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.
Project description:Azoarcus sp. BH72 is known to express nitrogenase genes endophytically in rice seedlings in gnotobiotic culture. Availability of fixed nitrogen is one of the important signals regulating the transcription of nitrogenase genes and hence nitrogen fixing activity. NifA is the essential transcription activator of nif genes. RNA isolated from the nifA knockout mutant of strain BH72 was compared with the transcriptome of wild type under nitrogen fixing condition using a global genome wide microarray approach and the differences in the gene expression profile were monitered.
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.
