Plant growth-promoting rhizobacteria (PGPR) can protect plants against pathogenic microbes through a diversity of mechanisms including competition for nutrients, production of antibiotics, and stimulation of the host immune system, a phenomenon called induced systemic resistance (ISR). In the past 30 years, the Pseudomonas spp. PGPR strains WCS358, WCS374 and WCS417 of the Willie Commelin Scholten (WCS) collection have been studied in detail in pioneering papers on the molecular basis of PGPR-mediated ISR and mechanisms of biological control of soil-borne pathogens via siderophore-mediated competition for iron. The genomes of the model WCS PGPR strains were sequenced and analyzed to unearth genetic cues related to biological questions that surfaced during the past 30 years of functional studies on these plant-beneficial microbes. Whole genome comparisons revealed important novel insights into iron acquisition strategies with consequences for both bacterial ecology and plant protection, specifics of bacterial determinants involved in plant-PGPR recognition, and diversity of protein secretion systems involved in microbe-microbe and microbe-plant communication. Furthermore, multi-locus sequence alignment and whole genome comparison revealed the taxonomic position of the WCS model strains within the Pseudomonas genus. Despite the enormous diversity of Pseudomonas spp. in soils, several plant-associated Pseudomonas spp. strains that have been isolated from different hosts at different geographic regions appear to be nearly isogenic to WCS358, WCS374, or WCS417. Interestingly, all these WCS look-a-likes have been selected because of their plant protective or plant growth-promoting properties. The genome sequences of the model WCS strains revealed that they can be considered representatives of universally-present plant-beneficial Pseudomonas spp. With their well-characterized functions in the promotion of plant growth and health, the fully sequenced genomes of the WCS strains provide a genetic framework that allows for detailed analysis of the biological mechanisms of the plant-beneficial traits of these PGPR. Considering the increasing focus on the role of the root microbiome in plant health, functional genomics of the WCS strains will enhance our understanding of the diversity of functions of the root microbiome.
Plant roots interact with an enormous diversity of commensal, mutualistic, and pathogenic microbes, which poses a big challenge to roots to distinguish beneficial microbes from harmful ones. Plants can effectively ward off pathogens following immune recognition of conserved microbe-associated molecular patterns (MAMPs). However, such immune elicitors are essentially not different from those of neutral and beneficial microbes that are abundantly present in the root microbiome. Recent studies indicate that the plant immune system plays an active role in influencing rhizosphere microbiome composition. Moreover, it has become increasingly clear that root-invading beneficial microbes, including rhizobia and arbuscular mycorrhiza, evade or suppress host immunity to establish a mutualistic relationship with their host. Evidence is accumulating that many free-living rhizosphere microbiota members can suppress root immune responses, highlighting root immune suppression as an important function of the root microbiome. Thus, the gate keeping functions of the plant immune system are not restricted to warding off root-invading pathogens but also extend to rhizosphere microbiota, likely to promote colonization by beneficial microbes and prevent growth-defense tradeoffs triggered by the MAMP-rich rhizosphere environment.
Abstract Beneficial plant root-associated microorganisms carry out a range of functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming several challenges, including competition with neighboring microorganisms and host immunity. Forward and reverse genetics has led to the identification of mechanisms that are used by beneficial microorganisms to overcome these challenges such as the production of iron-chelating compounds, the formation of strong biofilms, or the concealment of characteristic microbial molecular patterns that trigger the host immune system. However, how such mechanisms arose from an evolutionary perspective is much less understood. To study bacterial adaptation in the rhizosphere, we employed experimental evolution to track the physiological and genetic dynamics of root-dwelling Pseudomonas protegens in the Arabidopsis thaliana rhizosphere under axenic conditions. This simplified binary one plant-one bacterium system allows for the amplification of key adaptive mechanisms for bacterial rhizosphere colonization. We identified 35 mutations, including single-nucleotide polymorphisms, insertions, and deletions, distributed over 28 genes. We found that mutations in genes encoding global regulators, and in genes for siderophore production, cell surface decoration, attachment, and motility accumulated in parallel, underlining that bacterial adaptation to the rhizosphere follows multiple strategies. Notably, we observed that motility increased in parallel across multiple independent evolutionary lines. Altogether these results underscore the strength of experimental evolution to identify key genes, pathways, and processes for bacterial rhizosphere colonization, and a methodology for the development of elite beneficial microorganisms with enhanced root-colonizing capacities that can support sustainable agriculture in the future. Importance Beneficial root-associated microorganisms carry out many functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming many challenges. Previously, diverse mechanisms that are used by beneficial microorganisms to overcome these challenges were identified. However, how such mechanisms have developed from an evolutionary perspective is much less understood. Here, we employed experimental evolution to track the evolutionary dynamics of a root-dwelling pseudomonad on the root of Arabidopsis. We find that mutations in global regulators, as well as in genes for siderophore production, cell surface decoration, attachment, and motility accumulate in parallel, underlining these strategies for bacterial adaptation to the rhizosphere. We identified 35 mutations distributed over 28 genes. Altogether our results demonstrate the power of experimental evolution to identify key pathways for rhizosphere colonization and a methodology for the development of elite beneficial microorganisms that can support sustainable agriculture.
Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.
Two fractions of agglutination activity towards fluorescent pseudomonads were detected in root washes of potato, tomato, wheat, and bean. High-molecular-mass (>10 6 Da) components in crude root washes agglutinated only particular saprophytic, fluorescent Pseudomonas isolates. Ion-exchange treatment of the crude root washes resulted in preparations of lower-molecular-mass (10 5 to 10 6 Da) fractions which agglutinated almost all Pseudomonas isolates examined. Also, components able to suppress agglutination reactions of pseudomonads with the lower-molecular-mass root components were detected in crude root washes of all crops studied. Pseudomonas isolates were differentially agglutinated by both types of root components. The involvement of these two types of root components in short-term adherence and in colonization was studied in potato, tomato, and grass, using Pseudomonas isolates from these crops. Short-term adherence of isolates to roots was independent of their agglutination with either type of root components. With agglutination-negative mutants, the high-molecular-mass components seemed to be involved in adherence of Pseudomonas putida Corvallis to roots of all crops studied. Short-term adherence to roots of four Pseudomonas isolates could be influenced by addition of both crude and ion-exchange-treated root washes, depending on their agglutination phenotype with these root wash preparations. Potato root colonization by 10 different isolates from this crop, over a period of 7 days, was not correlated with their agglutination phenotype. Agg - mutants of P. putida Corvallis were not impaired in root colonization. It is concluded that the root agglutinins studied can be involved in short-term adherence of pseudomonads to roots but do not play a decisive role in their root colonization.
ABSTRACT Mutants of Pseudomonas fluorescens WCS374 defective in biosynthesis of the fluorescent siderophore pseudobactin still display siderophore activity, indicating the production of a second siderophore. A recombinant cosmid clone (pMB374-07) of a WCS374 gene library harboring loci necessary for the biosynthesis of salicylic acid (SA) and this second siderophore pseudomonine was isolated. The salicylate biosynthesis region of WCS374 was localized in a 5-kb Eco RI fragment of pMB374-07. The SA and pseudomonine biosynthesis region was identified by transfer of cosmid pMB374-07 to a pseudobactin-deficient strain of P. putida . Sequence analysis of the 5-kb subclone revealed the presence of four open reading frames (ORFs). Products of two ORFs ( pmsC and pmsB ) showed homologies with chorismate-utilizing enzymes; a third ORF ( pmsE ) encoded a protein with strong similarity with enzymes involved in the biosynthesis of siderophores in other bacterial species. The region also contained a putative histidine decarboxylase gene ( pmsA ). A putative promoter region and two predicted iron boxes were localized upstream of pmsC . We determined by reverse transcriptase-mediated PCR that the pmsCEAB genes are cotranscribed and that expression is iron regulated. In vivo expression of SA genes was achieved in P. putida and Escherichia coli cells. In E. coli , deletions affecting the first ORF ( pmsC ) diminished SA production, whereas deletion of pmsB abolished it completely. The pmsB gene induced low levels of SA production in E. coli when expressed under control of the lacZ promoter. Several lines of evidence indicate that SA and pseudomonine biosynthesis are related. Moreover, we isolated a Tn 5 mutant (374-05) that is simultaneously impaired in SA and pseudomonine production.
Plant roots are associated with a diverse microbial community that consists of antagonistic, commensal and mutualistic organisms. While beneficial plant-microbe interactions are important for plant performance, direct evidence for the evolution of rhizosphere mutualism remains elusive. Here we experimentally show that initially plant-antagonistic Pseudomonas protegens bacterium can rapidly evolve into a mutualist in the rhizosphere of Arabidopsis thaliana only within six plant growth cycles (6 months). Using an in vivo experimental evolution system, we observed that P. protegens rapidly diversified into distinct phenotypes, with some having clearly positive effects on the plant performance. Crucially, this evolution of plant facilitation was also accompanied with increased bacterial fitness indicative of a mutualistic interaction. At the genetic level, P. protegens mutualism was associated with mutations in the GacS/GacA two-component global regulator system, which is known to affect several aspects of bacterial physiology including carbon metabolism, host-microbe signalling, and downregulation of phytotoxic secondary metabolites. At the phenotypic level, the evolution of mutualism could be explained via two mechanisms: improved competition for root exudates and enhanced capacity for activating the root-specific transcription factor gene MYB72, which is essential for the production and excretion of the antimicrobial scopoletin for which the mutualists evolved enhanced tolerance. Together, these results show that plant-bacteria mutualism can rapidly evolve at an agriculturally relevant evolutionary timescale in the rhizosphere.
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