Differential Effects of Rare Specific Flavonoids on Compatible and Incompatible Strains in the Myrica gale - Frankia Actinorhizal Symbiosis

Abstract

Plant secondary metabolites, and specifically phenolics, play important roles when plants interact with their environment and can act as weapons or positive signals during biotic interactions. One such interaction, the establishment of mutualistic nitrogen-fixing symbioses, typically involves phenolic-based recognition mechanisms between host plants and bacterial symbionts during the early stages of interaction. While these mechanisms are well studied in the rhizobia-legume symbiosis, little is known about the role of plant phenolics in the symbiosis between actinorhizal plants and Frankia genus strains. In this study, the responsiveness of Frankia strains to plant phenolics was correlated with their symbiotic compatibility. We used Myrica gale, a host species with narrow symbiont specificity, and a set of compatible and noncompatible Frankia strains. M. gale fruit exudate phenolics were extracted, and 8 dominant molecules were purified and identified as flavonoids by high-resolution spectroscopic techniques. Total fruit exudates, along with two purified dihydrochalcone molecules, induced modifications of bacterial growth and nitrogen fixation according to the symbiotic specificity of strains, enhancing compatible strains and inhibiting incompatible ones. Candidate genes involved in these effects were identified by a global transcriptomic approach using ACN14a strain whole-genome microarrays. Fruit exudates induced differential expression of 22 genes involved mostly in oxidative stress response and drug resistance, along with the overexpression of a whiB transcriptional regulator. This work provides evidence for the involvement of plant secondary metabolites in determining symbiotic specificity and expands our understanding of the mechanisms, leading to the establishment of actinorhizal symbioses.Over the course of evolution, plants have developed many strategies to adapt to their environment and manage their biotic interactions. The production of secondary metabolites is an important tool in many of these strategies. In particular, secondary metabolites are involved in plant-microbe interactions both as weapons in plant defense mechanisms and as early signals in mutualistic as well as pathogenic relationships (22). For example, sesquiterpenes and flavonoids are involved in the early steps of arbuscular mycorrhizal symbiosis (1, 62). Similarly, in the early stages of the rhizobia-legume symbiosis, recognition of host flavonoids enhances rhizospheric competitiveness and root colonization and is the basic mechanism through which rhizobia induce the transcription of nod genes, leading to root hair deformation and nodulation in the plant tissues (13,49,55).Actinorhizal symbiosis is a less well-known nitrogen-fixing interaction, resulting from the association between the actinobacterium Frankia and plants belonging to eight dicotyledonous families collectively called "actinorhizal": Betulaceae, Myricaceae, Rosaceae, Datiscaceae, Elaeagnaceae, Coriariaceae, Casuarinaceae, and Rhamnaceae (8). Due to the nitrogen-fixing...

Casuarina Root Exudates Alter the Physiology, Surface Properties, and Plant Infectivity of Frankia sp. Strain CcI3

“…These results would suggest that the root exudates influence Frankia physiology by affecting cellular components beyond total protein levels. Our results with aqueous root exudates confirm previous studies on flavonoid effects on the growth of Frankia with methanol extracts or purchased phenolics (25,29,37). Although aqueous root exudates alone did not support Frankia growth, plant compounds in the root exudates enhanced the growth of Frankia.…”

“…The soil-and axenically grown C. cunninghamiana root exudates contained similar levels of phenolic (2.0 to 4.9 g/ml) and flavonoid (9.9 to 18.6 g/ml) compounds (data not shown). Flavonoid compounds have been identified from extracts of Alnus glutinosa, Myrica gale, and Casuarina glauca (1,29). Although flavonoid compounds were detected in the C. cunninghamiana root exudates, we did not want to exclude other possible plant-signaling molecules and used aqueous extracts throughout this study.…”

“…Although previous studies on root exudates have used solventextracted samples, aqueous root exudates were chosen for use in this study (1,13,29). Root exudates from plants grown under nitrogen-deficient and nitrogen-sufficient conditions were collected from both 2-month-old soil-grown plants and 3 week-, 1 month-, and 2-month-old axenically grown plants.…”

Flavonoids in plant rhizospheres: secretion, fate and their effects on biological communication

“…Among the flavonoid-like compounds extracted from red alder (Alnus rubra), one possible flavanone enhanced nodulation, whereas two other compounds were shown to act as inhibitors (Benoit and Berry 1997). To assess the roles of flavonoids in Frankia-actinorhizal symbiosis, eight flavonoid compounds were extracted from the fruits of Myrica gale, with two dihydrochacone compounds found to enhance the growth and nitrogen fixation of compatible, but not incompatible, Frankia strains (Popovici et al 2010). Transcriptome analysis revealed that genes involved in flavonoid synthesis and auxin transport were activated in the roots of C. glauca, suggesting the important roles of flavonoids and auxin during the Frankia infection process and nodule organogenesis (Hocher et al 2011).…”

Significant Natural Product Biosynthetic Potential of Actinorhizal Symbionts of the Genus Frankia, as Revealed by Comparative Genomic and Proteomic Analyses

“…43 Mb for the narrow-host-range Frankia strain CcI3 to 7.50 Mb for the medium-host-range Frankia strain ACN14a (ACN) to 8.98 Mb for the broad-host-range Frankia strain EAN1pec (EAN). Since the elucidation of these Frankia genomes, bioinformatic approaches have illuminated codon usage patterns (47), predicted secretosome profiles (35), and led to genomeguided studies on the Frankia transcriptome (3,44) and proteome (1,5,33,34). Genome mining also provides an opportunity to identify important physiology and metabolic functions, including secondary metabolism.…”