Introduction
The rhizosphere, the narrow zone surrounding and influenced by plant roots, harbors a plethora of microorganisms that can have deleterious or beneficial effects on plant growth and health (Raaijmakers et al.2009; Mendes et al. 2011). Amongst the best-studied beneficial rhizosphere microbes are the Plant Growth-Promoting Rhizobacteria (PGPR). Their beneficial association is thought to be ancient and presumably has been shaped by co-evolutionary processes in the long-term interactions with their host plants (Lambers et al. 2009). PGPR can enhance plant growth through direct and indirect mechanisms (Lugtenberg & Kamilova 2009). The direct mechanisms involve facilitation of nutrient acquisition and modulation of phytohormones, whereas indirect effects involve suppression of biotic stress factors through parasitism, antibiosis, competition and the induction of systemic resistance (van Loon et al. 1998; De Vleesschauwer & Höfte 2009; Van der Ent et al. 2009; Pineda et al. 2010; Stringlis et al. 2018b).
PGPR can also have profound effects on the physiology and metabolism of their host plants, not only by enhancing the production of known secondary metabolites but also by inducing the biosynthesis of yet-unknown compounds (van de Mortel et al. 2012; Etalo et al. 2018). The plant secondary metabolites affected by PGPR reported to date include, among others, polyphenols, flavonoids and glucosinolates but also primary metabolites such as carbohydrates and amino acids (Etalo et al. 2018). So far, only few studies have provided mechanistic insights into the association between rhizobacteria-induced biochemical changes and plant growth and defense (Walker et al.2011; Weston et al. 2012; van de Mortel et al. 2012; Etaloet al. 2018; Stringlis et al. 2018a; Hu et al.2018).
PGPR can prime plant defense against pathogens and insect herbivores and at the same time promote plant growth. This is in contrast to the widely accepted trade-off between plant defense and plant fitness in general and plant growth in particular. Understanding the underlying plant metabolic networks and unraveling their interactions is essential to understand and optimize rhizobacteria-mediated growth promotion and ISR. Although some reports indicated that rhizobacteria change specific classes of plant metabolites (Mishra et al. 2006; Walker et al. 2011; Chamam et al. 2013), their overarching effects on the global metabolome of plants and in particular on core metabolite pathways co-occurring with growth promotion across plant species are not well understood. In this context, PGPR-mediated re-routing of the plant’s metabolism could give insight into the metabolic interplay between plant defense and growth. Furthermore, it is highly instrumental to understand the organization of metabolite routes and how rhizobacteria tilt a particular pathway towards a desired plant phenotype or the enhanced production of high value plant compounds (HVPC) (Etalo et al. 2018).
Here, we studied the impact of three strains of different rhizobacterial genera on the phenotype and shoot metabolome of three plant species:Arabidopsis thaliana (model plant), Brassica olearaceavar. italica (crop) and Artemisia annua (medicinal plant). The rhizobacterial strains Paraburkholderia graminis PHS1 (Pbg ) (Carrión et al. 2018) and Microbacterium EC8 (MB) (Cordovez et al. 2018) were originally isolated from the rhizosphere of sugar beet (Beta vulgaris ). Pseudomonas fluorescens SS101 (Pf SS101) was isolated from the wheat rhizosphere (De Souza et al. 2003). These rhizobacteria showed growth promoting and pathogen-protective effects in different plant species (Tran et al. 2007; van de Mortel et al. 2012; Cheng et al. 2017; Carrión et al. 2018; Cordovez et al. 2018; Jeon et al. 2021). Here, our primary goal was to identify core metabolic pathways that are targeted by rhizobacteria in different plant species and to investigate the metabolomic changes induced in rhizobacteria-plant interactions that lead to either plant growth promotion (effective partnership) or growth reduction (ineffective partnership). Integration of in vitro bioassays, untargeted metabolomics and screening of pathways-specific mutants showed that inoculation of the plant root system with each of the three rhizobacterial genera induced substantial changes in the shoot metabolome in a species-specific manner. The results pointed to the phenylpropanoid pathway as a central plant metabolome response associated with effective or ineffective partnerships. Subsequent analysis of twelve Arabidopsis transparent testa (tt ) mutants defective in different branches of the flavonoid pathway, revealed that the proanthocyanidin branch is key in rhizobacteria-mediated plant growth promotion.