“Benzoxazinoids stimulate CheZ gene expression, chemotaxis and acts as a signaling molecule in Azospirillum brasilense Ab-V5, showing adaptation to ectopic benzoxazinoids compared to Pseudomonas protegens Pf-5.”

Root colonization by plant growth-promoting bacteria (PGPB) involves recruiting symbiotic partners from a diverse biosphere. Among characterized PGPB, Azospirillum brazilense Ab-V5 and Psuedomonas protegens Pf-5 are two potent inoculants renowned for their growth enhancing capacity through production of phytohormones and nitrogen fixation, and by disease suppression respectively. Pf-5 was originally isolated from the cotton rhizosphere while Ab-V5 was isolated from the maize rhizosphere. Many cereals such as maize produce indole derived benzoxazinoids (BXs), specialized metabolites that have a profound effect on structuring the root associated microbiome. Considering the strong impact BXs have on bacterial behavior in the rhizosphere, we postulated that BXs may interfere with specific mechanisms related to plant root colonization in bacteria adapted to BX release in the soil. Therefore, we studied the influence of the relatively stable lactam BX derivative MBOA on the transcriptomes of Ab-V5 and Pf-5. From 72 hours old Ab-V5 and Pf-5 cultures amended with 0.00 mM, 0.05 mM and 0.50 mM MBOA, we performed RNA sequencing of total RNA extracts. In Ab-V5, we could reveal the upregulation of a chemotaxis regulatory gene in response to 0.05 mM MBOA while both 0.05 mM and 0.50 mM substantially impacted signal transduction, cellular respiration and energy metabolism. The absence of upregulated chemotaxis genes at 0.50 mM and large alterations in expression profiles of primary metabolism related genes, suggested surplus energy was directed towards metabolic adaptation. Interestingly, symbiosis-related gene downregulation occurred in both treatments, leading to reduced biofilm formation, impaired auxin efflux carriers, and varied nitrogen homeostasis. In contrast, Pf-5 showed very little alterations of gene expression profiles in 0.50 mM MBOA, while no significant differentially expressed genes were found in 0.05 mM MBOA. Biofilm formation and chemotaxis were validated by in vitro assays, which additionally uncovered a time dependent-effect of MBOA on Ab-V5 biofilm, showing how biofilm formation is slowed down. In line with the results from in vitro biofilm assays, we inferred from microscopic analysis that biofilm on Ab-V5 infected Arabidopsis roots was augmented in MBOA treatment and consider the interference of host factors. The amount of adhering Ab-V5 cells on Arabidopsis roots was not altered, nor was peroxidase activity influenced by MBOA. This means that nor is absorption during initial contact of bacteria to the root, neither firm attachment by biofilm promoted by MBOA. However, irrespective of MBOA treatment, peroxidase activity was augmented which may be indicative of induction of systemic resistance. In conclusion, we found very little effect of MBOA treatment on Pf-5 gene expression, a bacteria not native to BX-producing plants, but found that MBOA acts as a signaling molecule evoking large scale adaptation of Ab-V5 to the MBOA regime via modulating signal transduction, gene regulation, primary metabolism, transport mechanisms and energy homeostasis. Finally, through chemotaxis of Ab-V5, MBOA promotes acquisition of Ab-V5 in the rhizosphere and all considered, may cause the metabolic transition required for root colonization.