Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts

After a century of investigations, the function of the obligate betaproteobacterial endosymbionts accommodated in leaf nodules of tropical Rubiaceae remained enigmatic. We report that the α-d-glucose analogue (+)-streptol, systemically supplied by mature Ca.Burkholderia kirkii nodules to their Psychotria hosts, exhibits potent and selective root growth inhibiting activity. We provide compelling evidence that (+)-streptol specifically affects meristematic root cells transitioning to anisotropic elongation by disrupting cell wall organization in a mechanism of action that is distinct from canonical cellulose biosynthesis inhibitors. We observed no inhibitory or cytotoxic effects on organisms other than seed plants, further suggesting (+)-streptol as a bona fide allelochemical. We propose that the suppression of growth of plant competitors is a major driver of the formation and maintenance of the Psychotria–Burkholderia association. In addition to potential agricultural applications as a herbicidal agent, (+)-streptol might also prove useful to dissect plant cell and organ growth processes.

Dissecting Metabolism of Leaf Nodules in Ardisia crenata and Psychotria punctata

Root-microbe interaction and its specialized root nodule structures and functions are well studied. In contrast, leaf nodules harboring microbial endophytes in special glandular leaf structures have only recently gained increased interest as plant-microbe phyllosphere interactions. Here, we applied a comprehensive metabolomics platform in combination with natural product isolation and characterization to dissect leaf and leaf nodule metabolism and functions in Ardisia crenata (Primulaceae) and Psychotria punctata (Rubiaceae). The results indicate that abiotic stress resilience plays an important part within the leaf nodule symbiosis of both species. Both species showed metabolic signatures of enhanced nitrogen assimilation/dissimilation pattern and increased polyamine levels in nodules compared to leaf lamina tissue potentially involved in senescence processes and photosynthesis. Multiple links to cytokinin and REDOX-active pathways were found. Our results further demonstrate that secondary metabolite production by endophytes is a key feature of this symbiotic system. Multiple anhydromuropeptides (AhMP) and their derivatives were identified as highly characteristic biomarkers for nodulation within both species. A novel epicatechin derivative was structurally elucidated with NMR and shown to be enriched within the leaf nodules of A. crenata. This enrichment within nodulated tissues was also observed for catechin and other flavonoids indicating that flavonoid metabolism may play an important role for leaf nodule symbiosis of A. crenata. In contrast, pavettamine was only detected in P. punctata and showed no nodule specific enrichment but a developmental effect. Further natural products were detected, including three putative unknown depsipeptide structures in A. crenata leaf nodules. The analysis presents a first metabolomics reference data set for the intimate interaction of microbes and plants in leaf nodules, reveals novel metabolic processes of plant-microbe interaction as well as the potential of natural product discovery in these systems.

Proteomics Analysis of Psychotria Leaf Nodule Symbiosis: Improved Genome Annotation and Metabolic Predictions

Several plant species of the genus Psychotria (Rubiaceae) harbor Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is considered to be obligatory because plants devoid of symbionts fail to develop into mature individuals. The genome of ‘Candidatus Burkholderia kirkii’ has been sequenced recently and has revealed evidence of reductive genome evolution, as shown by the proliferation of insertion sequences and the presence of numerous pseudogenes. We employed shotgun proteomics to investigate the expression of ‘Ca. B. kirkii’ proteins in the leaf nodule. Drawing from this dataset and refined comparative genomics analyses, we designed a new pseudogene prediction algorithm and improved the genome annotation. We also found conclusive evidence that nodule bacteria allocate vast resources to synthesis of secondary metabolites, possibly of the C7N aminocyclitol family. Expression of a putative 2-epi-5-valiolone synthase, a key enzyme of the C7N aminocyclitol synthesis, is high in the nodule population but downregulated in bacteria residing in the shoot apex, suggesting that production of secondary metabolites is particularly important in the leaf nodule.

Identification of the bacterial endosymbionts in leaf nodules of Pavetta (Rubiaceae)

Three genera in the Rubiaceae (Pavetta, Psychotria and Sericanthe) harbour bacterial endosymbionts within leaf nodules or galls. The present paper identifies the bacterial endophytes in three leaf-nodulating Pavetta species. In order to reveal their identity and assess their phylogenetic position, 16S rRNA, recA and gyrB genes were sequenced from an extensive sampling of Burkholderia strains. This multigene approach results in a robust phylogeny, which places the bacterial endosymbionts of Pavetta at two distinct positions within the genus Burkholderia (class Betaproteobacteria), suggesting that leaf-nodulating endosymbionts within Pavetta have different origins. The endophytes of nodulated Psychotria species were recognized as the closest relatives to the Pavetta endosymbionts. Our results suggest that the endosymbionts of Pavetta represent novel species, which can be classified as ‘Candidatus Burkholderia hispidae’, ‘Candidatus Burkholderia rigidae’ and ‘Candidatus Burkholderia schumannianae’.