Investigating MarR Regulators of the Indole-3-Acetic Acid Degradation Locus in Plant Growth-Promoting Rhizobacteria

Abstract

Amidst a growing global population and the drive to utilize biofuels in lieu of fossil fuels, crop demands continue to increase. Therefore, the ability to bioengineer plant-microbe interactions to optimize crop yields is vital to be able to meet increasing demands for crop production. This research builds on previous research which identifies the indole-3-acetic acid degradation (iad) locus in Variovorax as a crucial component in the regulation of plant hormones in the rhizosphere microbiome. MarR-family repressors, specifically MarR_73 and MarR_50, are positioned both upstream and downstream of this operon, respectively. These repressors play an important role in regulating the genes that control the degradation of IAA. For successful growth and fitness, plants require optimized levels of hormones and nutrients. Bacteria located in the rhizosphere produce excess auxin which has negative consequences on plant growth. Other bacteria, which contain the iad locus, control auxin levels through degradation and catabolism of indole-3-acetic acid (IAA), a plant hormone that impacts root growth. Hence, an understanding of these plant-microbe interactions at the genetic level can help optimize plant growth. Here, we use a fluorescent eGFP reporter to test the expression of the iad locus in various Variovorax mutant strains. First, we examine the expression of a strain with a △MarR _73 deletion, a △MarR _50 deletion, and a △MarR _73 + △MarR _50 double deletion both in the presence and absence of IAA. Then, we investigate the effects of mutations of several amino acid binding sites in the MarR_73 regulator on IAA degradation as well as eGFP expression in the presence of various auxin compounds, including IAA. Finally, we create cloning vectors with codon optimized genes of the MarR_73 regulator as well as of the MarR_50. Ultimately, these results demonstrate that MarR_73 is likely the primary repressor of iad whereas MarR_50 acts as a secondary repressor. Additionally, binding sites Ser-28 and His-32 are identified to be very important for the interactions of MarR_73 with IAA.