Determination of factors involved in establishment of Azoarcus sp. strain BH72-rice interaction

Abstract

Endophytic bacteria are known to have many beneficial effects on plants and hence plant endophytic interactions are extensively investigated. To understand and further manipulate endophytic interactions for enhancement of plant growth and productivity, it is necessary to determine factors which are responsible for establishment of such an interaction. Azoarcus sp. strain BH72, our model organism, colonizes roots of rice in high numbers and is also capable of endophytic nitrogen fixation inside the roots. Very few factors involved in establishment of Azoarcus-rice interactions have been investigated till date. In this work, attempts were made to further identify factors which might be responsible for establishment of this interaction. The recently discovered type VI secretion system (T6SS) found in many plant associated bacteria is known to be involved in host interaction in many of these organisms. Two gene clusters (sci and imp) encoding the putative T6SS have also been detected in Azoarcus sp. strain BH72 and are speculated to play an important role in host interaction. Recent, RT-PCR based studies revealed expression of the imp cluster was nitrogen regulated. Experiments were performed to determine transcriptional regulators involved in expression of the imp cluster. This would lead to a better understanding of the molecular basis of plant-endophyte interaction. Compounds secreted by the plant as root exudates are also known to influence behavior of the interacting microbe by alteration of specific gene expression patterns in the microbe. To investigate if Azoarcus sp. strain BH72 is also influenced by root exudates of rice variety Nipponbare, oligonucleotide transcriptome microarrays were applied. The analysis revealed that expression of 4.4% of Azoarcus transcriptome was significantly affected in response to exudates. For few of the differentially expressed genes, functional analysis was performed to determine the rhizosphere competence and colonization capacity of the mutant, where the respective gene was inactivated, in comparison to wild type BH72. The candidate genes chosen for mutagenesis and functional analysis included previously uncharacterized genes like azo2916, encoding for a minor pilin PilX, azo2408 and azo1544, encoding proteins with GGDEF or GGDEF and EAL domains that are responsible for the turnover of a global secondary messenger cyclic-di-GMP and azo3888, encoding a serine threonine kinase responsible for assembly and activation of T6SS. The analysis revealed that these genes were relevant for rhizosphere competence and host colonization, thus suggesting that exudates prepare the organism for initiating an interaction with the host. To further explore the determinants required by Azoarcus sp. strain BH72 to colonize, adapt and proliferate in the host, transcriptomic analysis was performed using the Illumina Sequencing technology with RNA isolated from roots of Oryza sativa cv. Nipponbare infected with Azoarcus sp. strain BH72. Further studies based on the differentially regulated genes obtained from this analysis would certainly broaden our knowledge about the factors involved in plant-endophyte interactions. To summarize, the study provides valuable insight about many previously unknown factors responsible for successful Azoarcus-rice interaction and also opens new avenues to further expand our understanding of this field.