proteins involved in translation and vitamin biosynthesis were detected to be suppressed. Nonetheless, strain BH72 appears not to be content with N2-fixation but is primed for alternative economic N-sources, such as nitrate, urea or amino acids: we detected strong induction of machineries for uptake and assimilation.
Project description:Transcriptome profile of wild type Azoarcus sp. BH72 grown under microaerobic condition (0.3% oxygen) compared with that grown under aerobic condition (21% oxygen), both in presence of combined nitrogen source, respectively
Project description:Endophytic colonization is a very complex process which is not yet completely understood. Molecules exuded by the plants may act as signals which influence the ability of the microbe to colonize the host or survive in the rhizosphere. Here we investigated whether root exudates of the host might play a role in initiating the endophyte-rice interaction. The whole genome microarray approach was used to investigate the response of the diazotrophic model endophyte, Azoarcus sp. strain BH72, to exudates of O. sativa cv. Nipponbare in order to identify differentially regulated genes. Azoarcus sp. strain BH72 was grown in the presence or absence of root exudates of Oryza sativa cv. Nipponbare for two different time points, and differences in the gene expression profile were monitored.